Using the Winogradsky Column - CiteSeerX

o fully understand the workings of the biolog-ical world, it is important that students have a fun-damental sense of the natural cycles that provide thenutrients and energy that power life, as well as asense of how these systems evolved. Many teacherscover carbon cycles and emphasize microbialprocesses when reviewing the complexities of nitro-gen cycling, but often the sulfur cycle, if covered, isdone so briefly. There may be many reasons for this:time limitations, the element is less prevalent thanothers as a biological constituent, or the topic isthought to be too complex. However, teaching thesulfur cycle in conjunction with the classicWinogradsky column exercise presents the opportu-nity to cover several important topics simultaneous-ly. The exercise links microbial processes, conceptsof biodiversity, inorganic chemistry, biogeochemicalcycling, evolution, microbiology, and microbial ecol-

ogy to help meet the many demands and standardsthat are part of today’s science classes.

The Winogradsky column is a glass or clearplastic column, filled with enriched soil or sedi-ment. When developed, it has an anaerobic lowerzone and aerobic upper zone that allow growth ofmicroorganisms under conditions similar to thosefound in sediments and water rich in nutrients(Sylvia et al., 1998). Often teachers simply conveythe message that different microorganisms exist indifferent strata of the column and that some live inthe aerobic and some in anaerobic zones. However,this is really where the discovery begins rather thanends! Explaining the complexity that lies within thedepths of the ecosystem allows deeper insights intothe microbial world.

In the laboratory, the Winogradsky columndemonstrates how the metabolic diversity ofprokaryotes transforms sulfur, an essential con-stituent of living matter and an abundant element inthe Earth’s crust (Stanier et al., 1976), to differentforms with varying redox states, thus supplyingnutrients and/or energy to the organism. The micro-bial assemblage that develops in the column spatial-ly separates organisms into distinct layers several

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BRIAN ROGAN teaches at The New Jewish High School,Waltham, MA 02453. MICHAEL LEMKE is in the BiologyDepartment, University of Illinois at Springfield, Springfield,IL 62703-5407. MICHAEL LEVANDOWSKY and THOMAS GORRELL

work at Haskins Laboratory, Pace University, New York, NY10038-1598.

Exploring the Sulfur Nutrient Cycle

Using the Winogradsky Column

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348 THE AMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 6, AUGUST 2005

SULFUR NUTRIENT CYCLE 349

centimeters thick even though in the environmentestablishment of similar layers of different organismswould typically exist in a few millimeters of sediment.The Winogradsky column creates conditions thatexpand the volume of natural processes, allowing aclear view of naturally-occurring phenomena. Soil sam-ples are collected from wetland habitats, amended withsimple inorganic and organic materials, then exposed tolight as an external energy source. The results are a mul-ticolored column of soil and water, each color linked toa chemical or biological process. The defined zones ofmicrobes develop form according to concentration gra-dients of oxygen, sulfur, nutrients, and light. Each func-tional microorganism group is dependent on otherfunctional microbial groups for development.

The Winogradsky column was developed andnamed after Sergei Winogradsky (1856-1953), aRussian microbiologist. He studied the complex inter-actions between environmental conditions and micro-bial activities using soil enrichment to isolate pure bac-terial cultures (Madigan et. al, 2000). Louis Pasteur,Robert Koch, and other scientists isolated cultures forstudy, but Winogradsky’s work was one of the first tostudy microorganisms in mixed enrichment cultures.The fact that the exercise works under a wide range ofcircumstances is a testament to the near ubiquity of cer-tain functional groups of microbes.

The Winogradsky column may also be used todemonstrate aspects of the earliest, sulfur-based lifeforms found on Earth. In an article in Nature, Nisbet(2000) paints a picture of an environment of earlyorganisms in the Archean period 2.5 to 4 billion yearsago that are analogous to those found in hydrothermalvents. Hydrothermal vents were first observed by thesubmersible vehicle Alvin that explored the Mid-Atlanticspreading ridge where the North American andEuropean plates are inexorably moving apart. Thisobservation marked the discovery of a system that mayhave remained intact since its formation as an ancientbiotic system utilizing simple nutrient cycles as an ener-gy source. In a sense, by creating a Winogradsky col-umn, we are modeling ancient environments, thoughperhaps at lower temperatures.

