Submitted By:

Md. Hasan Masrur (545)

Submission date: 3rd March, 2014

An Assignment on

Biotechnological methods

In

Pollution abatement

Course no: Env. 420

Submitted by:

GROUP NO: 2

SHARMINE I (547)

Submitted to:

ABDUL KADIR IBNE KAMAL

Assistant Professor,

Department of Environmental

Sciences, Jahangirnagar

University, Savar, Dhaka-1342

Department of Environmental Sciences,

Jahangirnagar University

Savar, Dhaka- 1342

Table of content

Content Page no. Metal pollution and its bio

abatement

Introduction

Materials and methods

Biological markers (biomarkers or

bioindicators)

Fish as Metal Biomarker for Water

Pollution in Worldwide

1-5

1

1-2 3-5

5

Eutrophication in biological

phosphorus removal

Enhanced Biological Phosphorus

Removal (EBPR)

Cultivation of microorganisms in

wastewater

6-10

6-8

9-10

Cell immobilization as a tool

in waste treatment

Cell immobilization

Methods for immobilization of

microbial cells

Factors affecting microbial cell

adsorption

11-19

11-18

16-18

19

References

20-23

1 | P a g e

Metal pollution and its bio abatement

Introduction

Heavy metals are non-biodegradable pollutants whose release in the

environment is mainly related to industrial wastewaters discharged from

industrial and mining activities. The use of bacterially mediated sulphate

reduction in Permeable Reactive Barrier is an alternative technique for the

remediation of heavy metals polluted streams. SRB are known to grow using

small organic molecules, essentially small molecular weight compounds, like

acetate, lactate, propionate, butyrate, valerate, methanol, ethanol, glycerol,

glucose (Postgate, 1979). However, pure substrates as carbon source may not

be cost effective for this kind of treatment. Usually, organic mixtures are used

in biological PRB construction as electron donor in the sulphate reduction:

biodegradable materials are generally mixed with more recalcitrant ones to

ensure long term SRB growth (Cocos et al., 2002; Gibert et al., 2004). Full

scale applications of organic-carbon based sulphate reducing PRB are also

characterized by the addition of gravel to improve barrier permeability and

limestone to increase pH and stimulate SRB growth (Ludwig et al., 2002).

Batch experiments were preliminarily performed to determine the optimal

mixture for treating heavy metals in biological PRB. Selected mixture was then

tested in continuous fixed column experiments to simulate permeable reactive

barriers running. Ethanol was also tested as electron donor for the

sulphatereduction for several reasons, including a well-defined and “clean”

composition, ease of availability, low cost and the possibility to use

bioethanol. Finally, preliminary batch tests starch were conducted to test the

ability of SRB to grow on this substrate.

Materials and methods

Batch tests with solid reactive mixtures

Eight reactive mixtures were prepared consisting of three main functional

components: a mix of organic materials, a neutralizing agent (limestone), and

a non-reactive porous medium (silica sand or perlite). A sample (20 g) of each

mixture was added in flasks and filled with 80 mL of liquid C Medium

(Postgate, 1979). Flasks were sealed and 20mL inoculum of bacteria

2 | P a g e

cultivated in C Medium were added. Best performing reactive mixture (RM:

6% leaves, 9% compost, 3% Fe(0), 30% silica sand, 30% perlite, 22%

limestone) was further tested in presence and in absence of bacteria and

single organic components (compost and leaves) were also investigated for

sulphate removal without inoculum. All experiments were conducted at 37°C

under shacking condition. pH, Eh, SO42- and S2- production were monitored

for 22 days. Each test was performed twice and average values were

considered.

Column tests with solid reactive mixture M8

Column tests were performed in a fixed bed column (height 1 m; diameter 0.2

m; column volume, V=6.65*10-3 m3) made of Plexiglas with 10 equally distant

outputs along the axial length, numbered from the bottom to the top of the

column. It was packed with perlite (an expanded clay) and silica sand on the

bottom (10 cm length) followed by reactive mixture (RM) (80 cm) and topped

with perlite and silica sand (10cm) (pore volume V0 = 1.5 L) .SRB were

inoculated in the core of the column. Column was regularly fed with a solution

containing heavy metals (Cd 0.1 mM, Cr(VI) 0.1 mM, Cu 0.1 mM, Zn 0.1 mM

and As(V) 27μM) and sulphate (31 mM). Samples from three different outputs

(1, 5 and 9) were analyzed for pH, Eh and the residual amounts of sulphates

and metals.

