22 Anaerobic Metabolism and its Regulation

MICHAEL J. MCINERNEYNorman, OK, USA

1 Introduction 4562 Trophic Structure of Anaerobic Ecosystems 4563 Methanogens 458

3.1 Physiology of Methanogens 4583.2 Effect of Sulfate on Methanogenesis 461

4 Regulation of Fermentative Metabolism 4615 Regulation of Syntrophic Metabolism 4636 Environmental Factors 467

6.1 Temperature 4676.2 Effect of pH and Volatile Acid Toxicity 468

7 Types of Reactors 4697.1 Conventional Stirred Anaerobic Reactor 4697.2 Phase Separated Systems 4707.3 Fixed Film or Anaerobic Filter Reactors 4707.4 Fluidized/Expanded Bed Reactors 4707.5 Anaerobic Rotating Biological Contactor 4707.6 Anaerobic Baffle Reactor 4717.7 Upflow Anaerobic Sludge Blanket Reactor (UASB) 471

8 Concluding Remarks 4719 References 472

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1 Introduction

Methanogenesis is an important terminalelectron accepting process in many anaerobicenvironments where the supply of oxygen,nitrate, oxidized forms of sulfur, iron, andmanganese are limited (FERRY, 1993). Exam-ples of such environments include freshwaterand some marine sediments, flooded soils, wetwood of trees, tundra, landfills, and sewage di-gestors. In these environments, a complex mi-crobial community consisting of many inter-acting microbial species completely degradesnatural polymers such as polysaccharides, pro-teins, nucleic acids, and lipids to methane andcarbon dioxide. Because of the large amountsof organic matter that are degraded in the nat-ural environments, methanogenesis is an im-portant process in cycling of carbon and otherelements in nature and may be responsible forup to 60% of atmospheric methane (CONRAD,1996). The amount of energy released duringmethanogenesis is relatively low compared toother terminal electron accepting processes.For example, the conversion of hexoses tomethane and carbon dioxide releases only15% of the energy that would be released bythe aerobic mineralization of hexose (SCHINK,1997). Thus, the amount of biomass producedper unit of substrate degraded is much lessthan that of other terminal electron acceptingprocesses. For this reason, methanogenesis hasbeen used as the treatment of choice forsewage and other complex wastes since sludgeyields are low and most of the energy in theoriginal substrates is retained in the energy-rich fuel, methane. Anaerobic digestion is of-ten a net energy producer, resulting in sig-nificantly lower operating costs compared toaerobic treatment (LETTINGA, 1995).

Although the low cell yields associated withanaerobic digestion make it attractive forwastewater treatment, it is also one of its maindisadvantages because large reactor volumesand long retention times are needed to achievethe required treatment efficiency (MCCARTY,1971). Thus, only relatively high strengthwastes such as sewage are treated by anaerob-ic digestion. In addition, anaerobic digestion isoften perceived as being an unstable process(ANDERSON et al., 1982; MCCARTY, 1971;

SPEECE, 1983). However, great advances havebeen made in the past 20 years in our under-standing of the biochemistry and energetics ofanaerobic metabolism. This has allowed thedelineation of the most sensitive steps in theprocess and the development of strategies toenhance the operational stability of anaerobicreactors. The result has been the developmentof novel reactors where the slow growing or-ganisms are retained in the reactor even athigh volume loadings.With these advances, it isnow believed that almost any type of waste canbe treated (LETTINGA, 1995).

This chapter will provide a brief overview ofthe microbiology of anaerobic digestion andthe factors that regulate this process. The con-straints that thermodynamics places on keyreactions will be discussed to illustrate its rolein the control of the flow of carbon and energyduring anaerobic digestion. In addition, theproperties of microorganisms involved will bediscussed to understand how population dy-namics influences the process. There is a num-ber of excellent reviews and books wherethese issues are treated in greater detail(DOLFING, 1988; FERRY, 1993; HOBSON et al.,1974; LETTINGA, 1995; LOWE et al., 1993; MALI-NA and POHLAND, 1992; MCCARTY and SMITH,1986; MCINERNEY and BRYANT, 1981; SCHINK,1988, 1997; SPEECE, 1983).

