cellulose degradation in anaerobic...

Ann~ Rev. Microbiol. 1995. 49:399-426Copyright ~ 1995 by Annual Reviews Inc. All rights reserved

CELLULOSE DEGRADATIONIN ANAEROBICENVIRONMENTS

Susan B. LeschineDepartment of Microbiology, University of Massachusetts, Amherst, Massachusetts01003-5720

KEY WORDS: cellulase, cellulose fermentation, microbial interactions, cellulolyticmicroorganisms, carbon c~,cle

CONTENTS

INTRODUCTION ........................................................ 400

THE SUBSTRATE ....................................................... 400

CELLULOLYTIC ENZYME SYSTEMS OF ANAEROBES ...................... 401

CELLULOSE DEGRADATION IN SOILS, SEDIMENTS, AND AQUATICENVIRONMENTS .............................................. 405

The Environments of Free-Living Anaerobic Cellulolytic Bacteria ............... 405Diversity of Cellulose-Fermenting Microorganisms ........................... 407Interactions Among Microorganisms Engaged in Cellulose Degradation ......... 410

CELLULOSE DE£IRADATION IN ASSOCIATION WITH ANIMALS ............ 412The Rumen ........................................................... 412Diversity of Cellulolytic Microorganisms in the Rumen ....................... 413Microbial Interactions in Cellulose Degradation in the Rumen ................. 414Other Animal.Associated Environments .................................... 416

CONCLUDING REMARKS ................................................ 418

ABSTRACT

In anaerobic environments rich in decaying plant material, the decompositionof cellulose is brought about by complex communities of interacting microor-ganisms. Because the substrate, cellulose, is insoluble, bacterial and fungaldegradation occurs exocellularly, either in association with the outer cell en-velope layer or extracellularly. Products of cellulose hydrolysis are availableas carbon and energy sources for other microbes that inhabit environments inwhich cellulose is biodegraded, and this availability forms the basis of manymicrobial interactions that occur in these environments. This review discusses

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400 LESCHINE

interactions among members of cellulose-decomposing microbial communitiesin various environments. It considers cellulose decomposing communities insoils, sediments, and aquatic environments, as well as those that degradecellulose in association with animals. These microbial communities contributesignificantly to the cycling of carbon on a global scale.

INTRODUCTION

The development of microbial communities that effect the degradation ofcellulose and other abundantly produced plant cell wall polymers in anaerobicenvironments is one of the hallmarks of evolution. Indeed, these communitiesplay a key role in the cycling of carbon on the planet. They coevolved withorganisms that developed light-driven mechanisms to reduce carbon dioxide,and their essential role is to retum carbon to the atmosphere. The ecology ofcellulose degradation in anaerobic environments is very complex; it involvesnumerous, varied interactions of metabolically diverse microorganisms whoseactivities are influenced by a wide range of environmental factors.

Anaerobic environments rich in decaying plant material are prevalent andtremendously varied. Not surprisingly, a wide range of equally varied cellu-lose-degrading microbial communities has evolved. Rather than serving as acomprehensive review of the ecology of cellulose degradation, this articlecovers environments and microbes that have been studied most extensively inrecent years, focusing on those environments that contribute significantly tothe cycling of carbon on a global scale. Included is information on the physi-ology of ceilulolytic microorganisms, the enzyme systems they produce, andtheir interactions with other microbes in the degradation of cellulose. In addi-tion, the review attempts to draw attention to those topics that, in my opinion,deserve further study.

THE SUBSTRATE

Cellulose is the most abundantly produced biopolymer in terrestrial environ-ments. Each year photosynthetic fixation of CO2 yields more than 10~ tonsof dry plant material worldwide (! 47), and almost half of this material consistsof cellulose (39). Cellulose is a homopolymer consisting of glucose units joinedby [~-1,4 bonds. The disaccharide cellobiose is regarded as the repeating unitin cellulose inasmuch as each glucose unit is rotated by 180° relative to itsneighbor. The size of cellulose molecules (degree of polymerization) variesfrom 7000 to 14,000 glucose moieties per molecule in secondary walls ofplants but may be as low as 500 glucose units per molecule in primary walls(93, 139).

Cellulose molecules are strongly associated through inter- and intramolecu-


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ANAEROBIC CELLULOSE DEGRADATION 401

lar hydrogen-bonding and van der Waals forces that result in the formation ofmicrofibrils, which in turn form fibers. Cellulose molecules are oriented inparallel, with reducing ends of adjacent glucan chains located at the same endof a microfibril. These molecules form highly ordered crystalline domainsinterspersed by more disordered, amorphous regions. The degree of crystal-linity in native cellulose is 60-90%. Cellulose can take on at least four differentcrystalline forms (I to IV), as determined by X-ray crystallography. The pre-dominant native form is referred to as cellulose I. Generally, the percentage

and crystalline form of cellulose within a plant cell wall varies according tocell type and developmental stage. For example, cellulose constitutes about20-40% of wall dry weight in growing primary walls and increases to 40--60%in secondary walls. The secondary walls of cotton seed hairs are nearly 100%cellulose. Secondary cell wall microfibrils have a higher cellulose I crystal-linity and may be thicker than primary wall microfibrils (100, 139).

Cellulose almost never occurs alone in nature but is usually associated withother plant substances. This association may affect its natural degradation.Cellulose fibrils are embedded in a matrix of other polymers, primarily includ-ing hemicelluloses, pectin, and proteins. Cellulose imparts tensile strength tothe wall to resist turgot pressure. High compression strengths are achievedwhen lignin (a complex aromatic polymer) replaces water in the matrix of cellwalls. Lignification greatly increases bonding within the wall and producesrigid, woody tissues able to withstand the compressive force of gravity (139).Hemicelluloses (e.g. xylans, glucomannans) are composed of linear andbranched heteropolymers of o-xylose, L-arabinose, D-mannose, D-glucose, D-galactose, and D-glucuronic acid. Xylans, often the most abundant hemiceilu-loses, have a ~-l,4-1inked xylopyranose backbone with attached side groupsof acetate, arabinofuranose, and O-methyl glucuronic acid (1 I, 39). Inasmuchas hemicelluloses surround the cellulose microfibrils and occupy spaces be-tween fibrils (39), this polymer must be degraded, at least in part, beforecellulose in plant cell walls can be effectively degraded by cellulolytic bacteria(150, 151). Moreover, arabinofuranosyl groups may be esterified by aromaticacids such as ferulic and p-coumaric acid (55) and may participate in lignin-hemicellulose cross-linkages 046), further complicating the microbial degra-dation of cellulose.