Batch tests with ethanol

Glass reaction flasks (120 mL), containing a sampling port, were used for all

the experiments. 80 mL of modified C Medium (ethanol 6 g/L instead of

lactate) was added in flasks. Therefore the flasks were sealed and 20 mL

inoculum of bacteria cultivated in C Medium were added by a sterile syringe

through the sampling port. All experiments were conducted at 37°C under

shacking condition. pH, Eh, SO42- and S2- production were monitored for 30

Column tests with ethanol

Column tests were performed in two fixed bed column like that used in

column tests with mixture RM. Columns were filled with perlite (pore volume

V0 = 3.5 L), one inoculated by SRB and the other used as blank. Both columns

3 | P a g e

were regularly fed with a solution containing sulphate (31 mM) and ethanol

(65 mM). Samples from three different outputs (1, 5 and 9) were analyzed for

pH, Eh and the residual amounts of sulphates.

Batch tests with starch

Glass reaction flasks (120 mL), as those previously described, were used for

the experiments with starch. 80 mL of C Medium (without carbon sources)

and starch (100 g/L) were adder in flask. Therefore the flasks were sealed and

20 mL inoculum of bacteria cultivated in C Medium were added by a sterile

syringe through the sampling port. All experiments were conducted at 37°C

under shacking condition. pH, Eh, SO42- and S2- production were monitored

for 100 days. Each test was performed twice and average values were

considered.

Biological markers (biomarkers or bioindicators)

In the attempt to define and measure the effects and presence of pollutants

on aquatic system, biomarkers have attracted a great deal of interest. The

principle behind the biomarker approach is the analysis of an organism to

their metal contents in order to monitor the metal excess in their tissues.

Various aquatic organisms occur in rivers, lakes, seas and marines potentially

useful as biomarkers of metal pollutants, including fish, shellfish, oyster,

mussels, clams, aquatic animals and aquatic plants and algae.

Fish as biomarkers

Fish from Lakes: Nasser Lake

Tilapia nilotica is one of the aquatic organisms affected by heavy metals, so in

many cases, Tilapia nilotica was used as metal biological marker in

toxicological studies in which it was substantiated with the highest sensitivity

to toxic effect (Patin, 1984). Rashed (2001a, b) studied Co, Cr, Cu, Fe, Mn, Ni,

Sr, Pb, Cd and Zn in different tissues of fish (Tilapia nilotica) from Nasser lake

to assess both the water pollution with these metals and the lethal level of

these metals in fish. Fish samples were collected from two Kohrs in Nasser

Lake ( Kohr Kalabsha and Kohr El-Ramel) .The fish tissues includes muscle,

gill, stomach, intestine , liver, veritable column and scales .The fish ages were

4 | P a g e

1,1.5 , 2, 2.5 and 3 years. This study resulted in that fish scales exhibited the

highest concentrations of Cd, Pb, Co,Cr,Ni and Sr (0.088,0.95,0.29,0.30,0.25

and 3.21 μg/g DW respectively). Whole fish contains the higher

concentrations of the studied metals compared to the previous study by

Awadallah et al.(1985) in the same fish from Nasser Lake, and this mean the

increase in metal pollution in Lake water as the results of man activities (Table

1). This increasing in metal concentration was as the result of increasing

pollution loads to the Lake from agricultural wastes, which include chemical

pesticide and fertilizers. These agricultural wastes reached the Lake body

from the agricultural farms on the beach of the Lake. The source of Pb in the

Lake water and fish was resulted from gasoline contains Pb from the fishery

boats and tour ships travels from Aswan to Sudan (Mohamed et al.1990).

Fish from River Nile

River Nile is the main source for potable water and as the result of man

activities in and on the river body it become loaded by metal pollution. Fish

in the River Nile was used as biological marker for the River pollution by

metals. Mohamed et al. (1990) used Tilapia nilotica fish as a biomarker for

the Nile water pollution with metals at the discharge.Point of fertilizer factory

with the Nile. Ag, Au, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr and Zn were

determined in tilapia nilotica fish collected from the Nile area at the point of

fertilizer discharge to the Nile and south and north this point. The results

revealed that fish near the point of the factory discharge possess the highest

levels of metals as the result of pollution with metals. Other study for using

fish as biomarker for water pollution with metals was conducted (Khallaf et

al., 1994). Two species of fresh water fish (Tilapia nilotica, named Bolti, and

Karmout ) caught from River Nile at Hawamdia and Kafer El-Zayat , at North

Egypt and also from governmental fish farms (Abbassa and Barseik) were

used to detectthe presence of industrial wastes especially heavy metals as

environmental pollution in the river track and its accumulation in edible fish

tissues. The result reveals that heavy metals in different water samples except

Cu and Zn were more than the recommended permissible levels (Table2). Iron

level in Hawamdia and Kafer-El-Zayat tilapia nilotica samples (63.4 and 54.7

5 | P a g e

μg/g respectively) was more than its permissible levels, these may be due to

the discharge of the adjacent chemical factories that used Fe in their

processing.

