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DEGRADATION OF PLANT RESIDUES1

DEGRADATION OF PLANT RESIDUES

Part of plant tissue showing different types of cells and the action of an unnamed enzyme X on middle lamella. Individual cells released by the action of X. Some of the cells show broken estimates based on dilution-plating), protozoa, 1.0; nematodes, 2.0; earthworms, 12.0; Enchytraeidae, 4.0-; molluscs, 5.0; acari, 1.0; Collembola, 2.0; Diptera, 3.0; other arthropods, 6.0; total microflora, 461.5 and total microfauna, 36.0.

The relationship between organic matter and plant growth may be direct or indirect. Organic matter is a natural substrate for saprophytic microorganisms and provides nutrition to plants indirectly through the activity of soil microorganisms. It is essential for the formation of soil aggregates and hence soil structure which ultimately determines the extent of soil aeration and rooting habit of plants. Organic matter helps in the conservation of soil nutrients by preventing erosion and surface run-off of nutrients.

Soil can be defined as a natural medium for plant growth composed of minerals, organic materials and living organisms. While physical weathering of rocks caused by changes in temperature and the consequent chemical decomposition contributes largely to the formation of the soil, biological activities such as root growth and microbial metabolism in the soil contribute to its texture and fertility. Undoubtedly, the amount of organic matter present in any soil determines its natural suitability for plant cultivation.

Soil organic matter comprises residues of plant and animals at all stages of decomposition mediated by soil microorganisms. Various organic compounds which reach the soil by way of animal and plant residues are made up of complex carbohydrates, simple sugars, starch, cellulose, hemicelluloses, pectins, gums, mucilage, proteins, fats, oils, waxes, resins, alcohols, aldehydes, ketones, organic acids, lignin, phenols, tannins, hydrocarbons, alkaloids, pigments and other products. The size of particles in the organic matter, the nature and abundance of microorganisms involved, the extent of availability of C, N, P and K, the moisture content of soil, its temperature, pH and aeration, presence of inhibitory substances (such as tannins) etc. are some of the major factors which influence the rate of organic matter decomposition.

Plant residues contain 15-60 percent cellulose, 10-30 per cent hemicellulose, 5-30 per cent lignin, 2-15 per cent protein and 10 per cent sugars, amino acids and organic acids. Cellulose occurs in a semicrystalline form with a molecular weight of 106 and has glucose units with B(1-4) linkages. The individual chains of glucose are held together by hydrogen bonds. Cellulase enzyme complex decomposes cellulose into disaccharide cellobiose which is hydrolyzed by the enzyme cellobiase to glucose. Hemicelluloses are various polymers of hexoses, pentoses and sometimes uronic acids with commonly occurring monomers such as xylose and mannose. Pectin is an example of hemicelluloses and is an important constituent of the middle lamella of cell walls. Pectin is degraded by the enzyme pectinase which is a complex of several enzymes.

Lignin is much more complex than celluloses and is formed by chemical reaction involving phenols and free radicals without any specific order. Lignin gets encrusted on the cellulose and hemicellulose matrix. Compounds like caffeic acid and ferulic acid have structures similar to lignin and they have been used 'in studies on


degradation of lignin.The lignin molecule has only three elements carbon, hydrogen and oxygen. The molecule is a polymer of aromatic nuclei with either a single repeating unit or several similar units as building blocks. The repeating units range from about 200 to 1000 depending upon the origin of lignin and the methods used to determine the molecular weight.

Degradation of lignin is brought about by fungi mainly belonging to Basidiomycetes. The genera of fungi which degrade lignin as well as cellulose are Clitocybe, Collybia, Mycena, Marasmius, Polystictus, Armillaria, Polyporus, Stereum, Ganoderma, Pleurotus, Trametes, Fomes and Ustulina.

Bacteria constitute the most abundant group of microorganisms. In normal fertile soils, 10-100 million bacteria are present per g of soil. This figure may increase depending on the organic matter content of any particular soil. The bulk of soil bacteria are heterotrophic and utilize readily available source of organic energy from sugars, starch, cellulose and protein. On the other hand, autotrophic bacteria which occupy a smaller portion of the biomass in soil and use inorganic sources such as iron (Ferrobacillus) and sulphur (Thiobacillus) are not directly involved in organic matter decomposition.

The number of actinomycetes may be as high as 200 million per g of soil and may increase in manured soils. Thermophilic (tolerating 50 to 65°C) forms are not uncommon in compost piles. Ac¬tinomycetes grow on complex substances such as keratin, chitin and other complex polysaccharides and thus play-an active role in humus formation. Soil fungi are mostly heterotrophs and use organic residues easily but their numbers vary in soil depending on whether a species has a dominant vegetative or reproductive phase in the soil environment. Sporulating fungi such as Mucor, Penicillium and Aspergillus appear on agar plates rather profusely than non-sporulating ones. Soil algae in cultivated soils vary greatly in numbers and may contribute a small amount of organic matter through their biomass but they do not have any active role in organic matter decomposition. The microorganisms involved in the decomposItion of organic matter are listed in. The end products of decomposition are CO2, H2O, NO3, SO4, CH4, NH4, and H2S depending on the availability of air.

Cellulose Degradation

Cellulose is a prominent carbonaceous constituent of higher and lower plants. Degradation of cellulose is ecologically important. Plant tissues give rise to many waste products which have a high cellulose content e.g: waste paper, timber and waste such as saw dust & wood shavings.

Cellulose is a carbohydrate composed of glucose units bound together in a long, linear chains by β linkages at carbon atoms 1 7 4 of the sugar molecule. Usually cellulose molecules are not found individually but are linked together to form microfibrils. The separate molecules are linked by H-bonding into highly ordered crystalline structures. Some parts of the microfibrils have less ordered non crystalline structures and are referred to as amorphous regions. Cellulose is typically found in the walls of plants cells . Cell walls have primary and


secondary regions and are separated from each other by a middle lamella. .The primary walls and middle lamella have a low cellulose 9 20 – 30% ) and secondary cell wall is high in cellulose

(35-60 % ).

A number of polysaccharides are also associated with the cellulose of the plant cell wall. These include xylans, mannans and polyuraonides. The polysaccharides that are structurally linked with the cellulose of cell wall have been termed Cellulosans

Hard and soft wood contain about 43% cellulose, the rest being made up largely of pectin (1-4% ) , hemicelluloses ( 30 -40% ) and lignin (20 -30 %).

