mbh _ 203 environmental microbiology - THE IMPRINT

PROF. BALASUBRAMANIAN SATHYAMURTHY 2015 EDITION MBH–203 ENVIRONMENTAL MICROBIOLOGY

Contact for your free pdf & job opportunities [email protected] or 8050673426 of 219

FOR MSC MICROBIOLOGY STUDENTS

2014 ONWARDS

Biochemistry scanner

THE IMPRINT MBH – 203 ENVIRONMENTAL MICROBIOLOGY (THEORY)

As per Bangalore University (CBCS) Syllabus

2014 Edition

BY: Prof. Balasubramanian Sathyamurthy

Supported By:

Ayesha Siddiqui

Kiran K.S.

THE MATERIALS FROM “THE IMPRINT (BIOCHEMISTRY SCANNER)” ARE NOT FOR COMMERCIAL OR BRAND BUILDING. HENCE ONLY ACADEMIC CONTENT WILL BE PRESENT INSIDE. WE THANK ALL THE CONTRIBUTORS FOR ENCOURAGING THIS.

BE GOOD – DO GOOD & HELP OTHERS



DEDICATION

I dedicate this material to my spiritual guru Shri Raghavendra swamigal,

parents, teachers, well wishers and students who always increase my

morale and confidence to share my knowledge to reach all beneficiaries.

PREFACE

Biochemistry scanner ‘THE IMPRINT’ consists of last ten years solved question paper of Bangalore University keeping in mind the syllabus and examination pattern of the University. The content taken from the reference books has been presented in a simple language for better understanding.

The Author Prof. Balasubramanian Sathyamurthy has 15 years of teaching experience and has taught in 5 Indian Universities including Bangalore University and more than 20 students has got university ranking under his guidance. THE IMPRINT is a genuine effort by the students to help their peers with their examinations with the strategy that has been successfully utilized by them. These final year M.Sc students have proven their mettle in university examinations and are College / University rank holders. This is truly for the students, by the students. We thank all the contributors for their valuable suggestion in bringing out this book. We hope this will be appreciated by the students and teachers alike. Suggestions are welcomed.

For any comments, queries, and suggestions and to get your free copy write

us at [email protected] or call 8050673426.



CONTRIBUTORS:

CHETAN ABBUR ANJALI TIWARI

AASHITA SINHA ASHWINI BELLATTI

BHARATH K CHAITHRA

GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE

KAMALA KISHORE

KIRAN KIRAN H.R

KRUTHI PRABAKAR KRUPA S

LATHA M MAMATA

MADHU PRAKASHHA G D MANJUNATH .B.P

NAYAB RASOOL S NAVYA KUCHARLAPATI

NEHA SHARIFF DIVYA DUBEY

NOOR AYESHA M PAYAL BANERJEE

POONAM PANCHAL PRAVEEN

PRAKASH K J M PRADEEP.R

PURSHOTHAM PUPPALA DEEPTHI

RAGHUNATH REDDY V RAMYA S

RAVI RESHMA

RUBY SHA SALMA H.

SHWETHA B S SHILPI CHOUBEY

SOUMOUNDA DAS SURENDRA N

THUMMALA MANOJ UDAYASHRE. B

DEEPIKA SHARMA

EDITION : 2015

PRINT : Bangalore

CONTACT : [email protected] or 8050673426



BANGALORE UNIVERSITY SYLLABUS (REVISED 2014) M.SC MICROBIOLOGY II SEMESTER

MBH – 203 ENVIRONMENTAL MICROBIOLOGY UNIT – 1 AEROBIOLOGY: (10 hrs) Air spora in different layers of atmosphere, bioaerosol, assessment of air

quality using air sampler based on principles of sedimentation, impaction,

impingement, suction and filtration. Brief account of transmission of airborne

microbes; Microbiology of indoor and outdoor. Allergy: Causes and tests for

detection of allergy. Endotoxin in air and its quantification and hazards.

Molecular methods of air quality assessment.

UNIT – 2 AQUATIC MICROBIOLOGY: (12 hrs) Fresh and marine ecosystem (estuaries, mangroves, deep sea, hydrothermal

vents, salt pans, coral reefs). Zonation of water ecosystem; upwelling,

eutrophication; food chain in aquatic ecosystems. Role of methanotrophs in

ecosystem. Potability of water, microbial assessment of water, water

purification. Ground water types and their contamination. Biofilm.

Waste treatment: sewage and effulent treatment; primary, secondary and

tertiary treatment, Solid waste treatment. Solid wastes as sources of energy

and food.

UNIT – 3 SOIL MICROBIOLOGY: (10 hrs) Biotic and abiotic interactions, concepts of habitat and niche. Microbial

communities; nature, structure and attributes, levels of species diversity,

succession and stability, r and k selection, genetic exchange between

communities. Biodiversity management and conservation. Role of microbes in

organic solid waste treatment matter in various soil types, subterranean

microbes. Biogeochemical cycles of carbon, nitrogen, phosphorous and

sulphur.

UNIT – 4 DIVERSITY IN ANOXIC ECO SYSTEM: (4 hrs) Methanogens – reduction of carbon monoxide – reduction of iron, sulphur,

manganes, nitrate and oxygen. Microbial transformations of Carbon,

Phosphorus, Sulphur, Nitrogen and Mercury.



UNIT – 5 EXTREMOPHILES: (6 hrs) The domain Archaea, acidophilic, alkalophilic, thermophilic, barophilic and

osmophilic and radiodurant microbes – mechanisms and adaptation.

Halophilic – membrane variation – electron transport – application of

thermophiles and extremophiles. Extremozyme.

UNIT – 6 ROLES OF MICROBES IN BIODEGRADATION: (10 hrs) Biodegradation of Xenobiotics, hydrocarbon, pesticides and plastics.

Biodeteroration of wood, pulp and paper. Biosorption/ bioaccumulation of

heavy metals. Bioremediation of soil, air, and water; various methods,

advantages and disadvantages. Bioleaching of iron, copper, gold and uranium

References: 1. Jagbir Singh. (2010). Solid Waste Management. I.K. International Publishing

House Ltd. New Delhi.

2. Volodymgr Ivanov. (2010). Environmental Microbiology for Engineers. CRC

Press. US

3. A.L. Bhatia. (2009). Textbook of Environmental Biology. I.K. International

Publishing House Ltd. New Delhi.

4. Atlas, R.M., (2005). Handbook of media for environmental microbiology. CRC

press.

5. Patrick, K. Jjemba. (2004). Environmental microbiology: principles and

applications. Sceince Publishers.

6. Christon J Hurst, Ronald L Crawford, Guy R Knudsen Michael J McInerney,

Linda D Stetzenbach, (2002). Manual of Environmental Microbiology 2nd

Edition. ASM press.

7. Francis H Chapelle, (2000), Ground Water Microbiology and Geochemistry. 2nd

Edition. John Wiley & Sons.

8. Robert L Tate, (2000), Soil Microbiology2nd Edition. John Wiley & Sons.



9. Gabriel Bitton, (1999), Waste Water Microbiology. 2nd Edition. Wiley-Liss.

10. Robert S. Burlage, Ronald Atlas, David Stahl, Gill Geesey, Gary Sayler (1998)

Techniques in Microbial Ecology. Oxford University Press. New York.

11. Christopher S Cox, Christopher M Wathes, (1995), Bioaerosols Handbook.

Lewis Publishers.

12. Ian L. Pepper, Charles P Gerba, Jeffrey W (1995), Environmental Microbiology:

A Laboratory Manual. Academic Press.

13. Baker K.H. and Herson, D.S. (1994), Bioremediation. Mc Graw Hill Inc., New

Yor



UNIT – 1 AEROBIOLOGY: Air spora in different layers of atmosphere, bioaerosol, assessment of air quality using air sampler based on principles of sedimentation, impaction, impingement, suction and filtration. Brief account of transmission of airborne microbes; Microbiology of indoor and outdoor. Allergy: Causes and tests for detection of allergy. Endotoxin in air and its quantification and hazards. Molecular methods of air quality assessment.

AEROBIOLOGY Aerobiology has been defined as the study of aerosolization, aerial

transmission, and deposition of biological materials. AIR SPORA IN DIFFERENT LAYERS OF ATMOSPHERE, BIOAEROSOL

A collection of airborne biological particles is called a bioaerosol. Bioaerosols

are generated by a wide variety of natural and human-made processes

including coughing, sneezing, wave action, splashes, wind, cooling towers,

ventilation systems, etc. Inhalation, ingestion, and dermal contact are routes of

human exposure to airborne microorganisms, but inhalation is the

predominant route that results in adverse human health effects. Airborne

Legionella pneumophila, Mycobacterium tuberculosis, and some pathogenic

viruses are known to be transmitted by aerosols. Asthma, hypersensitivity

pneumonitis and other respiratory illnesses are also associated with exposure

to bioaerosols.

Deterioration of building materials, offensive odors, and adverse human health

effects are associated with microbial contamination of indoor environments,

such as residences, offices, schools, health care facilities, enclosed agricultural

structures (barns and crop storage areas) industrial facilities and recycling

facilities.

Sources: There are two basic sources of bioaerosol: Natural,

Related to human activities



Natural sources are mainly soil and water, from which microorganisms are

being lifted up by the movement of air, and from organisms such as fungi, that

produce gigantic amounts of spores that are dispersed by the wind. Therefore,

there are always a given number of microorganisms in the air, as a natural

background. It is estimated, that the air is considered to be clean, if the

concentration of bacteria and fungi cells does not exceed 1000/m3 and

3000/m3 respectively. This latter statement is only true when the

concentration of microorganisms consists of saprophytic organisms, not

pathogenic organisms. If the concentration of microorganisms in the air

exceeds the above values, or contains microorganisms dangerous to humans,

then such air is considered to be microbiologically polluted. From the hygienic point of view, living sources of bioaerosols related to

human activity, are more important than the natural sources. The emissions

from these sources are dangerous due to the following two reasons:

They may distribute pathogenic microorganisms,

They often cause a high increase of microorganisms in the air, significantly

exceeding the natural background.

The emission sources of biological aerosols can have a localized character (e.g.

aeration tank) or a surface character (e.g. sewage-irrigated field).

The most important sources of bioaerosol emission are:

Agriculture and farming-food industry,

Sewage treatment plants,

Waste management.

Agriculture and food/farming industry This is the biggest source of bioaerosol emission, which results from the

intensification of methods of farm production. Aerosols are created during

almost all types of agricultural work, e.g. harvest, transport, storing, plant and

animal material processing, and in animal breeding facilities.

The significant dangers to human health are brought about by a vast number

of microorganisms, products of their decomposition and of organic dust, which

have an allergic and toxic affect. The presence of infectious microbes is less



significant here. The most important components of the above-mentioned

aerosols are:

Mold fungus, Aspergillus, Penicillium and Cladosporium, Alternaria.

Gram-negative chromatobars, mainly of the genus Erwinia,

Thermophilic acitnomycetes,

Dust of biological origin (among other things: particles of skin, feathers,

droppings, plant dust).

Contact with such aerosols may often bring about chronic diseases of the

respiratory system, e.g. allergic inflammation of pulmonary alveoli (Alveolitis

allergica). It is said, that there is an exceptional health risk, when over 50% of

the aerosol belongs to the respirable fraction, and bioaerosol's concentration

exceeds 105 cfu/m3. The above quantity is often exceeded a hundred times (e.g.

in pigsties, broiler houses, granaries).

Wastewater treatment The size of bioaerosol emission depends on - among other factors - the make-

up of sewage, sewage-treatment-plant flow capacity, as well as the methods

and types of the equipment. Favourable conditions for bioaerosol formation are

at the time of sewage spouting, aeration, mixing and dispersing. The quality of

the air-microflora make-up is closely connected to the content of the treated

sewage.



In mechanical-biological sewage treatment plants, which utilize the activated-

sludge method, the biggest emission of microorganisms can be observed in the

mechanical section, where raw sewage is introduced (drainage area, grill, sand

trap) as well as in the vicinity of aeration tanks and sludge drying beds.

Moreover, a strong emission may be also observed during sewage purification

in soil and in sprinkling beds. Significant air pollution may occur in the areas

stated above, which may even exceed the level of background concentration.

The following are some of the characteristic microorganisms present in

bioaerosol and sewage:

Intestinal bacteria (Enterobacteriaceae), including those of the coli group,

Salmonella, Shigella,

Hemolysis bacteria, mainly streptococcus and staphylococcus,

Pseudomonas bacteria,

Yeast such as Candida albicans and Cryptococcus,

Dermatophites (surface-mycosis inducing fungus) of Microsporum and

Trichophyton genus,

Protozoa,

Eggs of worms,

Intestinal viruses: enteroviruses and reoviruses.

From the stated microorganisms, the intestinal bacteria and viruses are most

specific to sewage bioaerosols, and usually don't occur in the down-wind side

of the plant.

Consequently, they are considered to be indicator microorganisms that are

helpful in determining the effect of the sewage-treatment plant upon the

surrounding environment.

In addition, the air in the plant's facility also contains endotoxins, in some

cases at concentrations exceeding the maximum limit.

Bioaerosols that originate in biological sewage-treatment plants usually do not

disperse more than a few hundred meters; actually, the pollution level is

already much lower 50 meters away from the source of the emission. Thus,



they are dangerous, mainly to those, who are directly within the facility. Blood

tests of workers who are subject to aerosol inhalation indicated an increased

level of antibodies of gram-negative bacteria and intestinal viruses. The

condition has been described as 'the sewage worker's syndrome', which has a

viral origin and manifests itself with a despondency, overall weakness, catarrh

and fever. Moreover, sewage workers and those who live in the vicinity of a

treatment plant have higher morbidity with intestinal and respiratory system

illnesses.

Waste management Various forms of waste management are additional sources of bioaerosol

emission:

Waste storage and Composting.

Waste storage The air around storage yards contains bacteria found commonly in nature and

saprophytic fungi of soil and water origin, some of which are opportunistic

pathogens. It means that under favourable conditions (weakening of the

defence system, penetrating the body in large numbers) they may invoke

various diseases in humans. The following are the dominating genera of

bacteria: Bacillus, Pseudomonas, Enterobacter. The last two genera are Gram-

negative bacteria which produce endotoxins and their presence is often

observed around waste dumps. In close proximity to dumps, the concentration

of microorganisms often exceeds 105/m3 .

It is believed that within dump sites and similar communal facilities (e.g.

composting site), the total number of bacteria in air should not exceed 104/m3,

and of Gram-negative bacteria 103/m3.

Thermophilic (flourish in temperatures at 37oC) and keratinolytic (breaking

down keratin) fungi are favorable indicator microorganisms of a dump’s impact

upon the surroundings.



Waste dumps provide suitable conditions for development of these types of

fungi due to increased humidity, temperature and numerous particles

containing keratin – proteins which are difficult to decompose found among

other places, in hair, feathers, and claws.

The thermophilic species of mold fungus (Alternaria alternata, Mucor pusillus,

and Aspergillus ochraceus) and yeast-like fungi (Candida sp. and Geotrichum

candidum) are quite common. They may induce allergies and produce

mycotoxins (especially molds) as well as cause deep mycosis (e.g. in respiratory

system).

Keratinolytic fungi are typical soil microorganisms that include numerous

dermatophytes (mycosis inducing fungus, e.g. Microsporum racemosum). The

ability to assimilate keratin allows them to penetrate the skin layers and hair.

The range of bioaerosols spread by waste storage facilities is usually greater

than that of sewage-treatment plants and may often exceed 1000 meters.

Composting Composting also emits large amounts of microorganisms - especially bacteria.

Particularly large air pollution is created during waste sorting, when the

concentration of bacteria often exceeds the limit of 105 CFU/m3.



Among these, there are Gram-negative bacteria which are potentially harmful

to humans. Due to high temperatures (65-70oC) of the composting process

most of the above bacteria usually get neutralized, however their endotoxins

demonstrate a certain degree of thermostability thus, when released into air,

they can cause poisoning.

The common mold fungus, Aspergillus fumigates, whose spore concentration in

air can equal 106/m3, is a good indicator of the effect of the composting

facilities on its surroundings. The above species is a typical opportunistic

pathogen which, among other diseases, causes aspergillosis and allergic

diseases of the respiratory system (e.g. Alveolitis allergica).

ASSESSMENT OF AIR QUALITY USING AIR SAMPLER There are three principal methods used to quantify microorganisms in the air.

Impaction is the forced deposition of airborne particles on a solid surface

Impingement is the trapping of airborne practices in a liquid matrix

Filtration is the trapping of airborne particles by size exclusion BASED ON PRINCIPLES OF SEDIMENTATION

Gravity is a non-quantitative method used in which agar medium is exposed to

the environment and airborne microorganisms are collected primarily by

settling. This method is often used because it is inexpensive and easily

performed.



BASED ON PRINCIPLES OF IMPACTION The impaction method separates particles from the air by utilizing the inertia of

the particles to force their deposition onto a solid or semi-solid surface. The

collection surface is usually an agar medium. The Anderson sixstage impact or

sampler (Anderson Instruments Inc., Smyra, GA) consist of six stages with

decreasing nozzle diameters, so that successive stages collect progressively

smaller particles. Thus the six-stage sampler measures the cultivable

bioaerosol concentrations in specific particle size ranges. BASED ON PRINCIPLES OF IMPINGEMENT

A commonly used liquid impinger is the AGI-30 (ACE Glass, Vineland, NJ). The

AGI-30 operates by drawing air through the inlet and into a liquid.Any particles

in the air become trapped in the liquid, which can then be assayed for the

presence of microorganisms.The AGI-30 is usually operated at a flow rate of

12.5 liters per minutes. The AGI-30 is easy to use, inexpensive, portable,

reliable, easily sterilized, and has high biological sampling efficiency in

comparison with many other sampling devices. The usual collection volume is

20 ml, and the typical sampling time is about 20 minutes. Longer sampling

times result in too much evaporation of the liquid in the impinger, and the

inactivation or death of microorganisms in the liquid.

BASED ON PRINCIPLES OF SUCTION AND FILTRATION



Filtration techniques are used largely for the collection of fungi and bacterial

spores because they are desiccation resistant. Filters are usually held in

disposable (although they may be reused) plastic filter cassettes during

bioaerosol sampling. Membrane filters used for sampling are usually supplied

as disks of 37- or 47mm diameter. Because the pressure drop across a filter

increases with air velocity through the filter, the use of larger filters results in a

lower pressure drop for a given volumetric flow rate. The use of the smaller (37-

mm) filter concentrates the organisms onto a smaller total area, thus

increasing the density of particles per unit area of the filter.

This may be helpful for direct microscopic examination of low concentrations of

organisms. In areas of high concentration, the organisms may have to be

eluted, diluted, and then refiltered for microscopic examination or assay. For a

better quantitative measure of the air volume sampled, a limiting orifice may be

placed between the cassette and the vacuum source.

Procedure: First period: Materials



All glass impinger AGI-30

1 37-mm air monitoring cassette

20 ml of 0.1% peptone solution

1 500- or 1000-ml Erlenmeyer flask

Rubber or plastic tubing for connecting the impinger and cassette to the

vacuum source

1 vacuum pump or vacuum source

1 100 ml sterile graduated cylinder

1 dilution blank with 0.1% peptone or phosphate buffered saline

2 1-ml pipettes

1 10-ml pipette

4 sterile 0.45m pore, 47-mm diameter membrane filters

1 filter unit (same as in Experiment 10)

2 sterile 37mm 0.45mm pore filters

Vacuum pump or other source

Forceps

Gas burner

Pipette bulb

Vortex mixer

4 nutrient agar (NA)1 or trypticase soy agar (TSA)1 plates

4 Sabouraud dextrose agar (SDA)1 plates.

Air Sampling by Impingement 1. Set up the AGI-30 all glass impinger as shown (Figure 23-2).

2. Add 20ml of 0.1% peptone to the reservoir followed by 0.1ml of anti-foam.

3. Add 0.1 ml of anti-foam agent.

4. Turn the vacuum source on for 10 minutes.

5. With a 1-ml pipette remove 0.5 ml of fluid from the reservoir and place 0.1 ml

each on one agar plate of either NA or TSA and spread plate the samples. Place

another 0.1 ml on a plate of Sabouraud dextrose agar for detection of fungi.

6. With a 10 ml pipette remove 6 ml of liquid from the reservoir and pass 5 ml

through a 0.45mm membrane filter. Place the filter on an NA or TSA plate.



Repeat the procedure, but place the membrane filter on Sabouraud dextrose

agar.

7. Incubate the NA or TSA plates at 35°C for 24–48 hours.

8. Incubate the SDA for 2 to 7 days.

Air Sampling by Filtration 9. Connect the air-sampling cassette to the vacuum source.

10. Turn on the vacuum source for 10 minutes.

11. Remove the membrane filter from the cassette with a pair of flamed forceps (see

Experiment 10). And place on either a plate of NA or TSA.

12. Repeat the same procedure placing the membrane filter on plate of SDA.

13. Incubate the plates as described under impingement.

Second Period Materials incubated plates from the previous Period 1

Examine the agar plates and count the umber of bacteria (NA or TSA) and fungi

(SDA) colonies.

Calculations Calculate the number of bacteria and fungi per cubic meter.The AGI-30

limiting orifice at the end of the glass tube, which is submerged into the

collection liquid, limits the amount of air passing through the liquid to 12.5

liters per minute. The concentration of microorganisms is usually reported as

numbers per cubic meter of air, which is calculated as follows.



BRIEF ACCOUNT OF TRANSMISSION OF AIRBORNE MICROBES - MICROBIOLOGY OF INDOOR AND OUTDOOR

Bioaerosols may carry microorganisms other than those which evoke

respiratory system diseases. The intestinal microorganisms contained in

aerosols may, after settling down, get into the digestive system (e.g. by hands)

causing various intestinal illnesses.

Infectious airborne diseases The mucous membrane of the respiratory system is a specific type of a

'gateway' for most airborne pathogenic microorganisms. Susceptibility to

infections is increased by dust and gaseous air-pollution, e.g. SO2 reacts with

water that is present in the respiratory system, creating H2SO4, which irritates

the layer of mucous. Consequently, in areas of heavy air pollution, especially

during smog, there is an increased rate of respiratory diseases. Bioaerosols

may, among other things, carry microbes that penetrate organs via the

respiratory system. After settling, microbes from the air may find their way

onto the skin or, carried by hands, get into the digestive system (from there,

carried by blood, to other systems, e.g. the nervous system). Fungi that cause

skin infections, intestinal bacteria that cause digestive system diseases or

nervous system attacking enteroviruses are all examples of the above.

Viral diseases After penetrating the respiratory system with inhaled air, particles of viruses

reproduce inside the cuticle cells of both the upper and lower respiratory

system. After reproduction some of the viruses stay inside the respiratory

system causing various ailments (runny nose, colds, bronchitis, pneumonia),

whereas others leave the respiratory system to attack other organs (e.g.

chickenpox viruses attack the skin). The most noteworthy viruses are:

Influenza (orthomyxoviruses)

Influenza, measles, bronchitis, mumps and pneumonia among newborns

(paramyxoviruses)

German measles (similar to paramyxoviruses)

Colds (rhinoviruses and koronaviruses)



Cowpox and true pox (pox type viruses)

Chickenpox (cold sore group of viruses)

Foot-and-mouth disease (picorna type viruses)

Meningitis, pleurodynia (enteroviruses)

Sore throat, pneumonia (adenoviruses)

Bacterial diseases Similarly to viruses, some bacteria that find their way to the respiratory system

may also cause ailments of other systems. Especially staphylococcus infections

assume various clinical forms (bone marrow inflammation, skin necrosis,

intestinal inflammation, pneumonia). Often, a susceptible base for development

of various bacterial diseases is first prepared by viral diseases, e.g.

staphylococcus pneumonia is usually preceded by a flu or mumps. Bacterial

airborne diseases include:

Tuberculosis (Mycobacterium tuberculosis),

Pneumonia (staphylococcus, pneumococci, Streptococcus pneumoniae, less

frequently chromatobars of Klebsiella pneumoniae),

Angina, scarlet fever, laryngitis (streptococcus),

Inflammation of upper and lower respiratory system and meningitis

(Haemophilus influenzae),

Whooping cough (chromatobars of Bordetella pertussis),

Diphtheria (Corynebacterium diphtheriae),

Legionnaires disease (chromatobars of Legionella genus, among others

L.pneumophila),

Nocardiosis (oxygen actinomycetes of Nocardia genus).

Fungal diseases Many potentially pathogenic airborne fungi or the so-called saprophytes live in

soil. They usually have an ability to break down keratin (keratinolysis) -

difficult to decompose proteins found in horny skin formations, e.g. human or

animal hair, feathers, claws. Some of the keratinolytic fungi, the so-called

dermatophytes, cause mycosis of the outer skin (dermatosis), as the break



down of keratin enables them to penetrate the epidermis. Other fungi, after

penetrating the respiratory system, cause deep mycosis (organ), e.g. attacking

lungs. The following are examples of airborne fungi diseases:

Mycosis (Microsporum racemosum),

Deep mycosis: aspergillosis (Aspergillus fumigatus), cryptococcus (Cryptococcus

neoformans).

Protozoan diseases Some protozoa, which are able to produce cysts that are resistant to

dehydration and solar radiation, may also infect humans by inhalation. The

most common example of the above is: Pneumocystis carinii which causes

pneumonia.

Dangers connected with pathogenic bioaerosols do not concern only human

diseases.

Other significant diseases are those that attack cultivated plants or farm

animals. The following are examples of the above:

Blight - grain disease caused by Puccinia graminis, and

Aphthous fever - very infectious disease that attacks artiodactylous animals.

ALLERGY Allergy is a changed, hypersensitive reaction of the person or animal to some

substances called allergens (gr. allos - other, ergon - action). CAUSES

Actually, it's an immunologic reaction, in which a needless production of

antibodies by B lymphocytes (mainly IgE and IgG immunoglobulins) occurs as

a hypersensitive response to penetration of antigens (called the allergen).

Excessively produced immunoglobulins combine with allergens, which cause

among other things:

A release of various compounds (e.g. histamines) from mast cells.

The released compounds induce inflamed reactions in the form of bronchus

asthma or hay fever,



Cause damaged tissue at the place of contact, - allergic pulmonary alveoli

inflammation (e.g. the so-called farmer's lung, or mushroom breeder's lung).

Many microbes exist as allergens. Besides these, there are other allergenic

factors such as anemophilous pollens (e.g. grass, nettle, comose), small

arachnids (mites) as well as biological dust (e.g. particles of feathers, hair or

droppings). Microorganisms differ in their allergenic influences. The strongest

allergens are mold fungi, thermophilus actinomycetes, as well as Gram-

negative chromatobars. The strength of allergenic bioaerosols depends not only

on the type of microorganisms but also on their A type of allergic reaction

induced by biological aerosols depends on the type of allergens that cause it as

well as, to a large extent, the size of its particles as it determines the degree of

penetration into the respiratory system:

Particles larger than 10μm, held in the nasal cavity, cause hay fever (e.g. fungi

spores of Alternaria, grass pollen)

Particles of diameter between 4-10μm, held in bronchi, cause asthma (e.g.

fungi spores of Cladosporium)

Particles larger than 4μm that penetrate alveoli, besides asthma induce allergic

inflammation of pulmonary alveoli (fungi spores of Aspergillus and Penicillium).

TESTS FOR DETECTION OF ALLERGY

Endotoxins Detecting endotoxins includes the following stages:



Air filtration through a membrane filter (made of glass fibre or polyvinyl

chloride),

Reactions of a series of dilutions of filtered cells with a Limulus lysate blood

preparation with an addition of a chromogenic substance,

Measurement of the luminescence formed.

Limulus Polyphemus is a marine arthropod related to arachnids, which lives of

the shores of North America. It is best known for its peculiar immunologic

system. Its blood cells (amebocyte), after coming into contact with cell walls of

Gram-negative bacteria (containing endotoxins) release an enzyme that causes

coagulation of particular blood proteins and formation of a clot that

immobilizes the bacteria.

The activation of the coagulation-by-endotoxin enzyme is a very sensitive

reaction.

Produced on a large scale Limulus Amebocyte Lysate (LAL) with the addition of

a chromogenic substance that demonstrates luminescence in cases of clot

formation (in the presence of endotoxins) is widely used in air testing. The

measurement of luminescence allows the determination of the amount of

endotoxins in the air that is being tested.

Other toxins and allergens Their detection often requires meticulous testing and is based on:

Immunologic reactions that use antibodies used against well known Antigens

(e.g. allergens),

Chromatographic testing (e.g. mycotoxins).

ENDOTOXIN IN AIR AND ITS QUANTIFICATION AND HAZARDS Poisoning/intoxication are caused by toxins that are produced by some

microorganisms.

Endotoxins and mycotoxins are the most significant types of toxins in polluted

air.

Endotoxins are the components of Gram-negative bacterial cell walls (A lipid

fragment of lipopolysaccharides LPS outer membrane).



They demonstrate toxic (and allergenic) effects on mammals. After being

inhaled into the lungs, they cause acute inflammation of the lungs.

Mycotoxins are produced by various mold fungi. The most common ones are

aflatoxins produced by Aspergillus flavus. These compounds (there are several

types of them) demonstrate strong toxic, mutagenic, carcinogenic, teratogenic

(cause malformation in a fetus) actions. Most often they lead to food

poisonings, however it has also been indicated, that inhaling dusts which

contain aflatoxins may bring about tumours of the liver and the respiratory

system. MOLECULAR METHODS OF AIR QUALITY ASSESSMENT.

Evaluation of air pollution includes both quantity and quality aspects, and

depends on the type of air evaluated. Different criteria are used for atmospheric

air and air inside various rooms. The values of safe concentration vary from

author to author.

According to the norms assumed in Poland the atmospheric air is clean when

the concentration of bacterial cells does not exceed 1000 cfu/m3 - fungi 3000

cfu/m3. Of course only when the organisms are saprophytic not pathogenic.

Inside a building the total number of bacteria should not exceed 2000 cfu/m3 -

or fungi 300 cfu/m3. When the concentration of microbes exceeds the above

norms, or when the aerosol contains harmful microbes, then such air is

considered to be microbiologically polluted.



Norms for other rooms depend on its pre-determined use, e.g. surgery room

may not contain any fungus and the number of bacteria cannot exceed 100

cfu/m3, whereas in a pigsty it is 200 000cfu/m3 of bacteria and 10 000

cfu/m3 of fungi.

Very important from a hygienic point of view is the knowledge about the

particle size distribution of the bioaerosol. The greater the proportion of small

particles in the bioaerosol, which can enter the alveoli (size about 1μm), the

greater the health hazard of the air, even if there are no microorganisms

causing infectious diseases in the aerosol. The inhalation of such air may

cause allergy, poisoning and dust diseases.

Qualitative examinations perforce must be limited to indicator microorganisms,

as the identification of pathogenic microbes is usually strenuous and

expensive. The indicator species need not be pathogenic, but their occurrence

points indirectly to a potential threat due to disease-causing microorganisms.

The following indicator microorganisms are used for microbial analysis of the

air:

Hemolytic staphylococci,

Mannitol fermenting and not-fermenting staphylococci,

Actinomycete bacteria,

Pseudomonas fluorescens.

Staphylococci are one of the most common bacteria in nature. They are not all

pathogenic; many of them appear on human skin and the mucous membranes

and do not cause diseases. Pathogenic staphylococci show a high metabolic

activity, which can be used to differentiate them from the non-pathogenic ones.

Pathogenic staphylococci cause:

Hemolysis of red blood cells (erythrocytes) on blood agar medium,

Acid fermentation of mannitol on mannitol salt agar medium (Chapman

medium).

The hemolysis consists of the destruction of erythrocytes by certain toxins

produced by bacteria, which results in the formation of a characteristic zone of

clearing around the colony.



The Chapman medium contains 10% NaCl, which ensures that mainly

staphylococci grow on this medium (they are resistant to high concentrations of

salt). The presence of mannitol in the medium is used to differentiate the

mannitol-fermenting staphylococci from the non-fermenting ones.

Determination of haemolysis and of the fermentation of mannitol increases the

probability that staphylococci detected are pathogenic.

Actinomycetes are typical soil bacteria. Their presence in the air can point to

the soil environment as the source of the pollution. Pseudomonas fluorescens is

a common water bacterium. Its presence in the air can point to the water

environment as the source of pollution.

In addition to the investigation of the microbiological air pollution emitter, one

uses typical species for this emission source.This can determine its impact on

the state of pollution of the air – the occurrence of the indicator

microorganisms will mark the border of the impact zone.



UNIT – 2 AQUATIC MICROBIOLOGY Fresh and marine ecosystem (estuaries, mangroves, deep sea, hydrothermal vents, salt pans, coral reefs). Zonation of water ecosystem; upwelling, eutrophication; food chain in aquatic ecosystems. Role of methanotrophs in ecosystem. Potability of water, microbial assessment of water, water purification. Ground water types and their contamination. Biofilm. Waste treatment: sewage and effulent treatment; primary, secondary and tertiary treatment, Solid waste treatment. Solid wastes as sources of energy and food.

FRESH AND MARINE ECOSYSTEM (ESTUARIES, MANGROVES, DEEP SEA, HYDROTHERMAL VENTS, SALT PANS, CORAL REEFS)

Ecology is the scientific study of relationships in the natural world. It includes

relationships between organisms and their physical environments

(physiological ecology); between organisms of the same species (population

ecology); between organisms of different species (community ecology); and

between organisms and the fluxes of matter and energy through biological

systems (ecosystem ecology).

Ecologists study these interactions in order to understand the abundance and

diversity of life within Earth's ecosystems—in other words, why there are so

many plants and animals, and why there are so many different types of plants

and animals. To answer these questions they may use field measurements,

such as counting and observing the behavior of species in their habitats;

laboratory experiments that analyze processes such as predation rates in

controlled settings; or field experiments, such as testing how plants grow in

their natural setting but with different levels of light, water, and other inputs.

Applied ecology uses information about these relationships to address issues

such as developing effective vaccination strategies, managing fisheries without

over-harvesting, designing land and marine conservation reserves for

threatened species, and modeling how natural ecosystems may respond to

global climate change.



Change is a constant process in ecosystems, driven by natural forces that

include climate shifts, species movement, and ecological succession. By

learning how ecosystems function, we can improve our ability to predict how

they will respond to changes in the environment. But since living organisms in

ecosystems are connected in complex relationships, it is not always easy to

anticipatehow a step such as introducing a new species will affect the rest of an

ecosystem.

Major Terrestrial and Aquatic Biomes Geography has a profound impact on ecosystems because global circulation

patterns and climate zones set basic physical conditions for the organisms that

inhabit a given area. The most important factors are temperature ranges,

moisture availability, light, and nutrient availability, which together determine

what types of life are most likely to flourish in specific regions and what

environmental challenges they will face.

Earth is divided into distinct climate zones that are created by global

circulation patterns. The tropics are the warmest, wettest regions of the globe,

while subtropical high-pressure zones create dry zones at about 30° latitude



north and south. Temperatures and precipitation are lowest at the poles. These

conditions create biomes— broad geographic zones whose plants and animals

are adapted to different climate patterns. Since temperature and precipitation

vary by latitude, Earth's major terrestrial biomes are broad zones that stretch

around the globe.

Each biome contains many ecosystems (smaller communities) made up of

organisms adapted for life in their specific settings. Land biomes are typically

named for their characteristic types of vegetation, which in turn influence what

kinds of animals will live there. Soil characteristics also vary from one biome to

another, depending on local climate and geology. compares some key

characteristics of three of the forest biomes.

Aquatic biomes (marine and freshwater) cover three-quarters of the Earth's

surface and include rivers, lakes, coral reefs, estuaries, and open ocean.

Oceans account for almost all of this area. Large bodies of water (oceans and

lakes) are stratified into layers: surface waters are warmest and contain most of

the available light, but depend on mixing to bring up nutrients from deeper

levels. The distribution of temperature, light, and nutrients set broad

conditions for life in aquatic biomes in much the same way that climate and

soils do for land biomes. Marine and freshwater biomes change daily or

seasonally. For example, in the intertidal zone where the oceans and land meet,

areas are submerged and exposed as the tide moves in and out. During the

winter months lakes and ponds can freeze over, and wetlands that are covered

with water in late winter and spring can dry out during the summer months.

There are important differences between marine and freshwater biomes. The

oceans occupy large continuous areas, while freshwater habitats vary in size



from small ponds to lakes covering thousands of square kilometers. As a result,

organisms that live in isolated and temporary freshwater environments must

be adapted to a wide range of conditions and able to disperse between habitats

when their conditions change or disappear.

Since biomes represent consistent sets of conditions for life, they will support

similar kinds of organisms wherever they exist, although the species in the

communities in different places may not be taxonomically related. For example,

large areas of Africa, Australia, South America, and India are covered by

savannas (grasslands with scattered trees). The various grasses, shrubs, and

trees that grow on savannas all are generally adapted to hot climates with

distinct rainy and dry seasons and periodic fires, although they may also have

characteristics that make them well-suited to specific conditions in the areas

where they appear.

ESTUARIES Estuaries. Estuaries are places where fresh and salt water mix. Typically they

occur where rivers enter the sea. EsteroLimantour, Drakes’Bay, Point Reyes

National Seashore.

Circulation and stratification in estuaries

Estuary circulation is governed by density stratification mainly driven by salt

concentration not temperature. The specific gravity of seawater is about 1.025.



In other words seawater weighs about 2.5% more than an equivalent volume of

fresh water. If you go back to the graph of water density as a function of

temperature in the introductory aquaticlecture, you’ll see that this is quite a bit

greater density difference than obtains due to temperature stratification. On

the other hand estuaries are often tidal and shallow, and river currents are

often strong. Hence salt and fresh water are often mixed in estuaries despite

strong density stratification. Left, salt wedge estuaries often occur at the

mouths of very strongly flowing rivers like the Mississippi and the Amazon. In

salt wedge estuaries most mixing takes place far from land. Deep estuaries (e.g.

fiords) have enough “shear”across the density gradient to mix a fair amount of

salt water into the fresher upper layer. This accelerates the upper flow and

hence the mixing. 0 =salinity of fresh water ~ 0 ppt. So = salinity ocean water ~

35 ppt.

Partially mixed estuaries Perhaps the commonest type. Most river valleys did not cut down that deeply in

the Pleistocene as sea levels were only ~ 100m lower than today.Plus, much

sediment has usually accumulated over the last 11,000 years. Fiords are an

exception because glaciers cut many of their valleys to depths well below

Pleistocene sea levels. In shallow estuaries,the “tidal prism”(The volume of

water that flows in and out of the estuary on the tidal cycle) creates strong

currents in the saltier layer because the volume of water in the tidal prism is

confined to a shallow layer. The tidal prism is influenced by tide amplitude and

area, not so much by depth of the water, especially depth below the low tide

line. Much salty water is mixed into the fresher surface water.