Fish as Metal Biomarker for Water Pollution in Worldwide

Arsenic as biomarker for water pollution was assessed by Takatsu and

Uchiumi (1998) in which the contents of the metal in the tissues of the fish,

Tribolodon hakonsis, from Lake Usoriko, located in Aomori Prefecture, Japan,

were examined. It was discovered that large amounts of As were accumulated

in the eye tissues. This might be partly related to the fact that the lake water

contains a relatively large amount of As. Mercury levels in muscle of some fish

species from Dique channel, Colombia was measured to assess the water

pollution with Hg (Olivero et al.1997). The highest values of Hg (105 μg/kg)

found in fish from the Dique channel were lower than those found in fish

species from the Lower Gallego and Cince Rivers in Spain (Raldua and

Pedrocchi, 1996). In the Tapajos River, an Amazon water body highly exploited

by gold mining activities, the average value for Hg in muscle of Carnivorous

fish was 690 μg/kg, almost ten times higher than those found in the Dique

channel (Malm et al., 1995). They also concluded that, however the highest

Hg concentration did not reach the limits level internationally accepted for

considering a fish not acceptable for human consumption (WHO, 1990).

6 | P a g e

Eutrophication in biological phosphorus removal

Enhanced Biological Phosphorus Removal (EBPR)

EBPR relies on the selection and proliferation of a microbial population

capable of uptaking orthophosphate in greater amounts than their normal

biological growth requirements.

Enhanced biological phosphorus removal is a process that uses alternating

anaerobic and aerobic zones to provide an environment that encourages the

growth of Phosphorus Accumulating Organisms (PAO). PAOs store excess

polyphosphate in their cell mass and phosphorus is removed with the waste

sludge.

Fig: A typical Enhanced Biological Phosphorus Removal configuration

7 | P a g e

Mechanism of biological phosphorus removal

The unique feature of EBPR is the anaerobic selector used in the treatment

process.

Fig: mechanism of biological phosphorus removal

The reactions that occur in the anaerobic zone

Phosphorus Accumulating Organisms (PAOs) use poly-phosphate and

glycogen stored in their cells as energy sources to enable them to uptake VFA.

VFA are converted to Polyhydroxyalkanoates (PHA) and stored in the cells of

PAOs. As they take up VFA, the PAOs release orthophosphate into the mixed

liquor. PAOs do not grow in the anaerobic zone but their ability to uptake

food in the form of VFAs gives them a competitive advantage over other

bacteria.

The reactions that occur in the aerobic zone

In the aerobic zone PAOs use PHA as a source of carbon and energy for

metabolism and cell growth. PAOs will also restore their supplies of glycogen

and poly-phosphate in the aerobic zone. To replenish their stored poly-

phosphate, PAOs will take up excess phosphate from the mixed liquor which

is the mechanism of biological phosphorus removal.

8 | P a g e

In the anaerobic zone PAOs will rapidly take up BOD (as VFAs) and release

orthophosphate into the mixed liquor. As the wastewater passes through the

anaerobic zone VFA will rapidly decrease and orthophosphate will increase.

In the aerobic zone BOD will continue to decrease. As PAOs restore their poly-

phosphate supplies in the aerobic zone the concentration of orthophosphate

in the mixed liquor will rapidly decrease.

Fig: the phosphorus and BOD profile through a treatment plant

Under anaerobic conditions PAOs take up VFA from the mixed liquor and

store it as PHA within their cells. To do this PAOs use the glycogen and poly-

phosphate as energy sources depleting their stores of these compounds.

Under aerobic conditions PAOs use up their stored PHA for metabolism and

growth and to restock their supplies of glycogen and poly-phosphate. To build

up their supply of polyphosphate PAOs will take up excess orthophosphate

from the mixed liquor in the aerobic zone.