Its high molecular weight and ordered tertiary structure make natural cellulose completely insoluble in water.

Biodegradation of Cellulose

Cellulose is susceptible to biodegradation by means of hydrolytic enzymes called cellulases which convert it to glucose and oligomeres. A wide variety of organisms are known to be able to produce celluloses. Among bacteria, both Gram –positive and Gram-negative species, aerobes and anaerobes, unicellular and mycelial

gliding, non motile and flagellated bacteria can degrade cellulose.

Among Fungi, cellulytic activity is shown by representatives of phycomycetes, ascomycetes basidomycetes fungi imperfecti.


Factors influencing Cellulose decomposition:

The rate at which cellulose is metabolized is governed by a number of environmental influences. The major environmental factors affecting the transformation are:

1. available nitrogen level2. temperature3. aeration4. moisture5. pH6. presence of other carbohydrates and relative proportion of lignin in the residue.

Modification in the physical and chemical characteristics of the habitat can alter either the composition of the microflora or the cellulose degrading activity of individual organisms.

1. Available nitrogen level: The application of inorganic nitrogen enhances cellulose breakdown in soil, either ammonium or nitrate salts serving as suitable sources of the element. The rate of decomposition is proportional to the concentration of nitrogen added, but at high application rates, cellulose decomposition does not respond to supplemental increments.

2. Temperature: It ranges between 5 to 65o C . Each of the variety of cellulytic organism is affected differently by temperature. Mesophiles dominate at moderate temperature while thermophiles micro flora adapted to hotter localities can degrade cellulose above 45 o C.

3. Aeration: The rate of cellulose decomposition in environment deficient in oxygen is significantly less compared to aerated habitats.

4. pH: In neutral and alkaline pH , many microbes are capable of growing and liberating the appropriate enzymes for the hydrolysis of the polysaccharide. In acidic condition, cellulose degradation is mediated largely by filamentous fungi. Although the process is rapid below pH 5 and occasionally below 4, soils with lower hydrogen ion concentration degrade cellulose more readily.

5. Presence of other carbohydrates: Addition of readily metabolizable substances to soils accelerates cellulose decomposition.

1. Aerobic Mesophilic Microflora:

Fungi are the main agents of cellulose degradation in humid soils.

Several protozoa in pure culture are cellulolytic. E.g.: Hartmanella and Schizopyranus.

2. Anaerobic Mesophilic Microflora:


Several microbes are capable of decomposing cellulose in the total absence of molecular oxygen and the polysaccharide disappears under anaeobiosis whether supplied as purified chemical or in the form of plant materials. Fungi and Actinomycetes are not significant decomposers in anaerobic environments.

The most common anaerobic cellulose fermenters in nature appear to be members of the genus Clostridia.

The principal cellulytic bacteria found in rumen are Bacteriodes, Succinogenes, Ruminococcus albus, R. flavifaciens, Butyrovibrio filarissolvens and Eubacterium cellulosolvens.

3. Thermophillic decomposers: Thermophilic cellulytic bacteria can be readily obtained from sol and manure . Both aerobic and anaerobic microorganisms function in thermophilic transformations. Clostridium thermocellum and Clostridium thermocellulaseum are two important cellulytic microbes. Thermophilic fungi include Chnetomium thermophile and Sporotrichum thermophile.

Biochemistry of Cellulose degradation:

Cellulose being an insoluble polymer of very large size can not permeate the microbial cell membrane. Fungi and bacteria degrade

cellulose extracellularly. In protozoa, degradation in intracellular as the particle are phagocytosed..

Fungi and bacteria degrade cellulose by means of extra cellular enzymes. Bacteria either secrete their cellulases as soluble extracellular enzymes or assemble them into large complexes called cellosomes. The cellulosome is a highly active cellulose system capable of completely degrading cellulose. It has more active against crystalline cellulose than against disordered cellulose. During the exponential phase of bacterial growth, the cellulosomes are primarily found attached to the cell surface. Large percentage of these complexes is released in to the medium when cells enter stationary phase.

As the microbial cells are impermeable to cellulose, organisms secrete extracellular enzymes. These extracellular enzymes catalyze hydrolytic conversion of insoluble sugars to soluble sugars making the molecules of sugar to enter the cell membrane. Inside the cell simple sugars are oxidized to provide energy for biosynthesis.

A cellulose system involves three types of enzymes

C1 enzyme

Cx or β 1 , 4 glucanase and

Fungi Bacteria Actinomycetes

Alternaria Achromobacter Micromonospora

Aspergillus Bacillus Nocardia

Fusarium Cellulomonas Streptomyces

Penicillium Clostridium Streptosporagium

Rhizoctonia Pseudomonas

Trichoderma Cytophaga

Verticillium Sporocytophaga


Β-glucosidase

Total degradation of cellulose involves all three enzymes. The C1 enzyme acts on native cellulose and does not appear to exhibit much action against partially degraded cellulose molecules.

Cx enzymes do not hydrolyze native cellulose but instead cleave partially degraded polymers.

There are two types of cleavages exhibited by Cx enzymes. Endo β 1,4 glucanases break the chain internally more or less a random resulting in the formation of cellulobiase and various oligomeres.

Exo β 1, 4 glucanase attack the polymers near the end of the chain, resulting principally in the formation of cellobiase. The degradation of cellobiase and other relatively small oligomeres is catalyzed by β glucosidase resulting in the formation of glucose.

* * * * *

Hemicellulose Degradation

Polysaccharides known as hemicelluloses are one of the major plant constituents added to soil, second only to cellulose and they consequently represent a significant source of energy and nutrients to the microflora.

Hemicelulose have no structural relationship to cellulose but rather are polysaccharides composed of various arrangements of pentoses such as xylose and arabinose, hexoses such as mannose, glucose and galactose and / or uronic acids such as glucoronic and galacturonic acids.E.g of hemicellulosic compounds are xylans, mannas and galactans.These residues are variably modified by acetylation or methylation. Hemicelluloses show a much lower degree of polymerization (fewer than 200 sugars residues) than cellulose. Hemicellulose can be extractable with dilute alkali. They are water insoluble polysaccharides. Upon hydrolysis with hot, dilute mineral acid they yield hexoses, pentoses and frequently uronic acids. Hemicelluloses are noncovalently associated with cellulose.

Hemicellulose can be divided into two categories: 1.Polyuronimide hemicelluloses –containing uronic acids and 2. Cellulosans hemicelluloses devoid of uronic acid units. Presence of uronic acid determines the type of hemicellulose.