Constructing a Winogradsky column from simplehomemade materials is only one of the exercises dis-cussed here. We also present a technique that uses therespiration of seed germination to allow the reciprocalprocess, anaerobic metabolism, to occur in a simpleglass baking dish. The objectives of this article are to:

1. describe the microbial ecosystem of theWinogradsky column as a tool for studying thecycling of sulfur

2. explain how use of the column may illustrate somefeatures of development of early life on Earth

3. discuss how organisms may be isolated andgrown in a homemade anaerobic chamber.

Useful laboratory and educational extensions of theexercise are also discussed.

Materials & MethodsMaterials needed for the construction of the

Winogradsky column are simple and common. Theyinclude:

• a clear glass or plastic container (e.g., a smooth-sided, quart plastic water bottle at least 15 cm inheight and 5 cm in diameter. Plastic bottles areflexible and can be manipulated to allow forextraction of species for culturing. Very tall con-tainers require longer development periods andthe bacteria may be more difficult to extract.)

• plastic film and a rubber band

• a wooden dowel

• a sulfur source (e.g., calcium sulfate, magnesiumsulfate, or egg yolk added at about 1-2% of thesoil weight)

• an inorganic carbon source (calcium carbonate[e.g., chalk or limestone] or baking soda may beadded to 1-2% of soil weight)

• hydric soil (e.g., pond mud or shallow river sedi-ment collected near the surface)

• cellulose (e.g., shredded paper towels)

• a 60-75 watt light source

• water from the same source as the sediment

Break up soil clumps and sieve out larger debris sothe column can be packed evenly. The muddy mixtureshould be stirred to gain a uniform consistency andshould include the sulfur and inorganic carbon sources.Place a 2-3 cm layer of the mud mixture in the column,add the source of cellulose, and stir and pack with thedowel. Add as much of the mixture as needed, 2-3 cm ata time, with gentle tamping with the dowel to force outtrapped air, until the tower of mud is about 5 cm fromthe top of the container. Your last layer should be 2-3cm of water. Cover the opening with plastic film andsecure with a rubber band. Place the column next to acontinuously-lit, low heat, moderate intensity lightsource, making sure the column does not overheat.

Examine the columns weekly for at least a month,recording changes in color as they occur. For theWinogradsky column to be successful, enough timemust be allowed for the cultures to develop. Thecolumns may show growth in a week, as indicated byformation of a black color near the bottom and disinte-gration of the cellulose (paper), but will probably notfully form and stabilize for four weeks or more. The

ideal situation would be for students to investigate thecolumn over the course of the year studying ecology,microbiology, biodiversity, evolution, and other biologi-cal themes.

VariationsConstruction and development of the Winogradsky

column incorporates several variables. With just a fewchanges, different columns can be created to comparegrowth rates, microbial populations, and ecologicaldiversity.

• Sulfur Source

To illustrate the importance of the type of sulfuras a substrate, sodium sulfide or elemental sulfurcan be added in place of a sulfate. This shouldreduce the growth of the sulfate-reducing bacte-ria and alter the composition of the microbialcommunity.

• Acidity

Acid affects the biotic component of our environ-ment and alters its function. Changing the pHcan affect which species grow and dominate.Many of the standard sulfur reducers are adaptedto pH 6-8 (Madigan et al., 2000). Creating a moreacidic or alkaline environment shifts the commu-nity composition and alters sulfur cycling.

• Heat

Thermophilic bacteria are adapted to higher heatthan most (i.e., mesophilic) bacteria. Mud fromsome sources (e.g., hot springs) may harbor ther-mo-tolerant or even thermophilic bacteria. If youput the column close to a light source that pro-duces heat, these may grow. This could lead to aclassroom discussion about thermal vent biolog-ical communities.

• Osmotic Stress or Marine Stimulation

Columns with different salt concentration canillustrate several principles. If you begin with afreshwater or wetland source, salt can be a stress-ing agent favoring halophilic and halotolerantbacteria. On the other hand, you can show thatnutrient cycles also occur normally in marineenvironments by simply collecting muds andwater from a marine source and letting the col-umn develop as described.

• Type of Light (Wavelength)

Fluorescent lights, incandescent lights, or lightfilters (i.e., colored cellophane) that remove partof the spectrum from a light source could beused to select for organisms with differentabsorptive pigments.