The importance of sludge age in the treatment system on biological

phosphorus removal

As with all activated sludge systems, sludge age determines how long the

biomass stays in the biological part of the treatment system. Because

biological phosphorus removal is compartmentalized into anaerobic, anoxic,

and aerated zones, all biological zones must be taken into consideration when

calculating sludge age. Selectors are usually much smaller than that of

9 | P a g e

aeration zones and should be taken into account when determining sludge

age.

The proper sludge age ensures the optimization of the biochemical reactions

that need to take place and for the biomass to uptake excess phosphates in

the aerobic basins (see key knowledge 1.3.5). Too short a sludge age and

insufficient treatment can occur with resultant poor effluent quality. As

sludge age increases in an activated sludge system, nitification becomes a

factor and the need for anoxic zones becomes critical for denitrification and

the removal of nitrates. Long sludge ages such as in extended aeration

systems, can lead to secondary release of phosphorus through biomass decay.

Long sludge ages can also result in biochemical reaction problems for PAOs.

Sludge ages of 5-30 days are likely to be observed in successful EBPR plants.

BOD5 (or COD) /Total P ratio: As discussed in key knowledge 1.2.1, the

influent BOD5 or COD to total phosphorus ratio is critical for PAOs to grow,

function, and take up phosphorus from solution. The influent BOD5 or COD

must be in a form that is readily available to PAOs, such as volatile fatty acids.

A minimum BOD5/total P ratio of 20:1 or a COD/total P ratio of 45:1 is

needed for successful bio-P removal.

Sludge Age (SA) and Hydraulic Retention Time (HRT): The contact time

between the influent and the bacteria (HRT) as well as the proper sludge age

(SA) for the microbial biochemical reactions to take place in the biomass are

critical for PAO growth, metabolism and selection. These parameters are

discussed further in key knowledges 2.2.3 and 2.2.4.

Temperature: Research shows that effective phosphorus removal has been

shown to occur between 5- 30°C as long as proper sludge ages are provided

for cold and warm weather temperatures. In fact, PAO’s may be at a selective

advantage in colder temperatures (5-10°C).

Internal Recycle Flows: Internal recycles are used in EBPR systems to

create favorable conditions for PAO selection and growth. Common internal

recycle flows include return activated sludge (RAS), anoxic zone to anaerobic

zone recycles, and aerobic to anoxic zone recycles.

10 | P a g e

Cultivation of microorganisms in wastewater

Biological removal of nutrients in bioreactors is, by definition, performed

bymicroorganisms. However, the species commonly used are non-specific and

environmentally enriched from the sludge by the incubation conditions, as

explained later for EBPR. Several attempts to intentionally use specific

microorganisms as cleaning agents were reported, as has been done in earlier

decades (for review of earlier cases: De la Nou¨e and De Pauw, 1988). These

include bacteria and microalgae.

Bacteria

The tropical cyanobacterium Phormidium bohneri in domestic wastewater

removed nitrogen and phosphate after growing 50 and 75h, respectively.

Adding monopotassium phosphate enhanced production of biomass by 56%,

but did not significantly affect the time for completely removing these

nutrients. This could not be repeated at the lower temperature.This failure

indicates that tertiary biological wastewater treatment at low temperatures

(51C) cannot be developed with the polar strains tested because they grow too

slowly under extreme cold. However, they may have potential at moderately

cool temperatures (about 151C and above), common from spring through fall

in northern climates (Chevalier et al., 2000).

Bacteria cultivated for relatively short periods in wastewater present a feasible

alternative to the longer EBPR process (described later). Staphylococcus

auricularis,growninsequencingbatchreactorsunderrepeated anaerobic and

aerobic conditions, was able to remove

between5and50mgPL1,correspondingtomorethan 90% removal of

phosphorus. These results, achieved after a short incubation period,

demonstrated that a long adaptation time, one of the major problems in

starting an EBPR process, could be addressed by a shorter approach (Choi

and Yoo, 2000).

The purple photosynthetic non-sulfur bacterium Rhodobacter

capsulatus,immobilizedoncellulosebeads, removed organic carbon,

ammonium ions, and phosphateionsfromadilutedgrowthmediumoveraperiod

11 | P a g e

of 19–22 days with a residence time of 10h at 351C (Sawayama et al., 1998b).