Soft wood hemicelluloses: There are three major soft wood hemicelluloses: Glucomannan, Galactogluco mannan, and Arabinoglucuronoxylan.

The two mannose containing polymers differ greatly in galactose content.

Hard wood hemicelloses: The major hardwood hemicellulose is glucuronoxylan. This polymers back bone consists of 1 4 linked β-D-xylopyrannose units, the majority of which are acetylated at C -2 or C-3 . About one 4 –O methyl ά –D glucoronic acid, attached to the backbone at C-2 I present for every 10 xylose units.


The polyuronides can be viewed as polysaccharides containing uronic acid residues in greater or lesser quantities as part of the structure of the molecule. Hydrolysis of polyuronide hemicellulose with hot dilute mineral acid cleaves the molecule and sugars and uronic acids. Products of hydrolysis contain two major polysaccharide types, both containing a pentose sugar and an uronic acid moiety. Xylose and Glucuronic acid in one and arabinose and gactcturonic acid are repeating constituents. The major ratio Xylose and Glucuronic acid of moieties varies according to the natural material.

Microorganisms

Many microbes of the soil utilize hemicellulose for growth and cell synthesis. Fungi, Actinomycetes and both aerobic and anaerobic bacteria are represented in the active populations.

Bacteria Substrate Anaerobes

Bacillus Xylan Clostridium thermocellum

Bacillus, Achromobacter Oat hemicellulose Clostridium thermosaccharolyticum

Pseudomonas Wheat pentoses Thermoanaerobium brocki

Cytophaga,sporocytophaga Hemicellulose Thermobacteroides

Lactobacillus Xylan

Vibrio Mannan,Xylan

Actinomycetes

Streptomyces Wheat pentosan,Oak hemicellulose

Fungi

Alternaria, Fusarium, Trichothecium, Helminthosporium.

Hemp polyuronide

Aspergillus,Rhizopus,Zygorhynchus Galactan,Mannan,Xylan

Chaetomium, Helminthosporium, Pencillium

Wheat pentose


The metabolism of hemicellulose is influenced by physical and chemical characteristics of the habitat .pH and temperature and age of plants has major role in degradation of hemicellulose.

Biochemistry of Hemicellulose Decomposition

Hemicellulose is converted into simpler compounds as they have high molecular weight and fail to pass through the cell membrane.

The enzymes catalyzing hemicellulose breakdown are broadly termed as hemicellulases.Xylan is the major hemicellulose. The complete conversion of glucuronoxylan into its building blocks requires the combined action of endo β 1, 4 xylanase and β –xylosidase as well as ά-L-arabinofuranosidase and an acetyl xylan esterase.

Many enzymes are involved in the degradation of hemicelluloses include endo enzymes. Degradation product includes Carbon dioxide, water, cell biomass and variety of small carbohydrates.

* * * * * *

Degradation of Lignin

Lignin is second to cellulose in terms of plant biomass. It protects cellulose and hemicellulose from enzymatic attack. Lignin is also an important structure component of plants. It provides structural rigidity, resistance to

compression and bending, resistance to pathogens. Lignin is a cementing agent in wood.

Lignins are three dimensional amorphous and highly branched unlike cellulose, starch or hemicellulose. They do not have identical linkages repeated at regular intervals. Lignins undergo more or less random reactions that are both chemical and enzymatic and yield polymers with no defined structure. They are richest source of aromatic compounds in nature and the bonds that lignin contain are difficult to cleave. This makes it difficult to extract

lignin in an unmodified state.

Lignin is found in the cell walls of higher plants, gymnosperms, and angiosperms, ferns and club mosses, predominantly in the vascular tissues specialize for liquid transport, but in combination with polysaccharides.

It is not found in mosses, lichens and algae that have no trachids. The increased mechanical strength that ligninification confers on woody tissues enables huge trees several hundred feet tall to remain upright.

Lignification is the process whereby growing lignin molecules fill up the spaces between the preformed cellulose fibrils and hemicellulose chains of the cell wall.

Chemistry of Lignin

Lignin is a complex polymer. The basic subunit is an aromatic ring with a three carbon side chain. So the basic subunit is called a phenyl propanoid. Both the phenyl group and the propanoid groups are modified in the lignin


itself. The R side chains differ among different plant species and the relative proportion of units derived from each of these three basic building blocks is different in different plants, plant tissues and plant ages.

Based on lignin precursors wood is divided into soft wood and hard wood.

A typical soft wood (Gymnosperms), lignin contains building blocks originating mainly from coniferyl alcohol, some from P-coumaryl alcohol but none from sinapylacohol.

Hard wood (Angiosperm) lignin is composed of equal amounts of (46%) each of conifeyl and sinapyl units and minor amounts of p-hydroxyphenyl propane units. The lignin polymer is formed by polymerization of varying

proportions of coumaryl, coniferyl and sinapyl subunits and their derivatives.

The monomers are not linked in the same way .With in one type of lignin, these are C-C bonds and C-O ether bonds .Some monomers may be involved in several linkages and others only one or more monomers at different

positions. The monomers have hydroxyl and methoxy substituents, some of which are conserved in the polymer.

Overall lignins are rather heterogeneous and much branched polymers of indefinite structure and size with estimates of molecular weight of lignin derivatives varying from a few thousand to over a million.

Decomposition

Lignin is highly resistant to biodegradation, much more so than other biopolymers. As lignin is heterogeneous in bond type and positioning and also most of the bonds are not amenable to hydrolytic cleavage. Added to this

lignin is insoluble and difficult to wet.

The decomposition of lignin proceeds either in the presence or in the absence of oxygen, but the rate of loss in both circumstances is characteristically less that observed for cellulose, hemicellulose and other carbohydrates.

Factors Affecting Lignin Decomposition

The rate and extent of lignin decomposition is affected by

1. Temperature

2. Availability of Nitrogen

3. Anaerobiosis

4. Constituent of plant residue.

Temperature has profound influence upon the rate and extent of breakdown. Little loss occurs at 7 ° C and progressively higher temperature favor the active Microflora. At 37° C, the oxidation is extensive and

appreciable quantities disappear. Even the thermophile bacteria degrade the lignin relatively at higher


temperature; unfortunately the thermophile organisms involved in the oxidation of lignin have not been isolated in pure culture.

The presence of an accessible source of energy can provide nutrients to support a larger Microflora that could bring about greater losses.