Isolation & Culturing ofOrganisms from theWinogradsky Column

When pigmented patches are visible in the column(Figure 1), one can attempt to isolate some of the organ-isms. Sampling can occur at weekly intervals to checksuccession or can be done at the end of the project tosee the final flora of bacteria that develops. Samplingmay be done using a standard bacteriological nichromewire loop or hypodermic needle (pierced through theside of the plastic container), however distinctionbetween the microbes and mud is often difficult. Lookfor Beggiatoa (Figure 2A) or Thiobacillus in the water-sediment interface. Flagellated organisms from the col-umn, such as Rhodopseudomonas (Figure 2B) or thosewith sulfur inclusion bodies, like Chromatium andThiospirillium (Figure 2D) are more easily identified ifyou carefully adjust the contrast on a standard lightcompound microscope or have a phase-contrast micro-scope. The green sulfur (Figure 2C) and sulfate-reduc-ing (Figure 2E) bacteria are more difficult to see.Another method is to plunge a microscope slide into thesoil and allow growth of adherent biofilms to form onthe glass, which is then easily examined under magnifi-cation (Anderson & Hairston, 2000).

Growing microorganisms isolated from the diverseconditions of the Winogradsky column will be chal-lenging, yet students interested in the complexity of themechanisms underlying the fundamental processes ofnature will want to explore this process. Several meth-ods for culturing microorganisms from the column havebeen described and include pipetting mud from eachcolored zone to individual tubes for incubation withlight (Benson, 2002) or separating the mud, layer bylayer, and drawing off the liquid just above the layer forculturing or microscope observation (Atlas & Bartha,1998). If the latter method is part of your plan, con-structing a Winogradsky column out of a clear, smooth-sided cylinder is very helpful. After removing the plasticwrap or plugs from the ends, the mud can be pushedout in a single unit and sampling can be easily done. Inaddition, specific microbiological media recipes (e.g.,Atlas, 1995; DIFCO Laboratories, 1984) are available toestablish enrichment cultures of some of the organisms.

Although one can buy jars (e.g., GasPak system) andother materials (e.g., Brewer’s Petri plate, Wright’s Tube;Harley & Prescott, 1999) designed specifically for anaer-obic culture, we present here a method for culturinganaerobic organisms that can work well under class-room conditions using simple household items. Themethod is also intrinsically interesting for studentsbecause of the irony of pitting one biological process(i.e., seed respiration) to provide conditions for another


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process (i.e., anaerobic respira-tion). This gives the teacher yetanother chance to test the crit-ical thinking of the student.

The medium has the fol-lowing components: 0.01%NaS•9H2O (i.e., 0.01 g/100 mlwater), 0.05% yeast extract,0.05% sodium malate, 0.05%L-cysteine, and 1.5% agar insolution. This is most easilyprepared by adding 0.01 gNaS•9H2O, 0.05 g yeastextract, 0.05 g sodium malate,0.05 g L-cysteine, and 1.5 gagar to 100 ml water. Adjustthe pH to 7.3 using dilute acidor base (i.e., < 0.1 M NaOH orHCl) and bring to boil, thendispense ingredients in cleanPetri dishes. Isolate mud fromthe red or green pigmentedanaerobic or microaerophilicareas of the column andinnoculate each to differentplates by streaking.

Place 1-2 cm layer of liveoats or other seeds, such asgrass seed, in a glass casseroledish, add enough water tomoisten well, and cover withmoist paper towels. Place theinoculated (streaked) Petridishes on this layer. Cover thewhole tray with a glass or plas-tic plate sealed around theedges with Vaseline to make anoxygen seal. Add a light sourceabove the chamber to give lightfor photosynthesis (Figure 3).As the seeds germinate, theyrespire, and oxygen is depletedin the sealed incubation tray,and carbon dioxide is pro-duced, making an appropriateatmosphere for anaerobic pho-tosynthesizers.

The media and chamberwork as follows: the presenceof sodium sulfide and cys-teine (an amino acid with anexposed sulfhydryl group)helps maintain reducing con-ditions; it is also a key com-ponent for anoxygenic photo-

Figure 2.Drawings and photomicrographs of some representative organisms found in a typical Winogradskycolumn. Photomicrographs shown in E used with permission from N. Pfennig.