Partially mixed estuaries are nutrient traps. Nutrients enter with the river and

salt water. Sinking particles from the fresher surface layer drop into the saltier

subsurface flow that carries them towards them back of the estuary. The deep

salty current then mixes into the upper layer laden with mineralized plant

nutrients. Thus estuaries are generally very productive.



Bar Built Estuaries When the coastal ocean is shallow, longshorecurrents often deposit long linear

sand islands that dam up a considerable area of water seaward of the drowned

river valleys. Cape Hatteras, North Carolina.

Columbia River Estuary Estuary flows are on a large enough scale to be influenced by Coriolis’Force.

The incoming salty current hugs the south bank of the Columbia while the

fresher surface current hugs the north bank. See block diagram below. With

tilted isolinesof equal salinity.

Negative estuaries In arid areas “negative estuaries”may form. If evaporation exceeds freshwater

input, the back of the estuary becomes a source of dense water saltier than

seawater. Now seawater enters at the surface and saltier water from the back of

the estuary flows out below. The Mediterranean Sea is a giant negative estuary.

What do you suppose the consequences of this mixing pattern are for primary

production?

During WWII German and Italian submarines used these currents tosneak past

the British naval base at Gibraltar. The excellent submarine flick DasBoot’s

obligatory depth charging scenes take place during the sub’s exit from the



Mediterranean in the deeper saltier current. The idea was to cut power entirely

and drift silently through the Strait in the appropriate current.

Estuaries are tough environments Organisms in estuaries are subject to tremendous osmotic stress. Organisms

adapted to fresh water have relatively low salt concentrations in their body

fluids. When immersed in salt water, the greater osmotic potential of seawater

sucks water out of them until their tissues become saltier. Marine organisms

immersed in fresh water draw fresh water into their tissues. Some organisms

can regulate their osmotic state by using powerful kidneys to excrete salt or

water as needed to maintain osmotic homeostasis. The

anadromoussalmonidfishes are an example. See middle and bottom panels at

right. Not only do estuaries have regular tidal fluctuations in salinity, but the

have salinity crises during floods (when normally salty reaches of the estuary

become very fresh) and droughts (when salty ocean water penetrates far back

into the estuary). In the top panel notice that diversity tends to be quite low in

estuaries, reaching a minimum where estuary specialist brackish water species

are most numerous. Note that estuaries also tend to be isolated from one

another by long stretches of ocean. This makes them very like islands. They are

very vulnerable to invasion by weedy pests from other estuaries.

While diversity is low, high productivity leads to large populations of a few

species of fish and shellfish. Fishers seek finfish, crabs, clams, and scallops.



Many bivalves like Scallops are filter feeder that pump water over surfaces

covered with mucous to trap an eat plankton and detritus. Since they are low

on the food chain, the productivity of this population can be large.

Many marine animals have complex life cycles with different stages adapted to

different ecological circumstances. Our commercially important Dungeness

crab’s post-larval stage user productive estuaries up and down the coast as

nurseries

MANGROVES

Mangroves are a diverse group of unrelated trees, palms, shrubs, vines and

ferns that share a common ability to live in waterlogged saline soils subjected

to regular flooding. They are highly specialised plants that have developed

unusual adaptations to the unique environmental conditions in which they are

found.

There are around 80 species of mangroves found throughout the world. Most

commonly they occur within tropical and subtropical sheltered coastal areas

subjected to tidal influences. An area influenced by tide can be interpreted to

mean a shoreline inundated by the extremes of tides, or it can more widely

refer to river-bank communities where tides cause some fluctuation in water

level but no change in salinity.

Therefore, mangroves can be found not only inhabiting extensive tidal mud

flats but also along freshwater riverbanks. Mangroves can be divided into two



distinct groups: exclusive and non-exclusive. Exclusive mangroves are the

largest group, comprising around 60 species (Saenger et al., 1983). These

mangroves are confined to intertidal areas and have not been found to exist

within any other type of vegetation community. The remaining 20 plant species

considered to be mangroves are referred to as non-exclusive. These plants are

not restricted to the typical mangrove environment and are often found within

drier, more terrestrial areas. Examples include Hibiscus tiliaceus and

Barringtonia acutangula.

Mangrove Distribution and Requirements Mangroves are commonly found throughout the world between latitudes 32°N

and 38°S. The upper and lower limits of this range are determined by

temperature, while rainfall and the level of protection from wind and wave

energy effect forest extent and diversity.

Temperature Mangrove communities most commonly occur in areas where the average

temperature of the coldest month is higher than 20°C and where the seasonal

range does not exceed 10°C. Temperatures of around 5°C and frosts also limit

mangrove distributions.

Rainfall Areas, which have a great variety of mangrove species, are found along coasts

that receive high rainfall, heavy run off and seepage into the intertidal zone

from the hinterland. Such areas are commonly subject to extensive

sedimentation, which provides a diverse range of substrate types and nutrient

levels, which in turn are favourable for mangrove growth.

Protection Mangrove establishment requires protection from strong winds and wind

generated waves, as wave action prevents seedling establishment. As a

consequence, mangrove communities tend to be located within sheltered

coastal areas, surrounding highly

indented estuaries, embayments and offshore islands protected by reefs and

shoals.



Essential Functions of Mangroves Ecological Values From an ecological perspective, mangroves are a unique and significant

ecosystem. They support a diverse range of plants including palms, trees,

shrubs and even ferns, which have developed unusual adaptations to the

prevailing environmental conditions. In fact these plants have been so

successful in their development that mangroves are among the most productive

natural systems found throughout the world.

Productivity Productivity is a concept used to describe the ecological value or function of a

vegetation community. Notably, productivity can be estimated by gaining

measurements of the amount of living material (ie. leaves, branches, stems and

roots) that is produced by a mangrove community over a specified time.

Mangrove productivity is important because it has direct impact on the health

and function of the marine food chain.

Like other plants, mangroves convert energy from the sun into organic matter

through the process of photosynthesis. When the leaves and branches of a

mangrove fall to the ground they provide a wide variety of aquatic animals such

as molluscs,

crabs and worms with a primary source of food. These primary level consumers

in turn support an array of secondary consumers, including small fish and

juvenile predators such as barramundi which, when mature, become third level

consumers.

In general, high levels of organic matter, or high productivity, means that a

larger number and more diverse array of animals can be supported within a

particular ecosystem.

Measuring mangrove productivity is not easy, and will probably never be

achieved in absolute terms. It is, however, possible to measure changes in a

particular element of a mangrove community, which can then be used as a

guide to productivity.

Leaf Litter



Many studies have used the collection and measurement of leaf litter fallen

from a mangrove area over a particular period of time as one such guide.

Habitat Mangroves also provide important permanent and temporary habitats for a

large number and range of marine and terrestrial fauna. Marine fauna

commonly found in mangroves includes molluscs, crustaceans (such as crabs

and prawns), a wide range of fish and of course, the saltwater crocodile.

Economic Values Commercial Fishing

Foreshore Protection Mangroves play an important role in coastal protection by acting as a natural

buffer to water erosion from both the land and the sea. DEEP SEA

For almost all of its vast extent, the deep sea is a very dark and cold

environment. Just how deep is the “deep sea”? It is incredibly deep; the

Mariana Trench is about 11,000 meters (about 36,000 feet) deep, 2000 meters

deeper than Mount Everest is tall!

Although about half of the Earth’s surface lies 3,000 or more meters below

water, the deep ocean is a fairly new area of study for scientists. In 1977, the

ALVIN, a specially designed submersible, first entered this area of permanent

darkness. ALVIN can dive to 4,000 meters (about 13,000 feet). For a point of

reference, large submarines dive to about 1,000 meters (about 3,280 feet),

while scuba divers have gone to a record depth of 133 meters (about 436 feet).

For an idea of what these depths mean, consider this: The TITANIC is under

about 4,000 meters of water. In order to explore the TITANIC, ALVIN had to

descend for about two and a half hours! The pressure of the water is so great at

depths below 1,000 meters that it would crush a regular submarine. All of the

animals that live in the deep sea must contend with these incredible pressures.

Beside being a “high pressure” environment, the deep sea is a very dark and

very cold environment. (The deep sea thermal vents found along areas of



extreme geologic activity are an exception to this statement with regard to

temperature.)

Most of the life in the ocean, then, is ultimately dependent on sunlight.

Phytoplankton and algae capture solar energy and then are food for

zooplankton, fish and so many other animals of the sea.

It would seem, then, that because of the lack of light, there would be no

organisms to begin food chains, no source of energy and no flow of nutrients.

This is almost true, no plants live in the deep sea. Without sunlight for

phytoplankton or seaweeds, it seemed there could be no life at all in the deep

sea. Scientists long thought of these regions as dark and lifeless. This made

sense to everybody...except that every once in awhile, fishermen trawling for

their catch or oceanographers on research vessels would dredge up bizarre fish

from deep waters. What could these animals be eating?

The answer is found nearer the surface. When surface organisms die, their

bodies decompose. The resulting decayed organic material, called detritus (dee-

try-tus), sinks. One can see this falling detritus in videos filmed on

submersibles exploring the deep sea. It looks so much like falling snow that

biologists call it “marine snow”. Detritus is like “slightly used food”. There’s still

some energy and some nutrients stored in that marine snow. The animals that

live in the dark waters of the deep ocean make a living feeding on the marine

snow.

Adapted to the dark, pressure, cold, and scarcity of food of the deep sea

environment, many of these animals look as if they could be in a science-fiction

movie. At first, people thought the strange appearances of the fish might be a

result of bringing them to the surface; the fish never survived the drastic

temperature and pressure changes of the trip up from the depths. Now, with

the development of submersibles, biologists have visited these creatures in

their own habitat and brought back footage of bizarre beings that inhabit the

deep, dark, cold waters.

In particular, deep sea fish have caught our imagination. Though the deep sea

fish tend to look fierce, most, but not all, are quite small. Their fierce



appearance comes from adaptations that allow them to eat anything that

comes their way in the sparsely populated waters. Many have sharp fang-like

teeth to hold prey. Many have greatly expandable stomachs and jaws that

unhinge to allow them to swallow prey larger than themselves. Some have

photophores, pockets of bioluminescent bacteria that they may use as fishing

lures to attract prey close to their hungry mouths. For example, the

lanternfish, which has rows of phosphorescent spots along its sides, looks like

a miniature ocean liner with all its portholes illuminated.

Photophores may also function as identification tags so fish can find others of

their species in order to reproduce. Special adaptations for reproduction must

be important in an unlighted sea where fellow fish may be few and far between.

The male anglerfish, for example, is much smaller than the female, and

attaches to the female, literally feeding off her blood. In return, he provides

sperm to fertilize her eggs.

The anglerfish also has a unique way of catching its prey. Attached to its head

just above the mouth, an anglerfish has a thread-like line with a light at the

end of it. The light dangles like a bit of shining bait. The anglerfish swims with

its mouth open, ready to snap up any small fish that is attracted to the shining

“bait”. The scarcity of food makes energy conservation a high priority for deep

sea fish. Since large fish require correspondingly large amounts of energy to

survive, most of the fish are small. Also, most of the deep sea fish are passive

hunters, waiting for a meal to swim by; active hunting consumes more energy. HYDROTHERMAL VENTS

Hydrothermal vents occur at ocean spreading centers, that is, at locations

where tectonic plates are pulling apart, creating new ocean floor as volcanic

material rises to fill in the space between the plates. At spreading centers ocean

water infiltrates the ocean floor and mixes with molten crust, after which

hydrothermal fluids rise back to the surface of the sea floor. As hydrothermal

fluids return to the ocean floor, they exit through narrow chimneys known as

white or black smokers (Metaxas, 2003). Exiting fluids range in tem peratures

between 300-400 degrees C and are rich in hydrogen sulfide, heavy metals,



and other elements. The high temperatures of vent fluids cause them to be

more buoyant than ocean water. As hydrothermal fluids escape into ocean

waters, they form buoyant plumes that rapidly mix with ambient seawater. The

plume rises until the fluids mix sufficiently to reach a state of neutral

buoyancy. At this point the plume spreads out horizontally: ocean currents

then dictate further mixing and movement.

The benthic zone surrounding hydrothermal vents is an extremely variable

environment. Within this area, vent fluids and oceanic waters mix. These two

water types possess very different physical and chemical properties.

Consequently, temperature and chemical gradients form within vent

environments. Small distances can make a big difference in the characteristics

of the water experienced by organisms that live near vents. As currents shift,

water properties can change dramatically in a matter of minutes or seconds.

Toxic substances precipitate from vent fluids. Living in such a unique physical

and chemical environment can require a considerable amount of adaptability.

Chemosynthesis The extremely productive nature of hydrothermal vent communities puzzled

scientists at first. Only certain organisms, known as primary producers, can

process energy from strictly inorganic sources. These primary producers

provide the organic compounds that other organisms need for growth and

energy. Sunlight usually provides the energy for the production of organic

compounds from inorganic compounds. Before hydrothermal vents were

discovered, prevailing opinion held that deep-sea communities relied on the

slow fallout of organic mater from the ocean’s surface. At the surface sufficient

sunlight penetrates to allow photosynthesis to occur, but even in the clearest

waters there is not enough light to fuel photosynthesis much below 100-200

meters deep.

Ecologists looked to photosynthetic processes as the ultimate source of energy

that fueled aquatic food webs. However, most of the organic compounds from

surface waters are consumed before they can penetrate to deep ocean waters.

Consequently, photosynthetic fallout could not account for the level of biomass



observed at venting sites. An alternative explanation was needed to account for

hydrothermal community productivity. Instead of relying on photosynthesis,

the vent food chain is built on the basis of chemosynthesis.

Hydrothermal vents have taught us that chemosynthetic microorganisms can

serve as primary producers without the aid of sunlight. At vents and methane

seeps, high concentrations of hydrogen sulfide or methane can provide

microorganisms with the chemical energy to synthesize organic compounds.

While scientists had recorded chemosynthetic processes as early as 1887, it

took the discovery of hydrothermal vents to highlight how important this

process could be. A particularly intriguing aspect of chemosynthesis at

hydrothermal vents is the symbiosis that exists between bacteria and some

vent organisms. A range of vent organisms, including tubeworms, mussels, and

clams, host symbiotic bacteria inside their bodies. Symbiotic bacteria living

within an organism are known as endosymbionts. Episymbionts, or symbiotic

bacteria living on the exterior of animals, are found on hydrothermal

polychaete worms and shrimp.

Organism adaptations to vent environments For the mussels or giant tubeworms living on an actively venting chimney,

environmental conditions vary dramatically from those experienced by

organisms just outside the range of the vent’s influence. Precipitating heavy

metals and other toxic substances can literally rain down on nearby animals.

Hydrogen sulfide, an essential component for the chemosynthetic processes

that provide energy for vent communities, can have effects similar to cyanide

on organisms not adapted to functioning at the high concentrations found at

vent sites. Thermally adapted bacteria and archaea may live at temperatures in

excess of 100 degrees C. It can take a great deal of adaptation to live in close

proximity to hydrothermal vents. Many of the 500 + animals that have so far

been discovered at vent sites seem to live exclusively in vent communities.

SALT PANS Saline and alkaline soils are commonly known as salt pans. For the purpose of

this report they are defined as sites with saline (conductivity >400 μS) or



alkaline (pH >7.0) soil horizons, or supporting flora and/or fauna known to be

halophytic. They formerly covered more than 40 000 ha of semi-arid land in the

Maniototo basin, mid-Manuherikia Valley, and upper Clutha Valley.

Mining, cultivation and irrigation have reduced the area of these soils to less

than 100 ha. There are about 30 sites remaining in Central Otago (McIntosh et

al. 1990, 1992). Although they have not been cultivated, all have been grazed,

most have also been modified by influences such as erosion and application of

fertiliser, and some by irrigation. Most are dominated by exotic plant species,

and some have no native plant species at all.

Of the few native plants that are found on inland saline soils, almost all also

occur in salt marsh and related communities on the coast, for example

Sarcocornia quinqueflora, Apium prostratum, Selliera cf. microphylla (col.) and

Atriplex buchananii. The exception is Lepidium kirkii, small cress considered

endangered and known only from about 200 plants at six localities in Central

Otago.

CORAL REEFS “Coral” is a general term for several different groups of cnidarians, only some

of which help build reefs.

In reef-building, or hermatypic, corals the polyps produce calcium carbonate

skeletons. Billions of these tiny skeletons build a massive reef. The most

important reef builders are a group known as scleractinian corals, sometimes

called the stony or “true” corals. Nearly all reef-building corals contain

symbiotic zooxanthellae that help the corals make their calcium carbonate

skeletons. Corals can produce their skeletons without zooxanthellae but only

very slowly, not nearly fast enough to build a reef. It is the zooxanthellae as

much as the corals themselves that construct the reef framework, and without

zooxanthellae there would be no reefs. Corals that do not help build reefs, or

ahermatypic corals, often lack zooxanthellae.

The Coral Polyp: You have to look closely to see the little polyps that build coral reefs. Coral

polyps are not only small but deceptively simple in appearance. They look



much like little sea anemones, consisting of an upright cylinder of tissue with a

ring of tentacles on top.

Like anemones and other cnidarians, they use their nematocyst-armed

tentacles to capture food, especially zooplankton. The tentacles surround the

mouth, the only opening to the sac-like gut.



Most reef-building corals are colonies of many polyps, all connected by a thin

sheet of tissue. The colony starts when a planktonic coral larva, called a

planula, settles on a hard surface. Coral larvae generally do not settle on soft

bottoms. Immediately after settling, the larva transforms, or metamorphoses, into a polyp. This single “founder” polyp, if it survives, divides over and over to

form the colony. The digestive systems of the polyps usually remain connected,

and they share a common nervous system.

A few reef-building corals consist of only a single polyp (Fig.).

Coral polyps lie in a cup-like skeleton of calcium carbonate that they make

themselves. The polyps continually lay down new layers of calcium carbonate,

building up the skeleton beneath them so that it grows upward and outward.

The skeleton forms nearly all of the bulk of the colony and can take many

different shapes.The actual living tissue is only a thin layer on the surface. It is

the calcareous coral skeletons that form the framework of the reef.

Coral Nutrition

Zooxanthellae nourish the host coral as well as help it deposit its skeleton.

They perform photosynthesis and pass some of the organic matter they make



on to the coral. Thus, the zooxanthellae feed the coral from the inside. Many

corals can survive and grow without eating, as long as the zooxanthellae have

enough light. Although corals get much of their nutrition from their

zooxanthellae, most eat when they get the chance. They prey voraciously on

zooplankton. The billions of coral polyps on a reef, along with all the other

hungry reef organisms, are very efficient at removing zooplankton brought in

by currents. Indeed, the reef has been called a “wall of mouths.” Coral polyps

catch zooplankton with their tentacles or in sheets of mucus that they secrete

on the colony surface. Tiny, hair-like cilia gather the mucus into threads and

pass them along to the mouth. Some corals hardly use their tentacles and rely

on the mucus method.

A few have even lost their tentacles altogether. Corals have still other ways of

feeding themselves. There are a number of long, coiled tubes called

mesenterial filaments attached to the wall of the gut.

The mesenterial filaments secrete digestive enzymes. The polyp can extrude the

filaments through the mouth or body wall to digest and absorb food particles

outside the body. Corals also use the mesenterial filaments to digest organic

matter from the sediments. In addition, corals can absorb dissolved organic matter (DOM)

ZONATION OF WATER ECOSYSTEM An aquatic ecosystem is an ecosystem in a body of water.

Communities of organisms that are dependent on each other and on their

environment live in aquatic ecosystems. The two main types of aquatic

ecosystems are marine ecosystems and freshwater ecosystems.

Marine Marine ecosystems cover approximately 71% of the Earth's surface and contain

approximately 97% of the planet's water. They generate 32% of the world's

net primary production. They are distinguished from freshwater ecosystems by

the presence of dissolved compounds, especially salts, in the water.

Approximately 85% of the dissolved materials

in seawater are sodium andchlorine. Seawater has an average salinity of



35 parts per thousand (ppt) of water. Actual salinity varies among different

marine ecosystems.

A classification of marine habitats. Marine ecosystems can be divided into many zones depending upon water

depth and shoreline features. The oceanic zone is the vast open part of the

ocean where animals such as whales, sharks, and tuna live. The benthic zone

consists of substrates below water where many invertebrates live.

The intertidal zone is the area between high and low tides; in this figure it is

termed the littoral zone. Other near-shore (neritic) zones can

include estuaries, salt marshes, coral reefs, lagoons and mangrove swamps. In

the deep water, hydrothermal vents may occur where chemosynthetic sulfur

bacteria form the base of the food web.

Classes of organisms found in marine ecosystems include brown algae,

dinoflagellates, corals, cephalopods, echinoderms, and sharks. Fishes caught

in marine ecosystems are the biggest source of commercial foods obtained from

wild populations.

Environmental problems concerning marine ecosystems include unsustainable

exploitation of marine resources (for example overfishing of certain

species), marine pollution, climate change, and building on coastal areas

Freshwater Freshwater ecosystems cover 0.80% of the Earth's surface and inhabit 0.009%

of its total water. They generate nearly 3% of its net primary



production. Freshwater ecosystems contain 41% of the world's known fish

species.

There are three basic types of freshwater ecosystems:

Lentic: slow moving water, including pools, ponds, and lakes.

Lotic: faster moving water, for example streams and rivers.

Wetlands: areas where the soil is saturated or inundated for at least part of the

time

Lentic Lake ecosystems can be divided into zones. One common system divides lakes

into three zones (see figure). The first, the littoral zone, is the shallow zone near

the shore. This is where rooted wetland plants occur. The offshore is divided

into two further zones, an open water zone and a deep water zone. In the open

water zone (or photic zone) sunlight supports photosynthetic algae, and the

species that feed upon them. In the deep water zone, sunlight is not available

and the food web is based on detritus entering from the littoral and photic

zones. Some systems use other names. The off shore areas may be called the

pelagic zone, and the aphotic zone may be called the profundal zone. Inland

from the littoral zone one can also frequently identify a riparian zone which has

plants still affected by the presence of the lake—this can include effects from

windfalls, spring flooding, and winter ice damage. The production of the lake as

a whole is the result of production from plants growing in the littoral zone,

combined with production from plankton growing in the open water.

Wetlands can be part of the lentic system, as they form naturally along most

lakeshores, the width of the wetland and littoral zone being dependent upon

the slope of the shoreline and the amount of natural change in water levels,

within and among years. Often dead trees accumulate in this zone, either from

windfalls on the shore or logs transported to the site during floods. This woody

debris provides important habitat for fish and nesting birds, as well as

protecting shorelines from erosion,

Two important subclasses of lakes are ponds, which typically are small lakes

that intergrade with wetlands, and water reservoirs. Over long periods of time,



lakes, or bays within them, may gradually become enriched by nutrients and

slowly fill in with organic sediments, a process called succession. When

humans use the watershed, the volumes of sediment entering the lake can

accelerate this process. The addition of sediments and nutrients to a lake is

known as eutrophication.

Ponds Ponds are small bodies of freshwater with shallow and still water, marsh,

and aquatic plants. They can be further divided into four zones: vegetation

zone, open water, bottom mud and surface film. The size and depth of ponds

often varies greatly with the time of year; many ponds are produced by spring

flooding from rivers. Food webs are based both on free-floating algae and

uponaquatic plants. There is usually a diverse array of aquatic life, with a few

examples including algae, snails, fish, beetles, water bugs, frogs, turtles, otters

and muskrats. Top predators may include large fish, herons, or alligators.

Since fish are a major predator upon amphibian larvae, ponds that dry up each

year, thereby killing resident fish, provide important refugia for amphibian

breeding. Ponds that dry up completely each year are often known as vernal

pools. Some ponds are produced by animal activity, including alligator holes

and beaver ponds, and these add important diversity to landscapes.

Lotic

The major zones in river ecosystems are determined by the river bed's gradient

or by the velocity of the current. Faster moving turbulent water typically

contains greater concentrations of dissolved oxygen, which supports greater

biodiversity than the slow moving water of pools. These distinctions form the

basis for the division of rivers into upland and lowland rivers. The food base of

streams within riparian forests is mostly derived from the trees, but wider

streams and those that lack a canopy derive the majority of their food base

from algae. Anadromous fish are also an important source of nutrients.

Environmental threats to rivers include loss of water, dams, chemical pollution

and introduced species. A dam produces negative effects that continue down

the watershed. The most important negative effects are the reduction of spring



flooding, which damages wetlands, and the retention of sediment, which leads

to loss of deltaic wetlands.

Wetlands Wetlands are dominated by vascular plants that have adapted to saturated

soil. There are four main types of wetlands: swamp, marsh, fen and bog (both

fens and bogs are types of mire). Wetlands are the most productive natural

ecosystems in the world because of the proximity of water and soil. Hence they

support large numbers of plant and animal species. Due to their productivity,

wetlands are often converted into dry land with dykes and drains and used for

agricultural purposes. The construction of dykes, and dams, has negative

consequences for individual wetlands and entire watersheds. Their closeness to

lakes and rivers means that they are often developed for human

settlement. Once settlements are constructed and protected by dykes, the

settlements then become vulnerable to land subsidence and ever increasing

risk of flooding. The Louisiana coast around New Orleans is a well-known

example;[8] the Danube Delta in Europe is another.

Functions Aquatic ecosystems perform many important environmental functions. For

example, they recycle nutrients, purify water, attenuate floods, recharge

ground water and provide habitats for wildlife. Aquatic ecosystems are also

used for human recreation, and are very important to the tourism industry,

especially in coastal regions. The health of an aquatic ecosystem is degraded

when the ecosystem's ability to absorb a stress has been exceeded. A stress on



an aquatic ecosystem can be a result of physical, chemical or biological

alterations of the environment. Physical alterations include changes in water

temperature, water flow and light availability. Chemical alterations include

changes in the loading rates of biostimulatory nutrients, oxygen consuming

materials, and toxins. Biological alterations include over-harvesting of

commercial species and the introduction of exotic species. Human populations

can impose excessive stresses on aquatic ecosystems. There are many

examples of excessive stresses with negative consequences.

UPWELLING Upwelling is an oceanographic phenomenon that involves wind-driven motion

of dense, cooler, and usually nutrient-rich water towards the ocean surface,

replacing the warmer, usually nutrient-depleted surface water. The nutrient-

rich upwelled water stimulates the growth and reproduction of primary

producers such as phytoplankton. Due to the biomass of phytoplankton and

presence of cool water in these regions, upwelling zones can be identified by

cool sea surface temperatures (SST) and high concentrations of chlorophyll-a

The increased availability in upwelling regions results in high levels of primary

productivity and thus fishery production. Approximately 25% of the total

global marine fish catches come from five upwellings that occupy only 5% of

the total ocean area. Upwellings that are driven by coastal currents or diverging

open ocean have the greatest impact on nutrient-enriched waters and global

fishery yields.

The major upwellings in the ocean are associated with the divergence of

currents that bring deeper, colder, nutrient rich waters to the surface. There

are at least five types of upwelling: coastal upwelling, large-scale wind-driven

upwelling in the ocean interior, upwelling associated with eddies,

topographically-associated upwelling, and broad-diffusive upwelling in the

ocean interior.

Coastal Coastal upwelling is the best known type of upwelling, and the most closely

related to human activities as it supports some of the most



productivefisheries in the world. Wind-driven currents are diverted to the right

of the winds in the Northern Hemisphere and to the left in the Southern

Hemisphere due to the Coriolis Effect. The result is a net movement of surface

water at right angles to the direction of the wind, known as the Ekman

transport. When Ekman transport is occurring away from the coast, surface

waters moving away are replaced by deeper, colder, and denser water.Normally,

this upwelling process occurs at a rate of about 5–10 meters per day, but the

rate and proximity of upwelling to the coast can be changed due to the strength

and distance of the wind.

Deep waters are rich in nutrients, including nitrate, phosphate and silicic acid,

themselves the result of decomposition of sinking organic matter (dead/detrital

plankton) from surface waters. When brought to the surface, these nutrients

are utilized by phytoplankton, along with dissolved CO2(carbon dioxide) and

light energy from the sun, to produce organic compounds, through the process

of photosynthesis. Upwelling regions therefore result in very high levels of

primary production (the amount of carbon fixed by phytoplankton) in

comparison to other areas of the ocean. They account for about 50% of global

marine productivity. High primary production propagates up the food

chain because phytoplanktons are at the base of the oceanic food chain.

The food chain follows the course of:

Phytoplankton → Zooplankton → Predatory zooplankton → Filter feeders →

Predatory fish → Marine birds, marine mammals

Equatorial Upwelling at the equator is associated with the Intertropical Convergence

Zone (ITCZ) which actually moves, and consequently, is often located north or

south of the equator. Easterly (westward) trade winds blow from the Northeast

and Southeast and converge along the equator blowing west to form the ITCZ.

Although there are no Coriolis forces present along the equator, upwelling still

occurs just north and south of the equator. This results in a divergence, with

denser, nutrient-rich water being upwelled from below, and results in the



remarkable fact that the equatorial region in the Pacific can be detected from

space as a broad line of high phytoplankton concentration.

Southern Ocean Large-scale upwelling is also found in the Southern Ocean. Here, strong

westerly (eastward) winds blow around Antarctica, driving a significant flow of

water northwards. This is actually a type of coastal upwelling. Since there are

no continents in a band of open latitudes between South America and the tip of

the Antarctic Peninsula, some of this water is drawn up from great depths. In

many numerical models and observational syntheses, the Southern Ocean

upwelling represents the primary means by which deep dense water is brought

to the surface. Shallower, wind-driven upwelling is also found in off the west

coasts of North and South America, northwest and southwest Africa, and

southwest Australia, all associated with oceanic subtropical high pressure

circulations (see coastal upwelling above).

Some models of the ocean circulation suggest that broad-scale upwelling

occurs in the tropics, as pressure driven flows converge water toward the low

latitudes where it is diffusively warmed from above. The required diffusion

coefficients, however, appear to be larger than are observed in the real ocean.

Nonetheless, some diffusive upwelling does probably occur.

Carbon Cycling in the Ocean Environment Microorganisms in the oceans can influence global carbon cycling and ocean-

atmosphere interactions. Most carbon processing occurs in the surface water

zone, with particulate organic carbon (POC), dissolved organic carbon (DOC),

and methane hydrate (in sediments) being major carbon pools. The ocean also

contains bicarbonate and dissolved CO2 (diss. CO2) that come from the

atmosphere and the degradation of organic carbon. Methane hydrate allows

microorganisms and associated animals such as ice worms to develop.



EUTROPHICATION

Eutrophication is the increase in the rate of production of carbon or the

accumulation of carbon in an aquatic ecosystem. Eutrophication is a term that

describes a complex of unfavourable symptoms connected with over-

fertilization. Urban sewage contains phosphates from human excrements,

washing detergents and liquids, food waste, food additives and other products.

Another significant source of phosphate pollution of water is sewage from the

agricultural industry. The presence of phosphorus in sewage introduced into

water along with nitrates and nitric dioxides causes increased development of

algae in both lotic and lentic waters. Increased eutrophication has been



considered to be hazardous to water reservoirs as a consequence of

uncontrollable growth of plant biomass.

The source of the increased organic carbon may come from within the system

(autochthonous) or from outside the system (allochthonous). Knowing the

sources and mechanisms of carbon accumulation is critical in developing

management strategies to reverse the process. Most lakes follow a path of

eutrophication over geologic time in transition from oligotrophic systems to

more eutrophic systems. When the addition of organic matter (e.g., from soil

erosion or increased inputs of nutrients from natural weathering of soils or

from human activities) proceeds at an increasing rate, the process of

eutrophication accelerates.

Eutrophication is not a trophic state but a process. A key element of

eutrophication is change. Upwelling systems cycle through phases of increased

nutrient availability, high primary and secondary productivity, and often,

oxygen depletion in the lower water column. The trophic status of upwelling

systems would be considered eutrophic [an organic carbon supply of 300 to 500

g of carbon per square meter per year, as defined by Nixon (1995)], but

upwelling systems are not undergoing eutrophication.

The causes of eutrophication should not be confused with the process itself.

The causes may include changes in physical characteristics of the system such

as changes in hydrology, changes in biological interactions such as reduced

grazing, or an increase in the input of organic and inorganic nutrients.

Although the causes may include direct natural or anthropogenic carbon

enrichment, eutrophication in the twentieth and twenty-first centuries is more

often caused by excess nutrients that would otherwise limit the growth of

phytoplankton. A variety of responses, such as noxious algal blooms, fish kills,

oxygen depletion, or seagrass habitat losses, should also not be confused with

the process of eutrophication.

The responses are multiple and may result in “increases” or “decreases” in

components of aquatic ecosystems, to which humans often ascribe beneficial or

detrimental values. More subtle responses of aquatic ecosystems to



eutrophication include shifts in phytoplankton and zooplankton communities,

shifts in the food webs that they support loss of biodiversity, changes in trophic

interactions, and changes in ecosystem functions and biogeochemical

processes.

FOOD CHAIN IN AQUATIC ECOSYSTEMS

ROLE OF METHANOTROPHS IN ECOSYSTEM

Chlorinated aliphatic hydrocarbons (e.g., trichloroethylene, dichloroethylene) in

aquifers undergo reductive dehalogenation under anaerobic conditions.

Under aerobic conditions, these compounds are degraded by methane-utilizing

bacteria called methanotrophs. These bacteria use methane as the sole source

of energy and as a major source of carbon. They can transform more than 50

percent of trichloroethene (TCE) into CO2 and bacterial biomass. High

conversion rates of TCE are obtained with methanotrophs with Vmax up to 290

nmol/min/mg of cells of Methylosinus trichosporium.

Potential pathways leading to complete detoxification of PCE in wetland environments, which typically contain anaerobic bulk soils and aerated zones within the rhizosphere. Certain strains within the genera listed can transform individual chlorinated ethenes metabolically via



dehalorespiration or, in the case of Polaromonas, via oxidation. Some methanotrophs can oxidize lesser chlorinated ethenes co-metabolically.

A methanotrophic biofilm reactor was shown to be capable of degrading TCE

and TCA in a continuous-flow operation for a period of 6 months. The

maximum degradation rate for TCE was 400 mg/L.h. The TCE was ultimately

converted to CO2 and CO by a microbial consortium comprised of a

metanotroph (Methylocystis sp.) and heterotrophic bacteria. The methanotroph

converts TCE to glycoxylic, dichloroacetic, and trichloroacetic acids, while

heterotrophic bacteria carry out the biotransformation to CO2 and CO.

A methanotrophic bacterium, isolated from groundwater, degraded

cometabolically TCE in the presence of methane or methanol used as primary

substrates. TCE can also be transformed to TCE epoxide by methane

monooxygenase produced by bacteria. The epoxide then breaks down

spontaneously to dichloroacetic acid and glycoxilic acid. The activity of the

soluble (i.e., nonmembrane-bound) methane monooxygenase (sMMO) is

associated with TCE biodegradation by pure cultures of methanotrophs

(Methylosinus trichosporium, Methylomonas methanica), and by microbial



isolates from groundwater. The synthesis of sMMO is stimulated only under

copper stress and is suppressed at copper concentration as low as 0.25 mM.

Mutants capable of producing methane monooxygenase in the presence of high

levels of copper (≤12 mM copper) have been isolated (Phelps et al., 1992). Genes

encoding subunits of this enzyme have been cloned and used as probes for the

detection of Methylosinus in bioreactors and other environmental samples.

Biodegradation of TCE in groundwater can also be enhanced by using phenol-

utilizing microorganisms.

A field demonstration of in situ biorestoration of an aquifer was undertaken at

Moffett Naval Air Station. Aquifer indigenous methanotrophic bacteria,

stimulated by addition of oxygen and methane, were able to metabolize

chlorinated aliphatic solvents such as TCE, cis- and trans-1, 2-dichloroethene

(DCE), and vinyl chloride (VC) under in situ conditions. The extent of

biotransformation was 20 percent for TCE, 40 percent for cis-DCE, 85 percent

for trans-DCE, and 95 percent for VC. Two field demonstration tests in the

Savannah River site, based on the in situ enhancement of methanotrophic

bacteria, showed the successful removal of TCE from aquifers.

Another field study using methane injection was conducted in Japan to clean

up groundwater contaminated with 220 mg/L of TCE. This experiment resulted

in a stimulation of methanotrophs from 10 - 104 cells/mL and in 10–20 percent

removal of TCE. Similar results were obtained by Semprini et al. (1990) as

regards in situ biostimulation using methane at a site with 36–97 mg/L of

TCE. POTABILITY OF WATER: MICROBIAL ASSESSMENT OF WATER

Potable water is water which is fit for consumption by humans and other

animals. It is also called drinking water, in a reference to its intended

use. Water may be naturally potable, as is the case with pristine springs, or it

may need to be treated in order to be safe. In either instance, the safety

of water is assessed with tests which look for potentially harmful

contaminants.



MICROBIAL ASSESSMENT OF WATER Monitoring and detection of indicator and disease-causing microorganisms are

a major part of sanitary microbiology. Bacteria from the intestinal tract

generally do not survive in the aquatic environment, are under physiological

stress, and gradually lose their ability to form colonies on differential and

selective media. Their die-out rate depends on the water temperature, the

effects of sunlight, the populations of other bacteria present, and the chemical

composition of the water. Procedures have been developed to attempt to

“resuscitate” these stressed coliforms before they are identified using selective

and differential media.

A wide range of viral, bacterial, and protozoan diseases result from the

contamination of water with human fecal wastes. Although many of these

pathogens can be

detected directly, environmental microbiologists have generally used indicator organisms as an index of possible water contamination by human pathogens.

Researchers are still searching for the “ideal” indicator organism to use in

sanitary microbiology. The following are among the suggested criteria for such

an indicator:

The indicator bacterium should be suitable for the analysis of all types of

water: tap river, ground, impounded, recreational, estuary, sea, and waste.

The indicator bacterium should be present whenever enteric pathogens are

present.

The indicator bacterium should survive longer than the hardiest enteric

pathogen.

The indicator bacterium should not reproduce in the contaminated water and

produce an inflated value.

The assay procedure for the indicator should have great specificity; in other

words, other bacteria should not give positive results. In addition, the

procedure should have high sensitivity and detect low levels of the indicator.

The testing method should be easy to perform.

The indicator should be harmless to humans.



The level of the indicator bacterium in contaminated water should have some

direct relationship to the degree of fecal pollution.

Coliforms, including Escherichia coli, are members of the family

Enterobacteriaceae. These bacteria make up approximately 10% of the

intestinal microorganisms of humans and other animals and have found

widespread use as indicator organisms.

They lose viability in fresh water at slower rates than most of the major

intestinal bacterial pathogens. When such “foreign” enteric indicator bacteria

are not detectable in a specific volume (100 ml) of water, the water is

considered potable, or suitable for human consumption.