CELLULOLYTIC ENZYME SYSTEMS OF ANAEROBES

One of the most important features of cellulose as a substrate for microorgan-isms is its insolubility. Bacterial and fungal degradation of cellulose and otherinsoluble polymers occurs exocellularly, either in association with the outercell envelope layer or extracellularly. This suggests that the assembly ofenzyme systems, which may be extremely complex, also occurs exocellularly.


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403 LE~CHINE

To function, these enzyme systems must be stable in the exocellular environ-ment; for example, they must be reasonably resistant to proteolytie attack.Also, the products of cellulose hydrolysis may be available as carbon andenergy sources for other microbes that inhabit environments in which celluloseis biodegraded, thereby forming the basis of many interactions between mi-croorganisms in these environments, as discussed below.

The enzymology of cellulose degradation has been an area of active researchfor more than 40 years. The focus of this research has frequently shifted. Forexample, the initial thrust to understand the mechanism of cellulose degrada-tion was spurred by a fungal attack on the cotton clothing and tents of troopsstationed in Southeast Asia during World War II. Research directed towarddeveloping ways to inhibit fungal cellulases was carried out at the US ArmyResearch and Development Command in Natick, Massachusetts. Under thedirection of Elwyn Reese and Mary Mandels, this work ultimately led to thedevelopment of seminal concepts related to the mechanism of cellulose deg-radation, including the role of synergism among components of the cellulasesystem (52, 137,138). With the energy crises of the 1970s, the focus of researchon cellulose biodegradation shifted to developing systems and procedures touse cellulose and other abundant plant polymers as a source of fuels andchemicals that could serve as a potential replacement for fossil hydrocarbons.The voluminous work that followed, aided by the development of recombinantDNA and nucleic acid sequencing techniques, has resulted in our currentunderstanding of the enzyme systems produced by diverse cellulolytic organ-isms. The enzymology of cellulose decomposition is described in severalexcellent recent reviews (6-8, 35, 41, 50, 51, 140, 186), It is also brieflyconsidered here, as an understanding of cellulolytic enzyme systems is centralto the picture of the overall process of cellulose degradation in natural envi-ronments.

The mechanism by which cellulases from anaerobic bacteria catalyze thedepolymerization of crystalline cellulose is poorly defined, despite numerousrecent investigations. However, this mechanism is clearly fundamentally dif-ferent from that of the cellulase systems of most aerobic fungi and bacteria(25). Because many of the early studies of bacterial cellulases employed thefungal system as a model, a brief description of the enzymology of that systemis presented here for clarity. Details may be found in recent reviews (186, 188).

The cellulase systems of fungi (e.g. Trichoderma reeseO comprise threemain activities: (a) endoglucanases, which randomly hydrolyze 1,4-I] bondswithin cellulose molecules, thereby producing reducing and nonreducing ends;(b) exoglucanases, which cleave cellobiose units from the nonreducing endsof cellulose polymers; and (c) ~3-glucosidases, which hydrolyze cellobiose andlow-molecular-weight cellodextrins, thereby yielding glucose. These enzy-marie components act synergistically in the hydrolysis of crystalline cellulose.


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Synergism has been explained by the proposal that endoglucanases attackamorphous regions of cellulose fibers, forming sites for exoglucanases whichcan then hydrolyze cellobiose units from more crystalline regions of the fibers.Finally, ~glucosidases, by hydrolyzing cellobiose, prevent the accumulationof this disaccharide, which is an inhibitor of exoglucanase activity. This modelfor the mechanism of action of the fungal cellulase system may be an over-simplification because it does not explain all types of observed synergism (75).

In contrast to cellulase systems of aerobic fungi described above, the cellu-lases of most anaerobic microorganisms are organized into large, multiproteincomplexes (6-8, 25, 35, 41, 81, 82; and references therein). In general, thesecomplexes efficiently catalyze the hydrolysis of cellulose as long as they retaintheir integrity, but even partial disassociation of the complexes as occurs underrelatively mild conditions causes loss of most activity against crystalline cel-lulose. Some of the proteins that result from the disassociation of the largecomplexes have endoglucanase, cellobiohydrolase, or xylanase activity; othersdo not appear to have enzymatic activity and may have a structural function(e.g. as a scaffolding protein) and/or may be involved in attachment of thecomplex to the substrate (22, 59, 76, 77, 83, 111, 112, 190). None of theindividual proteins in the complexes has been found to have significant activityagainst crystalline cellulose. However, the cellulase system of the thermophileClostridium stercorarium includes two enzymes designated avicelase I and II.[The avicelase assay measures the ability ofcellulase preparations to hydrolyzecrystalline cellulose (avicel) and is often considered a measure of exoglucanaseactivity.] Purified avicelase I is a monomeric 100,000 Mr protein that catalyzesthe hydrolysis of crystalline cellulose without requiring additional proteins orcofactors (15). It has been designated an endoglucanase, but differs fromendoglucanases produced by other cellulolytic anaerobes by its ability tohydrolyze crystalline cellulose (15).

The most thoroughly investigated cellulase complex, that of Clostridiurnthermocellum, was termed the cellulosome by Lamed and coworkers (81, 82,87). On the cell surface, these multiprotein, multifunctional enzymes appearas polycellulosomal aggregates and promote adherence of the bacterium tocellulose (5, 85, 104, 105, 177). Cellulosomes of C. thermocellum strainsrange in molecular weight from 2.0 to 6.5 x 106 (105, 190) and comprise14-26 polypeptide subunits (77, 86). Genes encoding several of the polypep-tides have been cloned and their nucleotide sequences determined. The largestsubunit, the cellulosome integrating protein CipA (also designated S! or SL),is a glycoprotein with a mass of 210-250 kDa (87, 189, 190). CipA has cellulose-binding domain and nine internal repeated sequences that bind thecatalytic subunits (45, 47, 132, 145, 162). Catalytic subunits contain conserved, duplicated segment that is believed to serve as a docking sequence(45, 47, 162). CipA functions as a scaffolding protein of the cellulosome,


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404 LESCHINE

aligning the catalytic subunits for efficient cellulose hydrolysis. Possibly, thecellulosome catalyzes multiple, nearly simultaneous cuttings of the glucanchain (41, 105). Fujino et al (46) proposed that a protein, ORF3p, encodedby a gene located downstream of the gene encoding CipA, binds to CipAand functions to anchor the cellulosome to the cell surface. However, morerecent results (143, 144) argue against this hypothesis and suggest that ORF3pmay bind individual cellulases and hemiceilulases to the cell surface. Cellu-losomes bind bacterial cells to cellulose (5, 81,177). Furthermore, the productsof cellulolysis may pass through the "fibrous contact corridors" (41) that areobserved between cells and cellulose. However, as discussed below, noncel-lulolytic commensal bacteria can grow in cellulose-degrading cocultures withC. thermocellum, which indicates that at least some soluble sugars are releasedfrom contact corridors or are otherwise available as growth substrate for othermicroorganisms.