A. Drawings by S.Winogradsky of the sulfur-oxidizing bacteria Beggiatoa.Fig. 1 shows drawings of the tip of a Beggiatoa alba filament that becomes depleted in sulfurglobules (dots inside filament) over time: a) B. alba grown with sulfides, b) same speciesgrown in low-sulfide water for 24 h, c) B. alba in low sulfide after 48 hours. Fig. 2: Beggiatoamedia bacterial filament. Fig. 3: tip of B. minima. Fig. 4: degenerated B. alba lacking sulfur(Winogradsky, 1949).

B. Non-sulfur purple bacteria Rhodopseudomonas (left) and Rhodospirilum (right) (Stanier et al.,1976).

C. Green sulfur bacteria Chlorobium limicola (Stanier et al., 1976).

D. Purple sulfur bacteria Chromatium (left) and Thiospirillium (right) (Stanier et al., 1976).

E. Sulfate-reducing bacteria Desulfovibrio desulfuricans (left) and Desulfonema limicola (right)(Madigan et al., 2000).

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synthesis in these microbes (i.e., a source of reducedsulfur or electron donor, for some of the green sulfurbacteria). Certain red anaerobic photosynthesizers canuse organic compounds, such as malic acid, as electrondonors, so this medium serves double duty for thistype of culture enrichment. The generalized anaerobicprocess is:

CO2 + 2H2X ____> (CH2O) + 2X + H2O

where X stands for some reducing agent (or none atall—some anaerobic bacteria can simply use H2 by itself).This metabolic reaction is thought to be ancestral in thesense of biochemical evolution to the familiar photo-synthetic process in modern oxygenic photosynthesis,in which X has become oxygen.

Discussion

Relating Microbiology to NutrientCycling in the Column

After a month to six weeks, the column should sta-bilize into three distinct zones and develop communi-ties of bacteria specific to their environmental require-ments (Figure 1).

Aerobic Zone

The top of the water column can contain large pop-ulations of diverse bacteria and protists. These are aer-obic organisms found in organic-rich freshwaterhabitats such as shallow ponds and polluted streams.The bacteria are often flagellated, allowing them tomigrate and establish themselves in new areas(Madigan et al., 2000). In addition, there may be largerprotozoa and invertebratesfrom the original water andmud source. At the very topof the zone the mud is themost oxygen-rich part of thecolumn, often colored a light-brown from iron-oxide pre-cipitate.

O x y g e n - p r o d u c i n gorganisms, such as the photo-synthetic cyanobacteria,often grow above the mud,forming a green zone. Theseare the only bacteria thathave photosynthesis like thatof plants. In fact, there is verystrong evidence that thechloroplasts of plants origi-nated from ancestralcyanobacteria that estab-

lished themselves as symbionts inside the cells of aprimitive eukaryote.

A little deeper in the column, hydrogen sulfide gas(H2S) is diffusing upward into the aerobic zone. Part ofthe sulfur cycle is evident here. The H2S gas has beenproduced by anaerobic microorganisms near the bot-tom of the column. These organisms reduced the sulfateoriginally mixed into the soil. Near the top of the col-umn, the H2S can be oxidized back to sulfate by the sul-fur-oxidizing bacteria, such as the genera Beggiatoa andThiobacillus (Atlas & Bartha, 1998). These bacteria arechemoautotrophs and gain energy from the oxidationof reduced sulfur to elemental sulfur or to sulfate andthey can also synthesize organic compounds autotroph-ically from CO2. Thiobacillus oxidizes sulfur whileBeggiatoa is sulfide-oxidizing.

Microaerophilic Zone

The diffusion of H2S from the sediment belowenables anaerobic photosynthetic bacteria (which typ-ically appear in brightly colored bands) to grow. Frombottom to top, green sulfur bacteria (GSB), such asChlorobium, create an olive-green color zone. Purplenon-sulfur bacteria (PNSB), such as Rhodospirilum andRhodopseudomonas, usually require a small amount ofoxygen and are located nearer to the top of column thanare the GSB. Growth of these organisms results in a darkred-rust color.