The coliform group includes E. coli, Enterobacter aerogenes, and Klebsiella

pneumoniae. Coliforms are defined as facultatively anaerobic, gram-negative,

nonsporing, rod-shaped bacteria that ferment lactose with gas formation within

48 hours at 35°C. The original test for coliforms that was used to meet this

definition involved the presumptive, confirmed, and completed tests, as shown

in figure. The presumptive step is carried out by means of tubes inoculated with three

different sample volumes to give an estimate of the most probable number (MPN) of coliforms in the water. The complete process, including the confirmed

and completed tests, requires at least 4 days of incubations and transfers.

Unfortunately the coliforms include a wide range of bacteria whose primary

source may not be the intestinal tract. To deal with this difficulty, tests have

been developed that allow waters to be tested for the presence of fecal coliforms. These are coliforms derived from the intestine of warm-blooded

animals, which can grow at the more restrictive temperature of 44.5°C. To test

for coliforms and fecal coliforms, and more effectively recover stressed

coliforms, a variety of simpler and more specific tests have been developed.

These include the membrane filtration technique, the presence-absence (P-A) test for coliforms and the related Colilert defined substrate test for detecting

both coliforms and E. coli.



The Multiple-Tube Fermentation Test. The multiple-tube fermentation technique has been used for many years for the sanitary analysis of water. Lactose broth tubes are inoculated with different water volumes in the presumptive test. Tubes that are positive for gas production are inoculated into brilliant green lactose bile broth in the confirmed test, and positive tubes are used to calculate the most probable number (MPN)



value. The completed test is used to establish that coliform bacteria are present. The membrane filtration technique, has become a common and often

preferred method of evaluating the microbiological characteristics of water. The

water sample is passed through a membrane filter. The filter with its trapped

bacteria is transferred to the surface of a solid medium or to an absorptive pad

containing the desired liquid medium. Use of the proper medium allows the

rapid detection of total coliforms, fecal coliforms, or fecal streptococci by the

presence of their characteristic colonies (Figure).

Coliform and Enterococcal Colonies.

Membrane filters made it possible to more rapidly test waters for the presence

of coliforms, fecal coliforms, and fecal enterococci by the use of differential

media. (a) Coliform reactions on an Endo medium. (b) Fecal coliform growth on

a bile salt medium (m-FC agar) containing aniline blue dye. (c) Fecal

enterococci growing on an azide-containing medium (KF agar) with TTC, an

artificial electron acceptor, added to allow better detection of colonies.

Samples can be placed on a less selective resuscitation medium, or incubated

at a less stressful temperature, prior to growth under the final set of selective

conditions. An example of a resuscitation step is the use of a 2 hour incubation

on a pad soaked with lauryl sulfate broth, as is carried out in the LES Endo

procedure. A resuscitation step often is needed with chlorinated samples,

where the microorganisms are especially stressed. The advantages and

disadvantages of the membrane filter technique are summarized. Advantages: Good reproducibility



Single-step results often possible

Filters can be transferred between different media

Large volumes can be processed to increase assay sensitivity

Time savings are considerable

Ability to complete filtrations on site

Lower total cost in comparison with MPN procedure Disadvantages: High-turbidity waters limit volumes sampled

High populations of background bacteria cause overgrowth

Metals and phenols can adsorb to filters and inhibit growth Membrane filters have been widely used with water that does not contain high

levels of background organisms, sediment, or heavy metals. More simplified

tests for detecting coliforms and fecal coliforms are now available. The

presence-absence test (P-A test) can be used for coliforms. This is a

modification of the MPN procedure, in which a larger water sample (100 ml) is

incubated in a single culture bottle with a triple-strength broth containing

lactose broth, lauryl tryptose broth, and bromcresol purple indicator. The P-A

test is based on the assumption that no coliforms should be present in 100 ml

of drinking water. A positive test results in the production of acid (a yellow

color) and constitutes a positive presumptive test requiring confirmation.

To test for both coliforms and E. coli, the related Colilert defined substrate test

can be used. A water sample of 100 ml is added to a specialized medium

containing o-nitrophenyl-β-Dgalactopyranoside (ONPG) and 4-

methylumbelliferyl- β-D-glucuronide

(MUG) as the only nutrients. If coliforms are present, the medium will turn

yellow within 24 hours at 35°C due to the hydrolysis of ONPG, which releases

o-nitrophenol, as shown in figure.



The Defined Substrate Test This much simpler test is now being used to detect coliforms and fecal coliforms in single 100 ml water samples. The medium uses ONPG and MUG (see text) as defined substrates. (a) Uninoculated control. (b)Yellow color due to the presence of coliforms. (c) Fluorescent reaction due to the presence of fecal coliforms. To check for E. coli, the medium is observed under long-wavelength UV light for

fluorescence. When E. coli is present, the MUG is modified to yield a fluorescent

product. If the test is negative for the presence of coliforms, the water is

considered acceptable for human consumption. The main change from

previous standards is the requirement to have waters free of coliforms and fecal

coliforms. If coliforms are present, fecal coliforms or E. coli must be tested for.

Molecular techniques are now used routinely to detect coliforms in waters and

other environments, including foods. 16 S rRNA gene-targeted primers for

coliforms have been developed. Using these primers, it is possible to detect one

colonyforming unit (CFU) of E. coli per 100 ml of water, if an eight-hour

enrichment step precedes the use of the PCR amplification. This allows the

differentiation of nonpathogenic and enterotoxigenic strains, including the

shiga-toxin producing E. coli O157:H7.

If unfiltered surface waters are being used, one coliform test must be run each

day when the waters have higher turbidities. Other indicator microorganisms

include fecal enterococci. The fecal enterococci are increasingly being used as

an indicator of fecal contamination in brackish and marine water. In salt water

these bacteria die back at a slower rate than the fecal coliforms, providing a

more reliable indicator of possible recent pollution. WATER PURIFICATION

Water purification is a critical link in controlling disease transmission in

waters. As shown in figure, water purification can involve a variety of steps,

depending on the type of impurities in the raw water source.



Several alternatives can be used for drinking water treatment depending on the initial water quality. A major concern is disinfection: chlorination can lead to the formation of disinfection byproducts (DBPs), including potentially carcinogenic trihalomethanes (THMs). For example, if the raw water contains large amounts of iron and manganese,

which will often precipitate when water is exposed to air, it may be necessary to

aerate the water and employ other methods to remove these ions early in the

purification sequence. Usually municipal water supplies are purified by a

process that consists of at least three or four steps. If the raw water contains a

great deal of suspended material, it often is first routed to a sedimentation basin and held so that sand and other very large particles can settle out. The

partially clarified water is then mixed with chemicals such as alum and lime

and moved to a settling basin where more material precipitates out. This

procedure is called coagulation or flocculation and removes microorganisms,

organic matter, toxic contaminants, and suspended fine particles. After these

steps the water is further purified by passing it through a filtration unit.



Physical filtration is an important step in drinking water treatment. This is a

cross section of a typical sand filter showing layers of sand and graded gravel

Rapid sand filters, which depend on the physical trap ping of fine particles

and flocs, are usually used for this purpose. This filtration removes up to 99%

of the remaining bacteria. After filtration the water is treated with a

disinfectant. This step usually involves chlorination, but ozonation is becoming

increasingly popular. When chlorination is employed, the chlorine dose must

be large enough to leave residual free chlorine at a concentration of 0.2 to 2.0

mg/liter. A concern is the creation of disinfection by-products (DBPs) such as

trihalomethanes (THMs) that are formed when chlorine reacts with organic

matter. Some of these compounds may be carcinogens.

The preceding purification process effectively removes or inactivates disease-

causing bacteria and indicator organisms (coliforms). Unfortunately, however,

the use of coagulants, rapid filtration, and chemical disinfection often does not

consistently and reliably remove Giardia lamblia cysts, Cryptosporidium

oocysts, Cyclospora, and viruses. Giardia, a cause of human diarrhea, is now

recognized as the most common identified waterborne pathogen in the United

States. The protozoan, first observed by Leeuwenhoek in 1681 has trophozoite

and cyst forms. The disease often is called “backpacker’s diarrhea” and is

transmitted primarily through untreated stream water or undependable

municipal water supplies. More consistent removal of Giardia cysts, which are

about 7 to 10 by 8 to 12 μm in size, can be achieved with slow sand filters. This treatment involves the slow passage of water through a bed of sand in



which a microbial layer covers the surface of each sand grain. Waterborne

microorganisms are removed by adhesion to the gelatinous surface microbial

layer. GROUND WATER TYPES AND THEIR CONTAMINATION

Groundwater, or water in gravel beds and fractured rocks below the surface

soil, is a widely used but often unappreciated water resource. In the United

States groundwater supplies at least 100 million people with drinking water,

and in rural and suburban areas beyond municipal water distribution systems,

90 to 95% of all drinking water comes from this source.

The great dependence on this resource has not resulted in a corresponding

understanding of microorganisms and microbiological processes that occur in

the groundwater environment. Increasing attention is now being given to

predicting the fate and effects of groundwater contamination on the chemical

and microbiological quality of this resource. Pathogenic microorganisms and

dissolved organic matter are removed from water during subsurface passage

through adsorption and trapping by fine sandy materials, clays, and organic

matter. Microorganisms associated with these materials—including predators

such as protozoa— can use the trapped pathogens as food. This results in

purified water with a lower microbial population.

This combination of adsorption-biological predation is used in home treatment

systems (figure). Conventional septic tank systems include an anaerobic

liquefaction and digestion step that occurs in the septic tank itself (the tank

functions as a simple anaerobic digester). This is followed by organic matter

adsorption and entrapment of microorganisms in the aerobic leach-field

environment where biological oxidation occurs.

The Septic Tank Home Treatment System - This system combines an anaerobic waste liquefaction unit (the septic tank) with an aerobic leach



field. Biological oxidation of the liquefied waste takes place in the leach field, unless the soil becomes flooded A septic tank may not operate correctly for several reasons. It will not function

properly if the retention time of the waste in the septic tank is too short.

Retention time decreases when the flow is too rapid or when excessive sludge

has accumulated in the septic tank. As a result undigested solids move into the

leach field, gradually plugging the system. If the leach field floods and becomes

anaerobic, biological oxidation does not occur, and effective treatment ceases.

When a suitable soil is not present and the septic tank outflow drains too

rapidly to the deeper subsurface, problems can occur. Fractured rocks and

coarse gravel materials provide little effective adsorption or filtration. This may

result in the contamination of well water with pathogens and the transmission

of disease. In addition, phosphorus from the waste will not be retained

effectively and may pollute the groundwater. This often leads to nutrient

enrichment of ponds, lakes, and rivers as the subsurface water enters these

environmentally sensitive water bodies.

Subsurface zones also can become contaminated with pollutants from other

sources. Land disposal of sewage sludges, illegal dumping of septic tank

pumpage, improper toxic waste disposal, and runoff from agricultural

operations all contribute to groundwater contamination with chemicals and

microorganisms. Deep-well injection of industrial wastes has raised questions

about the longer-term fate and effects of these materials.

Many pollutants that reach the subsurface will persist and may affect the

quality of groundwater for extended periods. Much research is being conducted

to find ways to treat groundwater in place—in situ treatment. Microorganisms

and microbial processes are critical in many of these remediation efforts BIOFILM

Biofilms consist of microorganisms immobilized at a substratum surface and

typically embedded in an organic polymer matrix of microbial origin. They

develop on virtually all surfaces immersed in natural aqueous environments,

including both biological (aquatic plants and animals) and abiological



(concrete, metal, plastics, stones). Biofilms form particularly rapidly in flowing

aqueous systems where a regular nutrient supply is provided to the

microorganisms. Extensive microbial growth, accompanied by excretion of

copious amounts of extracellular organic polymers, thus leads to the formation

of visible slimy layers (biofilms) on solid surfaces.

Most of the human gastrointestinal tract is colonized by specific groups of

microorganisms (the normal indigenous microbiota that give rise to natural

biofilms. At times, these natural biofilms provide protection for pathogenic

species, allowing them to colonize the host. Insertion of a prosthetic device into

the human body often leads to the formation of biofilms on the surface of the

device. The microorganisms primarily involved are Staphylococcus epidermidis,

other coagulase-negative staphylococci, and gram-negative bacteria. These

normal skin inhabitants possess the ability to tenaciously adhere to the

surfaces of inanimate prosthetic devices. Within the biofilms they are protected

from the body’s normal defense mechanisms and also from antibiotics; thus

the biofilm also provides a source of infection for other parts of the body as

bacteria detach during biofilm sloughing.

Some examples of biofilms of medical importance include:

Cystic fibrosis patients harboring great numbers of Pseudomonas aeruginosa

that produce large amounts of alginate polymers, which inhibit the diffusion of

antibiotics

Teeth, where biofilm forms plaque that leads to tooth decay.

Contact lenses, where bacteria may produce severe eye irritation,

inflammation, and infection

Air-conditioning and other water retention systems where potentially

pathogenic bacteria, such as Legionella species, may be protected from the

effects of chlorination by biofilms.

Microorganisms tend to create their own microenvironments and niches, even

without having a structured physical environment available, by creating

biofilms. These are organized microbial systems consisting of layers of



microbial cells associated with surfaces. Such biofilms are an important factor

in almost all areas of microbiology, as shown in figurea. Simple biofilms develop when microorganisms attach and form a monolayer of

cells.

Depending on the particular microbial growth environment (light, nutrients

present and diffusion rates), these biofilms can become more complex with

layers of organisms of different types (figure b).

A typical example would involve photosynthetic organisms on the surface,

facultative chemoorganotrophs in the middle, and possibly sulfate-reducing

microorganisms on the bottom. More complex biofilms can develop to form a

four-dimensional structure (X,Y, Z, and time) with cell aggregates, interstitial

pores, and conduit channels (figure c).

This developmental process involves the growth of attached microorganisms,

resulting in accumulation of additional cells on the surface, together with the

continuous trapping and immobilization of freefloating microorganisms that

move over the expanding biofilm. This structure allows nutrients to reach the

biomass, and the channels are shaped by protozoa that graze on bacteria.

These more complex biofilms, in which microorganisms create unique

environments, can be observed by the use of confocal scanning laser

microscopy (CSLM).



The diversity of nonliving and living surfaces that can be exploited by biofilm-

forming microorganisms is illustrated in figure.

These include surfaces in catheters and dialysis units, which have intimate

contact with human body fluids. Control of such microorganisms and their

establishment in these sensitive medical devices is an important part of

modern hospital care.

Microorganisms that form biofilms on living organisms such as plants or

animals have additional advantages. In these cases the surfaces themselves

often release nutrients, in the form of sloughed cells, soluble materials, and

gases. These biofilms also can play major roles in disease because they can

protect pathogens from disinfectants; create a focus for later occurrence of

disease, or release microorganisms and microbial products that may affect the

immunological system of a susceptible host.

Biofilms are critical in ocular diseases because Chlamydia, Staphylococcus, and

other pathogens survive in ocular devices such as contact lenses and in

cleaning solutions.



Depending on environmental conditions, biofilms can become so large that they

are visible and have macroscopic dimensions. Bands of microorganisms of

different colors are evident as shown in figure.

These thick biofilms, called microbial mats, are found in many freshwater and

marine environments. These mats are complex layered microbial communities

that can form at the surface of rocks or sediments in hypersaline and

freshwater lakes, lagoons, hot springs, and beach areas. They consist of

microbial filaments, including cyanobacteria. A major characteristic of mats is

the extreme gradients that are present.

Light only penetrates approximately 1 mm into these communities, and below

this photosynthetic zone, anaerobic conditions occur and sulfate-reducing

bacteria play a major role. The sulfide that these organisms produce diffuses to

the anaerobic lighted region, allowing sulfur-dependent photosynthetic

microorganisms to grow. Some believe that microbial mats could have allowed

the formation of terrestrial ecosystems prior to the development of vascular

plants, and fossil microbial mats, called stromatolites, have been dated at over

3.5 billion years old.

WASTE TREATMENT Selection of a treatment process is dependent on the nature of the wastewater

and the quality of the effluent desired. Hazardous components of the

wastewater may be either separated or converted to non-hazardous forms in

order to permit the disposal of the wastewater effluent by conventional

methods. Conversion processes can be done in one step or in multiple steps.



The hazkdous components which are separated from the wastewater must be

disposed of. This may take additional steps, e.g., sludge dewatering.

Liquids-Solids Separation Separation of suspended matter from wastewater can be accomplished by a

number of different processes. Large heavy solids are easier to remove than

finely divided light solids.

Screening devices are used to remove large pieces of solid matter that would

interfere with subsequent processing operations or would cause damage to

equipment such as pumps. Coarse screening devices may consist of parallel

bars, rods or wires, perforated plates, gratings, or wire mesh.

Gravity sedimentation

This process involves the containment of wastewater for a sufficient period of time to allow some or all of the suspended materials to either settle out or float

to the surface of the wastewater. In its simplest form as a batch process, a given volume of wastewater is transferred to a vessel and held there until

nearly all the settleable and floatable matter separates. The floating matter can

be skimmed off and the wastewater decanted for discharge or further

treatment. Sludge may be allowed to collect until several batches of wastewater

have been processed. Then it is removed. The vessel may have a conical bottom

so that the sludge can be removed via a valve.

Large settling ponds may be constructed which are drained periodically to

permit sludge removal. Solids-contact or sludge-blanket clarifiers are useful

where sludges are flocculent and of low density. They are designed with large

mixing and reaction zones that coupled with the sludge blanket account for

greater efficiency in solids removal. Gravity sedimentation works like the

clarifiers but more time is taken for the settling.

Dissolved-Air Flotation

It is useful for suspended matter that does not sink or float in a reasonable

period of time. Separation is brought about by the introduction of finely divided

gas bubbles which become attached to the particulate matter, causing it to

float to the surface where it is removed by skimming. Introduction of the gas



bubbles is usually accomplished by reducing the pressure of the wastewater

causing dissolved gases to be released. This is commonly used to separate

greasy or oily matter from industrial wastes. Granular-Media Filters or deep

bed filtration is a polishing step that removes small amounts of suspended

solids and produces highly clarified water. Chemical coagulation and

sedimentation typically precede this stage. Graded sand and pulverized coals

are commonly used in the filter beds. Conventional operation is usually by

downflow. The ability of the granular media filter beds to produce a clear

effluent results from the straining action and adhesion, which removes

particles finer than the pore space.

Surface Filters

It makes use of a fine medium such as a cloth or close mesh screen. In the

rotary vacuum filter,-the medium is in the form of a continuous belt and it

rotates over a perforated drum that is partially submerged in the slurry to be

filtered. Water is pulled through the filter cake that forms on the belt to the

inside of the drum, where it is transferred to the vacuum system.

Centrifugation is a useful alternative to filtration for sticky sludges that do not

dewater rapidly on a filter. They operate by a rapid rotation of a liquid

suspension, which induces a much greater force than gravity to hasten the

separation of the suspended matter.

Chemical Treatment Chemical treatment is a widely used process for the destruction or separation

of hazardous constituents in wastewater. This can be done by neutralization of

acidic or alkaline wastewater until a suitable pH is obtained.

Precipitation/Coagulation/Flocculation is used for the removal of heavy metals.

Precipitation refers to the formation of a solid phase, coagulation is where the

containment is trapped by the formation of a precipitate, and flocculation is the

agglomeration of a coagulating chemical.

Oxidation-Reduction or the redox processes are used for converting toxic

pollutants to harmless or less toxic materials that are more easily removed.

These processes involve the addition of chemical reagents to wastewaters,



causing changes in the oxidation states of substances both in the reagents and

in the wastewaters. In, order for one substance to be oxidized, another must be

reduced. Ozone is a powerful oxidizing agent that is usually oxygen at

temperatures and pressures up to 350°C and 180 atmospheres, respectively, to

treat organic wastes.

Ion exchange involves a change in the chemical form of a compound; the

exchange of ions in solution with other ions held by mixed anionic or cationic

groups or charges. Typically, a waste solution is percolated through a granular

bed of the ion exchanger, where certain ions in solution are replaced by ions

contained in the ion exchanger. If the exchange involves cations, the exchanger

is called a cation exchanger and correspondingly anion exchanger is one that

involves anion exchange.

Physical Methods There are several methods used for separating pollutants from wastewater:

activated carbon, steam stripping, evaporation, reverse osmosis, and solvent

extraction. The chemical and physical characteristics of the pollutant are

important in the selection of the physical removal method. Steam stripping is

effective for substances that have an appreciable vapor pressure at the boiling

point of water, whereas evaporation is effective for those chemicals that will not

volatilize. Soluble, small organic molecules are adsorbed by activated carbon

Large ions are separated by reverse osmosis. Activated Carbon Adsorption

Here the inorganic and organic chemicals are adsorbed onto activated carbon.

Usually hydrophobic chemicals are more likely to be removed. The degree of

adsorption is linked to the molecular weight, methanol-water coefficient, or

solubility (these are also linked to the recalcitrance and/or toxicity). The

smaller the size of the grain, the more surface area is available and so

equilibrium is reached quicker with powdered activated carbon compared to

the granular form. But then the powdered form needs more pumping to get the

wastewater through and hence the costs are increased. There are two principle

systems, one is downward flow through the bed (pressure or gravity flow) and

the other is upflow through a packed or expanded bed. Activated carbon



adsorption is applicable to the treatment of dilute aqueous wastes, but they

should be treated to remove suspended solids, oil, and grease. Temperature

and pH are also important for the different compounds to be treated. The

carbon is either disposed of or regenerated. Carbon has also been added

directly to biological treatment effluent in a contacting basin. The advantages of

this are that the sludge toxicity is reduced by selectively removing the toxic

organics from solution and that the carbon adsorption capacity is extended by

bioregeneration of the "biocompatible" species adsorbed on the surface.

For aqueous solvent waste containing contaminants in concentrations up to

10,000 mg/l, the activated sludge process has been proposed as a potential

applicable treatment. However these concentrations may be toxic to the sludge

or they may be easily stripped to the atmosphere, thereby creating another

hazard. The sludge may also contain recalcitrant waste, due to adsorption of

the contaminants and be difficult to dispose of.'

Evaporation is the process that heats the liquid, venting the vapors to the

atmosphere and concentrating the pollutants into slurry. Reverse osmosis--

Osmosis is the process where a solvent (e.g., water) moves from an area of low

concentration to high across a semipermeable membrane which does not allow

the dissolved solids to pass. In reverse osmosis, a pressure greater than the

osmotic pressure is applied so the flow is reversed. Pure water will then flow

through the membrane from the concentrated solution.

Solvent extraction is a process whereby a dissolved or adsorbed substance is

transferred from a liquid or solid phase to a solvent that preferentially dissolves

that substance. For the process to be effective, the extracting solvent must be

immiscible in the liquid and differ in density so that gravity separation is

possible and there is minimal contamination of the raffinate with the solvent.

The hydrophobic solutes are more likely to be extracted. Solvent extraction can

be performed as a batch process, or by the contact of the solvent with the feed

in staged or continuous equipment.

Steam stripping is where water vapor at elevated temperatures is used to

remove volatile components of a liquid. Countercurrent flow is generally used



to promote gas-liquid contact, thus allowing soluble gaseous organics from the

liquid waste to be continuously exchanged with molecules within the stripping

gas. Again, this is useful only for waste with low water solubilities.

Incineration Incineration is a high temperature oxidation process that converts the principal

elements (carbon, hydrogen, and oxygen) in most organic compounds to carbon

dioxide and water. Given with the problems of disposing on land, incineration

may take on a lead role in waste treatment. However, it is not without its

problems. There is fear among the general public about the nature of the stack

emissions but it is an efficient method. The destruction of the molecular

structure usually eliminates the toxicity of the chemical. But the existence of

other elements in a waste may result in the production of particulate pollutants

that require removal in off-gas treatment systems. There are several types of

incinerators available.

The liquid injection incinerators operate by spraying the combustible waste

mix with air into a chamber where flame oxidation takes place. The purpose of

spraying is to atomize the waste into small droplets which present a large

surface area for rapid heat transfer, thereby increasing the rate of vaporization

and mixing with air to promote combustion. Air is supplied to provide the

necessary mixing and turbulence. These incinerators are widely used for

destruction of liquid organic wastes.

Rotary-kiln incinerators are designed to process solids and tars that cannot

be processed in the liquid incinerator. The rotary-kiln is a cylindrical shell lined

with refractory material that is horizontally mounted at a slight incline. It is

rotated from 5 to 25 times at high temperatures, 1500 to 3000°F with excess

air, and the residence time varied depending on the nature of the waste. The

rotation causes a tumbling action that mixes the waste with air. The primary

function is to convert, through partial burning and volatization, solid wastes to

gases and ashhesidue. If the ash is free of dangerous levels of hazardous

wastes, it is put in a landfill.

Fume incineration



Large quantities of organic vapor fumes are produced by many industries,

including fat rendering, metal painting and varnishing, and various types of

printing. These vapors are generally mixes of hydrocarbons, alcohols, and

acetates. The mixes may not be acutely toxic but they do cause odor problems.

An integrated heatrecovery system demonstration at Case-Hoyt Company, near

Rochester, captures waste heat and simultaneously reduces plant air

emissions by oxidizing solvents into harmless gases as the heat is recovered.

Multiple hearth incinerators It is used for wastes that are difficult to burn or that contain valuable metals

that can be recovered. It consists of a refractory-lined circular steel shell, with

refractory hearths located one above the other. Solid waste or partially

dewatered sludge is fed to the top of the unit where a rotating plow rake plows

it across the hearth to dropholes. The uncombusted material falls to the next

hearth and the process is repeated until the combustion is complete.

Fluidized-bed incinerators (FBI)

These are applicable to the destruction of halogenated organic waste streams.

This type of incinerator consists of a vessel in which inert granular particles

are fluidized by a low velocity air stream which is passed through a distributor

plate below the bed. An FBI consists of a windbox (through which combustion

air is introduced to the reactor), and a reactor zone (containing a bed of sand,

waste injection, and removal ports). Temperatures are in the range of 1300 to

2100°F, gas residence times usually a few seconds. They have been used to

treat municipal sewage sludge, low quality fuels, pulp and paper effluents, and

food processing waste, refinery waste, radioactive waste, and miscellaneous

chemical waste.

A molten salt incinerator uses a molten salt such as a sodium carbonate as a

heat transfer and reaction medium. In the process, waste material along with

air is added below the surface of the bed so that any gases formed during

combustion are forced to pass through the melt. Reaction temperatures in the

bed range from 1500 to 2000°F and residence times are less than a second.



Any acidic gases formed are neutralized by the alkalinity of the bed. This can

change the fluidity of the bed so it needs replacement frequently.

Plasma arc incineration is based on the concept of reducing or pyrolyzing

waste molecules to the atomic state using a thermal plasma field. They system

uses very high energy at temperatures near to 10,000"C to break bonds of

hazardous waste chemical molecules down to the atomic state. An electrode

assembly ionizes air molecules which create a plasma field. Hazardous waste

mixtures interact with the field, forming simple molecules such as carbon

dioxide, hydrogen, hydrogen chloride, and other minor matrix compounds such

as acetylene and ethene. Westinghouse Electric has a mobile plasma arc unit

called Pyroplasma that reportedly treats liquid wastes at the rate of a 3

gal/min.'' The high temperatures decompose PCB and other wastes in an

oxygen-deficient atmosphere. Hydrogen chloride is treated with sodium to form

water and salt.

Lime or cement kiln incineration A cement kiln is basically a large rotary kiln in which raw materials are fed

countercurrent to combustion gas flow. The wet process kilns uses a 30%

water slurry feed and are the most suitable for hazardous waste destruction.

The products formed are alkaline and so act as a scrubber, removing acid

gases formed during combustion. This system operates at 2800°F resulting in

very efficient removal of wastes. A project" is in operation at Blue Circle Atlantic

Inc., a cement company in Ravena where it is evaluating the incorporation of

hazardous wastes into an existing cement-kiln operation. The facility is not yet

in operation but should be able to handle large amounts of waste and alleviate

some problems locally.

Wet Air Oxidation Wet air oxidation involves the aqueous phase oxidation of organic materials at

high temperature and pressure. A major advantage over other incineration

methods is that the water in the waste stream is kept in the liquid state. Water

is pumped into the reactor along with oxygen which is heated by the hot

effluent. Two types of reactors are used, a bubble tower reactor and a stirred



tank cascade reactor. This process is good for wastes that are too dilute to

incinerate but too toxic for biological methods. The products are usually acetic

acid and carbon dioxide.

Solidification Techniques There are several innovative non-thermal processes that have been developed

under the SITE program that immobilize wastes by vitrification or other types

of solidification." The SITE program is the Superfund Innovative Technology

Evaluation, a $20 milliodyear program which has been developed to encourage

the private development and demonstration of new technologies for cleaning up

hazardous wastes.

For example, the researchers at Battelle Pacific Northwest Laboratories have

developed an in-situ vitrification process (which was originally designed for the

containment of nuclear wastes) in which electrodes are sunk into a

contaminated area and attached to a diesel powered generator. The current

produced temperatures of about 3600°F which is much higher than the fusion

temperature of soil. An exhaust hood is placed over the site to collect and treat

any combustion products. The result is a massive glass-like product consisting

of completely immobilized organics, inorganics, steel drums, and other

components that are essentially locked up and inert. The time taken to

complete the process depends on the electrode depth and frequency.

Another solidification process uses a reagent called Urrichem that immobilizes

slurried hazardous components. The contaminated soil is excavated and

intimately mixed with the Urrichem off-site. After blending, the slurry is

pumped out of the mixer and hardens into a concrete like mass within 24

hours. Chemfix is a process developed by Chemfix Technologies (Metaire, LA).

In this technique, a proprietary blend of soluble silicates and additives is used

to convert high molecular weight organic and inorganic slurries into a cross-

linked, clay like matrix.

Alternative methods have also included landfills, deep well injection disposal,

and ocean dumping. Landfills were developed because it was believed that by

placing waste in designated ground areas, there would be a natural



decomposition over time. Unfortunately the water table rises in a landfill and

this mounding effect means that we get a leaching of the water containing

toxics since the water flow is always from areas of high to low head, i.e., there

is a gradient set up that favors water movement originating in the landfill and

away from it thus contaminating our groundwater supplies.

SEWAGE AND EFFULENT TREATMENT Biological treatment has been very successful in the removal of organic

pollutants and colloidal organics from wastewater. Activated sludge, biologicd

filters, aerated lagoons, oxidation ponds, and aerobic fermentation are some of

the methods available for wastewater biodegradation. In removal of toxic waste,

more care is needed since the bacteria are prone to destruction from shock

loading or increases of toxic material fed in without allowing time for the

population to grow large enough to deal with it.

Biodegradation occurs because bacteria are able to metabolize the organic

matter via enzyme systems to yield carbon dioxide, water, and energy. The

energy is used for synthesis, motility, and respiration. With simple dissolved

matter, it is taken into the cell and oxidized, but with more complex inorganics,

enzymes are secreted extracellularly to hydrolyze the proteins and fats into a

soluble form which can then be taken into the cell and oxidized. Hence the

more complex matter takes longer to process.

Some organic compounds are "refractory," they cannot be oxidized while others

are toxic to the bacteria at high concentrations. The purpose of biodegradation

is to convert the waste into the end products and material that will settle and

can be removed as sediment. Again, biodegradation may not be one hundred

percent, or toxic byproducts may be formed. Further treatment by chemical

methods or dilution may be needed to get the contaminant to a concentration

prescribed as safe.

Nitrogen and phosphorus are essential in the oxidation process for the

synthesis of new cells, and trace amounts of potassium and calcium are also

required. The former are sometimes deficient so nitrogen is added in the form

of ammoniacal nitrogen (nitrite and nitrate are not readily used by bacteria).



BOD or biochemical oxygen demand measures the strength of the organics

present and is defined as the amount of oxygen needed by the bacteria for

oxidation. The more concentrated the organic material the higher the BOD. A

B0D:N:P ratio of 100:5:1 is thought to be the optimum ratio of nutrients

needed by bacteria.

Activated Sludge Process This involves the generation of a suspended mass of bacteria in a reactor to

degrade soluble and finely suspended organic compounds. In this method the

wastewater with its organic compounds is fed into the aeration tank. This is

supplied with air and is vigorously mixed to allow maximum contact of bacteria

and waste. The contents, referred to as MLSS (mixed liquor suspended solids)

are then fed to a sedimentation tank where the treated solids settle to the

bottom and the top liquid layer is treated and discharged. Parts of the

biological solids are recycled back to the aeration tank to maintain the correct

mix; the remainder is waste. This method is flexible and can be used on almost

any type of biological waste. An industrial application has been demonstrated

for phenol degradation using a petroleum refinery wastewater: there was an

85-90% removal of phenol and cyanide in the steel industry.

Trickling Over Process Here the wastewater is distributed by a flow distributor over a fixed bed of

medium on which the bacteria grow forming a slime layer to which oxygen is

supplied. The wastewater flows down over the slime layer which absorbs

organic materials and nutrients, releasing the oxidized end products to the

drainage system underneath. Eventually some of the layer will detach with the

wastewater, and then some additional separation is necessary.

Stabilization This is a procedure where wastewater is stabilized by the actions of bacteria in

shallow ponds. There are basically two types of ponds, ones where there is a

natural supply of oxygen from algal photosynthesis (oxidation ponds) and

mechanically supplied oxygen (aerated lagoons). The bacteria metabolize the

wastes and the solids settle at the bottom as sludge. Also there is anaerobic



decomposition where the bacteria at the bottom will degrade the waste without

oxygen's presence. Or there can be a lagoon that has both aerobic and

anaerobic decomposition, with an interchange of products between the two

layers of bacteria in a symbiotic relationship.

PRIMARY, SECONDARY AND TERTIARY TREATMENT The water pollutants are discharge of sewage or waste water to the

environment is a matter of concern, as it poses serious threat to public health.

Treatment of water proceeds through four stages:

Preliminary Treatment

Primary Treatment

Secondary Treatment

Tertiary Treatment.

Preliminary Treatment; this process basically involves in removal of floating

materials such as leaves, papers, plastics etc ,settle able inorganic solids

(sand,grid)&fats and oils .

Three major types of equipment used;

Screeners-is a device with opening to remove the floating material sand

suspended particles.

Grit chambers-trough this heavy inorganic materials like sand, ash can be

removed.

Skimming tanks-several greasy &oily materials can be removed.

Primary Treatment- is aimed in removing of fine suspended organic solids

that cannot be removed in preliminary treatment.

Is involved in settling or sedimentation, in normal process sedimentation is

usually carried out twice –once before the secondary treatment referred as

primary sedimentation and then after secondary treatment is completed known

as secondary sedimentation.

Types of settling; 4 major types are;

Discrete settling

Flocculants settling

Zone settling



Compression settling.

Secondary or Biological Treatment; it is required for the removal of dissolved

and fine colloidal organic matter, it involves microorganisms (bacteria, algae,

fungi, protozoa, nematodes) it decomposes the unstable organic matter to

stable inorganic forms.

They are broadly classified as Aerobic & Anaerobic depending on the nature of

the microorganism used.

The biological processes are categorized as suspended growth system

&attached growth system.

Aerobic Suspended-most important biological system used is activated sludge

process, Activated sludge system, the effluent from primary treatment is

constantly agitated, aerated, and added to solid material remaining from earlier

water treatment. This sludge contains large numbers of aerobic organisms that

digest organic matter in wastewater.

However, filamentous bacteria multiply rapidly in such Systems and cause

some of the sludge to float on the Surface of the water instead of settling out.

This phenomenon, Called bulking, allows the floating matter to contaminate

the effluent. The sheathed bacterium which sometimes proliferates rapidly on

decaying leaves in small streams and causes a bloom can interfere with the

operation of sewage systems in this way. Its filaments clog filters and create

floating clumps of undigested organic matter. Sludge from both primary and

secondary treatments can be pumped into sludge digesters. Here, oxygen is

virtually excluded and anaerobic bacteria partially digest the sludge to simple

organic molecules and the gases carbon dioxide and methane.

The methane can be used for heating the digester and providing for other

power needs of the treatment plant. Undigested matter can be dried and used

as a soil conditioner.

In a trickling filter system; sewage is sprayed over a bed of rocks about 2 m

deep. The individual rocks are 5 to 10 cm in diameter and are coated with a

slimy film of aerobic organisms such as Sphaerotilus and Beggiatoa . Spraying

oxygenates the sewage so that the aerobes can decompose organic matter in it.



Such a system is less efficient but less subject to operational problems than an

activated sludge system.

It removes about 80% of the organic matter in the water.

Anaerobic suspended-Growth treatment processes; it basically involves the

decomposition of organic & inorganic matter in the absence of oxygen ,it is

important for treatment of sludge’s ,most commonly used are anaerobic

digestion process for treatment of sewage .

Anaerobic Digestion; the process is carried out in an air tight reactor, sludge

is introduced continuously or intermiltently,it takes about 15 days for process

to complete.

Tertiary Treatment or Advanced Treatment; The effluent from secondary treatment contains only 5% to 20% of the original

quantity of organic matter and can be discharged into flowing rivers without

causing serious problems. However, this effluent can contain large quantities of

phosphates and nitrates, which can increase the growth rate of plants in the

river.



Tertiary treatment is an extremely costly process that involves physical and

chemical methods. Fine sand and charcoals are used in filtration. Various

flocculating chemicals Precipitate phosphates and particulate matter.

Denitrifying bacteria convert nitrates to nitrogen gas.

Finally, chlorine is used to destroy any remaining organisms.

Water that has received tertiary treatment can be released into any body of

water without danger of causing eutrophication. Such water is pure enough to

be recycled into a domestic water supply. However, the chlorine-containing

effluent, when released into streams and lakes, can react to produce

carcinogenic compounds that may enter the food chain or be ingested directly

by humans in their drinking water. It would be Safer to remove the chlorine

before releasing the effluent,

But this is rarely done today, although the cost is not great. Ultraviolet lights

are now replacing chlorination as the final treatment of effluent it destroys

microbes without adding carcinogens to our streams and waters.

SOLID WASTE TREATMENT Solid waste is the unwanted or useless solid materials generated from

combined residential, industrial and commercial activities in a given area. It

may be categorized according to its origin (domestic, industrial, commercial,

construction or institutional); according to its contents (organic material, glass,

metal, plastic paper etc); or according to hazard potential (toxic, non-toxin,

flammable, radioactive, infectious etc). Management of solid waste reduces or

eliminates adverse impacts on the environment and human health and

supports economic development and improved quality of life. A number of

processes are involved in effectively managing waste for a municipality.These

includes monitoring, collection, transport, processing, recycling and disposal.

Reduce, Reuse, Recycle Methods of waste reduction, waste reuse and recycling are the preferred

options when managing waste. There are many environmental benefits that can

be derived from the use of these methods. They reduce or prevent green house



gas emissions, reduce the release of pollutants, conserve resources, save

energy and reduce the demand for waste treatment technology and landfill

space. Therefore it is advisable that these methods be adopted and

incorporated as part of the waste management plan.

Waste reduction and reuse Waste reduction and reuse of products are both methods of waste prevention.