Multiprotein cellulase complexes are produced by many diverse anaerobes,including Bacteroides cellulosolvens (84), ClostrMium cellulolyticum (2, 40,95), Clostridium cellulovorans (35, 149), Clostridiurn papyrosolvens C7 (21,22), Fibrobacter (formerly Bacteroides) succinogenes (54), Ruminococcusalbus (187), Rurninococcusflavefaciens (34), and the rumen fungus Neocalli-mastixfrontalis (180, 181). Several of these cellulolytic anaerobes (B. cellu-losolvens, C. cellulovorans, R. albus), as well as Acetivibrio cellulolyticus andClostridium cellobioparum, have cell-surface localized cellulosome-like struc-tures (85). In the rumen bacteria R. albus and F. succinogenes, activity againstcrystalline cellulose apparently results from cell-bound enzymes associatedwith the capsule (R. albus) (I 55) or outer membrane (F. succinogenes) (44).Enzymes associated with capsule or outer-membrane fragments may detachfrom cells, giving rise to a high-molecular-weight, sedimentable cellulasefraction (44). Generally, F. succinogenes seems to need to adhere to cellulose(78), and the properties of its cellulase system differ from those ascribed cellulosomes (43).

C. papyrosolvens C7, a mesophilic cellulolytic bacterium isolated from afreshwater sediment (89), lacks cellulosome clusters on its surface (21). Also,cells of this clostridium do not adhere to cellulose fibers (21). The cellulasesystem of this bacterium is found in culture supernatant fluids and comprisesat least seven distinct high-molecular-weight multiprotein complexes (M,500,000-660,000), each with different polypeptide composition and enzymaticand ultrastructural properties (130; M Pohlschrtider, SB Leschine & E Canale-Parola, unpublished data). Two of the complexes have xylanase activity, andthree others, avicelase activity. An avicelase-deficient mutant of C. papyrosol-vens C7, which cannot degrade crystalline cellulose, does not produce themulticomplex system (21, 22). Pohlschrtider and coworkers (130) found hydrolysis of crystalline cellulose by this multicomplex cellulase-xylanase


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system involves synergistic interactions among its multiprotein components.In some respects, cellulose hydrolysis by the multicomplex system of C.papyrosolvens C7 resembles that by the cellulosome of C. thermocellum. Forexample, this system requires Ca2. and a thiol reducing agent for activity (22),and it consists of particles, some of which are similar in ultrastructure to,although smaller than, the cellulosomes of C. thermocellum (M Pohlschrtider,SB Leschine & E Canale-Parola, unpublished data). However, it differs fromcellulosome systems in that it is a multicomplex rather than a unicomplexsystem. Also, components of the multicomplex system are never found asso-ciated with cells. As pointed out by Forsberg et al (44), cell-free cellulasecomplexes may be able to penetrate small spaces in cellulose fibers that thecell itself cannot enter, and thus the complexes gain access to a greater fractionof the cellulose. Examination of cultures of C. papyrosolvens C7 via lightmicroscopy showed that the bacterial cells do not adhere to cellulose fibers;rather, they accumulate near cellulose fibers. Hungate (62) described this samebehavior for cellulolytic bacteria he had isolated. Hsing & Canale-Parola (60)proposed that this behavior results from a chemotactic response toward prod-ucts of cellulose hydrolysis. Chemotactic behavior of this sort could play animportant role in the overall process of cellulose degradation in natural envi-ronments.

CELLULOSE DEGRADATION IN SOILS, SEDIMENTS,AND AQUATIC ENVIRONMENTS

The Environments of Free-Living Anaerobic CellulolyticBacteriaMost cellulose is degraded aerobically, but 5-10% is degraded anaerobically(38, 171). Thus, vast quantities of cellulose are degraded by cellulose-ferment-ing microorganisms in anaerobic environments. Anaerobic activity starts closeto the surface in soils and composts, as well as in freshwater, marine, andestuarine sediments, indicating that aerobic conditions normally prevail onlyin a thin crust at the atmospheric boundary (93). The anaerobic degradationof soil organic matter plays an extremely important role in the global cyclingof carbon. Soils are a huge reservoir of carbon: The top meter of soil containsabout twice as much carbon as is found in the atmosphere (133), and somerecent studies suggest that the rates of accumulation and turnover of below-ground carbon may be underestimated (135). Consequently, understanding theeffects of predicted global warming on decomposition of organic matter insoils and sediments is essential for modeling future atmospheric and climaticchanges (67).


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406 LESCHINE

A community of physiologically diverse microorganisms is responsible forthe anaerobic degradation of cellulose. This community structure contrastswith aerobic decomposition, which may be achieved through the activitiesof single species. For example, both carbohydrate and lignin components ofwood are completely decomposed to CO2 and H20 by single species of whiterot fungi (74). Anaerobic decomposition, on the other hand, requires mixedpopulations. The metabolic versatility of anaerobes arises largely becausethey can perform various fermentations and respirations, employing diverseelectron acceptors (e.g. carbon dioxide, inorganic sulfur compounds, inorganicnitrogen compounds) in place of oxygen (93, 128, 194; for a review of earlierliterature, see 63).