The photosynthetic bacteria, Rh. sphaeroides S, Rb. sphaeroides NR-3, and

Rhodopseudomonas palustris, immobilized in porous ceramic under aerobic

conditions, simultaneously removed COD, phosphates, nitrates, and H2S

from a synthetic sewage wastewater. In the batch treatment, 77% of the

phosphates were removed effectively within 48h. In semi-continuous

treatments, this removal took about one month (Nagadomi et al., 2000).

Microalgae

Unicellular microalgae Chlorella vulgaris and Scenedesmus dimorphus were

capable of removing upto 55% of the phosphates from dairy industry and pig

farming wastewaters(Gonzalezetal.,1997,forearlierstudiessee De la Nou¨e and

De Pauw, 1988). Another strain of Scenedesmus, grown in artificial

wastewater, also removed more than 50% of the phosphates (Voltolina et

al.,1999).Productionofstarchyieldedwastewaterwith a unique C:N:P ratio of

24:0.14:1. This effluent supported good growth of Spirulina platensis.

Reductionsinphosphatelevelsofthedigestedeffluentreached over 99% (Phang

et al., 2000). S. platensis could efficiently remove nitrates, ammonia, and

phosphates from synthetic wastewater (Ogbonna et al., 2000).

These studies employed microalgae in a suspension.

Theirapplicationisseverelylimitedbythedifficultiesof harvesting the enormous

microalgal population developedinthewateraftertreatment.Therefore,theideaof

entrapping microalgae for easy removal by sedimentation with spherical gels

gained some momentum. For example, C. vulgaris, immobilized in two natural

polysaccharide gels (carrageenan and alginate), was used to treat primary

domestic wastewater.

The most radical combination of microalgae and bacteria suggested so far is

the use of plant growthpromoting bacteria, used in agriculture, to enhance

the growth and nutrient removal capacity of microalgae from wastewater. The

bacteria of the genus Azospirillum are used as inoculants to promote the

growth and yield of numerous crop plants, mainly by affecting the hormonal

metabolism and mineral absorption of the plants (Bashan and Holguin, 1997).

The underlying hypothesis assumed that the bacteria will enhance the

12 | P a g e

performance of unicellular plants, like microalgae, and that the single-cell

plant will respond similarly to bacterial inoculation like a higher plant. Co-

immobilization of C. vulgaris and C. sorokiniana with A. brasilense, in small

alginate beads, significantly enhanced all the growth parameters of the

microalgae (Gonzalez and Bashan, 2000). Furthermore, these artificial

combinations (thus far not found in nature) profoundly changed many

cytological, physiological, and biochemical pathways and products within the

microalgal cells (Gonzalez- Bashan et al., 2000; Lebsky et al., 2001; de-

Bashan et al., 2002a). This co-immobilization, under semi-continuous

synthetic wastewater culture conditions, significantly increased the removal

of ammonium and soluble phosphate ions, compared to immobilization of the

microalgae alone (de-Bashan et al., 2002b). Recently, these combinations

were successful in significantly reducing ammonium and phosphate levels of

municipal wastewater (de-Bashan et al., 2004).

In summary, no new technology has emerged from decades of research on

intentionally using specific microorganisms for nutrient removal. Several

proposals, especially the entrapment of microorganisms in polysaccharide

gels and combinations of several organisms for simultaneous treatment of the

wastewater, have the best potential for commercial use. However, as yet, it is

a minor research avenue in the phosphate removal field in wastewater

treatment.

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Cell immobilization as a tool in waste treatment

Cell immobilization

Immobilization is a general term describing a wide variety of the cell or the

particle attachment or entrapment (Lopez et al., 1997). It can be applied to

basically all types of biocatalysts including enzymes, cellular organelles,

animal and plant cells. Currently, different kinds of immobilization have

found wide applications not only in the field of biotechnology, but also in

pharmaceutical, environmental, food and biosensor industries (Peinado et al.,

2005).

The cell immobilization emerged as an alternative for enzyme immobilization

(Cheetham et al., 1979; Parascandola and Scardi, 1980; Woodward, 1988).

Immobilization of cells containing specific enzymes has further advantages

such as elimination of long and expensive procedures for enzymes separation

and purification and it is vital to expand their application by enabling easy

separation and purification of products from reaction mixtures and efficient

recovery of catalyst (Junter and Jouene, 2004; Stolarzewicz et al., 2011). In

comparison with immobilized enzymes, immobilized cells provide new

possibilities since they can be used as natural, water-insoluble carriers of

required enzyme activities (Vojtisek and Jirku, 1983).