The metabolism of both GSB and PNSB provides anexcellent opportunity to draw comparisons between oxy-genic photosynthesis (oxygen producing, like greenplants) and anoxygenic photosynthesis (non-oxygen pro-ducing organism that pre-dated green plants). GSB andPNSB gain energy from light reactions and metabolize

CO2 in the same way as plantsdo. Yet, because they use H2Sinstead of water as the sourceof hydrogen (reducing power),they produce a more oxidizedsulfur product (Atlas &Bartha, 1998). Consider plantphotosynthesis (i.e., 6CO2 +6H2O => C6H12O6 + 6O2)expressed as CO2 + H2O ___>[CH2O] + O2. Then it is easyshow the parallel processesbetween photosynthesis andhydrogen sulfide oxidation(Atlas & Bartha, 1998) as:

CO2 + H2O __> [CH2O] + O2

(plant photosynthesis)

CO2 + H2S __> [CH2O] + S(bacterial anoxygenic photo-synthesis)

Figure 3.A simple, yet effective, anaerobic chamber can be constructedfrom common household items as depicted in this diagram.


Anaerobic Zone

Organisms that grow in anaerobic conditions fer-ment organic matter or perform anaerobic respiration.Fermentation is a process in which organic compoundsare degraded incompletely; for example, yeasts fermentsugars to alcohol. Anaerobic respiration is a process inwhich a substance other than oxygen is the terminalelectron acceptor.

Three primary strata form in the lower level of thecolumn. The uppermost anaerobic layer often containsGallionella and other iron-oxidizers. Enrich for these byadding a source of iron (i.e., a nail or piece of steelwool). If you isolate Gallionella and look after closingdown the condenser iris (or using a phase-contrastscope), you will see cells and stalks that appear as twist-ed threads. These organisms oxidize iron and produce arust-colored iron oxide layer.

Moving deeper into the column, purple sulfur bac-teria, such as Chromatium, may be found and these bac-teria produce a red-to-purple layer in the soil. Purple sul-fur bacteria reduce sulfates to sulfur; a type of metabo-lism that emerged on Earth early in the planet’s history.From an evolutionary standpoint, there is strong evi-dence that the mitochondria of present-day eukaryoteswere derived from the purple bacteria (Margulis et al.,1986).

In the deepest layers are obligate anaerobes thatscavenge and metabolize sulfur and carbon in ways wedo not often discuss in the science classroom. Sulfur-reducing bacteria like Desulfovibrio utilize fermentationproducts (see below) in anaerobic respiration, usingeither sulfate or other partly-oxidized forms of sulfur(e.g., thiosulfate) to produce the H2S gas that diffusesthrough the column. The H2S spontaneously complexeswith iron to form a black ferrous sulfide (FeS). This iswhy lake sediments (and our household drains) are fre-quently black.

Some of these organisms are anaerobic cellulose-degraders, such as Clostridium, that grow when oxygenis depleted in the sediment. Though Clostridia cells arekilled by exposure to oxygen, these organisms producespores that can survive aerobic conditions (Madigan etal., 2000). They degrade the cellulose to glucose andthen ferment the glucose to gain energy, producing arange of simple organic compounds (e.g., ethanol, aceticacid) as the fermentation end products. Sometimes, atthe very bottom of the column, depending on thesource of the mud, a pink layer will develop due to pur-ple sulfur bacteria with gas vesicles. A characteristicspecies is Amoebobacter, which also photsynthesizesusing H2S (Atlas & Bartha, 1998).

The Sulfur CycleSulfur is an assimilable, nutritional requirement for

most life, and yet represents a energy conduit, or dis-similatory pathway, for some microorganisms. As anutrient, it is a component of several amino acidsrequired for protein synthesis, and a number of otherimportant biochemical components of the cell. Itschemical versatility, or ability to exist in several oxida-tion states (Figure 4), makes it a significant part of theenergy cycles of many organisms. Because of this rangeof oxidation states, sulfur compounds can act as elec-tron acceptors and donors. As electrons move from onemolecule to another they also lose or gain energy. Thusthe many microbial transformations of sulfur com-pounds form a basic part of energy metabolism.

A number of species can use elemental sulfur anaer-obically as a terminal electron acceptor (the usual role ofoxygen in aerobic respiration), reducing sulfur to hydro-gen sulfide (H2S). Others can use thiosulfate or sulfate asan electron receptor. A number of sulfur pathways existin the column (Figure 4). There are numerous species inthis environment beyond those mentioned, and eachhas its own unique contribution to the sulfur cycle.