They eliminate the production of waste at the source of usual generation and

reduce the demands for large scale treatment and disposal facilities. Methods

of waste reduction include manufacturing products with less packaging,

encouraging customers to bring their own reusable bags for packaging,

encouraging the public to choose reusable products such as cloth napkins and

reusable plastic and glass containers, backyard composting and sharing and

donating any unwanted items rather than discarding them.

All of the methods of waste prevention mentioned require public participation.

In order to get the public onboard, training and educational programmes need

to be undertaken to educate the public about their role in the process. Also the

government may need to regulate the types and amount of packaging used by

manufacturers and make the reuse of shopping bags mandatory.

Recycling Recycling refers to the removal of items from the waste stream to be used as

raw materials in the manufacture of new products. Thus from this definition

recycling occurs in three phases: first the waste is sorted and recyclables

collected, the recyclables are used to create raw materials. These raw materials

are then used in the production of new products.

The sorting of recyclables may be done at the source (i.e. within the household

or office) for selective collection by the municipality or to be dropped off by the

waste producer at a recycling centres. The pre-sorting at the source requires

public participation which may not be forthcoming if there are no benefits to be

derived. Also a system of selective collection by the government can be costly. It

would require more frequent circulation of trucks within a neighbourhood or

the importation of more vehicles to facilitate the collection.



Another option is to mix the recyclables with the general waste stream for

collection and then sorting and recovery of the recyclable materials can be

performed by the municipality at a suitable site. The sorting by the

municipality has the advantage of eliminating the dependence on the public

and ensuring that the recycling does occur. The disadvantage however, is that

the value of the recyclable materials is reduced since being mixed in and

compacted with other garbage can have adverse effects on the quality of the

recyclable material.

Waste Collection Waste from our homes is generally collected by our local authorities through

regular waste collection, or by special collections for recycling. Within hot

climates such as that of the Caribbean the waste should be collected at least

twice a week to control fly breeding, and the harbouring of other pests in the

community. Other factors to consider when deciding on frequency of collection

are the odours caused by decomposition and the accumulated quantities.

Treatment & Disposal

Waste treatment techniques seek to transform the waste into a form that is

more manageable, reduce the volume or reduce the toxicity of the waste thus

making the waste easier to dispose of. Treatment methods are selected based



on the composition, quantity, and form of the waste material. Some waste

treatment methods being used today include subjecting the waste to extremely

high temperatures, dumping on land or land filling and use of biological

processes to treat the waste. It should be noted that treatment and disposal

options are chosen as a last resort to the previously mentioned management

strategies reducing, reusing and recycling of waste. Thermal treatment This refers to processes that involve the use of heat to treat waste. Listed below

are descriptions of some commonly utilized thermal treatment processes.

Incineration Incineration is the most common thermal treatment process. This is the

combustion of waste in the presence of oxygen. After incineration, the wastes

are converted to carbon dioxide, water vapour and ash. This method may be

used as a means of recovering energy to be used in heating or the supply of

electricity. In addition to supplying energy incineration technologies have the

advantage of reducing the volume of the waste, rendering it harmless, reducing

transportation costs and reducing the production of the green house gas

methane

Pyrolysis and Gasification Pyrolysis and gasification are similar processes they both decompose organic

waste by exposing it to high temperatures and low amounts of oxygen.

Gasification uses a low oxygen environment while pyrolysis allows no oxygen.

These techniques use heat and an oxygen starved environment to convert

biomass into other forms. A mixture of combustible and non-combustible gases

as well as pyroligenous liquid is produced by these processes. All of these

products have a high heat value and can be utilised. Gasification is

advantageous since it allows for the incineration of waste with energy recovery

and without the air pollution that is characteristic of other incineration

methods.

Open burning



Open burning is the burning of unwanted materials in a manner that causes

smoke and other emissions to be released directly into the air without passing

through a chimney or stack. This includes the burning of outdoor piles,

burning in a burn barrel and the use of incinerators which have no pollution

control devices and as such release the gaseous by products directly into the

atmosphere.

Openburning has been practiced by a number of urban centres because it

reduces the volume of refuse received at the dump and therefore extends the

life of their dumpsite. Garbage may be burnt because of the ease and

convenience of the method or because of the cheapness of the method. In

countries where house holders are required to pay for garbage disposal,

burning of waste in the backyard allows the householder to avoid paying the

costs associated with collecting, hauling and dumping the waste. Open burning

has many negative effects on both human health and the environment. This

uncontrolled burning of garbage releases many pollutants into the atmosphere.

These include dioxins, particulate matter, polycyclic aromatic compounds,

volatile organic compounds, carbon monoxide, hexachlorobenzene and ash. All

of these chemicals pose serious risks to human health. The Dioxins are capable

of producing a multitude of health.

Problems;

They can have adverse effects on reproduction, development, disrupt the

hormonal systems or even cause cancer. The polycyclic aromatic compounds

and the hexachlorobenzene are considered to be carcinogenic. The particulate

matter can be harmful to persons with respiratory problems such as asthma or

bronchitis and carbon monoxide can cause neurological symptoms.

The harmful effects of open burning are also felt by the environment. This

process releases acidic gases such as the halo-hydrides; it also may release the

oxides of nitrogen and carbon. Nitrogen oxides contribute to acid rain, ozone

depletion, smog and global warming. In addition to being a green house gas

carbon monoxide reacts with sunlight to produce ozone which can be harmful.



The particulate matter creates smoke and haze which contribute to air

pollution.

Dumps and Landfills Sanitary landfills Sanitary Landfills are designed to greatly reduce or eliminate the risks that

waste disposal may pose to the public health and environmental quality. They

are usually placed in areas where land features act as natural buffers between

the landfill and the environment. For example the area may be comprised of

clay soil which is fairly impermeable due to its tightly packed particles, or the

area may be characterised by a low water table and an absence of surface

water bodies thus preventing the threat of water contamination.

In addition to the strategic placement of the landfill other protective measures

are incorporated into its design. The bottom and sides of landfills are lined with

layers of clay or plastic to keep the liquid waste, known as leachate, from

escaping into the soil. The leachate is collected and pumped to the surface for

treatment. Boreholes or monitoring wells are dug in the vicinity of the landfill

to monitor groundwater quality.

These landfills present the least environmental and health risk and the records

kept can be a good source of information for future use in waste management,

however, the cost of establishing these sanitary landfills are high when

compared to the other land disposal methods.



Controlled dumps Controlled dumps are disposal sites which comply with most of the

requirements for a sanitary landfill but usually have one deficiency. They may

have a planned capacity but no cell planning, there may be partial leachate

management, partial or no gas management, regular cover, compaction in

some cases, basic record keeping and they are fenced or enclosed. These

dumps have a reduced risk of environmental contamination, the initial costs

are low and the operational costs are moderate. While there is controlled access

and use, they are still accessible by scavengers and so there is some recovery of

materials through this practice.

Bioreactor Landfills Recent technological advances have lead to the introduction of the Bioreactor

Landfill.

The Bioreactor landfills use enhanced microbiological processes to accelerate

the decomposition of waste. The main controlling factor is the constant

addition of liquid to maintain optimum moisture for microbial digestion. This

liquid is usually added by re circulating the landfill leachate. In cases where

leachate in not enough, water or other liquid waste such as sewage sludge can

be used. The landfill may use either anaerobic or aerobic microbial digestion or

it may be designed to combine the two. These enhanced microbial processes

have the advantage of rapidly reducing the volume of the waste creating more

space for additional waste, they also maximise the production and capture of

methane for energy recovery systems and they reduce the costs associated with

leachate management. For Bioreactor landfills to be successful the waste

should be comprised predominantly of organic matter and should be produced

in large volumes.

Biological waste treatment Composting Composting is the controlled aerobic decomposition of organic matter by the

action of micro organisms and small invertebrates. There are a number of

composting techniques being used today. These include: in vessel composting,



windrow composting, vermicomposting and static pile composting. The process

is controlled by making the environmental conditions optimum for the waste

decomposers to thrive. The rate of compost formation is controlled by the

composition and constituents of the materials i.e. their Carbon/Nitrogen (C/N)

ratio, the temperature, the moisture content and the amount of air.

The C/N ratio is very important for the process to be efficient. The micro

organisms require carbon as an energy source and nitrogen for the synthesis of

some proteins. If the correct C/N ration is not achieved, then application of the

compost with either a high or low C/N ratio can have adverse effects on both

the soil and the plants. A high C/N ratio can be corrected by dehydrated mud

and a low ratio corrected by adding cellulose.

Moisture content greatly influences the composting process. The microbes need

the moisture to perform their metabolic functions. If the waste becomes too dry

the composting is not favoured. If however there is too much moisture then it is

possible that it may displace the air in the compost heap depriving the

organisms of oxygen and drowning them.

A high temperature is desirable for the elimination of pathogenic organisms.

However, if temperatures are too high, above 75oC then the organisms

necessary to complete the composting process are destroyed. Optimum

temperatures for the process are in the range of 50-60oC with the ideal being

60oC.

Aeration is a very important and the quantity of air needs to be properly

controlled when composting. If there is insufficient oxygen the aerobes will

begin to die and will be replaced by anaerobes. The anaerobes are undesirable

since they will slow the process, produce odours and also produce the highly

flammable methane gas. Air can be incorporated by churning the compost.

Anaerobic Digestion Anaerobic digestion like composting uses biological processes to decompose

organic waste. However, where composting can use a variety of microbes and

must have air, anaerobic digestion uses bacteria and an oxygen free

environment to decompose the waste. Aerobic respiration, typical of



composting, results in the formation of Carbon dioxide and water. While the

anaerobic respiration results in the formation of Carbon Dioxide and methane.

In addition to generating the humus which is used as a soil enhancer,

Anaerobic Digestion is also used as a method of producing biogas which can be

used to generate electricity.

Optimal conditions for the process require nutrients such as nitrogen,

phosphorous and potassium, it requires that the pH be maintained around 7

and the alkalinity be appropriate to buffer pH changes, temperature should

also be controlled.

Integrated Solid Waste Management

Integrated Solid Waste Management (ISWM) takes an overall approach to

creating sustainable systems that are economically affordable, socially

acceptable and environmentally effective. An integrated solid waste

management system involves the use of a range of different treatment methods,

and key to the functioning of such a system is the collection and sorting of the

waste. It is important to note that no one single treatment method can manage

all the waste materials in an environmentally effective way. Thus all of the

available treatment and disposal options must be evaluated equally and the

best combination of the available options suited to the particular community

chosen. Effective management schemes therefore need to operate in ways

which best meet current social, economic, and environmental conditions of the

municipality.



SOLID WASTES AS SOURCES OF ENERGY AND FOOD. The latent energy present in its organic fraction can be recovered for gainful

utilisation through adoption of suitable Waste Processing and Treatment

technologies. The recovery of energy from wastes also offers a few additional

benefits as follows:

The total quantity of waste gets reduced by nearly 60% to over 90%, depending

upon the waste composition and the adopted technology;

Demand for land, which is already scarce in cities, for landfilling is reduced;

The cost of transportation of waste to far-away landfill sites also gets reduced

proportionately; and Net reduction in environmental pollution.

It is, therefore, only logical that, while every effort should be made in the first

place to minimise generation of waste materials and to recycle and reuse them

to the extent feasible, the option of Energy Recovery from Wastes be also duly examined. Wherever feasible, this option should be incorporated in the

over-all scheme of Waste Management.

BASIC TECHNIQUES OF ENERGY RECOVERY Energy can be recovered from the organic fraction of waste (biodegradable as

well as non-biodegradable) basically through two methods as follows:

Thermo-chemical conversion: This process entails thermal de-composition of

organic matter to produce either heat energy or fuel oil or gas; and

Bio-chemical conversion: This process is based on enzymatic decomposition

of organic matter by microbial action to produce methane gas or alcohol.

The Thermo-chemical conversion processes are useful for wastes containing

high percentage of organic non-biodegradable matter and low moisture content.

The main technological options under this category include Incineration and Pyrolysis/ Gasification. The bio-chemical conversion processes, on the other

hand, are preferred for wastes having high percentage of organic bio-

degradable (putrescible) matter and high level of moisture/ water content,

which aids microbial activity. The main technological options under this

category are Anaerobic Digestion, also referred to as Biomethanation.

The important physical parameters requiring consideration include:



Size of constituents

Density

Moisture content

Smaller size of the constituents aids in faster decomposition of the waste.

Wastes of the high density reflect a high proportion of biodegradable organic

matter and moisture. Low density wastes, on the other hand, indicate a high

proportion of paper, plastics and other combustibles.

High moisture content causes biodegradable waste fractions to decompose

more rapidly than in dry conditions. It also makes the waste rather unsuitable

for thermo-chemical conversion (incineration, pyrolysis/ gasification) for energy

recovery as heat must first be supplied to remove moisture.

The important chemical parameters to be considered for determining the

energy recovery potential and the suitability of waste treatment through bio

chemical or thermo-chemical conversion technologies include: -

Volatile Solids

Fixed Carbon content

Inerts,

Calorific Value

C/N ratio (Carbon/Nitrogen ratio)

Toxicity

The desirable range of important waste parameters for technical viability of

energy recovery through different treatment routes is given in the Table. The

parameter values indicated therein only denote the desirable requirements for

adoption of particular waste treatment method and do not necessarily pertain

to wastes generated / collected and delivered at the waste treatment facility. In

most cases the waste may need to be suitably segregated/ processed/ mixed

with suitable additives at site before actual treatment to make it more

compatible with the specific treatment method. This has to be assessed and

ensured before hand. For example, in case of Anaerobic digestion, if the C/N

ratio is less, high carbon content wastes (straw, paper etc.) may be added; if it



is high, high nitrogen content wastes (sewage sludge, slaughter house waste

etc.) may be added, to bring the C/N ratio within the desirable range.

Desirable range of important waste parameters for technical viability of energy recovery:

Waste Treatment Method

Basic principle Important Waste Parameters

Desirable Range

Thermo-chemical

conversion

-Incineration

-Pyrolysis

-Gasification

Decomposition of

organic matter by

action of heat.

Moisture content

Organic/

Volatile matter

Fixed Carbon

Total Inerts

Calorific Value (Net

Calorific Value)

< 45 %

> 40 %

< 15 %

< 35 %

>1200 k-cal/kg

Bio-chemical

Conversion

-Anaerobic

Digestion/

Bio-methanation

Decomposition of

organic matter by

microbial action

Moisture content

Organic /

Volatile matter

C/N ratio

>50 %

> 40 %

25-30

Assessment of Energy Recovery Potential A rough assessment of the potential of recovery of energy from MSW through

different treatment methods can be made from knowledge of its calorific value

and organic fraction, as under:

In thermo-chemical conversion all of the organic matter, biodegradable as well

as non-biodegradable, contributes to the energy output:

Total waste quantity: W tonnes

Net Calorific Value: NCV k-cal/kg.

Energy recovery potential (kWh) = NCV x W x 1000/860 = 1.16 x NCV x W

Power generation potential (kW) = 1.16 x NCV x W/ 24 = 0.048 x NCV x W

Conversion Efficiency = 25%



Net power generation potential (kW) = 0.012 x NCV x W

If NCV = 1200 k-cal/kg., then

Net power generation potential (kW) = 14.4 x W

In bio-chemical conversion, only the biodegradable fraction of the organic

matter can contribute to the energy output:

Total waste quantity: W (tonnes)

Total Organic / Volatile Solids: VS = 50 %, say

Organic bio-degradable fraction: approx. 66% of VS = 0.33 x W

Typical digestion efficiency = 60 %

Typical bio-gas yield: B (m3 )= 0.80 m3 / kg. of VS destroyed

= 0.80 x 0.60 x 0.33 x W x1000 = 158.4 x W

Calorific Value of bio-gas = 5000 kcal/m3 (typical)

Energy recovery potential (kWh) = B x 5000 / 860 = 921 x W

Power generation potential (kW) = 921 x W/ 24 = 38.4 x W

Typical Conversion Efficiency = 30%

Net power generation potential (kW) = 11.5 x W

In general, 100 tonnes of raw MSW with 50-60% organic matter can generate

about 1- 1.5 Mega Watt power, depending upon the waste characteristics.

Anaerobic Digestion (AD) In this process, also referred to as bio-methanation, the organic fraction of

wastes is segregated and fed to a closed container (biogas digester) where,

under anaerobic conditions, the organic wastes undergo bio-degradation

producing methane-rich biogas and effluent/ sludge. The biogas production

ranges from 50- 150m3/tonne of wastes, depending upon the composition of

waste. The biogas can be utilised either for cooking/ heating applications, or

through dual fuel or gas engines or gas / steam turbines for generating motive

power or electricity. The sludge from anaerobic digestion, after stabilisation,

can be used as a soil conditioner, or even sold as manure depending upon its

composition, which is determined mainly by the composition of the input

waste.



Fundamentally, the anaerobic digestion process can be divided into three

stages with three distinct physiological groups of micro-organisms:

Stage I: It involves the fermentative bacteria, which include anaerobic and

facultative micro-organisms. Complex organic materials, carbohydrates,

proteins and lipids are hydrolyzed and fermented into fatty acids, alcohol,

carbon dioxide, hydrogen, ammonia and sulfides.

Stage II: In this stage the acetogenic bacteria consume these primary products

and produce hydrogen, carbon dioxide and acetic acid.

Stage III: It utilizes two distinct types of methanogenic bacteria. The first

reduces carbon dioxide to methane, and the second decarboxylates acetic acid

to methane and carbon dioxide.



UNIT – 3 SOIL MICROBIOLOGY Biotic and abiotic interactions, concepts of habitat and niche. Microbial communities; nature, structure and attributes, levels of species diversity, succession and stability, r and k selection, genetic exchange between communities. Biodiversity management and conservation. Role of microbes in organic solid waste treatment matter in various soil types, subterranean microbes. Biogeochemical cycles of carbon, nitrogen, phosphorous and sulphur.

BIOTIC AND ABIOTIC INTERACTIONS Soils are one of the Earth's essential natural resources, yet they are often taken

for granted. Most people do not realise that soils are a living, breathing world

supporting nearly all terrestrial life. Soils and the functions they play within an

ecosystem vary greatly from ODe location to another as a result of many

factors, including differences ie. Climate, the animal and plant life living on

them, soil's parent material, the position of the soil on the landscape, and the

age of soil.

Composition of Soil Soils are composed of four main components:

Mineral particles of different sizes.

Organic materials from the remains of dead plants and animals.

Water that fills open pore spaces.

Air that fills open pore spaces.

The use and function of a soil depends on the amount of each component. For

example, a good soil for growing agricultural plants has about 45% minerals,

5% organic matter, 25% air, and 25% water. Plants that live in wetlands

require more water and less air. Soils used as raw material for bricks need to

be completely free of organic matter.

The Five Soil Forming Factors The properties of a soil are the result of the interaction between the five soil

forming factors. These factors are:

Parent Material:



The material from which soil is formed determines many of its properties. The

parent material of a soil may be bedrock, organic material, construction

material, or loose soil material deposited by wind, water, glaciers, volcanoes, or

moved down a slope by gravity.

Climate: Heat, rain, ice, snow, wind, sunshine, and other environmental forces break

down parent material, move loose soil material, determine the animals and

plants able to survive at a location, and affect the rates of soil forming

processes and the resulting soil properties.

Organisms: Soil is home to large numbers of plants, animals, and microorganisms. The

physical and chemical properties of a soil determine the type and number of

organisms that can survive and thrive in that soil. Organisms also shape the

soil they live in. For example, the growth of roots and the movement of animals

and microorganisms shift materials and chemicals around in soil profile. The

dead remains of soil organisms become organic matter that enriches the soil

with carbon and nutrients. Animals and microorganisms living in the soil

control the rates of decomposition for organic and waste materials. Organisms

in soil contribute to the exchange of gases such as carbon dioxide, oxygen, and

nitrogen between soil and the atmosphere. They also help soil filter impurities

in water. Human actions transform soil as well, as we farm, build, dam, dig,

process, transport, and dispose of waste.

Topography:

The location of a soil on a landscape also affects its formation and its resulting

properties. For example, soils at the bottom of a hill will get more water than

soils on the hillside, and soils on slopes that get direct sunlight will be drier

than soils on slopes that do not.

Time: The amount of time that the other 4 factors listed above have been interacting

with each other affects the properties of the soil. Some properties, such as

temperature and moisture content, change quickly, often over minutes and



hours. Others, such as Mineral changes, occur very slowly over hundreds or

thousands of years.

The soil profiles The five soil forming factors differ from place to place causing soil properties to

vary from one location to another. Each area of soil on a landscape has unique

characteristics. A vertical section at one location is called a soli profile. When

we look closely at the properties of a soil profile and consider the five soil

forming factors, the story of soil at that site and the formation of the area is

revealed.

These layers are known as horizons. Soil horizons can be as thin as a few

millimetres or thicker than a metre. Individual horizons are identified by the

properties they contain that are different from the horizons above and below

them

Some son horizons are formed as a result of the weathering of minerals and

decomposition of organic materials that move down the soil profile over time.

This movement, called illuviation, influences the horizon's composition and

properties. Other horizons may be formed by the disturbance of the soil profile

from erosion, deposition, or biological activity. Soils may also have been altered

by human activity. For example, builders compact soil, change its composition,

move soil from one location to another, or replace horizons in a different order

from their original formation.

Moisture in the Soil Moisture plays a major role in the chemical, biological and physical activities

that take place in soil. Chemically, moisture transports substances through the



profile. This affects soil properties such as colour, texture, pH, and fertility.

Biologically, moisture determines the types of plants that grow in soil and

affects the way the roots are distributed. For example, in desert areas where

soils are dry, plants such as cacti must store water, or send roots deep into soil

to tap water buried tens of metres below the surface.

Plants in tropical regions have many of their roots near the surface where

organic material stores much of the water and nutrients the plants need.

Agricultural plants grow best in soils w~ere water occupies approximately one-

fourth of the soil volume as vapour or liquid. Physically, soil moisture is part of

the hydrologic cycle. Water falls on the soil surface as precipitation. This water

seeps down into soil in a process called infiltration. After water infiltrates soil, it

is stored in the horizons, taken up by plants, moved upward by evaporation, or

moved downward into the underlying bedrock to become ground water. The

amount of moisture contained in a soil can change rapidly, sometimes

increasing within minutes or hours. In contrast, it might take weeks or months

for soils to dry out.

If a soil horizon is compacted, has very small pore spaces, or is saturated with

water, infiltration will occur slowly, increasing the potential for flooding in an

area. If the water cannot move down into soil fast enough, it will flow over the

surface as runoff and may rapidly end up in streams or other water bodies.

When soil is not covered by vegetation and the slope of the land is steep, water

erosion occurs. Deep scars are formed in the landscape as a result of the

combined force of the runoff water and soil particles flowing over the surface.

When a soil horizon is dry, or has large pore spaces that are similar in size to

tne horizon above, water will infiltrate the horizon quickly. If soil gets too dry

and is not covered by vegetation, wind erosion may occur.

Soil Temperature The temperature of a soil can change quickly. Near'th~ .surface, it changes

almost as quickly as the air temperature changes, but because soil is denser

than air, its temperature variations are less. Daily and annual cycles of soil



temperature can be measured. During a typical day, soil is cool in the morning,

warms during the afternoon, and then cools down again at night. Over the

course of the year, soil warms up or cools down with the seasons.

Because soil temperature changes more slowly than air temperature, it acts as

an insulator, protecting soil organisms and buried pipes from the extremes of

air temperature variations. In temperate regions, the surface soil may freeze in

winter and thaw in the spring, while in some colder climates, a permanent

layer of ice, called permafrost, is found below the soil surface. In either case,

the ground never freezes below a certain depth. The overlying soil acts as

insulation so that the temperature of the deeper layers of soil is almost

constant throughout the year. Temperature greatly affects the chemical and

biological activity in soil. Generally, the warmer the soil, the greater the

biological activity of microorganisms living in the soil.

Microorganisms in warm tropical soils break down organic materials much

faster than microorganisms in cold climate soils. Near the surface, the

temperature and moisture of soil affect the atmosphere as heat and water

vapour are exchanged between the land and the air. These effects are smaller

than those at the surfaces of oceans, seas, and large lakes, but can

significantly influence local weather conditions. Hurricanes have been found to

intensify when they pass over soil that is saturated with water. Meteorologists

have found that their forecasts can be improved if they factor soil temperature

and moisture into their calculations. CONCEPTS OF HABITAT AND NICHE

Microorganisms, as they interact with each other and with other organisms in

biogeochemical cycling, also are influenced by their immediate physical

environment, whether this might be soil, water, the deep marine environment,

or a plant or animal host. It is important to consider the specific environments

where microorganisms interact with each other, other organisms, and the

physical environment.



The Microenvironment and Niche The specific physical location of a microorganism is its microenvironment. In

this physical microenvironment, the flux of required oxidants, reductants, and

nutrients to the actual location of the microorganism can be limited. At the

same time, waste products may not be able to diffuse away from the

microorganism at rates sufficient to avoid growth inhibition by high waste

product concentrations. These fluxes and gradients create a unique niche, which includes the microorganism, its physical habitat, the time of resource

use, and the resources available for microbial growth and function.

The Creation of a Niche from a Microenvironment. As shown in this illustration, two nearby particles create a physical

microenvironment for possible use by microorganisms. Chemical gradients, as

with oxygen from the aerobic region, and sulfide from the anaerobic region,

create a unique niche. This niche thus is the physical environment and the

resources available for use by specialized aerobic sulfide-oxidizing bacteria.

This physically structured environment also can limit the predatory activities of

protozoa. If the microenvironment has pores with diameters of 3 to 6 μm, it will

protect bacteria in the pores from predation, while allowing diffusion of

nutrients and waste products. If the pores are larger, perhaps greater than 6

_m in diameter, protozoa may be able to feed on the bacteria. It is important to



emphasize that microorganisms can create their own microenvironments and

niches For example, microorganisms in the interior of a colony have markedly

different microenvironments and niches than those of the same microbial

populations located on the surface or edge of the colony. Microorganisms also

can associate with clays and form “clay hutches” for protection. MICROBIAL COMMUNITIES: NATURE, STRUCTURE AND ATTRIBUTES

Microbial Ecology is the science that specifically examines and determines the

relationship between microorganisms and their biotic and abiotic

environments.

Microbial community Species group microorganisms that can interact and breed together

Population is a group of organism that can interact with each other in an

integrated manner

Community is an integrated assemblage of microbial populations occurring and

interacting within a given location called a habitat. It is the highest biological

uit in an ecosystem.

Ecosystem is the totality of the biotic and abiotic components of an

environment.

Microorganism tends to live in a community, occupying their own niche in their

natural ecosystem. Generally, the species composition within an environment

may remain relatively stable provided there’s no drastic or unexpected change

in the environment. Any change in the environment/condition may lead to shift

in specie composition e..g in ripening banana, there’s an increase in glucose

concentration which causes yeast fermentation. Yeasts ferments carbohydrate

to alcohol. The alcohol is then oxidized to acetic acid which is required by a

fungus for its existence. Hence a microbial community succession is put in

place.

Indigenous/Autochthonous: They are the original inhabitant of an

environment. i.e. native organisms. e.g. E. coli in the GIT of man,

Staphylococcus albus on the skin, Streptococcus in the mouth.



Invaders/Allochthonous organisms: They usually originate from another place.

That is they are not native to the environment i.e. either in a vegetative resting

stage, spore, cyst, endospore. This include organism present on our skin, on

leaf surfaces which are derived and mainly from air. e.g. Puccinia graminis on

wheat, Mycobacterium tuberculosis in the lungs of man, Staphylococcus aureus

on the skin. Usually these alien organisms cannot cope with the biological

stress or the abiotic conditions in the environment.

Criteria proposed for isolation of indigenous organism Repeatedly isolate the organism many times from the same environ. Repeated

isolation indicates the presence of the organism in that very environment.

The organisms should be present in high population density in the

environment.

Such organism should be able to make use of the nutrients in the environ

when isolated in pure culture.

The organism should be able to tolerate environmental stress that is typical of

that particular environment.

LEVELS OF SPECIES DIVERSITY, SUCCESSION AND STABILITY Species diversity varies from one ecosystem to another; however the reasons for

the heterogeneity are not known.

Diversity is usually limited to their habitat because such organism found

would be limited to the environment.

Some habitat may be densely populated while others would have low number

of organism due to fluctuations of nutrient

Sewage water →Pond →Tap water →Distilled

It is not nutrient alone that determines the extent of diversity i.e. Diversity can

be low despite high nutrients and at the same time diversity can be low with

low nutrients.

Low species diversity characterized areas in the environment is due to one or

more ecological factors that approach the extreme Dispersal

Active method of dispersal

Passive method of dispersal



Dispersal is essential for the continuous existence of the microorganisms. The

microorganism either being a fastidious or obligate parasite will still need to be

dispersed from place to place. This is important so that the species may be able

to escape from detrimental or nutrient depleted environment, to continue its

existence in a more favourable environment. Microorganisms are usually

dispersed from centres of dispersals. This serves as the point from which the

propagules are disseminated e.g. Bacillus anthracis found in cattle. The greater

the efficiency of our dispersal mechanism, the smaller the number of

propagules that will be necessary for successful dissemination of

microorganisms e.g. during sneezing, a lot of microorganisms are released into

the air for respiratory infections e.g. Mycobacterium tuberculosis these are

expelled in large quantities. Puccinia graminis disperses large quantities of

spores. Algae also disperse large quantity of spore. Transmission can be

initiated as a result of some physical contact between the source of propagule

and the physical carrier/vector e.g. many of the propagules will not develop in

the soil unless it comes in contact with a suitable host e.g. Bacillus anthracis,

unless it comes in contact with a ruminant. Clostridum tetani which is a soil-

borne organism will not cause infection unless it comes in contact with an

open wound.

Active transmission This can be either by locomotion or taxis movement. Locomotion may be by

swarming, swimming or by the use of some locomotory organelles e.g.

swimming in the protozoans mastigotes, ciliates and the sarcodina. Presence of

flagella on some bacteria e.g. Salmonella sp. In fungi and algae, the production

of zoospores.

Taxis movement Response to light i.e. phototaxis exhibited by Rhodospirillim sp. It initiate

movement in the presence of light.

Chemotaxis: Movement due to the presence of chemical substance in the

environ e.g.



Phycomycutes.

Thigmotaxis: Movement initiated by avoiding obstacles on their way.

Thermotaxis Geotaxis Passive method Most microorganisms are dispersed mainly by the passive method of dispersal.

This is carried out through various media.

Air, Water, Soil, Biological vectors, Inanimate objects

Dispersal by air is very common among fungi, because many of the organisms

are blown by air, once in air they are propelled.

Features Possession of a means of being picked up i.e. they grow out of their

substratum, which render them vulnerable to the air.

They are resistance to fluctuations in temperature and humidity.

They possess spores or other features that maintain them in the air.

Release spores

The spores may be powdery e.g. puffballs

The type of organism in the air varies with altitude and climatic condition.

Some pathogenic organisms can be spread through the air, which may be

discharge through sneezing, coughing. e.g. Mycobacterium tuberculosis, etc

among the organisms that are carried in the dust, in human washes etc. For

plant derived microorganism they can be obtained by splash.

Soil/Water The dispersal of microorganism in water is diverse, a lot of microorganisms can

be encountered. Organisms can be transfered through water from far east to

the heart of Europe by the ship hull. In ground water, no photosynthesis

microorganisms in them but surface water can serve as an efficient

environment for photosynthesis microorganisms. For microorganism that can

penetrate the layer of the soil. They can be found in septic tanks polluted

water.



Factor affecting the distribution of microorganisms The amount/type of microorganisms found in water depends on the presence

of nutrients in the water.

Floating object present in water bodies may act as a vector of dispersal of

microorganisms form one place to another.

Lateral movement during rainfall may cause dispersal of microorganisms from

soil to water.

Discharge of industrial wastes and domestic sewage into water bodies may be a

source of transmission of microorganism from water to soil.

Inanimate It is possible when viable propagules comes in contact with an inanimate

object.

Inadequate cleaning of working equipment may lead to microorganism

dispersal which may affect the product.

Unhygienic handling may lead to contamination of the product e.g. using

unsterilized materials, contaminated hospital equipment, toilet seats,

agriculture implements, food and food products e.g. salmonella and shigella.

Milk and milk product may spread: Mycobacterium tuberculosis, Brucella,

Streptococcus sp Coxiella (indicator for pasteurization)

Biological Vectors Some microorganisms would not survive on their own in the environment

unless transported by biological vectors. They may act as a mean of transport

while others live on the biological vector as a parasite. The biological vector

may gain access into the host through the environment e.g. the honey bee may

act as a good vector to Botrytis arthrophilia (a fungus). From one organism to

the other, like can act as a vector for the transfer of Rickettsia, dog is the

carrier of the Rabies virus, earthworm can be a vector for many soil

heterotrophic microorganisms. snails – Phytophthora palmivora.

Colonization In any environs, that is colonized by microorganism, there are some that are

pioneer microorganism. They are the first to occupy a site or the group that



seems to appear first through they may not be sterile, but they are found to be

the dominant microorganism there. The very 1st group of microorganism can

withstand very hard environ are the algae. But to be able to survive in any

environment microorganism should posses some degree of invasiveness i.e.

being able to utilize the nutrients that are present in the environ e.g. the blue

green algae on bare rocks e.g. (Oscilltoria).

But in a rich environ, the heterotrophy are the usual colonizers. In the GIT of

neonates, it is expected to be sterile, but along the line it is occupied by some

organism ref to as the normal flora e.g. Streptococcus, Lactobacillus after taken

some food or milk.

Three are different barriers that can be overcome, e.g. the fatty acids on the

skin, enzymes in the blood, the body fluids and other barriers found on the

GIT. In plants, tree barks contains alkaloids which are antimicrobial in nature.

These are produced by plant to present colonization by microorganisms also,

resins, gum, quinines and quinones etc.

Succession and climax Environmental feedback is the situation whereby the organisms which are able

to colonize an environment may be subjected to shift in specie composition. It

is the modification of the habitat resulting from the presence of one or more

microbial population such feedbacks may affect: the size, activities as well as

survival. After the organisms have been dispersed by the air, they try to make

use of the materials in the environment. Once established, they tend to annexe

all the nutrients in the environment to provide for their metabolic activities.

When simple organism materials are introduced into the soil, there is always

the appearance of specialized groups of organisms and they are able to utilize

the materials present in the compound. The first group of the organisms may

be replaced by such organism that can make use of the waste products of the

pioneer organism.These organisms may not be new organisms coming in but

that they can utilize the product of the primary organisms. When an organic

matter is introduced into the environment (e.g. acetic acid). There’s a

progressive development from the pioneer community which contain few



species than a series of stages characterised by an increasing number of

organisms and to a phase that typified a high species diversity. e.g. Palm-wine

when from the tree contains sucrose mainly. After the tapping it derives

microorganisms from the air, gourd and during dripping. These

microorganisms aid the fermentation of it to Alcohol, which when left over or

for sometime it turns to Acetic acid (oxidation process) by acetic acid bacteria.

This is a good example of bacterial succession (Autogenic succession).

Autogenic: It is the one in which the indigenous alter their environment in

manner such that they are replaced by species better suited to the modified

environment.

Allogenic succession: This is one in which one type of community is replaced

by another because the habitat is altered by non-microbial factors. Such

factors include physical and chemical factors.

Factors that determines succession The provision by one community of nutrient that confers an ecological

advantage on that species constituting the next stage of succession. E.g. in

pioneer community that are able to utilize the nutrients that are present in the

environments. e.g. On a rock surface.

Making available of a population of nutrient present in sufficient supply to

allow for the growth of latter population such situation may occur when the

primary colonization excretes carbonaceous material that may be utilizable by

other specie present in the surrounding e.g. (a) Lactic acid bacteria action on

milk to produce acid. (b) Cellulose can be decomposed anaerobically by

Clostridium specie. (c) Algae during photosynthesis can produce some

intermediates products such as organic acid, simple CHO that may be ideal for

other organisms in the habitat. (d) Alteration in the colonization of an inorganic

nutrients, any influence succession e.g. sudden bloom in rivers and lakes e.g.

The Red sea which is due to the presence of Red algae in the sea.

Modification of the heterogenous substrates such that constituents favouring

the growth of the main species are exposed to attack while the initial



constitutes is lost to composition e.g. Phycomyces that are replaced by the slow

growing fungi, which will utilize the product of the Phycomyces.

Autointoxication: This is as a result of the fact that some organisms’ produces

toxins against themselves e.g. the Lactic acid bacteria in the wine help in

stabilizing the wine and destroy itself by its own high activity due to the high

acid level.

Elimination of microorganisms by physical means: Removal of a group of

microorganisms from their natural habitat, will affect the level of succession in

the environment.

Appearance of barriers with environmental feedbacks Usually possible in areas where there are antibodies, phytoalexins and

phagocytes. They are able to contribute to allogenic succession by selectively

destroying or retarding the growth of some microorganisms living in the host.

Such feedback mechanisms may provide a response resulting to the

displacement of the population that is sensitive to the foreign substances

Selective feeding by animals on microbial population E.g. fishes that feed mainly on algae and thus others organism will be more

dominant.

Change in temperature and light intensity Characteristics that aids/favours colonization and succession Presence at the colonizable site at the right time. It is only possible with good

dispersal mechanism and the organism is able to grow.

The organism should be able to survive for some time if conditions are not yet

suitable.

The organism should be able to develop when good condition set its.

They should be able to obtain all required nutrients from the ecosystem.

They should have the capacity to tolerate all the ecological abiotic factors in the

environment; pH, temperature, osmotic pressure, ox-red potential etc.

The organism should possess adequate mechanism to overcome or cope with

environmental resistance that may be caused by the inanimate components of

the environment.



Ability to overcome environmental resistance due to presence of other

microorganisms.

This is usually common in parasitic organisms. r AND k SELECTION - GENETIC EXCHANGE BETWEEN COMMUNITIES

r/K selection theory relates to the selection of combinations of traits in an

organism that trade off between quantity and quality of offspring. The focus

upon either increased quantity of offspring at the expense of

individual parental investment, or reduced quantity of offspring with a

corresponding increased parental investment, varies widely, seemingly to

promote success in particular environments.

In this context, r-selection makes a species prone to numerous reproductions

at low cost per individual offspring, while K-selected species expend high cost

in reproduction for a low number of more difficult to produce offspring. Neither

mode of propagation is intrinsically superior, nor in fact can they coexist in the

same habitat, as in rodents and elephants.

In r/K selection theory, selective pressures are hypothesised to

drive evolution in one of two generalized directions: r- or K-selection. These

terms, r and K, are drawn from standard ecological algebra, as illustrated in

the simplified Verhulst model of population dynamics.

where r is the maximum growth rate of the population (N), and K is

the carrying capacity of its local environmental setting, and the

notation dN/dtstands for the derivative of N with respect to t (time). Thus, the

equation relates the rate of change of the population, N, to the current

population size, and expresses the effect of the two parameters. As the name

implies, r-selected species are those that place an emphasis on a high growth

rate, and typically exploit less-crowded ecological niches and produce

many offspring, each of which has a relatively low probability of surviving to

adulthood (i.e., high r, low K).