In the absence of oxygen and certain other exogenous inorganic electronacceptors [e.g. nitrate, Mn(IV), Fe(llI), sulfate (94)], cellulose is decomposedby the anaerobic community into CH4, CO2, and H20 through a complexmicrobial food chain (109, 194) shown diagrammatically in Figure 1. Theprocesses are similar in most anaerobic soils and sediments [although perhapsnot all (see 14, 80)] and in anaerobic digestors. Cellulolytic microbes produceenzymes that depolymerize cellulose, thereby producing cellobiose, cellodex-trins, and some glucose. These sugars are fermented by cellulolytic and othersaccharolytic microorganisms. By keeping cellobiose concentrations low, andthus preventing inhibition of the cellulase system by this product of cellulosehydrolysis, noncellulolytic cellobioseofermenters may play a very importantrole in this process (93). These fermentations yield CO2, H~., organic acids (e.g.acetate, propionate, butyrate), and alcohols. Very little H~. escapes into theatmosphere because it is immediately consumed by methanogens or homoace-togens. Methanogens use H2 to reduce CO~. to CH4, and homoacetogens useH~ to reduce COs to acetate. Some methanogenic species use acetate producedby fermenters or by homoacetogens through the acetoclastic cleavage to CH4and COs (70, 97). Syntrophic bacteria play a key role in the conversion cellulose to CH,~ and CO2. These organisms ferment fatty acids such as propion-ate and butyrate, or alcohols, and produce acetate, CO2, and H2. They growonly in the presence of H_~-consuming organisms through interspecies H2 trans-fer. Syntrophic bacteria grow very slowly, and thus the fermentation of fattyacids is usually the rate-limiting step in the anaerobic decomposition of cellu-lose (109, 185).

Through the combined activities of several major physiological groups ofmicrobes, cellulose is completely dissimilated to CO2 and CH4. Thus, as asource of CO2 and CH~, the anaerobic decomposition of cellulose plays a majorrole in carbon cycling on the planet (91, 93). In marine environments, sulfateis plentiful and sulfate-reducing bacteria out-compete methanogens for H2.Thus, H2S is a major product of the anaerobic degradation of cellulose inmarine systems (93, 128).


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I

H2, C02

CELLULOSE

ICELLULOLYTICBACTERL4

I CELLOBIOSE, GLUCOSE, CELLODEXTRINS I

ICELLULOLYTICAND OTHERFERM EN TA TI ~ BACTERIA

I PROPIONATE, BUTYRATE

ATE

Figure I Diagrammatic representation of anaerobic cellulose degradation by microbialcommunities in soils and freshwater .sediments. Lactate, succinate, and ethanol are also producedby fermentative bacteria but usually do not accumulate. In environment,~ where nitrate, Mn(IV),Fe(lll), or sulfate are present, the final products of fermentation may differ.

Diversity of Cellulose-Fermenting Microorganisms

Although much of the work on cellulose degradation has involved thermo-philes such as C. thermocellurn, these bacteria probably play a minor role incellulose decomposition in most natural environments because they requireunusually high temperatures for growth and cellulose fermentation (106).Inasmuch as this article aims to review information on cellulose degradationas it occurs in common natural environments, this section focuses primarilyon mesophilic cellulose-fermenting microbes. Several mesophilic cellulolyticanaerobes have been isolated from soils, sediments, and composts fromgeographically widely separated locations. CIostridiurn papyrosolvensNCIB 11394 was isolated from estuarine sediments (96), and another strain this species, strain C7, was isolated from the sediment of a freshwater swamp(89, 130). Clostridiurn lentocellum was isolated from samples of the sameestuarine sediments fi’om which C. papyrosolvens NCIB11394 was derived(117). C. lentocellum differs from other mesophilic eellulolytie bacteria inseveral ways, but perhaps the most significant is the relatively slow rate ofcellulose degradation by cultures of this bacterium. Clostridium cellulofermen-


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408 LESCHINE

tans inhabits dairy farm soil (192). Cellulolytic anaerobes isolated from wet-wood of trees are similar in many respects to isolates from soil (173), andthese trees are probably infected by soil bacteria. Although Clostridium cello-bioparum was originally isolated from bovine rumen contents (62), this clos-tridium is reportedly also found in soil (152). In addition to the above-mentioned cellulolytic anaerobes, several other strains from soils and sedi-ments have been described (63, 89, 92, 102, 152; SB Leschine, E Canale-Pa-rola, E Monserrate & T Warnick, unpublished results).

All of the mesophilic strains from soils and sediments that have been ex-amined are motile rod-shaped organisms with a similar cell envelope ultras-tructure. Also, they have similar G+C contents. Cells of almost all strains staingram-negative, and most, but not all, form spores. All ferment not only cellu-lose, cellobiose, and glucose, but also components of the hemicellulosic portionof biomass (e.g. xylan, pentoses). Of those strains examined, all form acetate,ethanol, CO2, and H2 as primary products of fermentation. Other free-livingcellulose-fermenting mesophiles have been isolated from composts [e.g.C.cellulolyticum (129), Clostridium josui (161 )], sewage sludges [e.g. Acetivibriocellulolyticus (127), Bacteroides cellulosolvens (I 18), strain CM126 (120)],and anaerobic digestors [e.g. Clostridium aldrichii (191), Clostridium cel-erecrescens (125), C. cellulovorans (154), Clostridium populeti (I 53)]. Table1 lists some of the properties of these bacteria,

Phylogenetic relationships among species of mesophilic cellulolytic clos-tridia have been examined via 16S rRNA and rDNA sequence analyses (24,136). C. cellulovorans, along with two rumen cellulolytic bacteria, Clostridiumchartatabidum (72) and Clostridium "longisporum’" (64, 168), cluster withmembers of Clostridium group I [as defined by Johnson & Francis (69)], whichincludes the type species, Clostridium butyricum and many other noncellu-lolytic species (24, 136). C. lentocellum, C. celerecrescens, and C. populetiare members of Clostridium group II1 (69), which also includes many noncel-lulolytic species (136). C. papyrosolvens and most other mesophilic cellulolyticisolates from soil, C. cellulolyticum from composted grass (129), and A. cel-lulolyticus (127) and B. cellulosolvens from sewage sludge (118) form phylo-genetically coherent clusters within a more deeply branching group of cellu-lolytic clostridia that includes C. thermocellum ( ! 36; SB Leschine & R Seward,unpublished results). Thus far, this later group is defined solely by cellulolyticspecies. A point of interest, A. cellulolyticus and B. cellulosolvens were notclassified as clostridia because they stain gram-negative and spores were notobserved (118, 127). However, most cellulolytic clostridia stain gram-negativeand are oligosporogenous, and in some strains, spores are observed only rarely.Evidently, A. cellulolyticus and B. cellulosolvens are related to the cellulolyticclostridia in the above-mentioned group, and they probably share physiologicalproperties (e.g. cellulose fermentation characteristics) common to members


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this group. Similarities in the cellulase systems of A. cellulolyticus, B. cellu-losolvens, and C. thermocellum (84, 85) were noted above.