Additional QuestionsOther areas can be investigated as an activity with

students. The column is, in fact, almost limitless as asource of questions and projects

1. If iron is increased, how might that affect growthof the organisms in the column?

2. Are there methanogens (i.e., microorganisms thatproduce combustible methane) in the column?How would they be detected? (Hint: Use a light-ed match to check the head space or gas of an“old” column).

3. What would be the effects of manipulating thepH? Adding salt? Adding organic nutrients?Manipulating the temperature? Manipulating thespectral influx in the light source with filters?

ConclusionsThe Winogradsky column is a complex system and

an excellent example of an investigation that can spanthe level from guided inquiry all the way to open-endedprojects that can occupy students’ imaginations andstudies for months. It is also a window into the biodiver-sity of our world. The Winogradsky column is an excel-lent way to show students that not all bacteria arepathogens and they have an important role in the geo-chemical cycling of the biosphere, one they have been ful-filling since life first began nearly four billion years ago.


Figure 4.The sulfur cycle emphasizing the transition in oxidation state of the different sulfur compounds (after Fenchel et al., 1998) and the bacteria-mediated processes that occur in aerobic or anaerobic habitats.


AcknowledgmentsWe wish to extend a special thank you to Dr.

Norbert Pfennig for his permission to use his photomi-crographs in this publication and to Sherry Hutson(UIS) for graphic design assistance. We wish to thankthe 2000 Woodrow Wilson National FellowshipFoundation Summer Leadership Institute for Teachersthat focused on biodiversity for creating the forum forlearning and interaction; one of the many end productsbeing this article.

ReferencesAnderson, D.C. & Hairston, R.V. (2000). The Winogradsky

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Atlas, R. M. & Bartha, R. (1998). Microbial Ecology:Fundamentals and Application, 4th Edition. Menlo Park,CA: Addison Wesley Longman, Inc.

Benson, H. J. (2002). Microbiological Applications: LaboratoryManual in General Microbiology, 8th Edition. Madison, WI:McGraw Hill.

Burns, R. G. & Slater, J. H. (1982). Experimental MicrobialEcology. Boston, MA: Blackwell Scientific, Inc.

DIFCO Laboratories. (1984). DIFCO Manual: Dehydrated

Culture Media and Reagents for Microbiology, 10th Edition.Detroit, MI: DIFCO Laboratories.

Dixon, B. (1994). Power Unseen: How Microbes Rule the World.Spektrum, NY: W. H. Freeman.

Fenchel, T., King, G. M. & Blackburn, T. H. (1998). BacterialBiogeochemistry: The Ecophysiology of Mineral Cycling. SanDiego, CA: Academic Press.

Harley, J. P. & Prescott, L. M. (1999). Laboratory Exercises:Mirobiology, 4th Edition. Boston, MA: WCB McGraw-Hill.

Hudson, B. K. (1998). Microbiology in Today’s World, 2ndEdition. Dubuque, IA: Kendall-Hunt Publishing.

Madigan, M. T., Martinko, J. M. & Parker, J. (2000). BrockBiology of Microorganisms, 9th Edition. Upper Saddle River,NJ: Prentice-Hall.

Margulis, L. & Dorian, S. (1986). Microcosmos. Berkeley, CA:University of California Press.

Nisbet, E. (2000). The realms of Archaean life. Nature, 405,625-626.

Schlegel, H. G. (1993). General Microbiology, 2nd Edition.Cambridge, UK: Cambridge University Press.

Stanier, R. Y., Adelberg, E. A. & Ingraham, J. (1976). TheMicrobial World. Englewood Cliffs, NJ: Prentice-Hall, Inc.

Stanier, R. Y., Doudoroff, M. & Adelberg, E. A. (1963). TheMicrobial World. Englewood Cliffs, NJ: Prentice-Hall, Inc.

Sylvia, D. M., Fuhrman, J. J., Hartel, P. G. & Zuberer, D. A.(1998). Principles and Applications of Soil Microbiology.Upper Saddle River, NJ: Prentice Hall.

Winogradsky, S. (1949). Microbiologie du Sol: Problèmes etMéthodes. Paris, France: Masson et Cie Éditeurs.


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