2 Trophic Structure ofAnaerobic Ecosystems

In anaerobic digestion, organic matter iscompletely degraded to methane and carbondioxide in discrete steps by the concerted ac-tion of several different metabolic groups ofmicroorganisms (Fig. 1) (MCINERNEY andBRYANT, 1981). In the first step, fermentativebacteria hydrolyze the polymeric substratessuch as polysaccharides, proteins, and lipidsand ferment the hydrolysis products to acetateand longer-chain fatty acids, CO2, formate, H2,NH 4

c and HSP. Acetogeneic bacteria O-de-methylate pectins and low molecular weightligneous materials and ferment hydroxylated

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2 Trophic Structure of Anaerobic Ecosystems 457

and methoxylated aromatic compounds withthe production of acetate (DOLFING, 1988). Inthe next step, a group of organisms, usuallycalled syntrophic or proton reducing aceto-genic bacteria, degrades propionate andlonger-chain fatty acids, alcohols, and someamino acids and aromatic compounds to themethanogenic substrates, H2, formate, and ace-tate.The degradation of these compounds withH2 production is thermodynamically unfavor-able unless the concentration of H2 (or for-mate) is kept low by H2 using bacteria such asmethanogens (MCINERNEY and BRYANT, 1981).Because of the diverse number of organismsinvolved in these reactions and their ability toperform other types of metabolisms such asfermentation or sulfate reduction (SCHINK,1997; MCINERNEY, 1992), the organisms thatparticipate at this second step will be calledsyntrophic metabolizers. The final step in-volves two different groups of methanogens,the hydrogenotrophic methanogens which usethe H2 and formate produced by other bac-teria to reduce CO2 to CH4 and the aceto-

trophic methanogens which metabolize ace-tate to CO2 and CH4.

In the gastrointestinal tract of animals, or-ganic matter is only partially converted to CO2

and CH4 (WOLIN, 1982). Acetate and longer-chain fatty acids accumulate and are absorbedand used by the animal as energy sources. Onlyfermentative bacteria and H2 or formate usingmethanogens seem to be involved in this par-tial methane fermentation. In termite guts(BREZNAK, 1994), the cecum of rodents andthe large intestine of most humans (MILLER

and WOLIN, 1994), the reduction of carbondioxide to acetate by acetogenic bacteriaseems to be the dominant electron acceptingreaction. In these latter ecosystems, the com-munity structure appears to be similar to thatof the rumen, except that H2 using acetogenicbacteria replace the H2 using methanogens asthe terminal electron accepting group.

In ecosystems with high levels of sulfate,such as marine and estuarine sediments andpetroleum reservoirs, sulfate reduction ratherthan methanogenesis is the terminal electron

Fig. 1. Schematic representation ofthe different metabolic steps involvedin the complete degradation of organ-ic matter to methane and carbondioxide (modified from MCINERNEY

and BRYANT, 1981).

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accepting reaction. Organic matter is com-pletely oxidized to CO2 with the reduction ofsulfate to sulfide. Again, the degradation pro-cess involves the concerted efforts of severalmetabolic groups of bacteria with the sulfatereducing bacteria apparently performing thefunctions of the syntrophic metabolizers andthe hydrogenotrophic and acetotrophic meth-anogens (MCINERNEY, 1986; OUDE ELFERINK

et al., 1994; STAMS, 1994) since the degradationof propionate and longer-chain fatty acids tocarbon dioxide in marine sediments does notrequire interspecies H2 transfer (BANAT andNEDWELL, 1983). However, it is likely that theuse of H2 by sulfate reducers influences prod-uct formation of fermentative bacteria in amanner analogous to that found in methano-genic environments.

This brief survey of the trophic structure ofanaerobic ecosystems reveals that, in general,anaerobic metabolism proceeds in a stepwisemanner where several metabolic groups ofbacteria interact in the mineralization process.This is in contrast to metabolism under aerobicand denitrifying conditions where a single spe-cies is usually able to mineralize completely acompound when the electron acceptor (e.g.,oxygen or nitrate) is in excess. The degree ofmutual interdependence between the differenttrophic groups in anaerobic communities var-ies considerably depending on the genetic ca-pabilities of the organisms and the constraintsthat kinetics and thermodynamics place onkey reactions. For some interactions, energylimitations are such that neither partner canoperate without the activity of the other or-ganism. Perturbations may result in the accu-mulation of intermediates such as H2 and ace-tate that exceed the narrow limits that areneeded for the degradation of key intermedi-ates such as fatty acids to be thermodynami-cally favorable. Other perturbations may stim-ulate fermentative metabolism or enhance thegrowth of certain fermentative populations,resulting in the production of acetate or otherorganic acids at rates faster than the othertrophic groups can degrade these acids. Thus,the efficiency of anaerobic digestion will de-pend on the dynamics and kinetics of keypopulations within the reactor as well as onthermodynamic limitations. The challenge ofanaerobic digestion is to maintain the appro-

priate balance between the different groups ofbacteria so that rates of production of keyintermediates match consumption rates andthe pool sizes are and within the narrow limitsthat thermodynamics places on these reac-tions.