Age of the plant is another important variable. The lignin of young tissue disappears more rapidly than that in mature plants.

Anaerobic conversion of lignin does occur, rather slow though.

Microbiology of Lignin Degradation

Degradation by Fungi

The wood decaying fungi are the primary contributors to the degradation of wood in nature, secreting extracellular enzymes that degrade the polymeric components of the wood cell walls. They are classified as white rot, brown rot and soft rot fungi on the basis of morphological aspects of the decay. Once the fungus

manages to invade in the wood, it expands by growth of its hyphae in the lamina of parenchyma and vascular cells. The hyphae penetrate from one cell to the next either through pits or through cell walls. The hyphae of

some fungi grow within the middle lamella or the secondary walls.

White rots and brown rots are filamentous fungi that belong to the subdivision Basidomycetes. White rots typically cause bleaching of the wood, giving it a fibrons or spongy consistency. The white rot fungi degrade the

lignin and polysaccharides at a simililar rate and most of them prefer hard woods.

Brown rots preferentially degrade the polysaccharide component of the wood and cause little degradation of lignin. The decaying wood becomes brown and brittle. Most brown rot fungi attack soft woods.

Soft rot, a group of fungi belonging to ascomycetes and the fungi imperfecti area major cause of decay in the wood that is exposed to moisture. Soft rots degrade both polysaccharides and lignin but different rates that

depend on the fungal species. Soft rots are found in both soft wood and hard woods and their action leads t a softening of the wood.

White rot fungus: Phanaerochacte chrysosporium, Coriolus vessicolor, Polystictus, Armillacia, Pllyporus, Formes, etc. Other than wood softeners fungi such as strains of Pencillium, Fusarium and Aspergillus have been shown to

degrade lignin.

Large numbers of bacteria are known to degrade lignin. The notable species of bacteria involved in lignin degradation include Nocardia, Streptomyces, Bacillus, Pseudomonas, Flavobacterium, Aeromonas and

Xanthomonas.

Enzymology of Lignolysis:


Lignin peroxidase This extracellular enzyme with an acidic pH optimum catalyses the oxidative breakdown of lignin.

Lignin peroxidase catalyzes the cleavage of the arypropane side chains, ether bond cleavage, and aromatic ring opening and hydroxylation.

All these reactions can be adequately explained by a mechanism that begins with the catalyzed abstraction of an electron from aromatic nuclei in lignin to form unstable cation radicals. Subsequently various products are

formed by non enzymatic reaction of the radical cations with water, other nucleophiles and oxygen.

Other enzymes with roles in lignin breakdown:

In addition to lignin peroxidase lignolytic cultures produce other enzymes that function either in the breakdown of lignin or in modification of the breakdown products.

Mn(II) –dependent perxidases: Successful degradation of lignin requires attacks on both the non phenolic and phenolic lignin components.

The extracellular Mn (II) –dependent peroxidase oxidizes phenolic components of lignin, but they can not oxidize the non phenolc susbstrates of lignin peroxidase such as veratryl alcohol or the non phenolic compounds

of lignin substructure.

Quinone Reductases: Quinones are among the products of lignin degradation by the peroxidase and P.chrysosporim produces both intracellular and extracellular qunone reductases. The extra cellular cellobiose

quinine oxidoreductases are active only in the presence of cellulose. This enzyme uses cellulobiose as a hydrogen donor to reduce quinines to hydroquinones.ntracellular quinine reductases use NAD(P)H as cofactor.

Quinone reductases remove products of lignin degradation that might other wise repolymerize.

Metabolism of Lignin

Lignin

↓

Oxidative breakdown of lignocelluloses catalyzed by lignin peroxidase and MN (II) –dependent peroxidase and reductive mediators generated by these enzymes.

↓

Monosidi and oligolignols

↓


Further oxidative breakdown by the same mechanism as in lignin breakdown

↓ ↓ ↓ ↓

C1,C2,& C3 Fragments aromatic acids & aldehydes

Products of lignol ring Cleavage Quinones

↓

Hydroquinones

↓

Uptake by the fungus followed by degradation of the fragments through central metabolic pathways.

↓

Carbon dioxide and water

* * * *

Degradation of Pectin

Pectin is a major constituent of cereals, vegetables, fruits and fibers, is a complex high molecular weight heterogeneous and acidic structural polysaccharide. They are present in the middle lamella of plant cell wall as a thin layer of adhesive extracellular material attached with cellulose microfibrils surrounded by a matrix of hemicelluloses and proteins acting as a cementing agent.

Structure of pectin: The pectic carbohydrates are complex polysaccharides composed of galacturonic acid units bound to one another in a long chain. The carboxyl group of the galacturonic acid building block may be partially or completely esterified with methyl groups and may be partially or entirely neutralized by various cations. Sometimes the hydroxyl groups at C3 or C4 positions are also acetylated. Additionally rhamnose constitutes a minor component of pectin backbone and it is present as α-L rhamnosepyranose residue.

The complex pectic substances are:

1. Protopectin: It is water insoluble constituent of the plant cell wall.

2. Pectinic acids: These are colloidal polygalactoturonic acids with various amounts of methyl ester groups. Pectic acid has the unique property of forming gel with sugar and acid.


3. Pectin /Pectins: It is a water soluble polymeric material which has various degrees of esterification with methanol and can form gel with sugar and acid under suitable conditions.4.Pectic acids: The water soluble galacturonic acid polymers that are essentially devoid of methyl esters linkages..

Bacteria, Fungi and Actinomycetes are capable of acting upon and hydrolyzing pectin, Protopectin and Pectic acid using the polysaccharides as carbon and energy sources to support proliferation.

Bacteria: Bacillus,Erwinia,Pseudomonas,Clostridium, Thermoanarobacter,Xanthomonas.

Fungi: Fusarium, F.oxysporum.F.lycoperici,Veticillum ,Aspergillus

Actinomycetes: Streptomyces.

Pectin degrading enzymes:

The enzymes responsible for the degradation of pectin is widespread and have diverse modes of action.Pectolytic enzymes include a group of enzymes that are able to catalyze the breakdown of pectin containing substrates.Acidic pectinases have pH optima below 7 and are called acidic pectinases.

Alkaline pectinases have optima pH above.