By contrast, K-selected species display traits associated with living at densities

close to carrying capacity, and typically are strong competitors in such crowded

niches that invest more heavily in fewer offspring, each of which has a

relatively high probability of surviving to adulthood (i.e., lowr, high K). In

the scientific literature, r-selected species are occasionally referred to as

"opportunistic", while K-selected species are described as "equilibrium".

r-selection In unstable or unpredictable environments, r-selection predominates as the

ability to reproduce quickly is crucial. There is little advantage in adaptations

that permit successful competition with other organisms, because the

environment is likely to change again. Traits that are thought to be

characteristic of r-selection include: high fecundity, small body size, early

maturity onset, short generation time, and the ability to disperse offspring

widely.]

Organisms whose life history is subject to r-selection are often referred to as r-

strategists or r-selected. Organisms who exhibit r-selected traits can range

from bacteria and diatoms, to insects and weeds, to various semelparous

cephalopods and mammals, particularly small rodents.]

k-selection In stable or predictable environments, K-selection predominates as the ability

to compete successfully for limited resources is crucial and populations of K-

selected organisms typically are very constant and close to the maximum that

the environment can bear (unlike r-selected populations, where population

sizes can change much more rapidly).

Traits that are thought to be characteristic of K-selection include large body

size, long life expectancy, and the production of fewer offspring, which often

require extensive parental care until they mature. Organisms whose life history

is subject to K-selection are often referred to as K-strategists or K-selected.

Organisms with K-selected traits include large organisms such as elephants,

humans and whales, but also smaller, long-lived organisms such as Arctic

Terns



BIODIVERSITY MANAGEMENT AND CONSERVATION Biodiversity" is most commonly used to replace the more clearly defined and

long established terms, species diversity and species richness. Biologists most

often define biodiversity as the "totality of genes, species, and ecosystems of a

region". An advantage of this definition is that it seems to describe most

circumstances and presents a unified view of the traditional three levels at

which biological variety has been identified:

Species diversity

Ecosystem diversity

Genetic diversity

Biodiversity is not evenly distributed; rather it varies greatly across the globe as

well as within regions. Among other factors, the diversity of all living things

(biota) depends on temperature, precipitation, altitude, soils, geography and

the presence of other species. The study of the spatial distribution

of organisms, species, and ecosystems, is the science of biogeography.

The International Union for the Conservation of Nature (IUCN) has organized a

global assortment of scientists and research stations across the planet to

monitor the changing state of nature in an effort to tackle the extinction crisis.

The IUCN provides annual updates on the status of species conservation

through its Red List. The IUCN Red List serves as an international conservation

tool to identify those species most in need of conservation attention and by

providing a global index on the status of biodiversity. More than the dramatic

rates of species loss, however, conservation scientists note that the sixth mass

extinction is a biodiversity crisis requiring far more action than a priority focus

on rare, endemic orendangered species. Concerns for biodiversity loss covers a

broader conservation mandate that looks at ecological processes, such as

migration, and a holistic examination of biodiversity at levels beyond the

species, including genetic, population and ecosystem diversity. Extensive,

systematic, and rapid rates of biodiversity loss threatens the sustained well-

being of humanity by limiting supply of ecosystem services that are otherwise

regenerated by the complex and evolving holistic network of genetic and



ecosystem diversity. While the conservation status of species is employed

extensively in conservation management, some scientists highlight that it is the

common species that are the primary source of exploitation and habitat

alteration by humanity. Moreover, common species are often undervalued

despite their role as the primary source of ecosystem services.

While most in the community of conservation science "stress the importance"

of sustaining biodiversity, there is debate on how to prioritize genes, species, or

ecosystems, which are all components of biodiversity (e.g. Bowen, 1999). While

the predominant approach to date has been to focus efforts on endangered

species by conserving biodiversity hotspots, some scientists (e.g) and

conservation organizations, such as the Nature Conservancy, argue that it is

more cost effective, logical, and socially relevant to invest in biodiversity

coldspots. The costs of discovering, naming, and mapping out the distribution

every species, they argue, is an ill advised conservation venture. They reason it

is better to understand the significance of the ecological roles of species.

Biodiversity hotspots and coldspots are a way of recognizing that the spatial

concentration of genes, species, and ecosystems is not uniformly distributed on

the Earth's surface. For example, "[...] 44% of all species of vascular plants and

35% of all species in four vertebrate groups are confined to 25 hotspots

comprising only 1.4% of the land surface of the Earth."

Those arguing in favor of setting priorities for coldspots point out that there are

other measures to consider beyond biodiversity. They point out that

emphasizing hotspots downplays the importance of the social and ecological

connections to vast areas of the Earth's ecosystems where biomass, not

biodiversity, reigns supreme. It is estimated that 36% of the Earth's surface,

encompassing 38.9% of the world’s vertebrates, lacks the endemic species to

qualify as biodiversity hotspot. Moreover, measures show that maximizing

protections for biodiversity does not capture ecosystem services any better than

targeting randomly chosen regions. Population level biodiversity (i.e. coldspots)

are disappearing at a rate that is ten times that at the species level. The level of

importance in addressing biomass versus endemism as a concern for



conservation biology is highlighted in literature measuring the level of threat to

global ecosystem carbon stocks that do not necessarily reside in areas of

endemism. A hotspot priority approach would not invest so heavily in places

such as steppes, the Serengeti, the Arctic, ortaiga. These areas contribute a

great abundance of population (not species) level biodiversity and ecosystem

services, including cultural value and planetary nutrient cycling.

Those in favor of the hotspot approach point out that species are irreplaceable

components of the global ecosystem, they are concentrated in places that are

most threatened, and should therefore receive maximal strategic protections.

ROLE OF MICROBES IN ORGANIC SOLID WASTE TREATMENT MATTER IN VARIOUS SOIL TYPES

The soil contains a vast array of life forms ranging from submicroscopic (the

viruses), to earthworms, to large burrowing animals such as gophers and

ground squirrels. Microscopic life forms in the soil are generally called the "soil microflora" (though strictly speaking, not all are plants in the true sense of the

word) and the larger animals are called macrofauna.

Soil animals, especially, the earthworms and some insects tend to affect the

soil favorably through their burrowing and feeding activities which tend to

improve aeration and drainage through structural modifications of the soil

solum. In general, they affect soil chemical properties to a lesser extent though

their actions indirectly enhance microbial activities due to creation of a more

favorable soil environment.

Soil Microorganisms Soil microorganisms occur in huge numbers and display an enormous diversity

of forms and functions. Major microbial groups in soil are bacteria (including

actinomycetes), fungi, algae (including cyanobacteria) and protozoa.

Because of their extremely small cell size (one to several micrometers),

enormous numbers of soil microbes can occupy a relatively small volume,



hence space is rarely a constraint on soil microbes. Soil microbes can occur in

numbers ranging up to several million or more in a gram of fertile soil (a

volume approximately that of a red kidney bean). Note that the bacteria are

clearly the most numerous of the soil microbes. Perhaps more important than

the numbers of the various soils microbes is the microbial biomass contributed by the respective groups. It is the soil fungi which tend to

contribute the most biomass among the microbial groups. In fact, it is because

of their large contribution to the biomass that they are generally regarded as

being the dominant decomposer microbes in the soil. You might find it

surprising that there are literally "tons" of microbes beneath your feet as you

walk across a grassland in Africa or Australia or through a cornfield in the

American Midwest. Interestingly, a fungus discovered in the state of Michigan

may be one of the largest living organisms on the planet.

A fungus, Armillaria bulbosa, discovered in the U.S. in the state of Michigan,

could turn out to be earth's largest creature or at least among the largest.

Scientists discovered the fungus growing among the roots of hardwood trees in

a forest. The microscopic, branched filaments (called hyphae) of the fungus

occupy a 14.8 ha (37-acre) area of land. Careful genetic analysis has shown the

filaments constitute a single organism. Fungi generally radiate outward in a

circular pattern as they grow through the soil. In fact, the fairy rings of

mushrooms (named because ancient peoples thought they represented the

paths of fairies dancing in the night) often seen in lawns or on golf courses

actually represent the outer boundary of a developing fungus. Scientists

estimate that the portion of the Michigan fungus they have been able to identify

may weigh as much as 100 tons, slightly less than a blue whale. Imagine the

biochemical capacity of a soil microorganism this large!

The significance of these large amounts of microbial biomass in the soil lies not

only in their large biochemical capacity, but in the phenomenal diversity of

biochemical reactions attributed to the soil microbial population. It is worth

remembering that soil microbes not only interact with other members of their

own group, they also interact with other microbial groups. It is quite common



to find, for example, that degradation of plant materials occurs much more

quickly in the presence of the mixed soil population than it does when one or

more groups of soil microbes have been eliminated from the system.

Soil life can be divided into trophic (i.e. feeding) levels. At the base of the

trophic levels lies the soil microbial population which degrades plant, animal

and microbial bodies, and also serves as the food source for some of the levels

above it. For example, soil protozoa consume enormous numbers of bacteria

and even some fungal spores. These in turn are consumed by still larger soil

animals (nematodes, mites, etc.) which in turn are eaten by still larger animals

(e.g. worms and insects). Thus, nutrients flow through this microbial food web

which lies at the heart of controlling soil fertility and plant productivity in the

absence of external inputs such as fertilizers. In fact, the role of soil microbes

in degrading organic materials and thereby regenerating a supply of carbon

dioxide for plants is perhaps their most vital global function.

Nutrient Cycling by Soil Microbes Soil microbes exert much influence in controlling the quantities and forms of

various chemical elements found in soil. Most notable are the cycles for carbon,

nitrogen, sulfur and phosphorus, all of which are elements important in soil

fertility, and as we know today, may be involved in global environmental

phenomena. The mineralization (i.e. the conversion of organic forms of the

elements to their inorganic forms) of organic materials by soil microbes

liberates carbon dioxide, ammonium (which is rapidly converted to nitrate by

soil microbes), sulfate, phosphate and inorganic forms of other elements. This

is the basis of nutrient cycling in all major ecosystems of the world. John

Burroughs once said, "Without death and decay, how could life go on?" No

doubt, he was referring to the mineralization of nutrients from dead animals

and plants. We now know that soil microbes accomplish this task with

remarkable zeal and that in the process a substantial part (perhaps as much

as one third) of the decomposing materials are converted to the bodies of soil

microbes. This pool of microbial biomass constitutes a portion of the soil

organic matter which turns over (cycles) fairly quickly and therefore represents



a "fertility buffer" in the soil. Don't forget that the liberation of carbon dioxide

through microbial respiration makes possible the continued photosynthesis

(i.e. carbon dioxide fixation) by algae and green plants which in turn produce

more organic materials which may ultimately reach the soil, thereby

completing the cycle.

In the world's agricultural soils, the source of our food supply, mineralization of

nitrogen by soil microbes is a most important process. In those soils not

receiving external inputs of fertilizer nitrogen (e.g. most forested lands and

many grasslands) the liberation of ammonium from organic debris makes

possible the continued growth of new plant matter. Therefore, it is the soil

microbial population which controls the productivity of these soils if other

environmental factors (moisture, temperature) are suitable. In fact, fertilization

of a soil represents our attempt to balance the competition between plants and

soil microbes for available soil nitrogen. Nitrogen tied-up (assimilated into cell constituents) in microbial cells is not available for plants or other microbes

until that tissue has been decomposed by other microbes. In other words,

nitrogen contained in tissues is said to be immobilized. Microbes are the keys

for the remobilization of these nutrients. These mineralization/immobilization

phenomena are common to all the elements but typically they are only

agriculturally important for the macronutrients such as nitrogen, phosphorus

and sulfur.

Aside from their role in controlling the rates of production of inorganic forms of

nitrogen and sulfur, soil microbes, in particular soil bacteria, can control the

forms of the ions in which these nutrients occur. For example, ammonium

(NH4+) in the soil is usually rapidly oxidized by bacteria first to nitrite

(NO<sub<2< sub="">-) and then to nitrate (NO3-) which may readily leach

through soil. Ammonium is oxidized to nitrite and then to nitrate by the

bacteria Nitrosomonas and Nitrobacter, respectively. Thus, bacteria can

influence the form and, thereby, the retention of nitrogen in the soil. Similarly,

reduced sulfur compounds such as thiosulfate, elemental sulfur and even iron

pyrite (FeS2, "Fool's Gold") can be oxidized to sulfuric acid by soil bacteria. The



bacteria which accomplish the oxidation of reduced nitrogen and sulfur

compounds use these materials as energy sources to drive their metabolism.

Unlike the decomposer microbes which use organic carbon compounds from

organic matter for energy and to make cell matter (e.g. they are called

heterotrophs), these specialized bacteria called chemoautotrophs obtain their

carbon for cell synthesis from carbon dioxide or from dissolved carbonate.

</sub<2<> There are many genera of bacteria that can oxidize reduced sulfur

compounds. However, much of this activity, especially the oxidation of sulfur

and pyrite, can be attributed to bacteria of the genus Thiobacillus (thio = sulfur;

bacillus = rod-shaped bacterium). Thiobacillus thiooxidans can oxidize

elemental sulfur to sulfuric acid. Sulfur, therefore, can be used to decrease the

pH of an alkaline soil. Thiobacillus ferrooxidans attacks both the iron and

sulfur in iron pyrite, generating sulfuric acid and dissolved iron in the process.

This is also the basis of acid mine drainage associated with the mining of coal

throughout the world.

The long-term application of ammonium-based fertilizers can likewise result in

the acidification of agricultural soils through bacterial nitrification (the

conversion of ammonium to nitrate with the concurrent production of acidity).

Thus, we see that certain environmental problems can arise from the activities

of these chemoautotrophic soil bacteria.

Another important aspect of nutrient cycling is that under certain

circumstances nitrogen and sulfur may be converted to gaseous forms

(volatilized) and lost to the atmosphere. Nitrogen in the form of nitrate can be

converted to gases such as nitrous oxide (N2O) and dinitrogen (N2) through the

process of denitrification (the bacterial reduction of NO3- to N2O or N2) by soil

bacteria under anaerobic conditions. A consequence of denitrification is that

nitrogen, a precious nutrient for plants, is lost from the soil. On the other

hand, this process is a useful way to remove excess nitrate from wastewater.

Sulfur in the form of sulfate (SO4-2) is used by anaerobic bacteria like the

genus Desulfovibrio which convert it to hydrogen sulfide gas (H2S). Hydrogen

sulfide reacts with metal ions and forms very insoluble metallic sulfides like



pyrite (Fe2S). In fact, it is probable that the pyrites associated with coal seams

were deposited by the action of these bacteria eons ago. The black color of salt

marsh soils and the rotten egg smell associated with them are a result of the

activities of the sulfate-reducing bacteria in these habitats. They attest to the

occurrence of anaerobic conditions. Sulfur volatilization from soil represents

loss of a plant nutrient as well as a contribution of atmospheric sulfur which

may contribute to the phenomenon of acid precipitation.

We mentioned above that nitrogen can be lost from agricultural soils as well as

from other ecosystems. Fortunately, this "leak" in the terrestrial nitrogen cycle

can be at least partially replaced through another important biological process

called biological nitrogen fixation. In this process, which is unique to

bacteria and a few other microbes, notably the cyanobacteria (blue-green

algae), atmospheric dinitrogen (N2) is captured and converted to plant-available

forms. Biological nitrogen fixation is carried out by free-living bacteria and

cyanobacteria and by symbiotic microorganisms in a wide variety of

mutualistically symbiotic associations with higher plants.

The most useful and probably the most widely recognized example of symbiotic

nitrogen fixation is that of the Rhizobium - legume root-nodule symbiosis. Soil

bacteria belonging to the genera Rhizobium and Bradyrhizobium (and a few

others) are capable of inducing the formation of nodules on roots of specific

legumes (plants like peas, beans, peanuts, soybeans, alfalfa etc.) and fixing

large quantities of nitrogen in these structures. In the nodule, the bacteria are

supplied with carbon sources (photosynthate from the plant) that they need in

order to fix nitrogen. In return for this carbon, the bacteria fix atmospheric

nitrogen which is converted to amino acids used by the plant for growth. The

result of this unique plant-microbe partnership is that many legumes are self-

sufficient for nitrogen, that is, they are nearly independent of a supply of

nitrogen from the soil. It is no wonder that these plants are cultivated all over

the world as sources of food, fiber and forage. Nearly two-thirds of the world's

nitrogen supply is from biological nitrogen fixation. Legumes have been used

since the beginning of recorded history as "soil improving" crops known as



"green manures". Green manuring is the practice of growing a legume species

for the sole purpose of returning it to the soil to serve as a source of nitrogen

for an ensuing crop.

SUBTERRANEAN MICROBES Subterranean fauna are defined as fauna which live their entire lives (obligate)

below the surface of the earth. They are usually divided into two groups:

Stygofauna - aquatic and living in groundwater; and

Troglofauna - air-breathing and living in caves and voids.

Fauna that use a subterranean or cave environment for only part of the day

(e.g. soil-dwelling and burrowing species or cave-dwelling bats or birds) are not

defined as subterranean fauna. Subterranean fauna often display evolutionary

adaptations to underground life, particularly reduced pigment and reduced,

poorly functioning or non-existent eyes.

A community of bacteria that obtains its energy not from sunlight, but from

radioactive decay

Key taxonomic groups Examples of subterranean fauna are found within a large number of

invertebrate fauna groups, and invertebrate groups dominate the subterranean

fauna.

Crustacean groups including subterranean representatives are remipedes,

ostracods, isopods, copepods, syncarids, amphipods and decapods. Hexapod

groups include Blattodea (cockroaches), Orthoptera (crickets), Coleoptera

(beetles), Hemiptera (bugs), Thysanura (thrips), Diplura and Collembola

(springtails). Subterranean arachnid groups include Aranae (spiders),

pseudoscorpions, schizomids, Trombidiformes (mites), Opiliones (harvestmen),

and scorpions. Myriapod groups are also represented – diplopods (millipedes)

and chilipods (centipedes). Oligochaete, polychaete and aphanoneuran worms

are represented. Two main gastropod groups are known to include

subterranean fauna - Neotaeniglossa (family Hydrobiidae) and

Basommatophora (family Planorbidae). Stygofauna communities are often



dominated by crustaceans whereas troglofauna can include a wide range of

taxonomic groups which have adapted to underground life.

Subterranean communities share the following characteristics:

High endemism but low local diversity relative to regional diversity;

A relatively small number of genetic lineages resulting in species which look

dissimilar to related groups;

Many relicts from previous climatic conditions; and

Truncated food webs

Origins of subterranean fauna There are two main hypotheses regarding the origins of subterranean fauna

which account for the observed species diversity and vicariant distributions.

These hypotheses are not mutually exclusive:

Adaptive shift, where a surface species has pre-adaptations which allow it to

expand into subterranean environments thus inferring that subterranean

species are descended from previous surface or aquatic fauna. Changed

climatic conditions at the surface may cause significant changes to surface

fauna.

Subterranean environments acting as below ground “islands” providing a

mechanism for genetic isolation and evolutionary radiation.

Gene flow within subterranean habitats There are two main methods for describing gene flow within a subterranean

population (Gentile & Sbordoni 1998):

Direct - attempts to estimate gene flow by measuring dispersal of individuals;

and

Indirect - estimate gene flow from the spatial distribution of gene frequencies,

under the assumption that there is a balance between drift and gene flow.

BIOGEOCHEMICAL CYCLES OF CARBON Water is the source of oxygen for O2, and the aerobic atmosphere of Earth is

produced by continuous photosynthetic processes. When O2production initially

occurred on Earth, there were no aerobic organisms and molecular oxygen

accumulated. A robust collection of biological systems evolved to consume O2



through respiration, and currently there is equilibrium between O2 production

and O2 consumption. When compared to other elements, the oxygen cycle is

relatively simple. Large numbers of microorganisms interface with the oxygen

cycle through redox reactions.

The cycling of oxygen

Solid lines indicate reduction; dashed lines indicate oxidation. Heterotrophic

and chemolithotrophic microorganisms associated with the use of oxygen as

the terminal electron acceptor. Cyanobacteria were associated with the

photosynthetic release of molecular oxygen from water.

There is an interconnection between the oxygen and carbon cycles because

oxygen reserves are found in organic compounds and CO2.

With carbon as a principal element in cell systems, it would follow that

distribution, fluxes, and reserves of carbon would be of global interest.

Microorganisms have an important role in all aspects of the carbon cycle.

Primary producers are the organisms that fix carbon dioxide, and while higher

plants are important in terrestrial areas, algae and cyanobacteria account for

primary productivity in marine environments. Nonphotosynthetic fixation of

carbon dioxide by chemolithotrophic or heterotrophic bacteria accounts for

relatively small amounts of carbon transferred from the atmosphere to

biomass. Large quantities of carbon are dissolved in ocean water as

bicarbonate ion (HCO3-), and comparable quantities of primary productivity



occur in marine and terrestrial systems. Different groups of microorganisms

are used for the cycling of C under aerobic and anaerobic conditions

Microbial metabolism of carbon under anaerobic and aerobic conditions

A biogeochemical cycle involving major groups of microorganisms is shown in

Figure.

Soil is a great reservoir for carbon with appreciable quantities of complex

organic material known as humus is stable with a very low turnover rate.

Although carbon dioxide is released from plants and animals by respiration,

great quantities of carbon dioxide result from decomposition of organic matter

by microorganisms. Methane is produced by anaerobic archaea, and the

resulting methane hydrates produced may account for a considerable

percentage of total carbon on Earth.

Oxidation of methane with production of carbon dioxide is attributed to aerobic

bacteria. Carbon monoxide is produced by a few bacteria as a result of

decomposition, and this gas does not become toxic because aerobic bacteria

readily oxidize carbon monoxide to carbon dioxide.



Distribution of organic carbon on Earth; considerable variation in estimation of gas hydrates occurs because of the model used (OM = organic matter)

BIOGEOCHEMICAL CYCLES OF NITROGEN

Microorganisms require nitrogen to make up 10% of their cellular composition,

and the activities of bacteria also can have considerable influence on the

nitrogen cycle. The principal reservoir for nitrogen is the atmosphere.

According to the amount of organic nitrogen, land organisms contain about 5 ×

1015 g N, soil contains 6.5 × 1015 g N, and oceans contain about 8 × 1017 g N.

Approximately 1.7 × 1014 g N per year are converted to NH3 by nitrogen-fixing

prokaryotes as either free-growing or as symbiotic bacteria and in comparison,

lightning combustion accounts for about

1.9 × 1013 g N fixed per year.

When one considers the global cycle of nitrogen and the rates of conversions,

the amount of time that a nitrogen molecule would be present in the organic

form is about 370 years.

The role of microorganisms in the nitrogen cycle. Solid lines indicate reduction,



dashed lines indicate oxidation, and dotted lines indicate no change in oxidation state of N. Microorganisms associated with the following reactions: (1) many organisms; (2) Nitrosomonas; (3) Nitrobacter; (4) E. coli and many other bacteria; (5) assimilatory reduction by many organisms; (6) oxidative deamination; (7, 8) many organisms; (9) Bacillus, Paracoccus, and Pseudomonas; (10) aerobic—Azotobacter, cyanobacter; anaerobic—Clostridium and anaerobic photosynthetic bacteria; symbiotic—Rhizobium, Bradyrhizobium, and Frankia; (11) Brocadia sp.

Nitrogen Fixation One important aspect of the nitrogen cycle is biological nitrogen fixation, which

is the conversion of atmospheric N2 to ammonia by prokaryotes. The

enzymology for this reaction is unique in that the reduction of the triple bond

between nitrogen atoms requires nitrogenase, which is activated by an electron

donor (e.g., pyruvate). Electrons are transferred to the nitrogen atoms by a

special iron–molybdenum complex. At least 16 ATP is required to energize the

substrate–enzyme complex, making this an extremely energy-expensive

reaction.



A feature that is poorly understood is that nitrogenase releases one molecule of

H2 along with two molecules of NH3. Nitrogenase with the FeMo metal center is

the most common enzyme; however, some bacteria produce alternate enzymes

containing iron plus vanadium or only iron. These alternate enzymes are

produced when molybdenum or vanadium is limiting in the environment, and

this underscores the importance of nitrogen fixation to support prokaryotic

growth.

Bacteria that fix nitrogen are shown in Figure.

A phylogenic tree showing major groups of Bacteria; nitrogen fixation has been reported for bacteria in the groups indicated with an asterisk (*)

Nitrogen Assimilation While nitrogen fixation is limited to a few species of prokaryotes, most

microorganisms can readily assimilate NH3 into amino acids and other organic

compounds. The release of NH3 from organic nitrogen compounds, known as

mineralization, is attributed to enzymatic deamination reactions found in many

microbial cells. Nitrogen is used by living systems for the synthesis of amino

acids, nucleic acid bases, and various organic nitrogen compounds. The

nitrogen source for animals is amino acids; for plants it is nitrate; and for

bacteria it may be nitrate, ammonia, or dinitrogen. If ammonium ion is

available, microorganisms will assimilate ammonium to form amino acids. The



conversion of N2 to NH3 requires at least 16 mol ATP for each mole of N2 fixed,

and for each mole of l-glutamine formed, one mole of ATP is required. However,

if nitrate is the nitrogen source nitrate must first be reduced to ammonium,

and this reduction process is referred to as assimilatory nitrate reduction. There

are a series of interconversions of inorganic nitrogen compounds involving

ammonia, nitrite, and nitrate.

Nitrification Ammonia is oxidized to nitrate by aerobic organisms using a multistep process

as given below, and this process is especially important in soil because plants

use nitrate as the nitrogen source:

NH3 +2 O2 → NO2 − + 2H+ + H2O byNitrosomonas

NO2− + 0.5 O2 → NO3− by Nitrobacter

The steps in ammonia oxidation to nitrate proceed with numerous

intermediates as shown below:

Each step in the nitrification sequence shown above is mediated by a specific

enzyme.

The initial step in aerobic oxidation by ammonia monoxygenase (AMO) that is a

unique enzyme because it has three substrates: gaseous NH3 and O2 plus the

electron donor.

In anaerobic environments, denitrification occurs where nitrate is the electron

acceptor with the formation of dinitrogen (N2). While some bacteria can reduce

nitrate completely to nitrite, others are capable of reduction of nitrate only to

nitrite. Anaerobic bacteria, including Desulfovibrio and Clostridium, will

enzymatically reduce nitrite to ammonia. More recently there was a

demonstration of the anammox (anoxic ammonia oxidation) reaction where

bacteria convert ammonia and nitrate to dinitrogen by the following reaction:



Denitrification Denitrification is an important process in which nitrate serves as the electron

acceptor for anaerobic bacteria with the release of nitrogen from the

environment as N2. Strains of Pseudomonas are often associated with

denitrification reactions in the soil and contribute to the loss of nitrogen as a

plant nutrient in flooded fields. The pathway for nitrate reduction is a stepwise

reduction of the nitrogen atom with intermediates of nitrogen Each of these

steps in the reduction of nitrate to dinitrogen releases sufficient energy to

support bacterial growth on that specific reaction.

The denitrification pathway is also referred to as dissimilatory nitrate reduction

because nitrate or nitrogen oxides are final electron acceptors that enable

bacteria to grow. BIOGEOCHEMICAL CYCLES OF PHOSPHOROUS

Phosphorus is essential for microbial systems because it is a required

component for sugar phosphates, RNA, DNA and high energy molecules.

Because many of the inorganic salts of phosphate have a low solubility in

aquatic environments, phosphorus is commonly a limiting nutrient in natural

environments, and bacteria have adjusted to overcome this limitation. The

principal mechanism for bacterial acquisition of phosphorus is to use uptake

transport systems for inorganic phosphate. Since iron and calcium form

insoluble phosphate minerals, bacteria will solubilize phosphate by the

production of acidic end products of metabolism. Organic phosphorous

compounds in the form of phosphate esters are a product of biological material

decaying, and phosphatase enzymes will release inorganic phosphate. There



are two major groups of phosphatase enzymes: acid phosphatases and alkaline

phosphatases. This classification of phosphates reflects the pH for optimum

activity, and both types can use a variety of organic compounds with

phosphate esters as substrates. Generally, organisms in aquatic or soil

environments will use alkaline phosphatases, while intracellular organisms

growing inside vacuoles of host cells use acidic phosphatase.

Additionally, organic molecules containing phosphorus may have a direct C–P

bond; these molecules represent the phosphonates. Phosphonates are relatively

common in nature, and inorganic phosphate may be released following the

action of either a phosphonase or a C–P lyase. Bacterial utilization of

phosphate and phosphonate are summarized in the reactions shown in Figure.

Since inorganic phosphate may be limiting in the environment, many bacteria

will store phosphate as a dense granule inside the cell. During periods of

adequate levels of phosphate, it accumulates inside the cell as a polyphosphate

granule, and when extracellular phosphate becomes limiting the polyphosphate

reserve is utilized. For cultivation of microorganisms in the laboratory,

phosphate is commonly used as a buffer; however, in nature carbonate, and

not phosphate, functions to maintain the pH within the tolerance levels for

growth.

There is a sensitive regulatory process that controls the production of different

enzymes for phosphate utilization, and this is an important feature for

bacterial persistence in the environment. When inorganic phosphate is

available in adequate levels to support growth of bacteria, inorganic phosphate

is transported into the cell by a low-affinity transport system with the

repression of acid or alkaline phosphatase and phosphonase or a lyase.

If inorganic phosphate is limiting, bacteria will produce appropriate enzymes

for release of inorganic phosphate from phosphate esters or from

phosphonates.

Reactions summarizing phosphorus interactions with bacteria:



(A) Utilization of phosphonates and organophosphates as a substrate. The enzymes associated with release of phosphate from phosphonate would be either a lyase or a phosphonatase located between the outer membrane and the plasma membrane as indicated by the arrow. Phosphatase activity for release of phosphate from organophosphates would be functioning in the periplasmic region. Uptake of phosphate may be by the high-affinity Psi (Phosphate starvation-inducible) system. (B) Uptake of inorganic phosphate (Pi) by a low-affinity Pit (inorganic phosphate transporter) (PPG = polyphosphate granule in cytoplasm of cell). Phytic acid is a phosphate storage compound found in plant seeds and is an

excellent source of phosphate. It is inositol hexaphosphate, which is a hexose

with six phosphate residues esterified on the six-carbon sugar. Enzymatic

hydrolysis of phytic acid occurs by phytase according to the following reaction:

Phytase is an extracellular enzyme produced by several strains of bacteria and

fungi found in soil environments as well as in stomachs of ruminants (cows,

sheep, etc.). Monogastric animals such as humans, pigs, and chickens do not

have the appropriate microbial flora to produce phytase, so phytic acid is not



decomposed in the animal but is released with the intestinal waste. Phytic acid

is readily metabolized by microorganisms in the soil or in aquatic

environments. BIOGEOCHEMICAL CYCLES OF SULPHUR.

Sulfur, an element with multiple oxidation states, is required by biological

systems with one gram of bacteria requiring about 11 mg of sulfur for

synthesis of sulfur-containing amino acids. Earth’s surface contains about

0.1% sulfur, and the sulfur cycle reflects the turnover of sulfur compounds in

the environment.

It has been estimated that 90 million tons of sulfur enters the atmosphere from

biologically produced H2S, CS2, COS, and (CH3)2S, while 50 and 0.7 million

tons of sulfur are released into the atmosphere from burning fossil fuels and

volcanic activity, respectively.

H2S in the atmosphere is oxidized to SO2, and when it rains, sulfur dioxide

forms sulfurous acid (H2SO3). A small amount of H2S is oxidized to SO3 in the

atmosphere and returns to Earth as H2SO4. Some have estimated that each

year Earth may receive 1014g S as acid rain.

Organic Sulfur Metabolism Microorganisms and plants can synthesize all sulfur-containing amino acids

from sulfate, and this process is energy-dependent as shown.

In order for sulfate to be incorporated into cysteine, the sulfur atom must be

reduced from +6 to −2, and this process has been designated as assimilatory

sulfate reduction. Reduced sulfur (–SH or –S–S–) is the common form of sulfur



in cells but a few molecules of oxidized sulfur may be found in the form of

sulfate esters (C–O–SO3−) or sulfonates (C–SO3 −).

A special physiological group of prokaryotes use sulfate as the final electron

acceptor in respiration, and this process, resulting in the formation of H2S, is

termed dissimilatory sulfate reduction.

Inorganic Sulfur Metabolism A unique physiological group of anaerobic bacteria can obtain energy from the

reduction of sulfate to sulfide. Anaerobic sediments and mud often have an

odor of rotten eggs and this is attributed to the high levels of hydrogen sulfide

released by sulfate-reducing bacteria. With sulfate present in marine and

liminic environments at 28 mM, marine sediments will have appreciable

quantities of sulfide produced from dissimilatory sulfate reduction activities.

Various microorganisms can mineralize sulfur from organic sulfur compounds

with the release of H2S. Aquatic environments near neutrality will have

considerable levels of reduced sulfur in the form of HS−, and this ion can be

oxidized to elemental sulfur (S0) by chemolithotrophic bacteria or anaerobic

photosynthetic sulfur bacteria. Some sulfur bacteria will use S0 as an electron

acceptor for growth with the regeneration of hydrogen sulfide.

Chemolithotrophic bacteria will oxidize S0 to thiosulfate, sulfite, or sulfate.

Many different genera of microorganisms will oxidize thiosulfate or sulfite to

sulfate; however, there are relatively few bacteria that function as dissimilatory

sulfate reducers.

Microbial interactions with the sulfur cycle.



Reactions associated with reduction are indicated by solid lines; with oxidation,

by broken lines; and where the oxidation state of selenium does not change, by

a dotted line. The following microorganisms are associated with these

reactions: (1) phototrophic green and purple sulfur bacteria; (2) anaerobic

bacteria and archaea; (3) phototrophic green and purple sulfur bacteria; (4)

dismutation where four sulfite molecules produce three sulfate molecules and

one sulfide, Desulfovibrio sulfodismutans; (5) dissimilatory reduction by

Desulfovibrio, Desulfotomaculum, and other sulfate-reducing bacteria; (6)

metabolism by heterotrophic bacteria; (7) assimilatory reduction, most

microorganisms.



UNIT – 4 DIVERSITY IN ANOXIC ECO SYSTEM Methanogens – reduction of carbon monoxide – reduction of iron, sulphur, manganes, nitrate and oxygen. Microbial transformations of Carbon, Phosphorus, Sulphur, Nitrogen and Mercury.

METHANOGENS - REDUCTION OF CARBON MONOXIDE Methane-forming bacteria are known by several names Methanogenic bacteria,

Methanogens, Methane-forming bacteria or Methane-producing bacteria and

are a morphologically diverse group of organisms that have many shapes,

growth patterns, and sizes.The bacteria can be found as individual rods,

curved rods, spirals, and cocci or grouped as irregular clusters of cells, chains

of cells or filaments, and sarcina or cuboid arrangements.

The range in diameter sizes of individual cells is 0.1–15mm. Filaments can be

up to 200mm in length. Motile and nonmotile bacteria as well as spore-forming

and non-spore-forming bacteria can be found.



Methane-forming bacteria are some of the oldest bacteria and are grouped in

the domain Archaebacteria (from arachae meaning “ancient”). The domain

thrives in heat. Archaebacteria comprise all known methane-forming bacteria,

the extremely halophilic bacteria, thermoacidophilic bacteria, and the

extremely thermophilic bacteria. However, the methane-forming bacteria are

different from all other bacteria.

Methane-forming bacteria are oxygen-sensitive, fastidious anaerobes and are

free-living terrestrial and aquatic organisms. Although methane-forming

bacteria are oxygen sensitive, this is not a significant disadvantage. Methane-

forming bacteria are found in habitats that are rich in degradable organic

compounds. In these habitats, oxygen is rapidly removed through microbial

activity. Many occur as symbionts in animal digestive tracts. Methane-forming

bacteria also have unusually high sulfur content: Approximately 2.5% of the

total dry weight of the cell is sulfur. The methane-forming bacteria are

classified in the domain Archaebacteria because of several unique

characteristics that are not found in the true bacteria or Eubacteria.

These features include 1) a “nonrigid” cell wall and unique cell membrane lipid,

2) substrate degradation that produces methane as a waste, and 3) specialized

coenzymes. The cell wall lacks muramic acid, and the cell membrane does not

contain an ether lipid as its major constituent.

Cell wall of methane-forming bacteria

The cell wall of methane-forming bacteria (a) does not contain muramic acid, while the cell of other bacteria (b) contains varying amounts of muramic acid.



Coenzymes that are unique to methane-forming bacteria are coenzyme M and

the nickel containing coenzymes F420 and F430. Coenzyme M is used to

reduce carbon dioxide (CO2) to methane. The nickel-containing coenzymes are

important hydrogen carriers in methane-forming bacteria. The coenzymes are

metal laden organic acids that are incorporated into enzymes and allow the

enzymes to work more efficiently.The coenzymes are components of energy-

producing electron transfer systems that obtain energy for the bacterial cell

and remove electrons from degraded substrate.

Electrons (e) released from broken chemical bonds of substrates inside a bacterial cell are removed through the used of electron transport systems. These systems employ the use of proteins that contain co-enzymes such as metals and vitamins.

The unique chemical composition of the cell wall makes the bacteria “sensitive”

to toxicity from several fatty acids. Also, many methane-forming bacteria lack a

protective envelope around their cell wall.

Surfactants or hypotonic shock easily lyse methane-forming bacteria that do

not have this envelope.



Presence of an envelope on some methane-forming bacteria

Some methane-forming bacteria possess an envelope (a) that provides added protection for the bacterial cell. Methane-forming bacteria that do not possess an envelope (b) are easily lyzed in the presence of surfactants. All methane-forming bacteria produce methane. No other organism produces

methane. Methane-forming bacteria obtain energy by reducing simplistic

compounds or substrates such as carbon dioxide and acetate (CH3COOH).

Some methane-forming bacteria are capable of fixing molecular nitrogen (N2).

Methane-forming bacteria are classified according to their structure, substrate

utilization, types of enzymes produced, and temperature range of growth.There

are approximately 50 species of methane-forming bacteria that are classified in

three orders and four families.

Methane-forming bacteria grow as microbial consortia, tolerate high

concentrations of salt, and are obligate anaerobes. The bacteria grow on a

limited number of substrates. Methanobacterium formicium, for example, grows

on formate, carbondioxide, and hydrogen and is one of the more abundant

methane-forming bacteria in anaerobic digesters. Methanobacterium formicium

performs a significant role in sludge digestion and methane production.

Methanobacterium formicium and



Methanobrevibacter arboriphilus are two of the dominant methane-forming

bacteria in anaerobic digesters. The activity of these organisms and that of all

methaneforming bacteria is usually determined by measuring changes in

volatile acid concentration or methane production.