In the case of the immobilization of microbial cells, their field of application

spreads from industrial to environmental process. Microorganisms retained

on a carrier can be used in continuous and semi-continuous production

processes allowing for significant cost decrease, as the biocatalyst does not

need to be refilled (Wada et al., 1979; Park and Chang, 2000; Mrudula and

Shyam, 2012).

Cell immobilization has been defined as the physical confinement or

localization of viable microbial cells to a certain defined region of space in

such a way as to limit their free migration and exhibit hydrodynamic

characteristic which differ from those of the surrounding environment while

retaining their catalytic activities for repeated and continuous use (Dervakos

and Webb, 1991; Freeman and Lilly, 1998; Covizzi et al., 2007; Amim et al.,

14 | P a g e

2010). Since the early 70s, when Chibata’s group announced successful

operation of continuous fermentation of L-aspartic acid, numerous

research groups have attempted various microbial applications with

immobilized cells (Ramakrishna and Prakasham, 1999). Environmental

applications of immobilized microbial cells are reported by Bettmann and

Rehm (1984), Anselmo et al. (1985), Sahasrabudhe et al. (1988), Oreilly and

Crawford (1989), Beunink and Rehm (1990), Balfanz and Rehm (1991),

Stormo and Crawford (1992), Cassidy et al. (1996), Wang et al. (1997), Wang

et al. (2002), Wang et al. (2007), Zhang et al. (2007), Zhou et al. (2008), Bazot

and Lebeau (2009), Wang et al. (2010), Ahmad et al. (2012) and Nickzad et al.

(2012).

Support materials

The support selection is one of the crucial decisions to be made in the course

of preparation of the immobilization process (Zacheus et al., 2000). For

treatment of wastewater, support materials need to meet the following criteria:

insoluble, non-biodegradable, non-toxic, nonpolluting, light weight; flexibility

in overall shape, high mechanical and chemical stability, high diffusivity,

simple immobilization procedure, high biomass retention, minimal

attachment of other organisms and preferably a low cost price (Leenen et al.,

1996; Zacheus et al., 2000). Other criteria, such as physical characteristics

(porosity, swelling, compression, material and mean particle behavior), as well

as, possibility for microbial growth and solubility, are more application

specific (Górecka and Jastrzębska, 2011).

The carriers are classified as inorganic material (zeolite, clay, anthracite,

porous glass, activated charcoal, and ceramics) and organic polymers.

Inorganic carriers were selected to immobilize microorganisms because they

can resist microbial degradation and are thermostable (Cassidy et al., 1996;

Verma et al., 2006). The organic polymeric carriers are more abundant than

inorganic carriers and can be natural and synthetic polymeric carriers

(Cassidy et al., 1996). Several syn-thetic (acrylamide, polyurethane, polyvinyl,

resins) and natural polymer derivatives of algal polysaccharides (alginate,

carrageenan, agar, agarose), and chitosan, an amino polysaccharide derived

15 | P a g e

from chitin, has been experimentally used. The most commonly used

polymers are the natural polymers alginate and carrageenan but these

natural polymers are less stable in wastewater than synthetic polymers

(Bashan, 1998; Arica et al., 2004; Moreno-Garrido, 2008; Stolarzewicz et al.,

2011).

Alginates (polymers made of different proportions and sequences of

mannuronic and guluronic acids extracted from brown algae) are easy to

handle, nontoxic to humans, the environment, and the entrapped

microorganisms, legally safe for human use, available in large quantities, and

inexpensive.

Chitosan is inexpensive, non-toxic property and possesses potentially reactive

amino functional groups which can enhance the affinity of the carrier with

the microorganisms. However, the mechanical stability of the carrier would

decrease because of the biodegradability in the course of usage.

Other natural gels, such as agar, collagen andagarose, also can be used as

microbial encapsulation carriers (Zhou et al., 2008). Some natural polymers

are more vulnerable to environmental degra-dation by microbes. However,

diffusivity is higher in natural polymers and they are less hazardous to

produce (Leenen et al., 1996; Cassidy et al., 1996).

Synthetic polymeric supports are not easily biodegradable and have much

better mechanical performance compared with nature carrier. Materials, such

as polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethyleneglycol (PEG)

and polycarbamoyl sulphonate (PCS) were synthesized as encapsulation

carriers (Leenen et al., 1996).

In order to improve the stability of gel carrier, various synthetic plastics, for

example polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC), poly-

urethane (PU) and polyacrylonitrile (PAN) have been explored extensively as

immobilized microorganism carriers more recently (Zacheus et al., 2000).