Three classes of enzymes are concerned in the degradation of pectic substances

1. Esterases

2. Lyases

3. Protopectinases.

1. Esterases: Pectin esterase /Pectin petyl hydrolase/ polymethyl galacturonate esterase (PMGE).These deesterify the pectin molecule to pectic acid. The esterase preferentially acts only on the methyl ester groups.

Polygalacturonases/ polygalactosiduronate/Glycanolydrolase/Endopolygalacturonase: These cleave the glycosidic bonds of pectin by adding water molecules.

2. Lyases: These catalyze the breakdown of either pectic acid or pectin by β-elimination reaction. Two kinds of lyases are known.

Endo polygalacturonate lyases: These are a group of hydro lytic enzymes which randomely cleave α 4 glycosidic linkages by β-elimination in pectates. And pectic acids resulting in the rapid decrease in the viscosity. It causes random cleavage by transelimination process.

Exopolygalacturonate lyase: They attack preferentially on pectates than pectins and polymethygalacturonate.


Poly methyl galacturonate lyases: They are endo acting enzymes and therefore cause

decrease in the viscosity by catalyzing the β-elimination between 4 th and 5th carbon of pectin at the non reducing end.

3. Protopectinases: The insoluble protopectin is converted to highly polymerized water soluble pectin by the action of a group of enzymes known as protopectinase.

They are classified as A and B type depending on their mode of action. A type protopectin reacts with smooth regions in the protopectin which are composed of partially methylated galacturonic acid residues.

B-type protopectin reacts with the hardy regions which consist of rhamnogalacturonans and neutral sugars. They are considered glycohydrolases.

Polygalacturonase and pectin methyesterases are extra cellular enzymes.

Microbes produce a variety of pectin degrading enzymes which play an important role in the degradation of pectic substances and have extensive application in food processing.

* * * *

Starch Degradation

Starch serves as the plant as a storage products and as such it is the major reserve carbohydrates. This material occurs in large amounts in leaves carrying out photosynthesis but the polysaccharide is distributed in the xylem, phloem, cortex and pith of the stems of many species as well as in the tubers of bulbs, corns, rhizomes, fruits and seeds.

Typically the starch of higher plants accumulates in definite grains which may vary from 1 to 150 μm in diameter , the size depending upon the species. Microorganisms may also accumulate starch.

Plant starch usually contain two components amylose and amylopectin

Amylose: Linear polymer comprises of 200 to 500 or more glucose units linked together by an α 1-4 glycosidic bonding.

Amylopectin: The individual glucose units are likewise bound together by α 1-4 linkage but the molecule is branched and has side chains attached through the α 1-6 glycosidic linkage.


Starch commonly contain 70-80% amylopectin and 20-30% amylase but exceptions are not uncommon..E.g, the starch within the seeds of waxy corn contains no amylase, whereas that in wrinkled peas is essentially free of amylopectin.

In the highly branched structure of amylopectin the distance between branch points is approximately 5 – 8 glucose residues.

Microorganisms: Bacteria, Fungi, and Actinomycetes have the capacity to hydrolyze starch. Soil frequently contain 106 to 10 7 or more starch hydrolyser per gram . The organisms are particularly numerous in proximity to the plant root system, but their proportional incidence in the root zone is not significantly different from that in soil taken at a distance from the plant.

Microbial genera utilizing Starch

Achromobacter,Bacillus,Chromobacterium,Clostridium,Cytophaga,Flavobacterium,Micrococcus,Pseudomonas,Sarcina,Serratia.

Actinomycetes:Micromonospora,Nocardia,Streptomyces

Fungi:Aspergilus,Fomes,fusarium,Polyporus,Rhizopus.

Among bacteria are gram-positive, gram-negative, spore formers, non spore formers, aerobes, and obligate anaerobes as well as representatives of many physiologically different groups.

Starch is an excellent carbon source for most Actinomycetes and strains of Streptomyces. Nocardia and Micromonospora.

Many filamentous fungi are also capable of excreting the appropriate hydrolytic enzymes; yeasts on the other hand rarely attack the polysaccharides. Under the conditions of limiting oxygen, a fermentation occurs with the formation of appreciable lactic, acetic and butyric acids. The process of degradation goes on at a good pace even under total anaeobiosis.

Amylases

Amylases are characteristically extracellular and remain in the culture fluid after removal of the microorganisms. Two amylases are commonly concerned in the microbial breakdown of starch and are α –amylase and β-amylase.

β-amylases acts upon the both amylose and amylopectin, cleaving every second glucose -glucose bonds from the terminal end of the molecule. Hence maltose fragments are liberated from the straight chain of amylase and significant quantities of other intermediates do not accumulate.

β-amylase is incapable of catalyzing the hydrolysis of branch points of amylopectin.


In contrast the products formed by α –amylase in the depolymerization of amylopectin have a greater molecular weights than maltose.

Both the enzymes act upon the 1,4 linkage, but the hydrolysis is retarded once the amylopectin branch point is encountered. Several other microbial enzymes hydrolyze the branch point positions. The maltose that is formed is converted to glucose by mediation of the enzyme α- glycosidase so that starch is transformed ultimately to glucose.

Starch Amylase maltose α- glycosidase glucose

Starch hydrolyzing enzymes are usually adaptive , but the ability of microorganism to form amylolytic enzymes depends on the type of starch. Because amylases are extracellular the possibility exists that the enzymes are adsorbed and possibly inactivated by clays.

* * * *

HumusApart from the transient products in the decomposition of organic matter, a dark coloured and fairly stable soil organic matter called humus with known and unknown physical and chemical properties is also an intergral part

of the organic matter complex in soil. Humus can be defined as a ligno-protein complex or an amino acid-lignin complex containing approximately 45% lignin compounds, 35% amino acids, 11% carbohydrates, 4% cellulose, 7% hemicellulose, 3% fats, waxes and resins and 6% other miscellaneous substances including plant growth substances and inhibitors. However, the age and composition of the humus are dependent on its origin and environment. By radio carbon dating techniques, the age of humus in podzols has been estimated to be in the range of 1580 to 2860 years while that of chemozemic soils as 1000 years old. Among the fractions of humus, humic acids have been

regarded as the oldest and most persistent. Bacterial and algal protoplasm with their attendant biological constituents contribute in large measure to the nutritive value of humus. If decomposition by microorganisms is arrested by factors such as low temperature, anaerobiosis, low mineral content and the presence of microbial growth inhibitors such as phenolic compounds, the humus is known as raw humus. On the other hand, nutrient humus in fertile soils contains large amounts of sugars, starch and soluble energy material serving as substrates for microorganisms.