Many environments in which cellulose is degraded are deficient in combinednitrogen (for example, peat soils, agricultural and municipal wastes, and com-posts), and cellulose-fermenting bacteria that satisfy their nitrogen require-ments through the fixation of N2 should have a strong selective advantage overthose that require a source of combined nitrogen. Therefore, that severalcellulose-fermenting bacteria can satisfy their nitrogen requirement for growththrough the fixation of atmospheric N2 is not surprising (92). Nitrogenaseactivity has been demonstrated in C. papyrosolvens, C. cellobioparum, and inseveral other mesophilic cellulolytic strains from soils and sediments (12, 92;SB Leschine, T Chen & T Warnick, unpublished results). These findingssuggest that nitrogenase may be widespread among cellulose-fermenting bac-teria from soils. Thus, in the natural environments they inhabit, these bacteriamay contribute significant amounts of combined nitrogen to the growth ofother members of cellulose-degrading communities.

Although free-living anaerobic cellulolytic fungi similar to those found inthe rumen (124) have not yet been found, Durrant et al (37) reported isolation, using anaerobic culture conditions, of two strains of morphologicallyand physiologically distinct cellulose-fermenting fungi from soil. Both strainsgrow and utilize cellulose more rapidly when incubated under microaerophilicconditions, and one strain degrades cellulose most rapidly in well-aeratedcultures (37). Active cellulase and xylanase systems are produced by bothstrains, and enzymes are present in culture supernatant fluids (36). Clearly,fungi may play a significant role in the anaerobic degradation of cellulose insoils and sediments, and further studies are needed to explore their potentialcontributions.

Interactions Among Microorganisms Engaged in CelluloseDegradation

Microbial interactions appear to be important processes in the anaerobicdegradation of cellulose. The cellulose-fermenting bacterium makes a livingby degrading insoluble polymers in a highly competitive, nitrogen-poor en-vironment. To do so, it must export from the cell significant amounts ofprotein to catalyze the hydrolysis of cellulose, and then it must compete forsoluble sugars produced as a result of the activities of these enzymes. Becausethe products of the enzymatic attack on cellulose are available to otherbacteria, the activities of these other bacteria may in turn affect the activityof the cellulose-fermenting bacterium. For example, Cavedon & Canale-Pa-rola (20) found that C. papyrosolvens C7 grows in coculture with a noncel-lulolytic klebsiella in a chemically defined, vitamin-deficient mediumcontaining cellulose as the carbon and energy source. In this coculture, the


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extracellular cellulase system produced by the clostridium hydrolyzes cellu-lose to soluble sugars that are utilized as fermentable substrates for theklebsiella, and in turn, the klebsiella excretes vitamins required by the clos-tridium. In addition, the clostridium fixes N2, thus allowing growth of theklebsiella, which is not a nitrogen fixer. Mutualistic associations of this sortmay take place in natural environments where cellulose is degraded (20).Other mutually beneficial ifiteractions in cellulose-degrading eoeultures basedon the production of growth factors and the formation of fermentable solublesugars have been described (e.g. 62, 73, 114, 116).

Spirochetes are frequently observed in anaerobic cellulolytic enrichmentcultures, suggesting that free-living spirochetes associate and interact withcellulose-degrading bacteria in anaerobic environments (56). Recently, Pohl-schr6der et al (131) reported that Spirochaeta caldaria, a thermophilic spiro-chete from a freshwater hot spring, forms stable, cellulose-degrading cocul-tures with C. thermocellum. Cellulose is degraded more rapidly in coculturesthan in monocultures of C. thermocellum, which suggests that noncellulolyticspirochetes may enhance cellulose breakdown in natural environments. In thisstudy, the factors involved in the increased rate of cellulose degradation incocultures were not examined; however, a plausible explanation is that thespirochetes keep cellobiose concentrations low, thereby preventing cellobioseinhibition of the cellulase system of C. thermocellum (93). Similar results havebeen obtained in cultures of cellulolytic bacteria with other noncellulolyticsaccharolytic bacteria (42, 90, 114, 119).

Several studies have examined the effect of culturing a cellulose-fermentingmicroorganism with a noncellulolytic saccharolytic bacterium that producesethanol as the sole or primary nongaseous fermentation product. In all cases,ethanol production from cellulose is enhanced in cocultures as compared tomonocultures of the cellulolytic organism grown in the same medium (73, 90,114, 119, 142, 172). An interesting study carried out by Ng et al (119)demonstrated that stable cocultures of C. thermocellum and Clostridium ther-mohydrosulfuricum fermented a variety of cellulosic substrates, includingSolka Floc, and that ethanol yields exceeded those of C. thermocellum mono-cultures. The hemicellulosic portion of $olka Floc is not utilized by purecultures of either bacterium. C. thermocellum produces an enzyme system thatdepolymerizes hemiceilulose, but this bacterium cannot ferment pentosanproducts. In contrast, C. thermohydrosulfuricum ferments pentosans but cannotdepolymerize hemicellulose. In cocultures, hemicelluloses are fermentedthrough the combined activities of the two coculture members. Thus, bothcellulosic and hemiceilulosic portions of Solka Floc are fermented by cocul-tures, and ethanol is produced as a primary product.

The investigations described above suggest that, in natural environments,the soluble sugar products of cellulose hydrolysis are available as growth


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substrates for noncellulolytic commensal microorganisms, whose activitiesmay affect cellulose degradation in a variety of ways.