Among the various extensively used plastics carriers, polyurethane (PU) is one

kind of outstanding carrier for entrapping microorganisms in piloted

applications in practical wastewater treatment (Guimarães et al., 2002).

16 | P a g e

Martins et al. (2012) reported potential of the Gramnegative bacterium

Serratia marcescens and the yeast Candida rugosa to immobilization on

polyurethane foam.

Methods for immobilization of microbial cells

Immobilization of microbial cells in biological processes can occur either as a

natural phenomenon or through artificial process (Ramakrishna and

Prakasham, 1999). Different immobilization types have been defined: covalent

coupling/cross linking, capture behind semipermeable membrane or

encapsulation, entrapment and adsorption (Mallick, 2002). The types of

immobilization can be grouped as ‘‘passive” (using the natural tendency of

microorganisms to attach to surfaces-natural or synthetic, and grow on them)

and ‘‘active” (flocculant agents, chemical attachment and gel encapsulation)

(Cassidy et al., 1996; Cohen, 2001; Moreno-Garrido, 2008).

Covalent bonding/Cross linking: The mechanism involved in this method is

based on covalent bond formation between activated inorganic support and

cell in the presence of a binding (crosslinking) agent. For covalent linking,

chemical modification of the surface is necessary. Covalent attachment and

cross-linking are effective and durable to enzymes, but it is rarely applied for

immobilization of cells. It is caused mainly by the fact that agents used for

covalent bonds formation are usually cytotoxic and it is difficult to find

conditions.

Entrapment: Entrapment is an irreversible method, where immobilized cells

are entrapped in a support matrix or inside fibers. This technique creates a

protective barrier around the immobilized microbes, ensuring their prolonged

viability during not only processing but also storage.in polymers (Górecka and

Jastrzębska, 2011). Entrapment is the most method extensively studied in

cell immobilization. The matrices used are agar, alginate, carrageenan,

cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross-

linkable resins, polyacrylamide, polyester, polystyrene and polyurethane

(Lopez et al., 1997; Ramakrishna and Prakasham, 1999).

Entrapment of the microorganisms in porous polymer carrier was often used

to capture the microorganisms from suspended solution and then obtain the

immobilized microorganisms. The polymer matrix used in this method

confining microorganisms has porous structure, and thus the pollutant and

various metabolic products could easily diffuse through into the matrix. In

17 | P a g e

this method, a lot of porous polymers can entrap microorganisms under

ambient conditions (Verma et al., 2006).

As a rule, the entrapment methods are based on the inclusion of cells within

a rigid network to prevent the cells from diffusing into surrounding medium

while still allowing penetration of substrate. Entrapment of cells in alginate

gel is popular because of the requirement for mild conditions and the

simplicity of the used procedure. Several reports are available employing

alginate gel (Kierstan and Bucke, 1977).

Entrapment allows high mechanical strength, but contains some

disadvantages, such as, cell leakage, costs of immobilization, diffusion

limitations, and deactivation during immobilization and abrasion of support

material during usage. Another disadvantage is low loading capacity as

biocatalysts have to be incorporated into the support matrix (Krekeler et al.,

1991; Song et al., 2005; Gao et al., 2010; Stolarzewicz et al., 2011).

Encapsulation: Encapsulation is another irreversible immobilization method,

similar to entrapment. In this process, biocatalysts are restricted by the

membrane walls (usually in a form of a capsule), but free-floating within the

core space (Górecka and Jastrzębska, 2011). The membrane itself is semi-

permeable, allowing for free flow of substrates and nutrients (when cells are

used as a biocatalyst), yet keeping the biocatalyst inside. The factor

determining this phenomenon is the proper pore size of the membrane,

attuned to the size of core material. This limited access to the microcapsule

interior is one of the main advantages of microencapsulation, for it protects

the biocatalyst from the harsh environmental conditions. As most

immobilization method, it prevents biocatalyst leakage, increasing the process

efficiency as a result (Park and Chang, 2000).

The encapsulation method was used to enclose the microorganisms in a

polymer-gel by Jen et al. (1996) and is one of the most frequently used in

laboratory experiment up to now and there is far away engineering application

for wastewater treatment (Lozinsky and Plieva, 1998).

However, even though in encapsulation, high cell loading can be achieved,

but the capsules are still very weak (Song et al., 2005). The diffusion limitation

is one of the inevitable drawbacks associated with encapsulation method

(Lozinsky and Plieva, 1998).