INULIN

lnulin is a polysaccharide composed of fructose units, that is, a fructosan. The inulin molecule has approximately 25 to 28 fructose residues in the carbohydrate chain bound in (2 —>.l)—linkage. The substance occurs in a number of plants as the storage carbohydrate, replacing starch in this regard, and it has been reported in tubers, roots, stems, and leaves.

There is also a polysaccharide made up of fructose units in which the sugar units are bound together through the

number 2 carbon of one fructose and the number 6 carbon of the next sugar in the chain. This is a (2 —> 6)-fructosan. Many microorganisms utilize inulin. Bacteria of the genera Pseudomonas, Flavobacterium, Micrococcus, Cytop/zaga,

Ant/zrobacter, and Clostridium, many streptomycetes, and a heterogeneous group of fungi use the fructose

polysaccharide as carbon source for growth. The enzyme converting inulin to smaller fructose units is called inulinase. Little attention has been given to the transformation despite the large population active on the carbohydrate, but it has

been shown that inulinase, an extracellular catalyst, is highly active in several fungi and bacteria. As a rule, inulinases are exo enzymes and remove single fructose units from the end of the molecule, but the product of exo inulinase of some organisms may be a disaccharide containing two fructose units . On the basis of the rapid turnover exhibited in vitro, it is likely that fructosans are readily

transformed in soil. - Certain microorganisms possess two fructosanases-—inulinase, which by the structure of its substrate is a (2 —> 1)-fructosanase, and a (2 —> 6)-fructosanase. These enzymes are both extracellular. Some fungi produce both the (2 —+ 1)-and the (2 —> 6)-fructosanases while others produce only one or the

other of the polysaccharide-splitting enzymes. The enzymes are generally not formed when the organisms are grown on simple sugars but are excreted in appreciable amounts in the presence of the fructosan. The

mechanism of action, however, issomewhat different for the two catalysts. The (2 —> 6)-fructosanase of a species of Streptomyces leads to the accumulation of levanbiose, a‘ sugar containing two fructose units. The

hydrolysis of fructosan by the (2 ——> 1)-fructosanase (inulinase) of Aspergillmfumzgatus, on the other hand, results in the accumulation of slightly longer chains of fructose units. In contrast, the fructosanases of Fu.s

¢m'um monolyforme and P. funiculosum transform the fructosan entirely to fructose with no accumulation of significant quantities of intermediary compounds. Apparently, there are several end products of fructosanase

action—short fructose polymers, the disaccharide, or free fructose. Neverthe- less, the ultimate fate of the large molecule is an enzymatic degradation to units small enough to enter the cell.


Humus

While soil microorganisms in general take part in humus formation, some fungi such as Penicillium, Aspergillus and also actinomycetes produce dark humus-like substances (amino acids, peptides and polyphenols) which serve as structural units for the synthesis of humic substances. Extracts of spores of Aspergillus niger possess properties similar to those of humic acids. Thus in recent years, considerable evidence has come forth to show that humus is not only a biochemically derived material but also a synthetic microbial product.

Humic AcidsAn understanding of the biochemistry of humus degradation has posed problems mainly due to the procedural difficulties in solvent extraction of organic matter complexes. Further, bio-degradation products of lignin are closely linked with those of humus substances. In spite of these inherent difficulties, fulvic acid, humin and humic acid have been recognised as the three major fractions by subjecting humus complexes to solvent ex-traction procedures. Fulvic acid is the alkali as well as acid soluble part of soil organic matter and contains carbohydrates and proteins.

Humin is resistant to cold alkali and is a chemically heterogenous fraction. Humic acids form the bulk of the humus complex and are regarded as polymers of aromatic compounds. A wide variety of phenolic degradation products can be obtained from humic acids. Fungi and bacteria are known to decompose humic acids. Some of the fungi, mostly basidiomycetes and ascomycetes, capable of decomposing lignin, can also decompose humic acids.

Beneficial Role of Humic AcidsThe benefits of humic substances are reflected in improved seed germination, root growth, uptake of minerals by plants and other physiological effects on plant growth. Optimum levels of sodium humate are known to increase the percentage of germination of seeds in wheat, maize, gram, peas and beans. Root growth in tomato plants has been shown to be increased by the application of humic and fulvic acids at low concentrations. There are several reports to show that mobilization of N, P and K from the soil into the root system is increased in the presence of humus substances. The application of humic acid to soil is also known to decrease phosphorus fixation in soil, particularly in calcareous soil.

The uptake of trace elements by plants is increased by the application of humus substances since the latter is known to effectively chelate with trace metals, especially iron. A combination of fulvic acid and iron is known to be more effective in increasing lateral root formation in plants than iron alone. The chelating ability of humates suggests that they playa role similar to EDTA (ethylene-diamino tetraacetic acid), the well-known synthetic chelator.


Enzyme actions involved in plant metabolism have been linked with humus complexes since humic acids function as hydrogen acceptors. The growth stimulatory activity of humus complexes on wheat roots has been attributed to the noticeably increased cytochrome oxidase activity in the root system.

Genera of Microorganisms Capable of Utilising Different Components of Organic Matter

Nature of substrate in organic matter

Genera of microorganisms

Cellulose F. Alternaria, Aspergillus, Chaetomium, Coprinus, Fomes, Fusarium, Myrothecium, Penicillium,

Polyporus, Rhizoctonia, Rhizopus, Trametes, Triclwderma, Trichothecium, Verticillium, Zygorynchus

B. Achromobacter, Angiococcus, Bacillus, Cellfalcicula, Cellulamonas, Cellvibrio, Clostridium,

Cytophaga, Polyangium, Pseudomonas,

Sorangium, Sporocytophaga, Vibrio

A. Micromonopora, Nocardia, Streptomyces, Streptosporangium

Hemicellulose F. Alternaria, Fusarium, Trichothecium, Aspergillus, Rhizopus, Zygorynchus, Chaetomium,