In nature, methane-forming bacteria perform two very special roles.They

participate in the degradation of many organic compounds that are considered

biorecalcitrant, that is, can only be degraded slowly, and they produce methane

from the degradation of organic compounds. Methane is poorly soluble in

water, inert under anaerobic conditions, non-toxic, and able to escape from the

anaerobic environment.

Methane-forming bacteria are predominantly terrestrial and aquatic organisms

and are found naturally in decaying organic matter, deep-sea volcanic vents,

deep sediment, geothermal springs, and the black mud of lakes and swamps.

These bacteria also are found in the digestive tract of humans and animals,

particularly the rumen of herbivores and cecum of non-ruminant animals.

The rumen is a special organ in the digestive tract in which the degradation of

cellulose and complex polysaccharides occurs. Cows, goats, sheep, and deer

are examples of ruminant animals. The bacteria, including methane-forming

bacteria, that grow in the digestive tract of ruminant animals are symbionts

and obtain most of their carbon and energy from the degradation of cellulose

and other complex polysaccharides from plants. Ruminants cannot survive

without the bacteria. The bacteria and substrates produced by the bacteria

through their fermentative activities provide the ruminants with most of their

carbon and energy.

Methane-forming bacteria grow well in aquatic environments in which a strict

anaerobic condition exists. The anaerobic condition of an aquatic environment

is expressed in terms of its oxidation-reduction potential or ORP.

Methane-forming bacteria grow best in an environment with an ORP of less

than –300mV. Most facultative anaerobes do well in aquatic environments with

an ORP between +200 and –200mV.



There are Gram-negative and Gram-positive methane-forming bacteria that

reproduce slowly. Gram stain results (negative, positive, and variable) are

different within the same order of methane-forming bacteria because of their

different types of cell walls.

The reproductive times or generation times for methane-forming bacteria range

from 3 days at 35°C to 50 days at 10°C. Because of the long generation time of

methane-forming bacteria, high retention times are required in an anaerobic

digester to ensure the growth of a large population of methane-forming bacteria

for the degradation of organic compounds. At least 12 days are required to

obtain a large population of methane-forming bacteria.

Methane-forming bacteria obtain their energy for reproduction and cellular

activity from the degradation of a relatively small number of simple substrates



These substrates include hydrogen, 1-carbon compounds, and acetate as the

2-carbon compound. One-carbon compounds include formate, methanol,

carbondioxide, carbon monoxide (CO), and methylamine.The most familiar and

frequently acknowledged substrates of methane-forming bacteria are acetate

and hydrogen. Acetate is commonly split to form methane while hydrogen is

combined with carbondioxide to form methane. The splitting of acetate to form

methane is known as aceticlastic cleavage. Each methane-forming bacterium

has a specific substrate or group of substrates that it can degrade.

Hydrogen can serve as a universal substrate for methane-forming bacteria, and

carbon dioxide functions as an inorganic carbon source in the forms of

carbonate (CO32–) or bicarbonate (HCO3–). Carbon dioxide also serves as a

terminal acceptor of electrons released by degraded substrate. Other 1-carbon

compounds that can be converted to substrates for methaneforming bacteria

include dimethyl sulfide, dimethylamine, and trimethylamine. Several alcohols

including 2-propanol and 2-butanol as well as propanol and butanol may be

used in the reduction of carbon dioxide to methane.

The majority of methane produced in an anaerobic digester occurs from the use

of acetate and hydrogen by methane-forming bacteria. The fermentation of

substrates such as acetate (aceticlastic cleavage) results in the production of

methane (Equation 1), and the reduction of carbon dioxide also results in the

production of methane (Equation.2).

CH3COOH → CH4 + CO2 (1)

CO2 + 4H2 → CH4 + 2H2O (2)



Aceticlastic cleavage of acetate and reduction of carbon dioxide are the two

major pathways to methane production. Fermentation of propionate

(CH3CH2COOH) and butyrate (CH3CH2 CH2COOH) are minor pathways to

methane production. However, the fermentation of propionic acid to methane

requires two different species of bacteria and two microbial degradation steps

(Equations.3 and 4). In the first reaction, methane and acetate are produced

from the fermentation of propionate by a volatile acid-forming bacterium

(Syntrophobacter wolinii) and a methane-forming bacterium. In the second

reaction, methane is produced from the cleavage of acetate by a methane-

forming bacterium.

These reactions occur only if hydrogen and formate are kept low (used) by

methane-forming bacteria. Accordingly, the accumulation of propionate is a

common indicator of stress in an anaerobic digester.

4CH3CH2COOH + 2H2O → 4CH3COOH + CO2 + 3CH4 (3)

4CH3COOH → 4CH4 + 4CO2 (4)

Butyrate also is degraded to methane through two microbial degradation steps

(Equations 5 and 6).The degradation steps again are mediated by two different

bacteria. In the first reaction, methane and acetate are produced from the

fermentation of butyrate by a volatile acid-forming bacterium and a methane-

forming bacterium.

In the second reaction, methane is produced from the cleavage of acetate by a

methane-forming bacterium. Because butyrate can be used indirectly by

methane-forming bacteria, its accumulation is an indicator of stress in an

anaerobic digester.

CH3CH2CH2COOH + 2H2O → 4CH3COOH + CO2 + CH4 (5)

4CH3COOH → 4CH4 + 4 CO2 (6)

No species of methane-forming bacteria can utilize all substrates. Therefore,

successful fermentation of substrates in an anaerobic digester requires the

presence of not only a large number of methane-forming bacteria but also a

large diversity of methane-forming bacteria.



There are three principal groups of methane-forming bacteria. These groups

are 1) the hydrogenotrophic methanogens, 2) the acetotrophic methanogens,

and 3) the methylotrophic methanogens. The term “trophic” (from trophe¯,

“nourishment”) refers to the substrates used by the bacteria.

GROUP 1 HYDROGENOTROPHIC METHANOGENS The hydrogenotrophic methanogens use hydrogen to convert carbon dioxide to

methane (Equation 7). By converting carbon dioxide to methane, these

organisms help to maintain a low partial hydrogen pressure in an anaerobic

digester that is required for acetogenic bacteria.

CO2 + 4H2 → CH4 + 2H2O (7)

GROUP 2 ACETOTROPHIC METHANOGENS The acetotrophic methanogens “split” acetate into methane and carbon dioxide

(Equation 8). The carbon dioxide produced from acetate may be converted by

hydrogenotrophic methanogens to methane (Equation 7). Some

hydrogenotrophic methanogens use carbon monoxide to produce methane

(Equation 9).

4CH3COOH → 4CO2 + 2H2 (8)

4CO + 2H2O → CH4 + 3CO2 (9)

Hydrogenotrophic methanogens and are adversely affected by the accumulation

of hydrogen. Therefore, the maintenance of a low partial hydrogen pressure in

an anaerobic digester is favorable for the activity of not only acetate-forming

bacteria but also acetotrophic methanogens. Under a relatively high hydrogen

partial pressure, acetate and methane production are reduced.

GROUP 3 METHYLOTROPHIC METHANOGENS The methylotrophic methanogens grow on substrates that contain the methyl

group (–CH3). Examples of these substrates include methanol (CH3OH)

(Equation 10) and methylamines [(CH3)3–N] (Equation 11). Group 1 and 2

methanogens produce methane from CO2 and H2. Group 3 methanogens

produce methane directly from methyl groups and not from CO2.

3CH3OH + 6H → 3CH4 + 3H2O (10)

4(CH3)3 – N + 6H2O → 9CH4 + 3CO2 + 4NH3 (11)



The use of different substrates by methane-forming bacteria results in different

energy gains by the bacteria. For example, hydrogen-consuming methane

production results in more energy gain for methane-forming bacteria than

acetate degradation.

Although methane production using hydrogen is the more effective process of

energy capture by methane-forming bacteria, less than 30% of the methane

produced in an anaerobic digester is by this method. Approximately 70% of the

methane produced in an anaerobic digester is derived from acetate. The reason

for this is the limited supply of hydrogen in an anaerobic digester. The majority

of methane obtained from acetate is produced by two genera of acetotrophic

methanogens, Methanosarcina and Methanothrix. Reproduction of methane-

forming bacteria is mostly by fission, budding, constriction, and fragmentation.

Methane-forming bacteria reproduce very slowly. This slow growth rate is due

to the relatively small amount of energy obtained from the use of their limited

number of substrates. Therefore, a relatively large quantity of substrates must

be fermented for the population of methaneforming bacteria to double, that is,

a relatively small quantity of cells or sludge is produced for a relatively large

quantity of substrate degraded. Therefore, anaerobic digesters produce

relatively small quantities of bacteria cells or sludge (solids).

Under optimal conditions, the range of generation times of methane-forming

bacteria may be from a few days to several weeks.Therefore, if solids retention

time (SRT) is short or short-circuiting or early withdrawal of digester sludge

occurs, the population size of methane-forming bacteria is greatly reduced.



These conditions decrease the time available for reproduction of methane-

forming bacteria, that is, the bacteria are removed from the digester faster than

they can reproduce. This results in poor digester performance or failure of the

digester.

With increasing retention time the production of new methane-forming bacteria

gradually decreases as a result of increased energy requirements of the cells in

order to maintain cellular activity (more degradation of substrate). Therefore,

increasing retention time of a properly operated anaerobic digester results in

decreased sludge production. Increasing retention time produces a large

consumption of substrate by slowing reproducing bacteria as an energy

requirement of old cells (sludge) for the maintenance of cellular activity. Most

methane-forming bacteria are mesophiles or thermophiles, with some bacteria

growing at temperatures above 100°C.

Mesophiles are those organisms that grow best within the temperature range of

30–35°C, and thermophiles are those organisms that grow best within the

temperature range of 50–60°C. Some genera of methane-forming bacteria have

mesophilic and thermophilic species.

It is difficult to grow methane-forming bacteria in pure culture. Standard

laboratory enumeration techniques are not suitable for methane-forming

bacteria. This difficulty is caused by 1) their extreme obligate anaerobic nature



and the probability that they are killed rapidly by relatively short time

exposures to air compared with other anaerobes and 2) their limited number of

substrates. To correct for the oxygen sensitivity of methane-forming bacteria in

laboratory experiments with pure cultures, the “Hungate” technique is used.

Growth or cell masses of methaneforming bacteria may be gray, green, greenish

black, orange-brown, pink, purple, yellow, or white. REDUCTION OF IRON

The iron cycle (figure) includes several different genera that carry out iron

oxidations, transforming ferrous ion (Fe2+) to ferric ion (Fe3+). Thiobacillus

ferrooxidans carries out this process under acidic conditions, Gallionella is

active under neutral pH conditions, and Sulfolobus functions under acidic,

thermophilic conditions.

Much of the earlier literature suggested that additional genera could oxidize

iron, including Sphaerotilus and Leptothrix. These two genera are still termed

“iron bacteria” by many nonmicrobiologists. Confusion about the role of these

genera resulted from the occurrence of the chemical oxidation of ferrous ion to

ferric ion (forming insoluble iron precipitates) at neutral pH values, where

microorganisms also grow on organic substrates. These microorganisms are

now classified as chemoheterotrophs.

Recently microbes have been found that oxidize Fe2+ using nitrate as an

electron acceptor. This process occurs in aquatic sediments with depressed

levels of oxygen and may be another route by which large zones of oxidized iron

have accumulated in environments with lower oxygen levels.

Iron reduction occurs under anaerobic conditions resulting in the

accumulation of ferrous ion. Although many microorganisms can reduce small

amounts of iron during their metabolism, most iron reduction is carried out by

specialized iron-respiring microorganisms such as Geobacter metallireducens,

Geobacter sulfurreducens,

Ferribacterium limneticum, and Shewanella putrefaciens, which can obtain

energy for growth on organic matter using ferric iron as an oxidant.



In addition to these relatively simple reductions to ferrous ion, some

magnetotactic bacteria such as Aquaspirillum magnetotacticum transform

extracellular iron to the mixed valence iron oxide mineral magnetite (Fe3O4) and

construct intracellular magnetic compasses. Furthermore, dissimilatory

ironreducing bacteria accumulate magnetite as an extracellular product.

Magnetite has been detected in sediments, where it is present in particles

similar to those found in bacteria, indicating a longerterm contribution of

bacteria to iron cycling processes. Genes for magnetite synthesis have been

cloned into other organisms, creating new magnetically sensitive

microorganisms. Magnetotactic bacteria are now described as magneto-aerotactic bacteria, due to their using magnetic fields to migrate to the

position in a bog or swamp where the oxygen level is best suited for their

functioning.

A simplified iron cycle with examples of microorganisms contributing to these oxidation and reduction processes. In addition to ferrous ion (Fe2+) oxidation and ferric ion (Fe3+) reduction, magnetite (Fe3O4), a mixed valence iron compound formed by magnetotactic bacteria is important in



the iron cycle. Different microbial groups carry out the oxidation of ferrous ion depending on environmental conditions. In the last decade new microorganisms have been discovered that use ferrous

ion as an electron donor in anoxygenic photosynthesis. Thus, with production

of ferric ion in lighted anaerobic zones by iron-oxidizing bacteria, the stage is

set for subsequent chemotrophic-based iron reduction, such as by Geobacter

and Shewanella, creating a strictly anaerobic oxidation/reduction cycle for

iron. REDUCTION OF SULPHUR

Microorganisms contribute greatly to the sulfur cycle, a simplified version of

which is shown in figure. Photosynthetic microorganisms transform sulfur by

using sulfide as an electron source, allowing Thiobacillus and similar

chemolithoautotrophic genera to function. In contrast, when sulfate diffuses

into reduced habitats, it provides an opportunity for different groups of

microorganisms to carry out sulfate reduction. For example, when a usable organic reductant is present, Desulfovibrio uses

sulfate as an oxidant. This use of sulfate as an external electron acceptor to

form sulfide, which accumulates in the environment, is an example of a

dissimilatory reduction process and anaerobic respiration. In comparison,

the reduction of sulfate for use in amino acid and protein biosynthesis is

described as an assimilatory reduction process. Other microorganisms have

been found to carry out dissimilatory elemental sulfur reduction. These include

Desulfuromonas, thermophilic archaea, and also cyanobacteria in hypersaline

sediments. Sulfite is another critical intermediate that can be reduced to

sulfide by a wide variety of microorganisms, including Alteromonas and

Clostridium, as well as Desulfovibrio and Desulfotomaculum.

Desulfovibrio is usually considered as an obligate anaerobe. Recent research,

however, has shown that this interesting organism also respires using oxygen,

when it is present at a dissolved oxygen level of 0.04%. In addition to the very

important photolithotrophic sulfur oxidizers such as Chromatium and



Chlorobium, which function under strict anaerobic conditions in deep water

columns, a large and varied group of bacteria carry out aerobic anoxygenic photosynthesis. These aerobic anoxygenic phototrophs use bacteriochlorophyll a and

carotenoid pigments and are found in marine and freshwater environments;

they are often components of microbial mat communities. Important genera

include Erythromonas, Roseococcus, Porphyrobacter, and Roseobacter. “Minor”

compounds in the sulfur cycle play major roles in biology. An excellent example

is dimethylsulfoniopropionate (DMSP), which is used by bacterioplankton

(floating bacteria) as a sulfur source for protein synthesis, and which is

transformed to dimethylsulfide (DMS), a volatile sulfur form that can affect

atmospheric processes.

When pH and oxidation-reduction conditions are favorable, several key

transformations in the sulfur cycle also occur as the result of chemical

reactions in the absence of microorganisms. An important example of such an

abiotic process is the oxidation of sulfide to elemental sulfur. This takes place

rapidly at a neutral pH, with a half-life of approximately 10 minutes for sulfide

at room temperature.

The Basic Sulfur Cycle.



Photosynthetic and chemosynthetic microorganisms contribute to the environmental sulfur cycle. Sulfate and sulfite reductions carried out by Desulfovibrio and related microorganisms, noted with purple arrows, are dissimilatory processes. Sulfate reduction also can occur in assimilatory reactions, resulting in organic sulfur forms. Elemental sulfur reduction to sulfide is carried out by Desulfuromonas, thermophilic archaea, or cyanobacteria in hypersaline sediments. Sulfur oxidation can be carried out by a wide range of aerobic chemotrophs and by aerobic and anaerobic phototrophs.

REDUCTION OF MANGANESE The importance of microorganisms in manganese cycling is becoming much

better appreciated. The manganese cycle (figure) involves the transformation of

manganous ion (Mn2+) to MnO2 (equivalent to manganic ion [Mn4_]), which

occurs in hydrothermal vents, bogs, and as an important part of rock

varnishes. Leptothrix, Arthrobacter, Pedomicrobium, and the incompletely

characterized “Metallogenium” are important in Mn2+ oxidation. Shewanella,

Geobacter, and other chemoorganotrophs can carry out the complementary

manganese reduction process.

The Basic Manganese Cycle



Microorganisms make important contributions to the manganese cycle. Manganous ion (2+) is oxidized to manganic oxide (valence equivalent to 4+). Manganous oxide reduction is noted with a maroon arrow. Examples of organisms carrying out these processes are given.

REDUCTION OF NITRATE Several important aspects of the basic nitrogen cycle will be discussed: the

processes of nitrification, denitrification, and nitrogen fixation (figure). It

should be emphasized that this is a “basic” nitrogen cycle. Although not

mentioned in the figure, the heterotrophs can carry out nitrification, and some

of these heterotrophs combine nitrification with anaerobic denitrification, thus

oxidizing ammonium ion to N2O and N2 with depressed oxygen levels. The

occurrence of anoxic ammonium ion oxidation

(anammox is the term used for the commercial process) means that

nitrification is not only an aerobic process. Thus as we learn more about the

biogeochemical cycles, including that of nitrogen, the simple cycles of earlier

textbooks are no longer accurate representations of biogeochemical processes.

Nitrification is the aerobic process of ammonium ion (NH4+) oxidation to nitrite

(NO2_) and subsequent nitrite oxidationto nitrate (NO3-). Bacteria of the genera

Nitrosomonas and Nitrosococcus, for example, play important roles in the first

step, and Nitrobacter and related chemolithoautotrophic bacteria carry out the

second step. Recently Nitrosomonas eutropha has been found to oxidize

ammonium ion anaerobically to nitrite and nitric oxide (NO) using nitrogen

dioxide (NO2) as an oxidant in a denitrification- related reaction. In addition,

heterotrophic nitrification by bacteria and fungi contributes significantly to

these processes in more acidic environments.

The process of denitrification requires a different set of environmental

conditions. This dissimilatory process, in which nitrate is used as an oxidant in

anaerobic respiration, usually involves heterotrophs such as Pseudomonas

denitrificans. The major products of denitrification include nitrogen gas (N2)

and nitrous oxide (N2O), although nitrite (NO2_) also can accumulate. Nitrite is



of environmental concern because it can contribute to the formation of

carcinogenic nitrosamines. Finally, nitrate can be transformed to ammonia in

dissimilatory reduction by a variety of bacteria, including Geobacter

metallireducens, Desulfovibrio spp., and Clostridium.

Nitrogen assimilation occurs when inorganic nitrogen is used as a nutrient and

incorporated into new microbial biomass. Ammonium ion, because it is already

reduced, can be directly incorporated without major energy costs. However,

when nitrate is assimilated, it must be reduced with significant energy

expenditure. In this process nitrite may accumulate as a transient

intermediate.

Nitrogen fixation can be carried out by aerobic or anaerobic procaryotes and

does not occur in eucaryotes. Under aerobic conditions a wide range of free-

living microbial genera (Azotobacter, Azospirillum) contribute to this process.

Under anaerobic conditions the most important free-living nitrogen fixers are

members of the genus Clostridium. Nitrogen fixation by cyanobacteria such as

Anabaena and Oscillatoria can lead to the enrichment of aquatic environments

with nitrogen.

In addition, nitrogen fixation can occur through the activities of bacteria that

develop symbiotic associations with plants. These associations include

Rhizobium and Bradyrhizobium with legumes, Frankia in association with many

woody shrubs, and Anabaena, with Azolla, a water fern important in rice

cultivation.

The nitrogen-fixation process involves a sequence of reduction steps that

require major energy expenditures. Ammonia, the product of nitrogen

reduction, is immediately incorporated into organic matter as an amine.

Reductive processes are extremely sensitive to O2 and must occur under

anaerobic conditions even in aerobic microorganisms. Protection of the

nitrogen-fixing enzyme is achieved by means of a variety of mechanisms,

including physical barriers, as occurs with heterocysts in some cyanobacteria.

O2 scavenging molecules, and high rates of metabolic activity.



As shown in figure, microorganisms have been isolated that can couple the

anaerobic oxidation of NH4+ with the reduction of NO2-, to produce gaseous

nitrogen, in what has been termed the anammox process (anoxic ammonia

oxidation). This may provide a means by which nitrogen can be removed from

sewage plant effluents to decrease nitrogen flow to sensitive freshwater and

marine ecosystems. It has been suggested that chemolithotrophic members of

the planctomycetes play a role in this process.

The Basic Nitrogen Cycle.

Flows that occur predominantly under aerobic conditions are noted with open arrows. Anaerobic processes are noted with solid bold arrows. Processes occurring under both aerobic and anaerobic conditions are marked with cross-barred arrows. The anammox reaction of NO2_ and NH4+

to yield N2 is shown. Important genera contributing to the nitrogen cycle are given as examples.

REDUCTION OF OXYGEN The oxygen relationships for the use of these substrates also are of interest,

because most of them can be degraded easily with or without oxygen present.

The exceptions are hydrocarbons and lignin. Hydrocarbons are unique in that

microbial degradation, especially of straight-chained and branched forms,



involves the initial addition of molecular O2. Recently, anaerobic degradation of

hydrocarbons with sulfate or nitrate as oxidants has been observed.

With sulfate present, organisms of the genus Desulfovibrio are active. This

occurs only slowly and with microbial communities that have been exposed to

these compounds for extended periods. Such degradation may have resulted in

the sulfides that are present in “sour gases” associated with petroleum. Lignin,

an important structural component in mature plant materials, is a complex

amorphous polymer based on a phenylpropane building block, linked by

carbon-carbon and carbon-ether bonds. It makes up approximately 1/3 of the

weight of wood. This is a special case in which biodegradability is dependent on

O2 availability.

There often is no significant degradation because most filamentous fungi that

degrade native lignin in situ can function only under aerobic conditions where

oxidases can act by the release of active oxygen species. Lignin’s lack of

biodegradability under anaerobic conditions results in accumulation of lignified

materials, including the formation of peat bogs and muck soils. This absence of

lignin degradation under anaerobic conditions also is important in

construction. Large masonry structures often are built on swampy sites by

driving in wood pilings below the water table and placing the building footings

on the pilings. As long as the foundations remain water-saturated and

anaerobic, the structure is stable. If the water table drops, however, the pilings

will begin to rot and the structure will be threatened. Similarly, the cleanup of

harbors can lead to decomposition of costly docks built with wooden pilings

due to increased aerobic degradation of wood by filamentous fungi. Rumen

function provides a final example of the relationship between lignin degradation

and oxygen. The rumen, being almost free of oxygen, does not allow significant

degradation of lignin present in animal feeds. The use of sugars and

carbohydrates in the rumen leaves an inactive residue that can improve soils

more effectively than the original feeds.

Patterns of microbial degradation are important in many habitats. They

contribute to the accumulation of petroleum products, the formation of bogs,



and the preservation of valuable historical objects. The presence or absence of

oxygen also affects the final products that accumulate when organic substrates

have been processed by microorganisms and mineralized either under aerobic

or anaerobic conditions. Under aerobic conditions, oxidized products such as

nitrate, sulfate, and carbon dioxide (figure) will result from microbial

degradation of complex organic matter. In comparison, under anaerobic

conditions reduced end products tend to accumulate, including ammonium

ion, sulfide, and methane. These oxidized and reduced forms, if they remain in

the aerobic or anaerobic environments where they were formed, will usually

only serve as nutrients. If mixing occurs, oxidized species might be moved to a

more reduced zone or reduced species might be moved to a more oxidized zone.

Under such circumstances, additional energetic possibilities (linking of

oxidants and reductants) will be created, leading to succession and further

nutrient cycling as these mixed oxidants and reductants are exploited by the

microbial community. The Influence of Oxygen on Organic Matter



Decomposition. Microorganisms form different products when breaking down complex organic matter aerobically than they do under anaerobic conditions. Under aerobic conditions oxidized products accumulate, while reduced products accumulate anaerobically. These reactions also illustrate commensalistic transformations of a substrate, where the waste products of one group of microorganisms can be used by a second type of microorganism.

MICROBIAL TRANSFORMATIONS OF CARBON Carbon can be present in reduced forms, such as methane (CH4) and organic

matter, and in more oxidized forms, such as carbon monoxide (CO) and carbon

dioxide (CO2). The major pools present in an integrated basic carbon cycle are

shown in figure. The Basic Carbon Cycle in the Environment Reductants (e.g., hydrogen, which is a strong reductant) and oxidants (e.g., O2)

influence the course of biological and chemical reactions involving carbon.

Hydrogen can be produced during organic matter degradation especially under

anaerobic conditions when fermentation occurs. If hydrogen and methane are

generated, they can move upward from anaerobic to aerobic areas. This creates

an opportunity for aerobic hydrogen and methane oxidizers to function.

Methane levels in the atmosphere have been increasing approximately 1% per

year, from 0.7 to 1.6 to 1.7 ppm (volume) in the last 300 years. This methane is

derived from a variety of sources. If an aerobic water column is above the

anaerobic zone where the methanogens are located, the methane can be

oxidized before it reaches the atmosphere. In many situations, such as in rice

paddies without an overlying aerobic water zone, the methane will be released

directly to the atmosphere, thus contributing to global atmospheric methane

increases. Rice paddies, ruminants, coal mines, sewage treatment plants,

landfills, and marshes are important sources of methane. Anaerobic

microorganisms such as Methanobrevibacter in the guts of termites also can

contribute to methane production.



Carbon fixation can occur through the activities of photoautotrophic and chemoautotrophic microorganisms. Methane can be produced from inorganic substrates (CO2 + H2) or from organic matter. Carbon monoxide (CO)—produced by sources such as automobiles and industry—is returned to the carbon cycle by CO-oxidizing bacteria. Aerobic processes are noted with blue arrows, and anaerobic processes are shown with red arrows. Carbon fixation occurs through the activities of cyanobacteria and green algae,

photosynthetic bacteria (e.g., Chromatium and Chlorobium), and aerobic

chemolithoautotrophs. In the carbon cycle depicted in figure, no distinction is

made between different types of organic matter that are formed and degraded.

This is a marked oversimplification because organic matter varies widely in

physical characteristics and in the biochemistry of its synthesis and

degradation. Organic matter varies in terms of elemental composition,

structure of basic repeating units, linkages between repeating units, and

physical and chemical characteristics.

The degradation of this organic matter, once formed, is influenced by a series of

factors. These include (1) nutrients present in the environment; (2) abiotic

conditions (pH, oxidationreduction potential, O2, osmotic conditions), and (3)



the microbial community present. The major complex organic substrates used

by microorganisms are summarized in table. Of these, only previously grown microbial biomass contains all of the nutrients

required for microbial growth. Chitin, protein, microbial biomass, and nucleic

acids contain nitrogen in large amounts. If these substrates are used for

growth, the excess nitrogen and other minerals that are not used in the

formation of new microbial biomass will be released to the environment, in the

process of mineralization. This is the process in which organic matter is decomposed to release simpler,

inorganic compounds (e.g., CO2, NH4+, CH4, H2). The other complex substrates

in table contain only carbon, hydrogen, and oxygen. If microorganisms are to

grow by using these substrates, they must acquire the remaining nutrients

they need for biomass synthesis from the environment; in the process of

immobilization

MICROBIAL TRANSFORMATIONS OF PHOSPHORUS

Phosphorus is usually added to the soil in the form of various plant and animal

residues (in stable and green manures), in certain organic fertilizers (tankage,

bone meal, guano), and in various inorganic fertilizers. Phosphorus is present

in the latter either as insoluble rock phosphate, the chief constituent of which

is Ca3(P04)2, or as superphosphate, which, consists of rock phosphate



previously treated with sp.lfuric acid to make it soluble, or as various other

basic phosphates included in slag.

In the plant and animal residues, as well as in'stable and green manures,

phosphorus is present in the form of such compounds as phytin,

phospholipids, of which lecithin is the best known representative, and

nucleoproteins. Before the phosphorus can be utilized again by higher plants,

these organic complexes have to be decomposed by various soil organisms.

These processes of decomposition can be illustrated somewhat as follows:

The phytic acid originates in the decomposition of phytin. Lecithin contains

both phosphorus and nitrogen; it is first hydrolyzed to cholin (a nitrogen

complex), glycerophosphoric acid, and various fatty acids. Nucleoproteins are

compounds of one or more protein molecules with nucleic acid; the latter

contains the phosphorus. Nucleic acids themselves are also very complex in

composition, such as C36H48030N14P4. On decomposition, they give, in addition

to phosphoric acid, various carbohydrates and organic - bases (adenine,

guanine, cytosine). The decomposition processes of some of these substances

are frequently very complicated, involving a number of reactions, but they all

lead to the formation of phosphoric acid.

The phosphorus in organic combination may make up from 20 to 35 per cent of

the total phosphorus of many soils. Organic 'phosphorus may also compose a

large portion of the phosphorus occurring in solution in soils. In a study of

twenty-one soils of Southern and Central United States, the soluble organic

phosphorus (calculated as P2 05) was present in 0.176 parts per million of soil

solution, while the inorganic phosphorus was found in only 0.034 parts per

million.

The microbial cells which are constantly formed in the soil are also very rich in

phosphoric acid; the ash of the microbes frequently contains 50 per cent or

more phosphate, calculated as P205.



Largely as a result of this consumption of phosphorus by microbes and its use

in cell synthesis, the organic matter of the soil contains a definite amount of

phosphorus. The ratio of the carbon content of the soil to the organic

phosphorus content is more or less constant, just as is the case in the ratio of

the carbon to nitrogen.

When the organic matter of the soil or that added to the soil is decomposed,

there is a continuous liberation of phosphorus in an inorganic form, and the

more rapidly the organic matter is decomposed the more rapidly the

phosphorus is liberated. In the presence of an abundance of available organic

food material containing little or no phosphorus, the microbes will reassimilate

part of this phosphorus in the synthesis of their cell substance, and a

deficiency of phosphorus available for plant growth may be created. The

amount of food material used by microorganisms for growth, the amount of

microbial cell substance synthesized from the food, and the quantities of both

nitrogen and phosphorus which are assimilated in these processes are all in

definite ratios to one another. In other words, a definite amount of microbial

cell substance is formed per unit of organic food material consllmed, and quite

definite quantities of nitrogen and phosphorus are built into these cells.

Various fungi and bacteria are capable of liberating phosphorus from organic

complexes in an inorganic form. Some bacteria are particularly active in this

connection since they are capable of attacking the phosphorus-bearing

complexes more readily.

Processes similar to those causing reduction of nitrates and sulfates may be

concerned in the reduction of phosphates. Nitrates are quite readily reduced;

sulfates, with somewhat more difficulty; and phosphates, least readily of all.

Under anaerobic conditions, if organic food materials are available, if

phosphates are present in abundarlce and if the necessary bacteria are active,

phosphates may be reduced to phosphite (H3P03), hypophosphite (H3PO4),and

phosphine (PH3). The reactions may be illustrated in a general way as follows,

where C refers to carbon in organic combination:



The possible significance and importance of these reactions in the movements

of phosphorus in arable soils are unknown. However, since these processes are

favored only by rather extreme reduction conditions, it seems likely that

phosphate reduction is not responsible for very extensive changes under most

soil conditions. The extent of reduction of phosphates to the various

substances by bacteria under conditions favorable to the changes is shown in

Table.

The insoluble inorganic calcium phosphate may be present in the soil in the

native rock constituents or occur there subsequent to the addition of various

fertilizer mixtures. This form of phosphate is available to plants only in ·very

limited amounts. However, as a result of interaction with the various organic

and inorganic acids formed by microbes, the phosphate becomes changed. into

di- and mono-basic phosphates, which are more soluble and consequently

more readily available to plants. In view of the fact that the formation of the

inorganic and organic acids takes place in the soil constantly as a result of the

decomposition and oxidation processes, insoluble phosphates tend to become



gradually soluble, especially when accompanied by processes of active

decomposition of organic matter and oxidation of ammonia and incompletely

oxidized sulfur compounds. When the soil contains free carbonates and the

reaction is neutral or alkaline, the acids will be neutralized by the carbonates

in preference to the phosphates, and the latter will not go into solution so

readily.

The amount of phosphoric acid made available to plants at any given time is

thus found to be a result of various complex biological and chemical processes

which tend either to b, ring the phosphoric acid into solution (when the

decomposition processes are predominant) or take it out of solution (when the

synthesizing microbial processes are active and the amount of available

phosphate is limited). MICROBIAL TRANSFORMATIONS OF SULPHUR

Sulfur exists in the soil and is added to it constantly in plant and animal

residues, rain-water, and fertilizing materials, in several distinctly different

forms, namely, as organic sulfur compounds, inorganic sulfates, sulfides, and

elementary sulfur. In the organic matter of the soil, the sulfur is generally

bound up in forms resistant to decomposition and only after long periods of

time liberated in appreciable amounts as sulfate. The addition of elementary

sulfur or sulfate in fertilizer materials may be responsible for relatively large

amounts of sulfate in the soil solution from time to time, but, generally, sulfate

makes up a small portion of the total sulfur content of the soil. More commonly

from 80 to 90 per cent of the sulfur is present in organic combination, and only

10 to 20 per cent exists as sulfate.

The plant residues commonly added to the soil contain sulfur largely in organic

combination, varying from 0.05 to 1.0 per cent. Alfalfa hay, for example,

contains 0.29 per cent sulfur; turnip tops, 0.9 per cent; and wheat straw, 0.12

per cent. The sulfur in the organic compounds of such plant residues becomes

liberated as sulfate more rapidly than from the so-called humic matter of the

soil, just as ammonia is formed more rapidly from fresh organic matter rich in

nitrogen than from the residual organic matter in the humus.



Under natural conditions, many agents, both biological and non-biological,

carry the element through its course of -changes. Microbial effects are largely

oxidations and reductions of the inorganic sulfur compounds, and formation

and decomposition of organic compounds containing sulfur. Since sulfur

enters into the composition of all living cells, all forms of life become associated

with its transformation. In the decomposition of the organic residues by

microbes, the sulfur present in organic combination is changed, after several

transformations, into inorganic forms such as hydrogen sulfide and sulfate.

Some microorganisms utilize sulfate to satisfy their requirements for the

element. In time, the microbial cells become destroyed and mineralized. The

assimilation of sulfate is more pronounced when the organic matter which

serves as food contains little or no sulfur.

Table shows the rapid disappearance of sulfate when an organism (Aspergillus

niger) is feeding on a sugar.

Sulfur is present in the organic matter largely in the protein molecule. This

molecule contains an amino acid, cystine, which is .the sulfur carrier. When

proteins are hydrolyzed by microorganisms, the cystine is first liberated. When

the cystine molecule is decomposed by microorganisms in the soil, the sulfur is

usually liberated in the form of hydrogen sulfide:



Other organic compounds containing sulfur are frequently introduced into the

soil, but generally in very small amounts. These include taurine, among the

animal products, and certain glucosides, among the plant residues.

The nature of the compoun.d as well as the environmental conditions, in which

it is decomposed, whether aerobic or anaerobic, will determine to a large extent

the resultant form of the sulfur when it appears in tIle inorganic state. Under

anaerobic conditions sulfides are frequently produced in considerable

quantities. This is the case in water-logged soils, ditches, stagnant pools, and

seas that receive appreciable contributions of organic materials and sulfates.

Coloration of soils may be determined by sulfides of iron under anaerobic

conditions where organic matter is not so abundant as to conceal this

coloration. These sulfides are formed from the decomposition of organic

compounds containing sulfur and from reduction of sulfates, sulfur, or other

inorganic, incompletely oxidized sulfur compounds.

Direct evidence is still lacking to indicate that microorganisms convert sulfur,

associated in organic compounds, directly to sulfate. Some sulfur may be

liberated in this way, but a greater portion is first changed to sulfide and

appears as sulfate only after secondary attack by organisms able to oxidize

inorganic sulfur compounds.

Wherever protein materials undergo decomposition by the agency of bacteria,

sulfide is formed. Under anaerobic conditions it is not transformed further, but

under aerobic conditions it soon disappears and is oxidized to sulfate, as may

be represented by the following equation:

Various specific bacteria (largely of the Thiobacillus group) aye capable of

bringing about these processes in the soil.



The hydrogen sulfide is first oxidized to elementary sulfur. In the case of some

bacteria, this sulfur actually accumulates within or without the cells. Other

bacteria, however, immediately oxidize the sulfur to sulfuric acid. This acid

interacts with the soil bases:

Sulfide may be considered as an intermediate decomposition product. It is

unavailable to plants as such, is injurious rather than beneficial to the activity

of most soil microorganisms, and its presence in soils in any appreciable

amounts is indicative of partial anaerobic conditions llnsuited for plant growth.

Sulfide, like any other decomposition product such as ammonia,· is a "Taste

product left by the microorganisms as uIlnecessary in their further

development. Its formation as a product of decomposition of organic matter

indicates that the compounds from which it was produced contained more

sulfur than was required by the organisms in their growth while using this

organic matter as food. As hydrogen sulfide it represents a source of

considerable potential energy which will become liberated during its

transformation to stllfate.

After elementary sulfur is added to soil, it is oxidized directly to sulfuric acid if

the proper organisms are present. The oxidation proceeds particularly rapidly

where some of the specific sulfur bacteria are concerned. These organisms are

similar in their nutrition to the nitrifying bacteria in that they are autotrophic,

but they use the sulfide or sulfur as their specific foods, oxidizing them to



sulfate. Even in the absence of these organisms, sulfates are formed,

presumably in some way associated with the develop ment of various

heterotrophic organisms including both bacteria and fungi. The process of

sulfur oxidation can be utilized for increasing the acidity of the soil, and in view

of the fact that certain organisms producing plant diseases cannot thrive at

certain acid concentrations, it may be desirable to create such acid conditions

as are inhibitive to their development. This is brought about by adding sulfur

to soils. In the soil, it is oxidized to sulfuric acid and brings about the desired

effects as considered in more detail elsewhere. This process can also be utilized

for reducing the alkalinity of black alkali soils. The sulfuric acid formed by

theoxidation of sulfur interacts with the sodium carbonate, giving sodium

sulfate. The latter salt of sodium is much less injurious to plants than the

former; it has less undesirable effects on the physical condition of the soil and

it can also be more readily removed by irrigation and drainage waters. The

oxidation process is further utilized in transforming the insoluble phosphate of

the rock phosphate to more soluble form.s. When sulfur is mixed and

composted with rock phosphate and soil, in the proper proportions, the sulfur

becomes oxidized to sulfuric acid and changes the phosphate to the di-calcium

and mono-calcium forms. The application of such material to soils gives much

the same effects as the application of "superphosphate." Similar solution of

potassium occurs when certain insoluble minerals containing potassium are

substituted for the rock phosphate in these composts.