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Adsorption: The immobilization passive or adsorption natural of

microorganisms onto porous and inert support materials is similar to the

adsorption of colloid particles (Araujo et al., 2010). Apparently, it is the first

example of cell immobilization and probably is the simplest method of

reversible immobilization (Monsan et al., 1987; Klein and Ziehr, 1990).

This technique is based on the physical interaction between the

microorganism and the carrier surfaces, while frequently reversible is simple,

cheap and effective. The immobilization of microorganisms on properly chosen

adsorbents stimulates microbial metabolism, protects cells from unfavorable

agents, and preserves their physiological activity (Nikovskaya, 1989; Kozlyak

et al., 1991, 1993).

Different from the inherent problems associated with cell entrapment, cell

immobilization through adsorption provides a direct contact between

nutrients and the immobilized cells thus, eliminating such concerns

(Braschler et al., 2005). This cell immobilization technique involves the

transport of the cells from the bulk phase to the surface of support (porous

and inert support materials), followed by the adhesion of cells, and

subsequent colonization of the support surface (Kilonzo and Bergougnou,

2012).

Adsorption is based on weak forces, however, still enabling an efficient

binding process. Usually in bonds formation, several forces are involved: van

der Waals forces, ionic and hydrophobic interactions and hydrogen bonds.

Both electrostatic and hydrophobic interaction controlling the cell

immobilization on the support (Hsu et al., 2004, Górecka and Jastrzębska,

2011).

In contrast to ceramics, wood chips and straw, fibrous matrices provide

adequate supporting surfaces for cell adsorption (Talabardon et al., 2000;

Chu et al., 2009) due to their high specific surface area, void volume,

mechanical and permeability, low pressure drop, diffusion problems and

toxicity, maximum loading, biodegradability and durability and low cost and

high availability (Huang and Yang, 1998). Their natural configuration also

allows them to trap more cells than other materials (Yang and Shu, 1996;

Yang and Lo, 1998).

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Polyurethanes foams for immobilization by adsorption:

Polyurethanes (PU) are one of the most versatile materials in the world today.

They are known for being a perfect material for footwear, machinery industry,

coatings and paints, rigid insulation, elastic fiber, soft flexible foam, medical

devices (Romaškevič et al., 2006). Some time ago PU was found to be

applicable in the biochemical and biotechnological fields and flexible

polyurethane foams have gained relevance as microbial carriers for their good

mechanical properties, high porosity, large adsorption surface, resistance to

organic solvents and microbial attack, easy handling, regenerability and cost

effectiveness (Patil et al., 2006). In general, the high rates of sorption of

polyurethane foam in removing lubricants boats, while Silva et al. (2006) have

described that the immobilization of bacteria in polyurethane foam increased

resistance to high concentrations of sulphate.

Factors affecting microbial cell adsorption

There are many factors (such as the age and the physiological state of cells)

that influence the sorption of microbial cells. The surface structures of

bacterial cells (flagella and other appendages), superficial charges and

hydrophobicity also play an important part in the cell adherence to solid

surfaces (Donlan, 2002; Chae et al., 2006; Oulahal, et al., 2008). The

composition of the medium, its pH, and environmental conditions

considerably influence the adsorption of cells by changing their electrokinetic

potential (Stanley, 1983; Fletcher and Pringle, 1986; Kilonzo and Bergougnou,

2012).

The surface properties of adsorbents also affect the process of cell

immobilization (Busalmen and Sanchez, 2001, Ubbink and Schar-

Zammaretti, 2007). The degree of cell immobilization depends on the

structure and the size of adsorbent pores (Arinbasarova et al., 1982). The

nature of adsorbents is also important. Organic adsorbents are chemically

stable and show a great variety of surface properties and pore structures,

whereas inorganic adsorbents are resistant to biological degradation are

affordable, and can be easily regenerated. The positive charge and

hydrophobic character of the polyurethane foam, allow interaction with most

microbial cell surfaces (Afghan et al., 1984; Wang et al., 2009). They are

inexpensive and easily regenerated by extraction or washing with solvents

(Belyakova and Schevchenko, 1986).

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The microbial immobilization in polyurethane, combined with the use of

bioreactors improved significantly the biodegradation process of phenols and

derivatives (Pai et al., 1995). The highest efficiency in the degradation of

ophthalate by cells Bacillus-spp. immobilized in polyurethane foam, in

relation to alginate.

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