3 Methanogens

3.1 Physiology of Methanogens

Methanogens are a taxonomically and phy-logenetically diverse group of microorganisms(BOONE et al., 1993; JONES et al., 1987; ZINDER,1993) that all gain energy for growth from thereactions that lead to the production of meth-ane.As a group, methanogens use a small num-ber of compounds, H2 or one-carbon atomcompounds (BOONE et al., 1993; ZINDER,1993). This specialization makes methanogensdependent on other organisms for the supplyof substrates in most anaerobic environments.Without methanogens, effective degradationof organic matter would cease due to the accu-mulation of nongaseous products of fermenta-tion which have almost the same energy con-tent as the original substrate.

The ability of methanogens to use H2 plays akey regulatory role that controls the types ofproducts made by fermentative bacteria andsets the thermodynamic conditions requiredfor the degradation of fatty and aromatic acids.The favorable thermodynamics of H2 use bymethanogens (Tab. 1) allows them to metabo-lize H2 to very low partial pressures. Methano-gens are able to metabolize H2 down to H2

partial pressures ranging from 3–7 Pa (LOVLEY,1985; CORD-RUWISCH et al., 1988). Methano-gens also have a high affinity for H2 use, withapparent KM values in the range of 5–13 mM(670–1,700 Pa) (Tab. 2) (ROBINSON and TIED-JE, 1984; WARD and WINFREY, 1985; ZINDER,1993). With H2 partial pressures ranging from2–1,200 Pa in digestors (CORD-RUWISCH et al.,1997; GUWY et al., 1997; MOSEY and FERNAN-DEZ, 1989; STRONG and CORD-RUWISCH, 1995),this means that the methanogens are under-saturated with respect to H2 use. Thus, in-

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3 Methanogens 459

creases in H2 production may not result inhigher H2 partial pressures because the H2

consumption rate by methanogens would alsoincrease (KASPAR and WUHRMANN, 1978a;ROBINSON and TIEDJE, 1982; SHEA et al., 1968;

STRAYER and TIEDJE, 1978). Because of thelarge capacity for H2 use by methanogens, H2

concentrations are normally very low in well-operated anaerobic digestors, even thoughlarge amounts of H2 are produced. As will be

Tab. 1. Reactions Involved in Syntrophic Metabolisma

Reaction DG0b

[kJ per reaction]

Methanogenic reactions4H2cHCO 3

PcHc ] CH4 c3 H2O P135.6AcetatePcH2O ] CH4cHCO 3

P P 31.0

Syntrophic reactions without H2 use by methanogensLactatePc2H2O ] AcetatePcHCO 3

PcHcc2H2 P 4.2EthanolcH2O ] AcetatePcHcc2H2 c 9.6ButyratePc2 H2O ] 2 AcetatePcHcc2 H2 c 48.3PropionatePc3 H2O ] AcetatePcHCO 3

PcHcc3H2 c 76.1BenzoatePc7H2O ] 3 AcetatePcHCO 3

Pc3Hcc3H2 c 70.6b

AcetatePc4 H2O ] 2 HCO 3PcHcc4 H2 c104.6

Syntrophic reactions with H2 use by methanogens2 LactatePcH2O ] 2 AcetatePcHCO 3

PcCH4cHc P143.62 EthanolcHCO 3

P ] 2 AcetatePcH2OcHccCH4 P116.42 ButyratePcHCO 3

Pc H2O ] 4 AcetatePcHccCH4 P 39.44 PropionatePc3 H2O ] 4 AcetatePcHCO 3

PcHcc3 CH4 P102.44 BenzoatePc19H2O ] 12 AcetatePcHCO 3

Pc9Hcc3CH4 P124.4

a Calculated from the data in THAUER et al. (1977).b Calculated using the free energy of formation for benzoate given in KAISER and HANSELMANN (1982).

Tab. 2. Selected Kinetic Data for the Use of Hydrogen, Formate and Acetate by Methanogenic Bacteria

Substrate Organisms Apparent Km Reference Threshold Reference

Hydrogen most methano- 3–13 mM KRISTIJANSSON et al. 3–12 Pa CORD-RUWISCH et al.gens (1982), ROBINSON (1988), LOVLEY (1985),

and TIEDJE (1984) LOVLEY et al. (1984)

Formate many methano- 5–580 mM LOVLEY et al. (1984), 15–26 mM SCHAUER et al. (1982)gens SCHAUER et al. (1982)

Acetate Methano- 3.0–4.5 mM SCHÖNHEIT et al. 0.6–1.2 mM WESTERMANN et al.sarcina sp. (1982), WESTERMANN (1989), FUKUZAKI et al.

et al. (1989) (1990a)Methano- F0.1–1.2 mM HUSER et al. (1982), 5–75 mM WESTERMANN et al.saeta sp MIN and ZINDER (1989), MIN and

(1989), OHTSUBO ZINDER (1989),et al. (1992), AHRING AHRING and WESTER-and WESTERMANN MANN (1987a),(1987a) JETTEN et al. (1990)

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discussed in Sects. 4 and 5, the ability of meth-anogens to maintain low levels of H2 affectsthe types of products formed by fermentativebacteria and is essential for the degradation offatty and aromatic acids by syntrophic associa-tions.