Numerous studies have examined the effects on cellulose degradation ofcoculturing cellulose-fermenting bacteria with syntrophic and/or H2-consum-ing microorganisms (reviewed in 93, 128). In general, utilization of H~ methanogens in cocultures with cellulose-fermenting bacteria results in anincrease in acetate production relative to ethanol. For example, Laube & Martin(88) showed that cellulose-fermenting monocultures of A. cellulolyticus pro-duce ethanol, acetate, H2, and COz, and that cocultures of this bacterium withMethanosarcina barkeri produce more acetate and less ethanol. M. barkeriutilizes acetate, H~, and CO2 for methane production, and in cocultures, itutilizes acetate after a lag. In an A. cellulolyticus-M, barkeri-Desulfovibriosp. triculture, the H~ partial pressure is maintained at a level low enough forDesulfovibrio sp. to utilize ethanol. Thus, in tricultures, cellulose is rapidlyfermented to CO2 and CH4. Furthermore, a greater fraction of cellulose de-composes more rapidly in tricultures than in monocultures of the cellulolyticbacterium (88). In this example, Desulfovibrio sp. is a H2-producing bacteriumthat apparently grows syntrophically with the methanogen. If sulfate werepresent, Desulfovibrio sp. presumably would function instead as a H~-consum-ing bacterium in the reduction of sulfate to H~S, out-competing the methanogenfor H2.

CELLULOSE DEGRADATION IN ASSOCIATION WITHANIMALS

The Rumen

Vast amounts of cellulose are degraded anaerobically in the gastrointestinaltracts of herbivorous and wood-eating animals, Herbivores lack th~ enzymatic

capacity needed to degrade plant polysaccharides, particularly cellulose, andinstead rely on communities of microorganisms that have this capability. Anenlarged section of the gastrointestinal tract of herbivores functions as a fer-mentation vessel in which slowly degraded plant materials are retained longenough to be degraded by the microbes. This specialized portion of the gas-trointestinal tract is separated from the highly acidic pyloric region of the gutand is well buffered. Herbivores have evolved as either pregastric (foregut) postgastric (hindgut) fermenters, and their anatomy and evolutionary develop-ment have been described in detail (29, 61, 167).

Ruminants are cloven-hoofed herbivorous mammals that degrade plant ma-terials in a specialized foregut organ, the rumen. Several recent reviews (30,58, 98) and the classic work by Hungate (64) have described this extensivelystudied microbial ecosystem. Plant materials, mixed with saliva containing


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bicarbonate, enter the rumen and are mechanically ground into smaller particlesthrough the rotary motion of the rumen. Gradually the food and microbial masspasses into the reticulum, another organ of the foregut, where it forms clumpsor cuds, which are regurgitated into the mouth for additional chewing. Thecud mixed with saliva is swallowed and passes to the omasum and then to theabomasum, an acidic organ that is more like a true stomach. Chemical digestiveprocesses begin in the abomasum and continue in the small and large intestine.

While food remains in the rumen (-9-12 h), plant polysaccharides arehydrolyzed to oligo- and monosaccharides. These carbohydrates are then fer-mented, producing volatile fatty acids, including primarily acetic, propionic,and butyric acids, that are readily absorbed and serve as essential nutrients forthe ruminant. Resultant carbon dioxide and methane are eliminated by eruc-tation. In addition to carrying out this degradative process, the microbialcommunity prbduces other essential nutrients, such as vitamins, for the animal.Furthermore, when microbial cells pass through the gastrointestinal tract, theyam subjected to digestive processes similar to those of nonruminants. Thus,the rumen microbiota serves as a protein source for the animal as well.

In the rumen, cellulose is not completely converted to CO2 and CH4 as it isin environments such as soils and sediments. Instead, acetate, propionate, andbutyrate are significant products of cellulose fermentation. Longer turnovertimes in soils, sediments, and other natural environments allow these fattyacids to be converted to CO2 and CH4 by the activities of microorganisms, asdescribed above. Thus, in these complete bioconversion systems, acetate, aswell as H~ and COs, are primary substrates for methanogens, whereas in therumen, the predominant substrates for methanogens are H: and CO2 (109).

Diversity of Cellulolytic Microorganisms in the Rumen

The microbiology of the rumen has been the subject of studies for more than45 years, and the literature describing microbes from this environment hasbeen reviewed many times (e.g. 16, 23a, 30, 31, 57, 58, 64, 65, 98, 124, 158,179, 182, 184). Bacteria are generally believed to provide most of the cellu-Iolytic activity in the rumen, but rumen fungi, and to a lesser extent rumenprotozoa may make a significant contribution. Based on determinations ofrelative numbers in the rumen and the ability to degrade purified and intactforage cellulose, the principal cellulolytic bacterial species appear to be F.succinogenes, R. albus, and R. flavefaciens (17-19, 28, 30, 148, 158, 160,165). Also, some strains of the rumen bacterium Butyrivibriofibrisolvens arecellulolytic (165) and may play an important role in cellulose degradation.Other cellulolytic species found sporadically or in low numbers in the rumeninclude Eubacterium (formerly Cillobacterium) cellulosolvens (19, 134, 164),Micromonospora ruminantium (99), Micromonospora propionici (62a), andclostridial species (62, 64, 72, 168). Although these later species are believed


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414 LESCHINE

to have a limited role in cellulose degradation in the rumen, some may be veryactively eellulolytie under certain conditions. For example, Varel (168) foundthat the rate and extent of alfalfa cell wall degradation by C. longisporumexceeded that of principal rumen cellulolytic species. In contrast, barley strawwas poorly degraded. These results indicate that rumen cellulolytic microbesmay have a high degree of substrate specificity.

Other members of the rumen microbial community are the anaerobic fungi(reviewed in 30, 124). Because they are found in large numbers attached plant fragments removed from the mmen, and they ferment the major plantpolysaccharides, they are believed to take part in decomposing cellulose in therumen. Five genera of fungi have been described, Neocalliraastix, Caecomyces(formerly Sphaeromonas), Piromyces (formerly Piromonas), Orpinomyces,and Ruminomyces. Pure cultures of rumen fungi can solubilize a high propor-tion of plant fragments, and they seem to have the ability to penetrate plantcell walls and gain access to plant polysaccharides not available to cellulolyticbacteria (124). Thus, it seems likely that the rumen anaerobic fungi play significant role in fiber degradation in the rumen.

The existence of protozoa in the rumen has been known for more than 150years (see 64). These anaerobic microbes ferment components of plant material(64). Most are ciliates that, taxonomically, fall into two groups, the holotrichprotozoa and the entodiniomorphs (179). The holotrichs are generally believedto utilize nonstructural polysaccharides and soluble sugars (178, 179). On theother hand, all of the entodiniomorphs, except one species, contain cellulase.However, this activity may result from cellulase production by intracellularbacteria (179). Entodiniomorphid protozoa engulf and digest cellulose and usethe products for the synthesis of intracellular polysaccharide (64). The ciliateprotozoa may be responsible for a third or more of the microbial degradationof fiber in the rumen (32, 179).