Large amounts of sulfide may be produced in nature by the reduction of

sulfates as well as elementary sulfur itself. These processes consume

appreciable amounts of energy which must be supplied from other sources,

such as the oxidation of organic compounds. Further, the reaction proceeds

only under anaerobic conditions. In such an environment the reactions take

place somewhat as follows:



In these reductions the sulfur and sulfate act as oxidizing substances, and

serve, under these anaerobic conditions, in much the same capacity as oxygen

itself does in an aerobic environment.

In case conditions are changed, after the formation of sulfide, so as to permit

the entrance of free oxygen, thus creating aerobic conditions, the sulfide does

not persist but is oxidized again to sulfate. In certain lakes and seas, as well as

in a number of curative muds, the two processes, namely, the oxidation of

hydrogen sufide to sulfate and the reduction of sulfate to hydrogen sulfide, may

go on side by side. In the lower layers of the lake or sea where free oxygen is

scarce, the reduction process predominates. The hydrogen sulfide once formed

moves upward in the convection currents or as bubbles of gas, and at or near

the surface of the lake, on coming in contact with the oxygen of the air, it is

oxidized to sulfate by specific oxidizing bacteria. The sulfate may diffuse

downward again, where it is again reduced to sulfide under the anaerobic

conditions.

The reduction of sulfur to sulfide may be brought about by a great number of

different bacteria, but the reduction of sulfate is limited to very few organisms,

although these are widely distributed. Spirillum desulfuricans is the name

generally applied to the reducing form which is found in fresh water and soils.

As this short discussion indicates, there are striking similarities between" the

transformations of various compounds of sulfur and nitrogen through the



agency of microorganisms. Organic compounds of nitrogen decompose to form

ammonia, while the sulfur compounds lead to hydrogen sulfide. Ammonia is

changed to nitrite and nitrate, while sulfide is oxidized to sulfur, sulfate, and

numerous incompletely oxidized inorganic substances. These oxidations are all

the result of the action of specific autotrophic bacteria. Both nitrates and

sulfates ma)T be reduced by anaerobic bacteria, leading to a variety of products

including ammonia and" hydrogen sulfide. Although a process like nitrogen

fixation is not found in sulfur transformations, the reactions associated with

oxidation and reduction of elementary sulfur are similar in certain respects.

Inorganic and organic nitrogen compounds furnish different microorganisms

with their requirements for this element, and similar sulfur compounds serve a

like purpose in microbial metabolism. In fact, most of the nitrogen

transformations have very close counterparts in changes of sulfur compounds.

MICROBIAL TRANSFORMATIONS OF NITROGEN Ammonia, the end product of the reactions just considered, becomes the raw

material for the process of nitrate formation. Very few species of soil organisms

are concerned in this process. These organisms are conveniently divided into

two groups, in each of which a limited number of bacterial species is known.

One group oxidizes ammonia to nitrous acid or nitrite, and the other

transforms nitrous acid to nitric acid or nitrate. The process of conversion of

ammonia to the more highly oxidized inorganic compounds of nitrogen as

nitrite and nitrate is frequently referred to as nitrification.

None of the bacteria belonging to either group is able to transform ammonia

directly to nitrate, but each is confined to merely one stage of the reaction. It

seems possible that quite a variety of other microorganisms besides the specific

nitrifying bacteria may be concerned in the production of some nitrite or nitrate

from ammonia, but information concerning the relationship of these organisms

to the oxidation process is indefinite and little more than suggestive.



Nitrification by the specific bacteria is proportionally rapid and considered to

be of major importance in the formation of nitrate in soils.

The conditions suitable for the formation of nitrite and nitrate by nitrifying

bacteria are quite simple and include merely an inorganic medium containing

salts ofammonia and several nutrient elements, a neutral reaction, and aerobic

conditions. If soil is introduced into such a medium, active transformation of

ammonia results, first giving- rise to nitrite; when a large part of the ammonia

has disappeared, nitrate is formed in increasing amounts at the expense of the

nitrite. The two groups of bacteria concerned in the transformation are thus

quite distinct -from one another, but they are alike physiologically in that they

are both autotrophic, that is, they do not use organic substances as food but

can use the energy liberated in the specific oxidation processes for their growth

and development. The carbon necessary for the synthesis of their cells is taken

from the carbon dioxide of the atmosphere and used in a manner similar to its

utilization by green plants.

The organisms forming nitrite from ammonia have been designated as

Nitrosomonas or Nitrosococcus, depending upon their morphology. The

organisms producing nitrate from nitrite are called Nitrobacter. These forms are

all very similar in morphology being spherical or short oval cells, either motile

or non-motile. The difficulties involved in isolating these organisms in pure

culture have delayed the description of more than a few species. The fact that

very few species have been obtained is of less significance than the fact that

nitrifying organisms exist in practically all soils. Since Winogradsky first

obtained the organisms in 1891, they have been found to be active practically

everywhere, with the exception of certain acid forest and peat soils. After

drainage, cultivation, and liming, even these soils can be made a favorable

medium for nitrification. The optimum reaction for the activities of the



organisms is about pH 7.0 to 8.0, but some strains will still grow at as high

acidity as pH 3.5 and at an alkalinity of pH 10.0. Under conditions of

increasing acidity or alkalinity there develop certain acid-tolerant or alkali-

tolerant strains which have different reaction requirements from those strains

commonly encountered in most arable soils. However, at acidity greater than

pH 3.5, the nitrifying bacteria practically cease functioning. Acidity is but one

of the factors inhibiting development of nitrifying organisms in acid peat bogs.

The limited supply of oxygen in the water saturated environment is insufficient

to satisfy the requirements of these strictly aerobic bacteria. The addition of

lime to an acid soil, or the drainage of a water-logged soil, will, therefore,

materially favor the development of nitrifying organisms and the process of

nitrification. The organisms may be found at some depth in soils, but naturally

are much less abundant and little active in the lower layers, on account of the

less aerobic conditions and the lack of materials upon which to feed. In arid

soils these bacteria are more abundant in deeper layers than in soils of humid

regions, since plant roots penetrate to greater depths and moisture conditions

are more favorable at considerable distances below the surface.

The numbers of nitrifying organisms in soils vary greatly, depending upon

a'number of factors; in cultivated soils they have been found to range from a

few hundred to more than a million cells per gram.

The data in Table show that with increasing acidity there is a pronounced

decrease in the abundance of these organisms. In many cases the reaction of

the soil affects nitrifying organisms only indirectly by modifying plant

development, which, in turn, affects the soil organisms. Because of the limited

number of organisms associated with the process of nitrification and the



similarity of their characteristics of growth, the soil conditions under which

nitrate accumulates are much more restricted than for ammonia formation.

Nitrification is affected much more by modification of soil reaction, aeration,

moisture, and salt concentration than the process of ammonia formation is

altered by such changes. The treatment of soils with steam or volatile

antiseptics, known as processes of partial sterilization of soils, eliminates most

of the nitrite- and nitrate-forming organisms, while leaving many of the

ammonia forming organisms uninjured. This results in the accumulation of

ammonia in partially sterilized soils, since once the ammonia is forlned, it

persists as such, because of the lack of organisms capable of further

transforming it to nitrate.

In acid soils, such as raw-humus forest soils and certain acid peats which have

been drained and not limed liberally, the decomposition of the nitrogenous

organic substances is associated with the ·formation and accumulation of

nitrogen as ammonia, without its being changed to nitrate. In the case of forest

soils this process is of greatest importance in modifying the very nature of the

forest vegetation. In the" raw-humus" types of soil, only those trees develop

which can utilize .ammoniacal nitrogen and are capable of forming mycorrhiza

with special fungi, which decompose the organic matter. However, in the case

of soils favorable for nitrate formation, the "mull" types are produced. These are

considered richer soils; they bring about a more rapid regeneration of the

young forest, and the trees grow rapidly without having to depend upon the

formation of mycorrhiza. In the case of the" raw-humus" soils, the organic

matter is attacked largely by fungi that allow an abundant accumulation of

organic matter which is characteristically brown; the nitrogen is liberated as

ammonia. In the case of the" mull" soils, decomposition is more rapid and is

brought about by a varied microbial population including bacteri~,

actinomyces, and fungi, which· give rise to a black instead of a brown soil;

nitrogen is liberated as nitrate.

Undoubtedly some plants utilize nitrogen in forms other than nitrate, but many

plants use nitrate almost exclusively, and some use other forms than nitrate



principally during young stages of development. The marked effect of the

nitrification process on plant growth is shown in Table.

Nitrate formation is controlled or affected by numerous factors, chief among

which are presence of ammonia, soil reaction, aera tion, amount and kind of

organic matter, temperature, moisture, and concentration of inorganic

substances. MICROBIAL TRANSFORMATIONS OF MERCURY.

In addition to metals such as iron and manganese, which are largely nontoxic

to microorganisms and animals, there are a series of metals that have varied

toxic effects on microorganisms and homeothermic animals. Microorganisms

play important roles in modifying the toxicity of these metals (table).

The “metals” can be considered in broad categories. The “noble metals” tend

not to cross the vertebrate blood-brain barrier but can have distinct effects on

microorganisms. Microorganisms also can reduce ionic forms of noble metals to

their elemental forms.

The second group includes metals or metalloids that microorganisms can

methylate to form more mobile products called organometals. Some

organometals can cross the blood-brain barrier and affect the central nervous

system of vertebrates.



Organometals contain carbon-metal bonds. These bonds are their unique

identifying characteristics. The mercury cycle is of particular interest and

illustrates many characteristics of those metals that can be methylated.

Mercury compounds were widely used in industrial processes over the

centuries. One has only to think of Lewis Carroll’s allusion to this problem

when he wrote of the Mad Hatter in Alice in Wonderland. At that time mercury

was used in the shaping of felt hats. Microorganisms methylated some of the

mercury, thus rendering it more toxic to the hatmakers. A devastating

situation developed in southwestern Japan when large-scale mercury poisoning

occurred in the Minamata Bay region because of industrial mercury released

into the marine environment. Inorganic mercury that accumulated in bottom

muds of the bay was methylated by anaerobic bacteria of the genus

Desulfovibrio. Such methylated mercury forms are volatile and lipid soluble,

and the mercury concentrations increased in the food chain (by the process of

biomagnification).

The mercury was ultimately ingested by the human population, the “top

consumers,” through their primary food source—fish—leading to severe

neurological disorders.

A similar situation has occurred in many of the freshwater lakes in the north-

central United States and in Canada, where mercury compounds were used to

control microbial growth in pulp mills. Even decades later the fish in lakes

downstream from these pulp mills cannot yet be used for food, and fishing is

only for recreation.

The third group of metals occurs in ionic forms directly toxic to

microorganisms. The metals in this group also can affect more complex

organisms. However, plasma proteins react with the ionic forms of these metals

and aid in their excretion unless excessive long-term contact and ingestion

occur. Relatively high doses of these metals are required to cause lethal effects.

At lower concentrations many of these metals serve as required trace elements.

The differing sensitivity of more complex organisms and microorganisms to



metals forms the basis of many antiseptic procedures developed over the last

150 years.

The noble metals, although microorganisms tend to develop resistance to them,

continue to be used in preference to antibiotics in some medical applications.

Examples include the treatment of burns with silver-containing antimicrobial

compounds and the use of silver-plated catheters.

The Mercury Cycle

Interactions between the atmosphere, aerobic water, and anaerobic sediment

are critical. Microorganisms in anaerobic sediments, primarily Desulfovibrio,

can transform mercury to methylated forms that can be transported to water

and the atmosphere. These methylated forms also undergo biomagnification.

The production of volatile elemental mercury (Hg0) releases this metal to

waters and the atmosphere. Sulfide, if present in the anaerobic sediment, can

react with ionic mercury to produce less soluble HgS.



UNIT – 5 EXTREMOPHILES: The domain Archaea, acidophilic, alkalophilic, thermophilic, barophilic and osmophilic and radiodurant microbes – mechanisms and adaptation. Halophilic – membrane variation – electron transport – application of thermophiles and extremophiles. Extremozyme.

THE DOMAIN ARCHAEA MICROBES , ACIDOPHILIC, ALKALOPHILIC, THERMOPHILIC, BAROPHILIC AND OSMOPHILIC AND RADIODURANT

MICROBES – MECHANISMS AND ADAPTATION As a group the Archaea [Greek archaios, ancient] are quite diverse, both in

morphology and physiology. They can stain either gram positive or gram

negative and may be spherical, rod-shaped, spiral, lobed, plate-shaped,

irregularly shaped, or pleomorphic. Some are single cells, whereas others form

filaments or aggregates. They range in diameter from 0.1 to over 15 μm, and

some filaments can grow up to 200 _m in length. Multiplication may be by

binary fission, budding, fragmentation, or other mechanisms. Archaea are just

as diverse physiologically. They can be aerobic, facultatively anaerobic, or

strictly anaerobic. Nutritionally they range from chemolithoautotrophs to

organotrophs. Some are mesophiles; others are hyperthermophiles that can

grow above 100°C.

Archaea often are found in extreme aquatic and terrestrial habitats. They are

often present in anaerobic, hypersaline, or hightemperature environments.

Recently archaea have been discovered in cold environments. It appears that

they constitute up to 34% of the procaryotic biomass in coastal Antarctic

surface waters. A few are symbionts in animal digestive systems.

Archaeal Cell Walls Although archaea can stain either gram positive or gram negative depending on

the thickness and mass of the cell wall, their wall structure and chemistry

differ from that of the bacteria. There is considerable variety in archaeal wall

structure. Many gram-positive archaea have a wall with a single thick

homogeneous layer resembling that in gram-positive bacteria and thus stain

gram positive (figurea).



Gram-negative archaea lack the outer membrane and complex combined in

various ways to yield membranes of different rigidity and thickness. For

example, the C20 diethers can be used to make a regular bilayer membrane. A

much more rigid monolayer membrane may be constructed of C40 tetraether

lipids (figure 20.5b). Of course archaeal membranes may contain a mix of

diethers, tetraethers, and other lipids. As might be expected from their need for

stability, the membranes of extreme thermophiles such as Thermoplasma and

Sulfolobus are almost completely tetraether monolayers.

Metabolism Some archaea are organotrophs; others are autotrophic. A few even carry out

an unusual form of photosynthesis. Archaeal carbohydrate metabolism is best

understood. The enzyme 6-phosphofructokinase has not been found in

archaea, and they do not appear to degrade glucose by way of the Embden-

Meyerhof pathway. Extreme halophiles and thermophiles catabolize glucose

using a modified form of the Entner-Doudoroff pathway in which the initial

intermediates are not phosphorylated.

The halophiles have slightly different modifications of the pathway than do the

extreme thermophiles but still produce pyruvate and NADH or NADPH.

Methanogens do not catabolize glucose to any significant extent. In contrast

with glucose degradation, gluconeogenesis proceeds by a reversal of the



Embden-Meyerhof pathway in halophiles and methanogens. All archaea that

have been studied can oxidize pyruvate to acetyl-CoA. They lack the pyruvate

dehydrogenase complex present in eucaryotes and respiratory bacteria and use

the enzyme pyruvate oxidoreductase for this purpose. Halophiles and the

extreme thermophile Thermoplasma do seem to have a functional tricarboxylic

acid cycle. No methanogen has yet been found with a complete tricarboxylic

acid cycle. Evidence for functional respiratory chains has been obtained in

halophiles and thermophiles.

Mechanisms of Autotrophic CO2 Fixation. The reductive tricarboxylic acid cycle. The cycle is reversed with ATP and reducing equivalents [H] to form acetyl-CoA from CO2. The acetyl-CoA may be carboxylated to yield pyruvate, which can then be converted to glucose and other compounds. This sequence appears to function in Thermoproteus neutrophilus.



The synthesis of acetyl-CoA and pyruvate from CO2 in Methanobacterium thermoautotrophicum. One carbon comes from the reduction of CO2 to a methyl group, and the second is produced by reducing CO2 to carbon monoxide through the action of the enzyme CO dehydrogenase (E1). The two carbons are then combined to form an acetyl group. Corrin-E2 represents the cobamide-containing enzyme involved in methyl transfers



ACIDOPHILIC MICROBES - MECHANISMS AND ADAPTATION Acidophiles or Acidophilic organisms are those that thrive under highly

acidic conditions (usually at pH 2.0 or below). These organisms can be found in

different branches of the tree of life, including Archaea, Bacteria,

and Eukaryotes.

A partial list of these organisms include:

Archaea

Sulfolobales, an order in the Crenarchaeota branch of Archaea,

Thermoplasmatales, an order in the Euryarchaeota branch of Archaea,

Acidianus brierleyi, A. infernus, facultatively anaerobic thermoacidophilic

archaebacteria , Metallosphaera sedula, thermoacidophilic

Bacteria

Acidobacterium, a phylum of Bacteria

Acidithiobacillales, an order of Proteobacteria e.g. A.ferrooxidans, A. thiooxidans

Thiobacillus prosperus, T. acidophilus, T. organovorus, T. cuprinus

Acetobacter aceti, a bacterium that produces acetic acid (vinegar) from the

oxidation of ethanol.

Alicyclobacillus, a genus of bacteria that can contaminate fruit juices

Mechanisms of adaptation Most acidophile organisms have evolved extremely efficient mechanisms to

pump protons out of the intracellular space in order to keep the cytoplasm at

or near neutral pH. Therefore, intracellular proteins do not need to develop acid

stability through evolution. However, other acidophiles, such

as Acetobacter aceti, have an acidified cytoplasm which forces nearly all

proteins in the genome to evolve acid stability.

For this reason, Acetobacter aceti has become a valuable resource for

understanding the mechanisms by which proteins can attain acid stability.

Studies of proteins adapted to low pH have revealed a few general mechanisms

by which proteins can achieve acid stability. In most acid stable proteins (such

as pepsin and the soxF protein from Sulfolobus acidocaldarius), there is an



overabundance of acidic residues which minimizes low pH destabilization

induced by a buildup of positive charge. Other mechanisms include

minimization of solvent accessibility of acidic residues or binding of metal

cofactors. In a specialized case of acid stability, the NAPase protein

from Nocardiopsis alba was shown to have relocated acid-sensitive salt

bridges away from regions that play an important role in the unfolding process.

In this case of kinetic acid stability, protein longevity is accomplished across a

wide range of pH, both acidic and basic

ALKALOPHILIC MICROBES – MECHANISMS AND ADAPTATION Alkaliphiles are a class of extremophilic microbes capable of survival in alkaline

(pH roughly 8.5-11) environments, growing optimally around a pH of 10. These

bacteria can be further categorized as obligate alkaliphiles (those that require

high pH to survive), facultative alkaliphiles (those able to survive in high pH,

but also grow under normal conditions) and haloalakliphiles (those that require

high salt content to survive).

Adaptation: Alkaliphiles maintain cytosolic acidification through both passive and active

means. In passive acidification, it has been proposed that cell walls contain

acidic polymers composed of residues such as galacturonic acid, gluconic acid,

glutamic acid, aspartic acid, and phosphoric acid. Together, these residues

form an acidic matrix that helps protect the plasma membrane from alkaline

conditions by preventing the entry of hydroxide ions, and allowing for the

uptake of sodium and hydronium ions. In addition, the peptidoglycan in

alkaliphilic B. subtilis has been observed to contain higher levels of

hexosamines and amino acids as compared to its neutrophilic counterpart.

When alkaliphiles lose these acidic residues in the form of induced mutations,

it has been shown that their ability to grow in alkaline conditions is severely

hindered.

However, it is generally agreed upon that passive methods of cytosolic

acidification are not sufficient to maintain an internal pH 2-2.3 levels blow that

of external pH; there must also be active forms of acidification. The most



characterized method of active acidification is in the form of

Na+/H+ antiporters. In this model, H+ ions are first extruded through the

electron transport chain in respiring cells and to some extent through

an ATPase in fermentative cells. This proton extrusion establishes a proton

gradient that drives electrogenic antiporters—which drive intracellular Na+ out

of the cell in exchange for a greater number of H+ ions, leading to the net

accumulation of internal protons. This proton accumulation leads to a lowering

of cytosolic pH. The extruded Na+ can be used for solute symport, which are

necessary for cellular processes. It has been noted that Na+/H+ antiport is

required for alkaliphilic growth, whereas either K+/H+ antiporters or Na+/H+

antiporters can be utilized by neutrophilic bacteria. If Na+/H+ antiporters are

disabled through mutation or another means, the bacteria are rendered

neutrophilic. The sodium required for this antiport system is the reason some

alkaliphiles can only grow in saline environments.

ATP production: In addition to the method of proton extrusion discussed above, it is believed

that the general method of cellular respiration is different in obligate

alkaliphiles as compared to neutrophiles. Generally, ATP production operates

by establishing a proton gradient (greater H+ concentration outside the

membrane) and a transmembrane electrical potential (with a positive charge

outside the membrane).

However, since alkaliphiles have a reversed pH gradient, it would seem that

ATP production—which is based on a strong proton motive force—would be

severely reduced. However, the opposite is true. It has been proposed that

while the pH gradient has been reversed, the transmembrane electrical

potential is greatly increased. This increase in charge causes the production of

greater amounts of ATP by each translocated proton when driven through an

ATPase.

THERMOPHILIC MICROBES – MECHANISMS AND ADAPTATION A thermophile is an organism — a type of extremophile — that thrives at

relatively high temperatures, between 45 and 122 °C (113 and 252 °F). Many



thermophiles are archaea. Thermophilic eubacteria are suggested to have been

among the earliest bacteria.

Unlike other types of bacteria, thermophiles can survive at much hotter

temperatures; where as other bacteria would be damaged and sometimes killed

if exposed to the same temperatures.

As a prerequisite for their survival, thermophiles contain enzymes that can

function at high temperatures. Some of these enzymes are used in molecular

biology (for example, heat-stable DNA polymerases for PCR), and in washing

agents.

Thermophiles are classified into obligate and facultative thermophiles: Obligate

thermophiles (also called extreme thermophiles) require such high

temperatures for growth, whereas facultative thermophiles (also called

moderate thermophiles) can thrive at high temperatures, but also at lower

temperatures (below 50°C). Hyperthermophiles are particularly extreme

thermophiles for which the optimal temperatures are above 80°C.

Bacteria within the Alicyclobacillus genus are acidophilic thermophiles, which

can cause contamination in fruit juice drinks.

Thermophiles, meaning heat-loving, are organisms with an optimum growth

temperature of 50°C or more, a maximum of up to 70°C or more, and a

minimum of about 40°C, but these are only approximate. Some extreme

thermophiles (hyperthermophiles) require a very high temperature (80°C to

105°C) for growth. Their membranes and proteins are unusually stable at these

extremely high temperatures. Thus, many important biotechnological processes

use thermophilic enzymes because of their ability to withstand intense heat.

Many of the hyperthermophiles Archea require elemental sulfur for growth.

Some are anaerobes that use the sulfur instead of oxygen as an electron

acceptor during cellular respiration. Some are lithotrophs that oxidize sulfur

to sulfuric acid as an energy source, thus requiring the microorganism to be

adapted to very low pH (i.e., it is an acidophile as well as thermophile). These

organisms are inhabitants of hot, sulfur-rich environments usually associated

with volcanism, such as hot springs, geysers, and fumaroles



BAROPHILIC MICROBES – MECHANISMS AND ADAPTATION A piezophile (also called a barophile) is an organism which thrives at high

pressures, such as deep sea bacteria or archaea. They are generally found

on ocean floors, where pressure often exceeds 380 atm (38 MPa). Some have

been found at the bottom of the Pacific Ocean where the maximum pressure is

roughly 117 MPa. The high pressures experienced by these organisms can

cause the normally fluid cell membrane to become waxy and

relatively impermeable to nutrients. These organisms have adapted in novel

ways to become tolerant of these pressures in order to colonize deep

sea habitats. One example, xenophyophores, has been found in the deepest

ocean trench, 6.6 miles (10,541 meters) below the surface.

Barotolerant bacteria are able to survive at high pressures, but can exist in less

extreme environments as well. Obligate barophiles cannot survive outside of

such environments. For example, theHalomonas species Halomonas

salaria requires a pressure of 1000 atm (100 MPa) and a temperature of

3 degrees Celsius. Most piezophiles grow in darkness and are usually very UV-

sensitive; they lack many mechanisms of DNA repair

OSMOPHILIC MICROBES – MECHANISMS AND ADAPTATION Osmophilic organisms are microorganisms adapted to environments with high

osmotic pressures, such as high sugar concentrations. Osmophiles are similar

to halophillic (salt-loving) organisms because a critical aspect of both types of

environment is their low water activity, aW. High sugar concentrations

represent a growth-limiting factor for many microorganisms, yet osmophiles

protect themselves against this high osmotic pressure by the synthesis

of osmoprotectants such as alcohols and amino acids. Nearly all osmophilic

microorganisms are from theyeast genus.

Osmophile yeasts are important because they cause spoilage in the sugar and

sweet goods industry, with products such as fruit juices, fruit juice

concentrates, liquid sugars (such as golden syrup), honey and in some cases

marzipan. Among the most osmophillic are:



Saccharomyces rouxii - 0.62 aW

Saccharomyces bailii - 0.80 aW

Debaryomyces - 0.83 aW

Wallemia sebi - 0.87 aW

Saccharomyces cerevisiae - 0.90 aW

RADIODURANT MICROBES – MECHANISMS AND ADAPTATION Radiodurans is an extremophilic bacterium, one of the most radioresistant

organisms known. It can survive cold, dehydration, vacuum, and acid, and is

therefore known as a polyextremophile.

Eg. Deinococcus Radiodurans D. radiodurans is a rather large, spherical bacterium, with a diameter of 1.5 to

3.5 µm. Four cells normally stick together, forming a tetrad. The bacteria are

easily cultured and do not appear to cause disease. Colonies are smooth,

convex, and pink to red in color. The cells stain Gram positive, although its cell

envelope is unusual and is reminiscent of the cell walls of Gram negative

bacteria.

D. radiodurans does not form endospores and is nonmotile. It is an obligate

aerobic chemoorganoheterotroph, i.e., it uses oxygen to derive energy from

organic compounds in its environment. It is often found in habitats rich in

organic materials, such as soil, feces, meat, or sewage, but has also been

isolated from dried foods, room dust, medical instruments and textiles.

It is extremely resistant to ionizing radiation, ultraviolet light, desiccation, and

oxidizing and electrophilic agents.

Its genome consists of two circular chromosomes, one 2.65 million base pairs

long and the other 412,000 base pairs long, as well as a megaplasmid of

177,000 base pairs and a plasmid of 46,000 base pairs. It has about

3,195 genes. In its stationary phase, each bacterial cell contains four copies of

this genome; when rapidly multiplying, each bacterium contains 8-10 copies of

the genome.

Ionizing radiation resistance mechanisms



Deinococcus accomplishes its resistance to radiation by having multiple copies

of its genome and rapid DNA repair mechanisms. It usually repairs breaks in

its chromosomes within 12–24 hours through a 2-step process. First, D.

radiodurans reconnects some chromosome fragments through a process

called single-stranded annealing. In the second step, a protein mends double-

strand breaks through homologous recombination. This process does not

introduce any more mutations than a normal round of replication would. HALOPHILIC – MEMBRANE VARIATION – ELECTRON TRANSPORT

The halophilic (“salt-loving”) bacterium Halobacterium salinarum, an

archaebacterium derived from very ancient evolutionary progenitors, traps the

energy of sunlight in a process very different from the photosynthetic

mechanisms we have described so far. This bacterium lives only in brine ponds

and salt lakes (Great Salt Lake and the Dead Sea, for example), where the high

salt concentration—which can exceed 4 M—results from water loss by

evaporation; indeed, halobacteria cannot live in NaCl concentrations lower than

3 M. These organisms are aerobes and normally use O2 to oxidize organic fuel

molecules. However, the solubility of O2 is so low in brine ponds that

sometimes oxidative metabolism must be supplemented by sunlight as an

alternative source of energy.

The plasma membrane of H. salinarum contains patches of the light-absorbing

pigment bacteriorhodopsin, which contains retinal as a prosthetic group.

When the cells are illuminated, all-trans-retinal bound to the

bacteriorhodopsin absorbs a photon and undergoes photoisomerization to 13-

cis-retinal. The restoration of all-trans-retinal is accompanied by the outward

movement of protons through the plasma membrane. Bacteriorhodopsin, with

only 247 amino acid residues, is the simplest light-driven proton pump known.

The difference in the three-dimensional structure of bacteriorhodopsin in the

dark and after illumination (Fig.a) suggests a pathway by which a concerted

series of proton “hops” could effectively move a proton across the membrane.

Light-driven proton pumping by bacteriorhodopsin.



Bacteriorhodopsin (Mr 26,000) has seven membrane-spanning α helices (PDB

ID 1C8R). The chromophore all-trans-retinal (purple) is covalently attached via

a Schiff base to the Є-amino group of a Lys residue deep in the membrane

interior. Running through the protein are a series of Asp and Glu residues and

a series of closely associated water molecules that together provide the

transmembrane path for protons (red arrows). Steps 1 through 5 indicate

proton movements, described below.

The chromophore retinal is bound through a Schiff-base linkage to the Є-

amino group of a Lys residue. In the dark, the N of this Schiff base is

protonated; in the light, photoisomerization of retinal lowers the pKa of this

group and it releases its proton to a nearby Asp residue, triggering a series of

proton hops that ultimately result in the release of a proton at the outer

surface of the membrane (Fig.b).

The electrochemical potential across the membrane drives protons back into

the cell through a membrane ATP synthase complex very similar to that of

mitochondria and chloroplasts. Thus, when O2 is limited, halobacteria can use

light to supplement the ATP synthesized by oxidative phosphorylation.

Halobacteria do not evolve O2, nor do they carry out photoreduction of NADP+;

their phototransducing machinery is therefore much simpler than that of

cyanobacteria or plants. Nevertheless, the proton-pumping mechanism used by

this simple protein may prove to be prototypical for the many other, more

complexes, ion pumps.



In the dark (left panel), the Schiff base is protonated. Illumination (right panel)

photoisomerizes the retinal, forcing subtle conformational changes in the

protein that alter the distance between the Schiff base and its neighboring

amino acid residues. Interaction with these neighbors lowers the pKa of the

protonated Schiff base, and the base gives up its proton to a nearby carboxyl

group on Asp85 (step 1 in (a)).

This initiates a series of concerted proton hops between water molecules in the

interior of the protein, which ends with 2 the release of a proton that was

shared by Glu194 and Glu204 near the extracellular surface. 3 The Schiff base

reacquires a proton from Asp96, which 4 takes up a proton from the cytosol. 5

Finally, Asp85 gives up its proton, leading to reprotonation of the Glu204-

Glu194 pair. The system is now ready for another round of proton pumping.

APPLICATION OF THERMOPHILES AND EXTREMOPHILES The thermoalkaliphilic catalase, which initiates the breakdown of hydrogen

peroxide into oxygen and water, was isolated from an organism, Thermus

brockianus, found in Yellowstone National Park by Idaho National

Laboratory researchers. The catalase operates over a temperature range from

30°C to over 94°C and a pH range from 6-10. This catalase is extremely stable

compared to other catalases at high temperatures and pH. In a comparative



study, the T. brockianus catalase exhibited a half life of 15 days at 80°C and pH

10 while a catalase derived from Aspergillus niger had a half life of 15 seconds

under the same conditions. The catalase will have applications for removal of

hydrogen peroxide in industrial processes such as pulp and paper bleaching,

textile bleaching, food pasteurization, and surface decontamination of food

packaging. DNA modifying enzymes such as Taq DNA polymerase and

some Bacillus enzymes used in clinical diagnostics and starch liquefaction are

produced commercially by several biotechnology companies

EXTREMOZYME An extremozyme is an enzyme, often created by extremophiles that can

function under extreme environmental conditions such as very high pH, very

low pH, high temperature, high salinity, or other factors, that would otherwise

denature typical enzymes (e.g. catalase, rubisco, carbonic anhydrase). This

feature makes these enzymes of interest to a variety of biotechnical

applications.



UNIT – 6 ROLES OF MICROBES IN BIODEGRADATION Biodegradation of Xenobiotics, hydrocarbon, pesticides and plastics. Biodeteroration of wood, pulp and paper. Biosorption/ bioaccumulation of heavy metals. Bioremediation of soil, air, and water; various methods, advantages and disadvantages. Bioleaching of iron, copper, gold and uranium.

BIODEGRADATION OF XENOBIOTICS Many environmental contaminants are subject to chemical or photochemical

reactions. However, biological organisms—particularly microorganisms—play a

more important role in the removal of many hazardous organics from the

environment. Thermodynamically feasible contaminant transformations often

do not occur in the absence of a biological catalyst, due to kinetic limitations,

but are facilitated by microorganisms via enzymes, which lower the activation

energy that must be overcome for a reaction to proceed, and the investment of

biochemical energy to convert oxygen and other key coreactants to more

reactive forms.

Extent of Biodegradation Biodegradation is the general term used to describe the biological conversion of

organic contaminants to products that are generally lower in free energy.

This term is often used loosely and interpreted in various ways. However, it

does not imply anything about the extent of contaminant transformation or

detoxification. Thus, biodegradation refers to biotransformation reactions that

result in only minor changes in contaminant structures, as well as

mineralization, which is the conversion of organic compounds into their

inorganic constituents (e.g., H2O, CO2, NO3 −, SO4 2−, PO43−, and Cl−). In some

cases, biotransformation reactions generate products that have similar or

greater levels of toxicity than those of the parent contaminant. Examples

include the conversion of nitroaromatic compounds to more reactive and toxic

nitroso and hydroxylamino derivatives, and the anaerobic conversion of the

suspected carcinogen trichloroethene (TCE) to the known carcinogen vinyl

chloride (VC).



In contrast, the inorganic products of mineralization usually pose no health

risks at the concentrations produced by contaminant biodegradation in the

environment. Thus, care should be taken in interpreting simple observations of

parent compound removal in terms of hazard reduction, and biodegradation

products should be identified to ensure that bioremediation goals are being

met. BIODEGRADATION OF HYDROCARBON

Hydrocarbons—compounds consisting of carbon and hydrogen alone—are

common groundwater contaminants and often amenable to bioremediation.

Releases of gasoline and other refined petroleum products represent a major

source of hydrocarbon contamination.

The BTEX compounds are of particular concern and regulatory importance

because they constitute a significant fraction of gasoline and are relatively

soluble and highly toxic. Benzene is a known human carcinogen.

PAHs containing from two to five fused aromatic rings are also of significant

concern because of the mutagenicity and carcinogenicity of several of these

compounds and their tendency to bioaccumulate. PAHs are generated from the

incomplete combustion of organic matter. Extensive PAH contamination is

associated with coal gasification sites (manufactured-gas plants), as well as the

production and use of the coal tar creosote, a wood preservative. Methyl tert-

butyl ether (MTBE) is a fuel additive that was used originally as a replacement

for tetraethyllead to increase the octane rating of gasoline and prevent engine

knocking when leaded gasoline was phased out in the 1970s and 1980s. The

1990 Clean Air Act Amendments led to the addition of increased amounts of

MTBE to reformulated gasoline; however, MTBE is a problematic groundwater

pollutant and its use as a fuel additive is being phased out, due to concerns

about its potential carcinogenicity, taste and odor problems at very low MTBE

concentrations, and its tendency to migrate through the subsurface more

rapidly than do hydrocarbon co-contaminants such as the BTEX compounds.

BTEX Bacteria that can aerobically biodegrade BTEX compounds are

indigenous at nearly all contaminated sites. Aerobic biodegradation of a BTEX



compound is initiated by one of many different monooxygenases and/or

dioxygenases that can react with these compounds. Dioxygenase-mediated

attack of the aromatic nucleus of benzene yields catechol (1,2-

dihydroxybenzene)

Biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) via initial Oxygenase-catalyzed reactions that lead to the formation of catechols, which undergo ring cleavage and are ultimately converted to central metabolic pathway intermediates. Note that individual arrows frequently encompass multiple reactions. The aliphatic side chains and aromatic rings of toluene, ethylbenzene, or the

xylene isomers also serve as sites of initial attack by oxygenases, which

generally lead to formation of substituted catechols. The presence of hydroxyl

groups on adjacent carbons prepares the aromatic ring for further reaction



with ring cleavage dioxygenases through the insertion of two additional oxygen

atoms. Ring cleavage serves two important functions:

Regeneration of the NAD(P)H that was invested in activating the ring for

cleavage, and generation of metabolic intermediates that are used in synthesis

and energy generation.

The aromatic ring may be opened between the hydroxyl groups, via the ortho

cleavage (β-ketoadipate) pathway, or adjacent to the hydroxyl groups, via the

meta cleavage (TOL) pathway. The products of both ring fission reactions are

further degraded to form key intermediates in central metabolic pathways.

Field and laboratory studies have also demonstrated that biodegradation of

BTEX compounds can occur under different anaerobic terminal electron-

accepting processes (TEAPs), including Fe(III), nitrate, and sulfate reduction

and methanogenesis. Toluene is often the most readily degraded BTEX

compound under anaerobic conditions. The anaerobic biodegradation of the

other BTEX compounds has been observed at some contaminated sites, but not

others. In particular, benzene is frequently recalcitrant or biodegraded only

after lengthy lag periods. Thus, while growth of bacterial strains on benzene

under denitrifying conditions has been observed. Anaerobic benzene

degradation appears to be a highly site-specific process.

Anaerobic bacteria use several different strategies to initiate the biodegradation

of BTEX compounds; however, they all appear to direct the contaminants to

formation of benzoyl-CoA as a central biodegradation intermediate, analogous

to the formation of catecholic compounds during aerobic BTEX biodegradation.

The CoA (coenzyme A) substituent is analogous to the dihydroxy groups in

catecholic compounds in that it prepares the aromatic nucleus for subsequent

(reduction) reactions that lead to destabilization and cleavage of the ring under

anaerobic conditions.

The initial attack on toluene involves the formation of a new carbon–carbon

bond through the reaction of the methyl group with fumarate. The fumarate

addition reaction initiates toluene biodegradation under a broad range of

anaerobic TEAPs, and analogous reactions have been observed for other



methylated aromatics (including m-xylene) and methylene groups in aliphatic

compounds. Anaerobic ethylbenzene biodegradation is initiated by a

dehydrogenation reaction, an oxidation that results in the hydroxylation of the

aliphatic substituent but, unlike reactions mediated by oxygenases, uses water

rather than molecular oxygen as the coreactant.