Helminthosporium, Penicillium, Coriolus, Fames, Polyporus

B. Bacillus, Achromobacter, Pseudomonas, Cytophaga, Sporocytophaga,

Lactobacillus, Vibrio

A. Streptomyces

Lignin F. Clavaria, Clitocybe, Collybia, Flammula, Hypholoma, Lepiota,

Mycena, Pholiota, Arthrobotrys, Cephalosporium, Humicola

B. Pseudomonas, Flavobacterium

Starch F. Aspergillus, Fomes, Fusarium, Polyporus, Rhizopus

B. Achromobacter, Bacillus, Chromobacterium, Clostridium, Cytophaga

A. Micromonospora, Nocardia, Streptomyces

Pectin F. Fusarium, Verticillium

B. Bacillus, Clostridium, Pseudomonas

Inulin F. Penicillium, Aspergillus, Fusarium

B. Pseudomonas, Flavobacterium, Beneckea, Micrococcus, Cytophaga,

Clostridium

Chitin F. Fusarium, Mucor, Mortierella, Trichoderma, Aspergillus, Gliocladium,

Penicillium, Thamnidium, Absidia


B. Cytophaga, Achromobacter, Bacillus, Beneckea, Chromobacterium,

Flavobacterium, Micrococcus, Pseudomonas

A. Streptomyces, Nocardia, Micromonospora

Proteins and nucleic

acids Cutin

B. Bacillus, Pseudomonas, Clostridium, Serratia, Micrococcus

Cutin F. Penicillium, Rhodotorula, Mortierella B. Bacillus

A. Streptomyces

Tannin F. Aspergillus, Penicillium

Humic acid F Penicillium, Polystictus

Fulvic acid F. Poria

Mineralization and Immobilization ProcessesWhen microorganisms grow and multiply on organic debris, carbon is utilised for building the cellular material of microbial cells with the release of carbon dioxide, methane and other volatile substances. In this process, microorganisms also assimilate nitrogen, phosphorus, potassium and sulphur which get bound in the cell protoplasm. Therefore, the C/N, C/P, C/K or C/S ratios in soil are governed by the extent of organic matter utilised by soil microorganisms depending on the oxygen content and the microbial biomass at a particular stage in decomposition. Thus, three parallel processes go on during decomposition: (1) degradation of plant and animal remains by cellulases and other microbial enzymes, (2) the increase in the biomass of microorganisms which comprises polysaccharides and proteins and (3) the accumulation or liberation of end products. The term mineralization is used to designate the conversion of organic complexes of an element to its inorganic state which embodies the first of the three processes mentioned above.

Mineralization and Immobilization Process


Effects of Residues of Crops on Plant GrowthThe decomposition products of plant residues in soil may become toxic to growth of plants under certain conditions. The absence of satisfactory extraction procedures and bio-assay methods have come in the way of identifying the nature and extent of phytotoxic principles produced by plant remains which undergo decomposition. However, detrimental effects of plant residues have been detected through seed germination tests, growth of radicles and seedling injury under laboratory conditions which have been supported by field observations like stunted overall growth of plants, chlorosis, slow maturation, premature leaf abscising and failure of flowering and seed setting.

The oxygen status of the soil (aerobic or anaerobic) is the most important factor in determining the qualitative and quantitative aspects of microbially mediated bio-degradation of plant remains. Some of the phytotoxic com-pounds detected so far include methane, acetic, lactic, butyric, formic and other organic acids; phenolic compounds including syringaldehyde, vanillin, p-hydroxybenzaldehyde, ferulic, syringic, vanillic, p-hydroxybenzoic, p- methoxybenzoic and benzoic acids, various amino acids and many other unidentified products. These products seem to accumulate under waterlogged anaerobic surroundings whereas under normal arable soils the presence of toxic compounds is either rare or negligible


Soil SicknessApples, peaches, grapes, cherries and plums are prone to suffer from soil sickness if replanted in the same soil successively. They however, recover from sickness if replanted in new soils. The symptoms differ from plant to plant and the disease syndrome has been frequently reported from Germany, Canada and U.S.A. Such plant disorders may be attributed to nutritional deficiency, pathogenic microorganisms whose identity is yet to be established and phytotoxins secreted by roots of plants or by microbial decomposition of plant residues. A cumulative effect of one or more factors may also be responsible for the disease syndrome, in fruit orchards.

For example, the failure of peach cultivation in California and Ontario has been attributed to a phytotoxin produced by residues of a previous crop. The barks of roots of peach contain cyanogenic glycoside, amygdalin in relatively low amounts and as such is not toxic to peach plants. The glycoside is converted to toxic components like benzaldehyde and hydrogen cyanide through the mediation of microorganisms in soil supporting the growth of peach plants. The hydrolysis of amygdalin has also been attributed to a soil nematode (Pratylenchus penetrans) found in peach soils which secretes an enzyme.

In apple trees, one of the constituents of the bark is phloridzin which is broken down in soil to phloretin, phloroglucinol, p-hydroxyhydrocinnamic acid and p-hydroxybenzoic acid compounds which have been proved to be toxic to apple plants. Penicillium expansum, a normal fungal inhabitant of soil from apple plantations, produce patulin and an unidentified phenolic compound in media amended with apple residues. These compounds are also inhibitory to the growth of apple trees.

CompostingFarmyard manure is the oldest manure known to mankind and is made up of solid excreta or dung of animals, urine and plant remains which are allowed to decay with the help of soil microorganisms capable of decomposing complex organic debris into substances that the easily assimilated by plants. The manorial value of farmyard manure depends on the nature of raw materials used and the extent of decomposition by soil microorganisms.

Compositing farm residues and night soil has been practiced for long in China and India. In China, the compost pits dug in soil have usually dimensions of 35 m x 25m x 15m (L x B x H). The pits are filled layer by layer and each layer is about 15cm thick. The bottom layer (layer No.1) consists of green plants and aquatic weeds available on the farm followed by silt-straw mixture (layer No.2) and animal excreta (layer No.3). The layering is repeated until the pit is filled. Finally, a layer of mud is made on top of the pit in such a way that water of about 4 cm depth is maintained on the surface to create anaerobic conditions which helps to reduce losses of nitrogen.

In a time span of about 10 weeks, the mud plaster is dismantled and the contents of the pit are turned over or mixed with superphosphate and water (if necessary); At the end of 3 months, the compost is ready for use on the farm. The compost may have a CN ratio of 15-20 and organic matter content of 8-10 per cent. A compost is considered superior if the CN ratio is 20 or less and the organic matter content is around 30-60 per cent.


In India, the pit method is also practiced without any water logging in an elevated place often protected by a shed. The layering at the bottom is usually the urine-soaked bed in the cattle shed. The bed is made of farm materials such as vegetable wastes, fodder remnants, green matter etc.