Microbial Interactions in Cellulose Degradation in the Rumen

Interactions similar to those described above among free-living cellulose-fer-menting bacteria and noncellulolytic bacteria have been observed in studiesof mmen bacteria. For example, in cocultures of rumen cellulolytic bacteriaand methanogens, interspecies H2 transfer occurs. Consequently, the oxidativereactions of fermentation pathways are coupled to the reductive reactions ofmethanogenesis, and the flow of electrons shifts away from reduced products(e.g. lactate, succinate) of cellulolytic members of cocultures, resulting greater acetate yields (109, 184).

Competitive and cooperative interactions among various cellulolytic andother saccharolytic microorganisms may affect the degradation of plant fiberin the rumen (30). Interactions among different cellulose-fermenting rumenmicrobes have been examined using 16S rRNA-targeted oligonucleotide


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probes (121,122). R. albus 8 inhibits the growth of R. flavefaciens FD-1 whencocultures are grown with acid-swollen cellulose or with alkaline hydrogenperoxide-treated wheat straw as substrate (122), possibly because R. albus 8produces a bacteriocin-like substance (121). In cocultures of R. albus 8 andF. succinogenes grown on the above-mentioned substrates, competition appar-ently does not occur, but R. flavefaciens FD-1 out-competes F. succinogenesin cocultures grown on acid-swollen cellulose. Complex patterns of competi-tion among microbes occur in tricultures of R. albus 8, R. flavefaciens FD-I,and F. succinogenes (122). This study demonstrates the utility of molecularprobes in investigations of competition among cellulolytic microorganismsengaged in cellulose degradation.

Spirochetes are present in the mmen in very large numbers (156). Althoughcellulose-degrading spirochetes have not been found in the rumen, many spi-rochetes isolated from rumen fluid can grow at the expense of plant polymerssuch as xylan, arabinogalactan, pectin, polygalacturonate, and starch (56, 126).Also, many ferment cellobiose (126) and thus may be expected to interact withthe cellulolytie members of the rumen. Electron microscopy observations haveindicated that spirochetes associate with plant cell wall materials undergoingdegradation in the rumen (I, 23, 33, 159). Moreover, Kudo et al (79) observedspirochete cells firmly adhering to fibers of sterilized barley straw in prepara-tions inoculated with pure cultures of Treponema b~.antiL a cellobiose-fer-menting, succinate-producing tureen spirochete (157). In cocultures of succinogenes and T. bryantii, the rod-shaped cells of F. succinogenes appearedto colonize and erode the cellulosic substrata, while spirochete cells wereobserved on the same surface immediately juxtaposed to the cellulolytic cells(79). Stanton & Canale-Parola (157) reported that bryantii grew in coculturewith F. succinogenes in a medium that contained cellulose as the fermentablesubstrate. The spirochete enhanced cellulose breakdown by the rumen cellu-Iolytic bacterium when the two organisms were cocultured on agar media.These investigators suggested that T. b~.antii is chemotactically attracted bysoluble sugars resulting from the cellulolytic activity of F. succinogenes, andthat as a result of their incessant motion, the spirochetes may randomly pushthe nonmotile F. succinogenes cells toward the cellulose fibers and thus en-hance cellulose breakdown. Kudo et al (79) reported that in cocultures of bryantii with either F. succinogenes or R. albus, degradation of barley straw,as well as the production of volatile fatty acids, other organic acids, and ethanolwere enhanced. These authors concluded that cellulolytic bacteria interact withnoncellulolytic spirochetes to promote the digestion of cellulosic materials inthe rumen. They further suggested that enhanced cellulose degradation mayoccur after the cotransportation ofcellulolytic and spirochete cells, as describedabove (157), or through physiological interactions that might be related to theuse of cellobiose by the spirochete. If the spirochete utilizes this product of


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416 LESCHINE

cellulose hydrolysis, such use would relieve cellobiose’s inhibition of cellulaseactivity.

Interactions among rumen bacteria and fungi have been studied extensively.Cellulose degradation and fungal growth are stimulated when fungi are cocul-tured with methanogenic bacteria (4, 10, 68). Methanogens cause a shift in thefermentation products to more acetate and less lactate, succinate, and ethanol,as would be expected because methanogens maintain low H2 levels. Thismethanogenic activity facilitates production of H2 by fungi, and thus the flowof electrons moves away from the more reduced fungal products (lactate,succinate, ethanol) toward methane (4, 103, 115). Other fungus-bacteriumassociations, ranging from synergism to antagonism, have been reported (9,66, 141).

The activities of protozoa apparently have important effects on cellulose-decomposing bacterial and fungal communities. Protozoal predatory activityon rumen bacteria and fungi has been documented (64, 123, 179). In culturesof a rumen fungus, Piromyces sp., and of a mixed rumen protozoal population,cellulose degradation was decreased compared with monocultures of the fun-gus, owing at least in part to protozoal predation (113). However, the amountof cellulose degraded per unit of fungal biomass was larger in mixed cultures(113). Also, formate (which is used as an indicator of fungal growth) was detected; propionate and butyrate increased; and lactate decreased in mixedcultures compared with production of these organic acids in the fungal mono-cultures (113). A stimulatory effect of protozoa on bacterial cellulolysis hasbeen reported (193). Although the basis of this stimulation is unclear, hydro-lytic enzymes released from protozoal cells may play a role in augmentingplant-fiber degradation (179).

Other Animal-Associated Environments

Besides the ruminants, many other herbivorous and wood-eating animals har-bor complex communities of microorganisms and rely, at least to some extent,on the microbes’ capacity to degrade the structural polymers of plant cell walls.For example, the hoatzin, a tropical leaf-eating bird from South America, hasa foregut fermentation system that resembles the rumen (53). Also, nonrumi-nant mammals such as kangaroos, elephants, and monkeys have complexforestomachs in which plant material is degraded by microbial processes ap-parently similar to those that occur in the rumen (3, 64, 166). Other mammalssuch as humans, pigs, horses, and rodents are postgastric (hindgut) fermenters.Host digestion in the stomach precedes microbial fermentation of undigestedplant material in the colon or large intestine. These fermentations are carriedout by complex microbial communities including cellulolytic bacteria (169,176), but in contrast to ruminal fermentations, protozoa and fungi are notinvolved (109). Polysaccharides are degraded by schemes similar to those


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found in the rumen, producing acetate, propionate, butyrate, H2, and CO2 (183).