PAHs Under aerobic conditions, biodegradation of PAHs with two or three aromatic

rings such as naphthalene, anthracene, and phenanthrene often occurs readily

via reactions that are analogous to the biodegradation of the monoaromatic

BTEX compounds. Monooxygenase- or dioxygenase-catalyzed reactions lead to

the formation of catechols or o-phthalate (1,2-benzenedicarboxylate)

intermediates that can be attacked by dioxygenases leading to eventual ring

cleavage.

Similar reactions may also contribute to the biodegradation of four- and five-

ring PAHs; however, the solubilities of these higher-molecular-weight PAHs are

extremely low and limit the biodegradation of these compounds. Bacteria use

different trategies for increasing the limited bioavailability of PAHs, including

the formation of biofilms on PAH crystals and production of biosurfactants that

enhance their dissolution.

Evaluation of bioavailability and treatment strategies are discussed below.

Utilization of naphthalene as the sole carbon and energy source has been

demonstrated under denitrifying and sulfate-reducing conditions, and

biodegradation of several other PAHs under these TEAPs has been observed.

Under sulfate-reducing conditions, naphthalene is converted to 2-

methylnaphthalene, which is further transformed through the addition of

fumarate (as observed for toluene and m-xylene).

MTBE The MTBE molecule contains both a stable ether bond and bulky methyl

branching, structural features that are often resistant to biodegradation. Under

aerobic conditions, cometabolism of MTBE can be mediated by organisms

growing on short (C3–C5) normal and branched alkanes or other organic



compounds that may be present as co-contaminants due to their presence in

gasoline.

Aerobic growth on MTBE has also been observed. Metabolic and co-metabolic

biodegradation of MTBE under aerobic conditions is initiated by a

monooxygenase-mediated attack on the methoxy group and leads to the

formation of tert-butyl alcohol (TBA) plus either formaldehyde or formic acid,

depending on the degradation pathway. In some cases, TBA persists; however,

TBA can putatively be funneled into central metabolic pathways via several

routes and can be used by some aerobic microorganisms as the sole source of

carbon and energy. Nevertheless, MTBE and TBA frequently persist at

contaminated sites.

This suggests that microorganisms that can biodegrade MTBE and/or TBA are

not abundant in the environment. Another possibility is that the concentration

of MTBE present at contaminated sites is below the minimum substrate level or

threshold needed to sustain growth. There is evidence suggesting that

substantial amounts of MTBE can be biodegraded anaerobically at a number of

gasoline-contaminated sites, often after a lengthy lag period. However, in some

cases, anaerobic biodegradation of MTBE does not proceed past TBA, which is

an unacceptable bioremediation end product, due to its toxicity.

Chlorinated Aliphatic Hydrocarbons Chlorinated aliphatic hydrocarbons (CAHs), including chlorinated methanes,

ethanes, and ethenes, have a number of industrial uses. In particular, their

widespread use as solvents (e.g., in the removal of grease from metal, clothing,

and other materials) has led to frequent contamination of soil and groundwater

through spills and improper disposal, particularly at military and industrial

sites and dry-cleaning facilities. Contamination with CAHs is of concern

because of their toxicity to humans and, in many cases, known or likely

carcinogenicity.

As discussed above, polar carbon–halogen bonds may serve as the site of initial

attack for each of the three major biodegradation mechanisms—hydrolysis,

oxidation involving electrophilic oxygen, and reduction—although individual



CAHs vary with respect to their susceptibility to transformation via a given

mechanism. Hydrolysis is most commonly observed for CAHs with two or fewer

chlorine substituents on a given carbon.

Hydrolytic attack of dichloromethane and 1,2-dichloroethane by some

organisms allows them to use the CAHs as a source of carbon and energy, and

longer halogenated alkanes are also subject to hydrolysis. The selection of an

oxidative or reductive biodegradation-based cleanup approach depends on the

predominant redox conditions in the contaminated groundwater, the relative

susceptibility of the contaminant(s) to oxidation and reduction reactions, the

physiological capabilities of the indigenous microorganisms, and the

availability of co-substrates, including electron donors and/or oxygen. For

example, there are many challenges associated with the implementation of

bioremediation strategies based on co-metabolic reactions. As discussed above,

biodegradation of co-metabolic substrates occurs relatively slowly compared to

metabolic processes due to competition with growth substrates for key

enzymes, diversion of coreactants in metabolic reactions, and/or cellular

damage caused by transformation product toxicity. Nevertheless, if the

contaminated groundwater is aerobic and a potential source of carbon and

energy (e.g., toluene or methane) is present, it may be reasonable to select a

bioremediation strategy based on aerobic co-metabolism. Co-metabolic

oxidation has also been used successfully to bioremediate TCE-contaminated

groundwater through the careful addition of both oxygen and either phenol or

toluene as the source of carbon and energy.

Only the most highly chlorinated aliphatic hydrocarbons (e.g., carbon

tetrachloride, TeCA, and PCE) appear to be resistant to aerobic co-metabolic

transformations.

On the other hand, all chlorinated methanes, ethanes, and ethenes can

undergo reductive dechlorination reactions, although reductive dechlorination

of more highly chlorinated CAHs (e.g., PCE and TCE) tends to occur at faster

rates than does transformation of the less chlorinated analogs (e.g., DCE and

VC). Thus, efforts to bioremediate anaerobic groundwater systems



contaminated with highly chlorinated aliphatic hydrocarbons typically focus on

promoting reductive dechlorination processes. Often, this involves the addition

of electron donors, a practice known as biostimulation, due to the limited

availability of suitable electron donors at most CAH-contaminated sites. In

particular, biostimulation of reductive dechlorination reactions is an effective

bioremediation practice for CAHs that can serve as terminal electron acceptors

in dehalorespiration.

CAHs can serve as terminal electron acceptors, and their susceptibility to co-

metabolic biotransformations and utilization as an electron donor under

aerobic and anaerobic conditions. However, the biodegradation of CAHs is an

active area of research, and new information on the metabolic roles that these

compounds can fulfill is constantly emerging. For example, utilization of

chloromethane and cis-1, 2 – DCE as electron donors under anaerobic and

aerobic conditions, respectively, has been reported. Further, several

chlorinated ethanes, including 1,1,1-trichloroethane, 1,1,2-trichloroethane,

1,1-dichloroethane, and 1,2-dichloroethane, are now known to serve as

terminal electron acceptors for certain dehalorespirers.

Halogenated Aromatic Hydrocarbons Several categories of halogenated aromatic hydrocarbons are of environmental

significance, including polychlorinated biphenyls (PCBs) and polybrominated

diphenyl ethers (PBDEs). PCBs were widely used in electrical capacitors and

transformers and dielectric and hydraulic fluids until the 1970s, when PCB use

was banned in the United States. Spills and improper disposal practices led to

widespread and persistent contamination of the environment with PCBs. The

concern over PCBs is due primarily to their tendency to partition into organic

matter and thus bioaccumulate in ecological food chains. Unlike PCBs, PBDEs

break down with heat.

In the process, they release bromine radicals that help quench combustion

processes.

These properties have led to their extensive use as flame retardants in plastics

and textiles and their broad dissemination in the environment. PBDEs levels



are also increasing rapidly in animal tissues and human breast milk, which is

of concern due to the endocrine-disrupting action of some PBDEs.

PCBs Up to 209 distinct PCB molecules (or congeners) that differ with respect to the

numbers and positions of chlorine substituents on the biphenyl backbone can

be found.

Structure and numbering of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). However, PCBs were typically produced and sold commercially as mixtures of

60 to 90 congeners under the U.S. trade name Aroclor. The Aroclor product

numbers indicate the overall degree of chlorination in the mixture. For

example, Aroclor 1242 contains 12 carbon atoms and 42% chlorine by weight,

which corresponds to an average of approximately three chlorines per biphenyl

molecule. Microorganisms utilize strategies similar to those involved in the

biodegradation of CAHs and the BTEX compounds to biodegrade PCBs.

However, individual congeners vary with respect to their susceptibility to

various biotransformation mechanisms, which can complicate efforts to

detoxify Aroclors under a given set of conditions. Under aerobic conditions,

lightly chlorinated PCBs (generally those with three or fewer chlorines) are

converted to dihydroxylated intermediates by dioxygenases, reactions that are

analogous to the ring activation mechanisms in BTEX biodegradation. The

dioxygenases typically attack the 2 and 3 positions (Figure 8.5) on the more



lightly chlorinated ring and are hindered by chlorine substituents in these

positions. The activated ring undergoes meta cleavage, as in toluene

biodegradation, and leads to the formation of a chlorobenzoic acid, which is not

transformed by most PCB-degrading bacteria but generally can be mineralized

by chlorobenzoate-degrading populations. Other bacteria can dihydroxylate

certain PCBs in the 3 and 4 positions via a similar biodegradation pathway.

Bacteria that can aerobically degrade lightly chlorinated PCBs (either

metabolically or cometabolically while growing on biphenyl or another primary

substrate) appear to be fairly common in contaminated soils. Thus, under

aerobic conditions, the composition of an Aroclor mixture is expected to shift

toward more highly chlorinated congeners, which tend to persist under aerobic

conditions. Conversely, the more highly chlorinated PCB congeners are better

suited than the lightly chlorinated congeners for reductive dechlorination.

Several microbial reductive dechlorination processes that target chlorines in

different positions have been identified, although dechlorinations in the para

and meta positions (relative to the biphenyl linkage) generally are dominant.

Recently, a Dehalococcoides population and a related strain that can respire

PCBs as terminal electron acceptors have been identified.

PBDEs The nomenclature and number of PBDE congeners are the same as for the

PCBs.

Studies of the biodegradability of PBDEs have largely focused on the potential

for dechlorination of the fully brominated decabromodiphenyl ether and other

highly brominated congeners, due to their potential for biodegradation to less

brominated congeners, which are more toxic and bioavailable.

Microbial reductive dechlorination of highly brominated PBDEs has been

observed in both complex cultures and in pure dehalorespiring cultures

maintained on chlorinated electron acceptors. Up to five bromines were

removed by the dehalorespiring strains. At least two aspects of anaerobic PBDE

and PCB biodegradation are similar. First, dechlorination of the more highly

halogenated congeners occurred more slowly than did PBDEs containing fewer



bromine substituents, presumably due to the reduced bioavailability of the

more hydrophobic, highly brominated congeners. Second, removal of bromines

occurred predominantly in the para and Meta positions (relative to the ether

linkage). BIODEGRADATION OF PESTICIDES

According to the definition by the International Union of Pure and Applied

Chemistry, the term biodegradation is “Breakdown of a substance catalyzed by

enzymes in vitro or in vivo. This may be characterized for the purpose of hazard

assessment such as:

Primary Alteration of the chemical structure of a substance resulting in loss of a

specific property of that substance.

Environmentally acceptable Biodegradation to such an extent as to remove undesirable properties of the

compound. This often corresponds to primary biodegradation but it depends on

the circumstances under which the products are discharged into the

environment.

Ultimate Complete breakdown of a compound to either fully oxidized or reduced simple

molecules (such as carbon dioxide/methane, nitrate/ammonium and water).

It should be noted that the biodegradation products can be more harmful than

the substance degraded.”

Microbial degradation of chemical compounds in the environment is an

important route for the removal of these compounds. The biodegradation of

these compounds, i.e., pesticides, is often complex and involves a series of

biochemical reactions. Although many enzymes efficiently catalyze the

biodegradation of pesticides, the full understanding of the biodegradation

pathway often requires new investigations. Several pesticide biodegradation

studies have shown only the total of degraded pesticide, but have not

investigated in depth the new biotransformed products and their fate in the

environment.



Organochorine pesticides The organochlorine pesticides are known to be highly persistant in the

environment. This class of pesticides includes the chlorinated derivatives of

diphenyl ethane (dichlorodiphenyltrichloroethane - DDT, its metabolites

dichlorodiphenyldichloroethylene - DDE, dichlorodiphenyldichloroethane -

DDD, and methoxychlor), hexachlorobenzene (HCB), the group of

hexachlorocyclohexane (�-HCH, �-HCH, ϒ-HCH, �-HCH, or lindane), the

group of cyclodiene (aldrin, dieldrin, endrin, chlordane, nonachlor, heptachlor

and heptachlor-epoxide), and chlorinated hydrocarbons (dodecachlorine,

toxaphene, and chlordecone)

Figure shows some structures of organochlorine pesticides.

Microbial degradation of organochloride pesticides



The fate of pesticides in the environment is determined by both biotic and

abiotic factors. The rate at which different pesticides are biodegraded varies

widely. Some pesticides such as DDT and dieldrin have proven to be

recalcitrant. Consequently, they remain in the environment for a long time and

accumulate into food chains for decades after their application to the soil.

Most of the studies involving the biodegradation of organochlorine pesticides

are done in pure cultures. The culture is usually isolated from a soil sample,

generally contaminated with organochlorine pesticides. The strains are

characterized and tested with different concentrations of the pesticide studied.

DDT-metabolising microbes have been isolated from a range of habitats,

including animal feces, soil, sewage, activated sludge, and marine and

freshwater sediments.

The degradation of organochlorine pesticides by pure cultures has been proven

to occur in situ.

Biodegradation of DDT residues largely involves co-metabolism, that is, it

requires the presence of an alternative carbon source, in which

microorganisms growing at the expense of a substrate are able to transform

DDT residues without deriving any nutrient or energy for growth from the

process Under reducing conditions, reductive dechlorination is the major

mechanism for the microbial conversion of both the o,p'-DDT and p,p'-DDT

isomers of DDT to DDD.

The reaction involves the substitution of an aliphatic chlorine for a hydrogen

atom. Using metabolic inhibitors together with changes in pH and temperature,

Wedemeyer (1967) found that discrete enzymes were involved in the

metabolism of DDT by Aerobacter aerogenes. The suggested pathway for the

anaerobic transformation of DDT by bacteria is shown in Figure.

Degradation proceeds by successive reductive dechlorination reactions of DDT

to yield 2, 2-bis(p-chlorophenyl)ethylene (DDNU), which is then oxidised to 2,2-

bis(p-chlorophenyl)ethanol (DDOH). Further oxidation of DDOH yields bis

(pchlorophenyl) acetic acid (DDA) which is decarboxylated to bis (p-



chlorophenyl) methane (DDM). DDM is metabolized to 4,

4’dichlorobenzophenone (DBP) or, alternatively, may undergo cleavage of one of

the aromatic rings to form p-chlorophenylacetic acid (PCPA).

Under anaerobic conditions DBP was not further metabolized.

Table 1 presents some of the microorganisms that were able to degrade

organochlorine pesticides. Among microorganisms, bacteria comprise the major

group involved in organochlorine degradation, especially soil habitants

belonging to genera Bacillus, Pseudomonas, Arthrobacter and Micrococcus.

Organophosphate pesticides The organophosphorus pesticides (OP) are all esters of phosphoric acid and are

also called organophosphates, which include aliphatic, phenyl and heterocyclic

derivatives.

Owing to large-scale use of OP compounds, contaminations of soil and water

systems have been reported from all parts of the world. In light of this,

bioremediation provides a suitable way to remove contaminants from the

environment as, in most cases, OP compounds are totally mineralized by the

microorganisms. Most OP compounds are degraded by microorganisms in the

environment as a source of phosphorus and /or carbon.



Classification of Pesticides Thus, the OP pesticides can be hydrolyzed and detoxified by carboxylesterase

and phosphotriesterase enzymes. The organophosphorates possess an efficient

insecticide activity, due to its characteristic of irreversibly inhibiting the

enzyme acetylcholinesterase in the nervous system, which acts in both insects

and in mammal. In man, the organophosphates are absorbed through all

routes, reaching high concentrations in fatty tissues, liver, kidneys, salivary

glands, thyroid, pancreas, lungs, stomach, intestines and, at smaller

proportions, in the central nervous system (SNC) and muscles. However, the

organophosphates do not accumulate in the human organism, as it is readily

biotransformed in the liver. The excretion of these compounds and of their

metabolites is quite fast, taking place mostly in the urine and, at small

proportions, in the feces, usually within 48 h.

Microbial degradation of organophosphate pesticides Methyl parathion (O,O-dimethyl-O-(p-nitro-phenylphosphorothioate) is one of

the most used organophosphorus pesticides. This product is widely used

throughout the world and its residues are regularly detected in a range of fruits

and vegetables. Investigation of microbial degradation is useful for developing

insecticide degradation strategies using microorganisms. Bacteria with the

ability to degrade methyl parathion have been isolated worldwide.

Two bacteria identified as Pseudomonas putida and Acinetobacter

rhizosphaerae, able to rapidly degrade the organophosphate fenamiphos, were



isolated. Denaturating gradient gel electrophoresis analysis revealed that the

two isolates were dominant members of the enrichment culture. Clone libraries

further showed that bacteria belonging to α, β, γ, Proteobacteria and

Bacteroidetes were also present in the final enrichment, but were not isolated.

Both strains hydrolyzed FEN to fenamiphos phenol and ethyl hydrogen

isopropylphosphoramidate (IPEPAA), which was further transformed, only by P.

putida. The two strains were using FEN as C and N source. Cross-feeding

studies with other pesticides showed that P. putida degraded OPs with a P–O–C

linkage.

Thus, both bacteria were able to hydrolyze FEN, without prior formation of FSO

or FSO2, to FEN-OH which was further transformed only by P. putida (Figure),

suggesting elimination of environmentally relevant metabolites.

In addition, P. putida was the first wild-type bacterial isolate able to degrade

OPs. All the above characteristics of P. putida and its demonstrated ability to

remove aged residues of FEN highlight its high bioremediation potential.

Herein, it was shown that the construction of genetically engineered

microorganism (GEM) and the dual-species consortium has the potential to be

used in the degradations of different kinds of pesticides. These studies show

the benefits of bioremediation in multiple pesticidecontaminated environments



and mineralization of toxic intermediates in the environment, which can lead to

complete bioremediation of contaminated sites that have an adverse effect.

BIODEGRADATION OF PLASTICS Plastics are man-made long chain polymeric molecules. They are widely used,

economical materials characterized by excellent all-round properties, easy

molding and manufacturing. Traditionally plastics are very stable and not

readily degraded in the ambient environment. As a result, environmental

pollution from synthetic plastics has been recognized as a large problem.

Most of this plastic waste has been accumulating in landfills. Therefore, in

order to save capacity for plastic waste disposal, there is a growing interest

both in the development of newer, readily biodegradable plastics and in the

biodegradation of conventional plastic waste.

Types of plastics and usage Plastics are synthetic polymers. There are two main processes in the

manufacture of synthetic polymers. The first involves breaking the double bond

in the original olefin by additional polymerization to form new carbon-carbon

bonds, the carbon-chain polymers. For example, the fabrication of polyolefins,

such as polyethylene and polypropylene, is based on this general reaction. The

second process is the elimination of water (or condensation) between a

carboxylic acid and an alcohol or amine to form polyester or polyamide.

Polyurethane is also made by this general reaction.

Plastics are divided into two groups: thermoplastics and thermoset plastics.

Thermoplastics are the products of the first kind of general reaction mentioned

above. Thermoplastics can be repeatedly softened and hardened by heating and

cooling. In thermoplastics, the atoms and molecules are joined end-to-end into

a series of long, sole carbon chains. These long carbon chains are independent

of the others. This structure in which the backbone is solely built of carbon

atoms makes thermoplastics resistant to degradation or hydrolytic cleavage of

chemical bonds. Consequently, thermoplastics are considered non-

biodegradable plastics. Thermoset plastics are synthesized from the second

kind of general reaction stated above. They are solidified after being melted by



heating. The process of changing from the liquid state to the solid state is

irreversible.

Distinguished from the linear structure of thermoplastics, thermoset plastics

have a highly cross-linked structure. Since the main chain of thermoset

plastics is made of heteroatoms, it is possible that they are potentially

susceptible to be degraded by the hydrolytic cleavage of chemical bonds such

as ester bonds or amide bonds.

Main plastics and their applications

Biodegradable plastics

Degradation of thermoplastic polyolefins Generally speaking, synthetic polyolefins are inert materials whose backbones

consist of only long carbon chains. This characteristic structure makes



polyolefins non-susceptible to degradation by microorganisms. However, a

comprehensive study of polyolefin biodegradation has shown that some

microorganisms could utilize polyolefins with low molecular weight. This

biodegradation always follows photo-degradation and chemical degradation.

Although traditional polyolefins are non-biodegradable, their biodegradability is

enhanced when blending with starch or other polyesters. The degradation of

blends of low density polyethylene (PE) or isotactic polypropylene (PP) with

glycerol plasticized starch (GS). Glycerol mono-ethers, fatty alcohols or

epoxidized rubber were required as compatibilizers. The results showed that

the blends were subject to different kinds of degradation. However, the degree

of degradation was a function of the type of polymer and the blend

composition. The biodegradation of the polyolefin chain was clearly observed.

Therefore, with the development of starch-plastic as well as the discovery of

other additives added to synthetic plastics, biodegradable polyolefins provide

an attractive option for reducing plastic waste in the environment.

Biodegradable Polyolefins Traditionally, polyolefins are considered to be nonbiodegradable for three

reasons. First, the hydrophobic character of polyolefins makes this material

resistant to hydrolysis. Secondly, the use of anti-oxidants and stabilizers

during manufacture keeps polyolefins from oxidation and biodegradation.

Thirdly, polyolefins have high molecular weights36 of 4000 to 28,000.

Therefore, to make polyolefins biodegradable, these factors have to be

considered. The molecular weight of biodegradable polyolefins must be less

than 500. Accordingly, the principle of making biodegradable polyolefins

involves adding special additives to the synthetic polyolefins so that the

modified structures are susceptible to photo-degradation and chemical

degradation.

As a result, the long carbon chains are broken to shorter segments and their

molecular weights are reduced below 500. Microorganisms can then assimilate

the polyolefins monomeric and oligomeric breakdown products previously

derived from photo and chemical degradations.



As commercial products, synthetic polyolefins resist oxidation and

biodegradation because of the presence of anti-oxidants and stabilizers. The

use of pro-oxidant additives makes polyolefins oxo-bio-degradable. First, pro-

oxidant activities can change the polyolefins’ surface from hydrophobic

character to hydrophilic. Secondly, pro-oxidants can catalyze the breakdown

the long chain of polyolefins and produce lower molecular weight products

either during photolysis or thermolysis. The thermo-oxidative degradation of

polyethylene films during composting conditions and in the presence of pro-

oxidant additives. The metal combinations were the most active pro-oxidants.

To be active as catalysts, it is necessary that two metal ions of similar stability

be involved in the metal combinations, and also when the two metal ions are

oxidized by oxidants, the oxidation number of the metal ion must be only one

unit different from the one before oxidation.

For example, Mn (manganese) is a suitable metal participating in metal

combination for pro-oxidant activity. As an oxidation-reduction catalyst, two

Mn2+ ions with similar stability can form and would be oxidized to Mn3+ and

then later reduced to Mn2+. Thus, when polyolefins are exposed to the

environment, a free radical chain in the material can react with oxygen from

the atmosphere and produce hydro-peroxides that can, in turn, be hydrolyzed

and photolyzed. Also the pro-oxidant catalyzes the reaction of chain scission in

the polymer, producing low molecular mass oxidation products, such as

carboxylic acids, alcohols and ketones. Furthermore, peroxidation modifies the

material surface character from hydrophobic to hydrophilic. Consequently,

microorganisms can access the material surface, bio-assimilate the low

molecular mass, hydrophilic oxidant products and facilitate the biodegradation

process.

Starch can also be blended into the polymers for producing biodegradable

polyolefins. However, as mentioned earlier, without the addition of a suitable

pro-oxidant system, biodegradation will simply cause the removal of starch and

leave behind shorter chains of unmodified polyolefin. The amount of starch

required to be added to synthetic polyolefins needs to be optimized. If the



amount of starch is too high, the mechanical properties of the material may be

adversely affected. On the contrary, if the amount of starch is too low, the

material may not biodegrade.

Biodegradation of Polyethylene Since polyethylene (PE) is widely used as packaging material, considerable

research not only on biodegradable polyethylene but also on biodegradation of

polyethylene has been recently conducted. Biodegradation of polyethylene is

known to occur by two mechanisms: hydro-biodegradation and

oxobiodegradation. These two mechanisms agree with the modifications due to

the two additives, starch and pro-oxidant, used in the synthesis of

biodegradable polyethylene. Starch blend polyethylene has a continuous starch

phase that makes the material hydrophilic and, therefore, catalyzed by amylase

enzymes.

Microorganisms can easily access, attack and remove this part. Thus the

hydrophilic polyethylene with the matrix continues to be hydro-biodegraded.

For the biodegradable polyethylene synthesized by adding pro-oxidant additive,

biodegradation occurs following photo-degradation and chemical degradation.

The pro-oxidant is a metal combination. After transition metal catalyzed

thermal peroxidation, biodegradation of the low molecular weight oxidation

products occurs sequentially.

Degradation of Polyesters There are two kinds of polyesters: aliphatic and aromatic. Their

biodegradability is completely different. Pure aromatic polyesters are quite

insensitive to any hydrolytic degradation. It was observed that direct microbial

or enzymatic attack of pure aromatic polyester was not significant. However,

other research has recently claimed that aromatic polyester could be

disintegrated by microbial strains of Trichosporum andArthrobacter in a time

scale of weeks. Some growth of Aspergillus Niger was found on the surface of

aromatic polyesters.

Degradation of Polyurethane



Polyurethane (PUR) is commonly utilized as a constituent material in many

products including furniture, coating, construction materials, fibers, and

paints.

Structurally, PUR is the condensation product of polyisocyanate and polyol

having intramolecular urethane bonds (carbonate ester bond, -NHCOO-). The

urethane bond in PUR has been reported to be susceptible to microbial attack.

The hydrolysis of ester bonds in PUR is postulated to be the mechanism of PUR

biodegradation. The breakdown products of the biodegradation are derived

from polyester segment in PUR when ester bonds are hydrolyzed and cleaved.

Three types of PUR degradation have been identified in literature: fungal

biodegradation, bacterial biodegradation and degradation by polyurethanase

enzymes. For example, four species of fungi, Curvularia senegalensis, Fusarium

solani, Aureobasidium pullulans and Cladosporium sp, were obtained from soil

and found to degrade ester-based polyurethane.

Degradation of Polyhydroxyalkanoates Bacteria produce polyhydroalkanoates as energy storage materials. A good

example of this is polyhydroxybutyrate (PHB), which is made by numerous

microorganisms.26 PHAs are easily metabolized. The enzymes responsible for

the biodegradation, PHA depolymerases, have wide substrate specificity. PHAs

and PHBs are recently finding commercial interest. They may also find

applications as blends and additives similar to starch based plastics.

BIODETERORATION OF WOOD Blanchette (2000) described the microbial decay processes of historic wood in

detail.

Terrestrial decay occurs primarily through the action of fungal growth and

there are three mechanisms of deterioration. White rot fungi, a heterogeneous

group within the

Basidiomycota, degrade cell wall components and cause the characteristic

bleaching of wood. Brown rot fungi cause rapid depolymerization of cellulose

and degradation of cell wall carbohydrates, leaving behind a lignin-rich brown-

colored wood. The most destructive brown rot fungus is Serpula lacrymans. A



third type of terrestrial degradation, soft rot, is caused by fungi within the

Ascomycota and Deuteromycota and is characterized by degradation of the

secondary cell wall of the wood cells. In contrast, wood in aquatic environments

is attacked primarily by bacteria. Three different patterns of bacterial attack

have been characterized: erosion of the secondary wall, formation of pits

(cavitation) in the secondary wall, and tunneling through the secondary wall

and middle lamellae.

Microbial growth interacts with environmental factors in the degradation of

wood. In terrestrial environments, temperature and relative humidity regulate

microbial growth and deterioration. Sulfur dioxide in air pollution reacts on

wood to form gypsum, which can accumulate on the wood surface as well as

inside the wood.

During formation, the gypsum traps hydrocarbons generated in the urban

environment from burning fossil fuels, which then serve as a substrate for

microbial growth.

BIODETERORATION OF PAPER There are two separate processes of concern in the study of microbial

deterioration of paper, both of which are primarily the result of fungal growth.

The first is degradation of the paper itself through fungal hydrolysis of the

cellulose fibers.

The second and more interesting phenomenon is foxing, the appearance of

reddish-brown colored stains. The origin of fox spots on paper (so-called

because the reddish-brown to yellow stains may resemble the color of fox fur)

has historically been attributed to both metal (iron or manganese)

contamination and fungal growth. Questions about the role of fungi in foxing

arose from two problems.

First was the inability to determine if fungal growth was the actual cause of the

staining and not simply a secondary problem. Second, if one were to assume

that fungi were responsible, how would one determine if observed or cultured

organisms were the fungi responsible for discoloration? Solutions to each of



these problems were hampered by difficulties reproducing the foxing effect with

cultured fungi in the laboratory.

Evidence for fungal growth as the cause of foxing comes from several sources.

Fungi are invariably found associated with fox spots. Fox spots also appear to

occur in areas of low iron concentration on paper. Finally, the reddish-brown

color of fox spots appears to contain a variety of organic acids,

oligosaccharides, and amino acids (combinations of some of these components

can induce similar stains on paper, particularly, γ –aminobutyric acid, β-

alanine, glycine, ornithine, and serine). A wide variety of fungi have been

isolated from foxed paper, including Aspergillus, Chaotomium, Cladosporium,

Penicillium, and yeasts. Bacteria have also been found but rarely identified, and

it is not clear that they play a role in the discoloration.

Growth of fungi associated with foxing is dependent on the environmental

conditions in which the paper was stored. Arai (2000) found that a water

activity of 0.84 and temperatures above 25◦C were necessary to culture fungi

from fox spots. Due to the hygroscopic nature of paper, recommended storage

conditions are 16 to 20◦C and 40 to 60% relative humidity.

BIOSORPTION/ BIOACCUMULATION OF HEAVY METALS Biosorption is a physiochemical process that occurs naturally in certain

biomass which allows it to passively concentrate and bind contaminants onto

its cellular structure. Though using biomass in environmental cleanup has

been in practice for a while, scientists and engineers are hoping this

phenomenon will provide an economical alternative for removing toxic heavy

metals from industrial wastewater and aid in environmental remediation.



Pollution interacts naturally with biological systems. It is currently

uncontrolled, seeping into any biological entity within the range of exposure.

The most problematic contaminants include heavy metals, pesticides and other

organic compounds which can be toxic to wildlife and humans in small

concentration. There are existing methods for remediation, but they are

expensive or ineffective.

However, an extensive body of research has found that a wide variety of

commonly discarded waste including eggshells, bones, peat, fungi, seaweed,

yeast and carrot peels can efficiently remove toxic heavy metal

ions from contaminated water. Ions from metals like mercury can react in the

environment to form harmful compounds like methylmercury, a compound

known to be toxic in humans. In addition, adsorbing biomass, or biosorbents,

can also remove other harmful metals like: arsenic, lead, cadmium,

cobalt, chromium anduranium. Biosorption may be used as an

environmentally friendly filtering technique. There is no doubt that the world

could benefit from more rigorous filtering of harmful pollutants created by

industrial processes and all-around human activity.

The idea of using biomass as a tool in environmental cleanup has been around

since the early 1900s when Arden and Lockett discovered certain types of living

bacteria cultures were capable of recovering nitrogen and phosphorus from raw

sewage when it was mixed in an aeration tank.

This discovery became known as the activated sludge process which is

structured around the concept of bioaccumulation and is still widely used in

wastewater treatment plants today. It wasn't until the late 1970s when

scientists noticed the sequestering characteristic in dead biomass which

resulted in a shift in research from bioaccumulation to biosorption.

Though bioaccumulation and biosorption are used synonymously, they are

very different in how they sequester contaminants:

Biosorption: Biosorption is a metabolically passive process, meaning it does not require

energy, and the amount of contaminants a sorbent can remove is dependent on



kinetic equilibrium and the composition of the sorbents cellular

surface. Contaminants are adsorbed onto the cellular structure.

Bioaccumulation is an active metabolic process driven by energy from a living

organism and requires respiration.

Bioaccumulation: Bioaccumulation occurs by absorbing contaminants which are transferred onto

and within the cellular surface. Both bioaccumulation and biosorption occur

naturally in all living organisms however, in a controlled experiment conducted

on living and dead strains of bacillus sphaericus it was found that the

biosorption of chromium ions was 13–20% higher in dead cells than living

cells.

In terms of environmental remediation, biosorption is preferable to

bioaccumulation because it occurs at a faster rate and can produce higher

concentrations. Since metals are bound onto the cellular surface, biosorption is

a reversible process whereas bioaccumulation is only partially reversible

Uses: Even though the term biosorption may be relatively new, it has been put to use

in many applications for a long time. One very widely known use of biosorption

is seen in activated carbon filters. They can filter air and water by allowing

contaminants to bind to their incredibly porous and high surface area

structure. The structure of the activated carbon is generated as the result of

charcoal being treated with oxygen. Another type of carbon, sequestered

carbon, can be used as a filtration media. It is made by carbon sequestration,

which uses the opposite technique as for creating activated carbon. It is made

by heating biomass in the absence of oxygen. The two filters allow for

biosorption of different types of contaminants due to their chemical

compositions — one with infused oxygen and the other without BIOREMEDIATION OF SOIL, AIR, AND WATER; VARIOUS METHODS,

ADVANTAGES AND DISADVANTAGES Bioremediation is the use of microorganisms for the degradation of hazardous

chemicals in soil, sediments, water, or other contaminated materials. Often the



microorganisms metabolize the chemicals to produce carbon dioxide or

methane, water and biomass. Alternatively, the contaminants may be

enzymatically transformed to metabolites that are less toxic or innocuous.

It should be noted that in some instances, the metabolites formed are more

toxic than the parent compound. For example, perchloroethylene and

trichloroethylene may degrade to vinyl chloride.

There are at least five critical factors that should be considered when

evaluating the use of bioremediation for site clean up. These factors are:

Magnitude, toxicity, and mobility of contaminants. It is imperative that the site be properly investigated and characterized to

determine the (a) horizontal and vertical extent of contamination; (b) the kinds

and concentrations of contaminants at the site; (c) the likely mobility of

contaminants in the future, which depends in part on the geological

characteristics of the site.

Proximity of human and environmental receptors. Whether bioremediation is the appropriate cleanup remedy for a site is

dependent on whether the rate and extent of contaminant degradation is

sufficient to maintain low risks to human or environmental receptors.

Degradability of contaminants. The biodegradability of a compound is generally high if the compound occurs

naturally in the environment (e.g., petroleum hydrocarbons). Often, compounds

with a high molecular weight, particularly those with complex ring structures

and halogen substituents, degrade more slowly than simpler straight chain

hydrocarbons or low molecular weight compounds. Whether synthetic

compounds are metabolized by microorganisms is largely determined by

whether the compound has structural features similar to naturally occurring

compounds. The rate and extent to which the compound is metabolized in the

environment is often determined by the availability of electron acceptors and

other nutrients.

Planned site use.



A critical factor in deciding whether bioremediation is the appropriate cleanup

remedy for a site is whether the rate and extent of contaminant degradation is

sufficient to reduce risks to acceptable levels.

Ability to properly monitor. There are inherent uncertainties in the use of bioremediation for contaminated

soils and aquifers due to physical, chemical and biological heterogeneities of

the contaminated matrix. It is important to recognize that biological processes

are dynamic and, given current knowledge, often lack the predictability of more

conventional remediation technologies. Thus, it is important to insure that

unacceptable risks do not develop in the future. These risks may include

migration of contaminants to previously uncontaminated media and the failure

of bioremediation to achieve acceptable contaminant concentrations.

The remainder of this document will focus on the factors that influence the rate

and extent of contaminant degradation by microorganisms. These can be

broadly grouped into two classes of factors: (a) biological factors and (b)

environmental factors. The biological factors are primarily concerned with the

numbers of specific kinds of microorganisms present and the expression and

activity of metabolic enzymes, in other words, the amount of “catalyst” present.

The environmental factors include chemical and physical characteristics that

influence the bioavailability of contaminants, the availability of other nutrients,

the activity of biological processes (temperature and pH, for example), and

characteristics of the contaminants with respect to how they interact with the

site’s geochemical and geological characteristics.

Potential Advantages and Disadvantages of Bioremediation Technologies The use of intrinsic or engineered bioremediation processes offers several

potential advantages that are attractive to site owners, regulatory agencies, and

the public. These include:

Lower cost than conventional technologies.

Contaminants usually converted to innocuous products.

Contaminants are destroyed, not simply transferred to different environmental

media.



Nonintrusive, potentially allowing for continued site use.

Relative ease of implementation.

However, there are potential disadvantages to bioremediation as well, these

include:

May be difficult to control.

Amendments introduced into the environment to enhance bioremediation may

cause other contamination problems.

May not reduce concentration of contaminants to required levels.

Requires more time.

May require more extensive monitoring.

Lack of (hydraulic) control.

Dynamic process, difficult to predict future effectiveness. BIOLEACHING OF IRON, COPPER, GOLD AND URANIUM.

Bioleaching is the use of microorganisms, which produce acids from reduced

sulfur compounds, to create acidic environments that solubilize desired metals

for recovery. This approach is used to recover metals from ores and mining

tailings with metal levels too low for smelting. Bioleaching carried out by

natural populations of Leptospirillum-like species, Thiobacillus thiooxidans, and

related thiobacilli, for example, allows recovery of up to 70% of the copper in

low-grade ores. As shown in figure, this involves the biological oxidation of

copper present in these ores to produce soluble copper sulfate. The copper

sulfate can then be recovered by reacting the leaching solution, which contains

up to 3.0 g/liter of soluble copper, with iron. The copper sulfate reacts with the

elemental iron to form ferrosulfate, and the copper is reduced to the elemental

form, which precipitates out in a settling trench. The process is summarized in

the following reaction: CuSO4 + Fe0 →Cu0 + FeSO4

Bioleaching may require added phosphorus and nitrogen if these are limiting in

the ore materials, and the same process can be used to solubilize uranium.

It is apparent that nature will assist in bioremediation if given a chance. The

role of natural microorganisms in biodegradation is now better appreciated. An



excellent example is the recent work with the very versatile fungus

Phanerochaete chrysosporium.

Often biodegradation and biodeterioration have major negative effects, and it

becomes important to control and limit these processes by environmental

management. Problems include unwanted degradation of paper, jet fuels,

textiles, and leather goods. A global concern is microbial-based metal

corrosion.

Copper Leaching from Low-Grade Ores The chemistry and microbiology of copper ore leaching involve interesting

complementary reactions. The microbial contribution is the oxidation of ferrous

ion (Fe2+) to ferric ion (Fe3+). Leptospirillum ferrooxidans and related

microorganisms are very active in this oxidation. The ferric ion then reacts

chemically to solubilize the copper. The soluble copper is recovered by a

chemical reaction with elemental iron, which results in an elemental copper

precipitate.

All the best - THE IMPRINT TEAM

mbh _ 203 environmental microbiology - THE IMPRINT

Documents

Transcript of mbh _ 203 environmental microbiology - THE IMPRINT