The bed layer is sprinkled with a slurry of cowdung and mixed with well decomposed manure from the previous batch. This sort of layering and sprinkling with cowdung slurry is repeated until the pit is filled. The compost pit sits for a period of 2-3 months within which time the contents are turned over or stirred three times.

Composting can also be done by the heap method in which the base material on a hard ground consists of hardwood materials coming from cotton and pigeonpea stalks followed by layering with farm residues such as leaves, hay and garbage.

The heap can be rectangular in shape. After wetting with water, the heap is mud plastered and allowed to sit. Within a period of 2-3 months, the heap is broken, materials turned or stirred and again mud plastered. The final product becomes a heap of well decomposed organic matter.

High temperature composting is done in China by heaping alternate layers of night soil, urine, wage, animal dung and chopped plant residues. The base material consists of hard stalks of crops and this is followed by layering with other materials. Water is added at optimum levels. The entire heap is shaped finally with mud plaster taking care to insert bamboo or maize stalks into the mud covered heap all the way to the bottom of the heap.

After 24 hours, the bamboo poles or maize stalks are withdrawn to leave behind holes for ventilation. Within 4-5 days, the temperature in the heap reaches 60-70°C when the holes are closed and sealed with mud plaster.

Composting


After a period of 2 weeks the mud plaster is broken and the contents mixed followed by resealing with mud plaster. At the end of 2 months, the decomposed compost free from pathogens is ready for use on the farm.

The finished compost can be enriched with finely powdered rock phos¬phate and inoculated with non-symbiotic nitrogen fixers such as Azotobac¬ter, Azospirillum and phosphate dissolving bacterial or fungal species such as Pseudomonas, Micrococcus, Bacillus, Flavobacterium, Pencillium, Fusarium, Asppergillus etc.

Vermicomposting The use of earthworms in composting process is known as vermicomposting. One kg of earthworms can consume one kg of organic materials in a day and excrete as castings that are rich in nitrate, available phosphorus, potassium, calcium and magnesium. The castings encourage growth of bacteria and actinomycetes due to aeration in soil pores.

There are about 3000 species of earthworms in the world and about 500 in India alone. The earthworm is an aerator by making tunnels and by crushing and mixing of soil. By its enzymatic and biological activity, the earthworms stimulate soil microbiological activity. Vermicomposting began in Ontario, Canada in 1970 and today the USA, Japan and Phillippines are leading in vermicomposting both. quantitatively as well as qualitatively.

A moist compost heap of 204m x 1.2m x 0.6m deep can support 50,000 worms. A shallow well drained heap is ideal for worms to feed and multiply. Lumbricus rubellus (the red worm) and Eisenia foetida are thermotolerant and can stand the heat generated in the composting process. There are many other suitable species of worms available with breeders which can be had from agricultural universities and centres of agriculture research. The bedding for multiplying worms comprises of any moistened organic residue such as saw dust, cereal straw, rice husks, bagasse, card board and so on. This bedding material kept in boxes is covered with damp sack and left for 4 weeks followed by the addition of chicken manure, green matter and water hyacinth. The pH must be around 7.0 and temperature about 2D-27°C. The breeder earthworms are allowed to multiply in these boxes, taking care to avoid predators such as birds and frogs.

A series of compost pits of dimensions 3m x 4m x 1m deep are dug with sloping sides. Bamboo poles are placed at the bottom of the pits and lined with gunny sack to avoid the scape of worms. The pits are filled with moist


farm wastes, animal manure and leaves that are well chopped. The worms are picked by hand from the boxes and placed into the pit. By incubating the compost pit in the shade and keeping it moist but not water logged for 2 months, a good organic matter rich vermicompost can be prepared for use on the farm.

Green ManureThe practice of green manuring of soil is as old as agriculture itself. Many leguminous and non-leguminous crops a e grown and turned into the soil while they are still green to enrich soil nitrogen. When organic matter is decomposed, the nitrogen bound in the organic matter is released first as ammonia. The ammonia may be absorbed by the plant or converted to nitrate. Apart from enrichment of soil nitrogen, green manuring enriches the phosphorus, calcium, sulphur and other mineral content of soil. The foliage of following crops are used in India for green leaf manuring: Gliricidia maculata, Pongamia glabra, Calotropis gigantea, Tephrosia purpurea, T. candida, Indigofera teysmanni, Cassia tora, Sesbania speciosa and Ipomoea carnea. Many species of the following genera hold promise as potential green manure crops for rice cultivation: Aeschynomene, Cassia, Crotalaria, Cyamopsis, Desmodium, Indigofera, Lathyrus, Melilotus, Stizolobium, Phaseolus, Sesbania and Vigna.

The importance of stem nodulating Sesbania rostrata as a green manure plant in rice cultivation has already been mentioned in the chapter on Rhizobium and root nodulation.

Anaerobic Decomposition of Organic MatterUnder anaerobic conditions, decomposition of organic residues takes place by the activity of both mesophrk and thermophilic microorganisms resulting in the production of carbondioxide, hydrogen, ethyl alcohol and organic acids such as acetic, formic, lactic, succinic and butyric acids. Among the mesophilic flora, bacteria are more active than fungi or actinomycetes in cellulolytic activity. They belong to the genus Clostridium and are numerous in peaty soils and manure pits but rarely encountered in cultivated arable soils. In compost heaps, both mesophilic and thermophilic microorganisms (bacteria and actinomycetes) are important in the break down of cellulose substrates. As stated above, the primary microbial colonizers initially break down the complex carbohydrates and proteins into organic acids and alcohols. At a later stage, the methane bacteria which are strict anaerobes begin to act upon the secondary substrates chiefly lactic, acetic and butyric acids and ferment them into CH2 and CO2 whose ratio is variable depending on the nature of reactions. It is not easy to isolate pure cultures of methane bacteria. However, by enrichment culture technique, methane bacteria have been isolated and grouped under four genera: Methanobacterium, Methanobacillus, Methanosarcina and Methanococcus.

Experiments with pure cultures as well as mixed cultures of methane bacteria have shown that among the several types of reactions which can produce CH4, the following typical ones are important:

Anaerobic Decomposition of Organic Matter


I Non-methanogens II Methanogens

A. Clostridium acetobutylicum C1 H2 oxidizing methanogens

Eubacterium limosum Methanobacterium

Methanobrevibacter

Methanospirillum

B. Volatile fatty acid oxidizers C2 Aceticlastic methanogens

Syntrophomonas wolfeii Methanosoricina

Syntrophobacter wo

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