Formate is a common fermentation product,especially by bacteria that use pyruvate–for-mate lyase in their metabolism, and may be anessential intermediate for syntrophic metab-olism (THIELE and ZEIKUS, 1988; DONG et al.,1994). Many methanogens are able to useformate and it serves as a source of electronsfor methane formation equivalent to H2. Theaffinity for formate use varies for differentmethanogens (Tab. 2).The apparent KM valuesfor formate use was 5–26 mM for two rumenmethanogens, 580 mM for Methanobacteriumformicicum, and 0.22 mM for Methanospiril-lum hungatei (LOVLEY et al., 1984; SCHAUER etal., 1982). SCHAUER et al. (1982) reported thatthe lowest concentration of formate metabo-lized was 26 mM for M. formicicum and 15 mMfor M. hungatei.

Acetate is a major product of fermentativemetabolism and is quantitatively the most im-portant substrate for methane production.About 60–90% of the methane produced inanaerobic digestors are derived from themethyl group of acetate (BOONE, 1982;MACKIE and BRYANT, 1981; MOUNTFORT andASHER, 1978; SMITH and MAH, 1966). At ther-mophilic temperatures or at high ammoniumlevels, the oxidation of the methyl group ofacetate to H2 may be the predominant routefor acetate metabolism (see Sect. 5). Acetateusing methanogens include members of thegenera Methanosarcina and Methanosaeta(BOONE et al., 1993). Methanosarcina sp. havefaster growth rates, higher apparent KM valuesfor acetate use, and higher threshold acetatevalues than Methanosaeta sp. (Tab. 2) (ZINDER,1993). The differences in the apparent KM va-lues for acetate use have been attributed todifferences in respective enzymes used to acti-vate acetate (JETTEN et al., 1990). Since Me-thanosarcina sp. and Methanosaeta sp. havedifferent threshold values for acetate use butuse the same reaction for acetate metabolism(Tab. 1), the threshold values cannot representa thermodynamic limitation. Acetate thres-hold values may result when a critical or in-

hibitory concentration of undissociated aceticacid is reached which, for Methanosarcinca sp.,is between 4 and 7 mM (FUKUZAKI et al.,1990a).

Consistent with the known growth charac-teristics of the acetotrophic methanogens, adrop in acetate concentrations below 1 mMwas correlated with a displacement of Me-thanosarcina sp. by Methanosaeta sp. in a ther-mophilic digestor (ZINDER et al., 1984). Gener-ally, high-rate anaerobic sludge blanket reac-tors usually have granules composed of Me-thanosaeta sp. rather than Methanosarcina sp.(GROTENHUIS et al., 1991; WU et al., 1993).Interestingly, RASKIN et al. (1996) found thatMethanosaeta sp. and Methanosarcina sp. werepresent at approximately equal levels in glu-cose degrading anaerobic biofilm reactorseven though acetate levels were below thethreshold values for Methanosarcina sp. Themaintenance of Methanosarcina in these bio-films could not be attributed to the utilizationof other substrates such as methanol, suggest-ing that there may be Methanosarcina sp. thathave a lower threshold value than has been re-ported in the literature. STRAYER and TIEDJE

(1978) found that acetate was converted tomethane at or near the maximal rate ineutrophic lake sediments since the addition ofacetate to these sediments did not result in acorresponding increase in the rate of acetateutilization or methane production. This sug-gests that there is little additional capacity tometabolize acetate if the acetate productionrates increase. A similar conclusion wasreached by HICKEY and SWITZENBAUM (1991)for anaerobic sewage digestors. This is prob-ably the reason why acetate concentrations indigestors increase when organic or volumeloading rates increase.

Methanol, methylamine and trimethylaminealso serve as substrates for Methanosarcina sp. and other methylotrophic methanogens(BOONE et al., 1993).These compounds may beimportant substrates for methanogenesis inmarine systems (OREMLAND and POLCIN,1982). Some hydrogenotrophic methanogenscan oxidize secondary alcohols to ketones andprimary alcohols to carboxylic acids (WIDDEL,1986).Additional methanogenic substrates arediscussed by ZINDER (1993) and BOONE et al.(1993).

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