Most of the volatile fatty acids produced in hindgut fermentations are absorbedinto the blood and metabolized in tissues, and this process supplies about5-10% of dietary energy in humans and pigs and about 40% in rabbits andponies (108, 109).

Production of methane by human intestinal microbial communities occursonly in some individuals (110, 174), even though the kinds and amounts acids produced by methanogenic and nonmethanogenic intestinal fermenta-tions are very similar (175). Apparently, during nonmethanogenic fermenta-tions in humans, and in other mammals such as rats, guinea pigs, and rabbits,H2 is used to reduce CO2 to acetate (14, 109). Similarly, significant amountsof acetate, rather than CH4, are formed from CO2 in the guts of various termites(14).

Many insects, such as termites, feed on cellulose, which is degraded bycomplex microbial eomrnunities in the insect gut (reviewed in 13; see also26). Cellulolytic enzymes in insects may originate not only from the gutmicrobial community, but also from the insect itself or, in fungus-growingtermites, from the insect’s feed (101). In phylogenetically lower termites,cellulolytic flagellated protozoa are the major, and possibly the only, sourceof cellulolytic enzymes, and these protozoa appear to be essential for thesurvival of termites on wood or native cellulose diets (13). The phylogeneti-cally higher termites lack protozoa and depend largely on the activities of gutbacterial communities for the degradation of cellulose (13). Recently, Gijzenet ai (49) reported that cellulase activities in hindgut extracts of the cockroachPeriplaneta americana are high in cockroaches fed cellulose-rich diets. Thenumber of Nyctotherus ovalis, the major hindgut protozoan, is also relativelylarge in insects on these diets. As N. ovalis harbors endosymbiotic methano-gens (48), these findings suggest that this protozoan may be involved producing both cellulolytic and methanogenic activities in the hindgut ofcockroaches.

Herbivory in reptiles is most often associated with extinct members of thisclass, and in terms of biomass consumption and evolutionary longevity, themost successful vertebrates in the history of the Earth were the plant-eatingdinosaurs. Although relatively few extant reptiles subsist primarily on plants,some of those that do display adaptations for herbivory that are analogous tothose found in nonmminant mammals, with characteristic features of the di-gestive tract associated with processing high-fiber food and maintaining cel-lulose-degrading microbial communities (163, and references therein). McBee& McBee (107) have estimated that volatile fatty acid production satisfies30-40% of the energetic requirement of the green iguana (Iguana iguana),indicating that hindgut fermentation of fiber is a significant energy source inthis animal. Little information is available on the microorganisms responsible


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~ 18 LE$CHINE

for the hindgut fermentation. Culturing indicated that Leuconostoc and Clos-tridium spp. were dominant, and no species of Bacteroides, Fusobacterium,and Ruminococcus were found (107). Variable numbers of ciliated protozoanshave been observed in iguana hindguts (163). Testudines (turtles and tortoises)also possess a modified hindgut region in which fibrous plant material isdegraded by microbial communities, which include protozoa and bacteria innumbers comparable to those found in the rumen (163).

CONCLUDING REMARKS

This article has reviewed numerous studies that deal with various aspects ofthe degradation of cellulose in anaerobic environments. Microorganisms thatact as the biological catalysts in the anaerobic degradation of cellulose are ofenormous biotechnological interest for their hydrolytic enzymes and fermen-tation products. Great effort has been devoted to studying microbial cellulasesystems, resulting in a fair understanding of the enzymology of cellulosedegradation. However, fundamental questions remain unanswered. For exam-ple, the mechanism of cellulose hydrolysis by the cellulase systems of eitherbacteria or fungi remains largely unknown, and so it is nearly impossible todiscern similarities or differences in the molecular mechanisms employed bydifferent systems. Also, essentially nothing is known about the exo- or ex-tracellular assembly of complex cellulase systems. The biotechnologicai po-tential of cellulolytic microorganisms cannot be fully realized withoutknowledge of their genetics, which is virtually nonexistent at present, and animproved understanding of their physiology. Studies such as those carried outby D’Elia & Chesbro (27) are needed to appreciate the effects of growth rateand energy demands on cellulase production.

Other interest in anaerobic cellulose degradation involves the role of thisprocess in the nutrition of economically important animals, particularly rumi-nants. Numerous recent publications, including several reviews (e.g. see 71),discuss progress in this area. Although the rumen is one of the best knownmicrobial ecosystems, a greater understanding of interactions among popula-tions of different cellulolytic and noncellulolytic organisms is needed to accu-rately picture the overall process of plant-fiber degradation in the rumen. Inthis regard, the above-mentioned studies by Odenyo et al (121, 122) employmolecular tools that may prove generally useful in addressing fundamentalquestions related to microbial ecology. Finally, on a global scale, the anaerobicdegradation of cellulose has a huge impact on the carbon cycle, so there istremendous interest in learning how this process may be affected by predictedclimatic change.

Important aspects of cellulose degradation in anaerobic environments havebeen addressed infrequently in the literature and hence were given scant


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attention in this article. For example, facultatively anaerobic microorganismspotentially could play pivotal roles in cellulose decomposition in anaerobicsoils, sediments, and other natural environments, and their ecology deserves

greater attention. It is hoped that this review will stimulate additional researchaddressing the many unanswered questions and will lead to a better under-standing of the anaerobic degradation of cellulose.

ACKNOWLEDGMENTS

Research in the author’s laboratory on the diversity of cellulolytic anaerobeswas supported by a grant from the National Science Foundation, and studies

of their cellulase systems and interactions in cellulose degradation were sup-ported by a grant from the Department of Energy. I thank all former and presentmembers of the laboratory for their many important contributions, especiallyTsute Chen for computer assistance and Ercole Canale-Parola for introducingme to the cellulolytic microbial world and for his untiring support, encourage-ment, and friendship.

Any Annual Review chapter, as well as any article cited in an Annual Review chapter,may be purchased from the Annual Reviews Prcprints and Reprints service.

1-800-347-8007; 415-259-.$017; emaih [email protected]

Literature Cited

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