World Journal

of Microbiology

& Biotechnology

Temperature degradation

12.497-503

regulation of anaerobic of organic matter

P. Westermann Anaerobic degradation of organic matter follows similar pathways in digesters and anaerobic freshwater sediments. The responsible microorganisms are linked in a complex food web, where short chain fatty acids and H, are important intermediates. Degradation of short-chain fatty acids is endothermic under standard conditions and is only possible at low H, partial pressures maintained by exothermic methanogenesis. The coupling between these and hence sensitive to environmental changes such as endothermic and exothermic processes is delicate, temperature variations. The effect of temperature on thermodynamics and on kinetics of these and other anaerobic degradation processes with emphasis on freshwater ecosystems is discussed. Key words: Methanogenesis,

short chain fatty acids, syntrophism,

In temperate climates, temperature is one of the most important variables controlling the rate of microbial degradation of organic matter to methane, carbon dioxide, and water in anaerobic environments such as sediments and waterlogged soils (Conrad et al. 1987; Westermann & Ahring 1987). Because of atmospheric fluxes, methane is a major element of global carbon cycles and plays an important role in atmospheric chemistry and radiation transfer (Watson et al. 1990). The comprehension of the significance of methane emissions from wetlands to climate change issues has stimulated studies of detailed mechanisms of temperature regulation in these environments. In anaerobic digestion of organic matter for production of combustible biogas, temperature regulation is an integral part of the design and operation of the biogas plants, and temperature fluctuations are considered detrimental to optimal process operation (Buhr & Andrews 1977). The effect of temperature on metabolism is commonly presented as a temperature coefficient (Q,J which is the increase in the process rate for each 10°C rise in temperature. Alternatively In (metabolic rate or growth rate) is plotted against the reciprocal temperature (K) in an Arrhenius plot, from which the activation energy can be calculated. The temperature coefficient and the activation energy are

The author is with the Department of General Microbiology, Institute of Molecular Biology, University of Copenhagen, Solvgade 83 H, DK-1307 Copenhagen K. Denmark; fax: + 45 35 32 20 40. 01996

Rapid Science

thermodynamics.

commonly measured in fractions of an ecosystem exposed to laboratory manipulations (e.g. homogenization) or as maximum specific growth rates in axenic cultures of bacteria. In well balanced ecosystems, the substrate concentrations are generally several times lower than those required to saturate the bacterial enzymes involved, and therefore reaction rates are highly sensitive to environmental changes in substrate concentrations. Only a few investigations have been carried out to relate temperature to metabolism or growth at substrate concentrations relevant to environmental conditions. This review will summarize and discuss these more complex relationships between temperature and microbial activity in mainly anaerobic freshwater ecosystems.

Anaerobic

Degradation

of Organic

Polymers

The microbial degradation of organic matter under aerobic conditions is most commonly carried out by single organisms, capable of hydrolyzing and oxidising the biological polymers completely to CO, and H,O (Figure I). Under anaerobic conditions no organism is capable of mineralizing biological polymers to CO, and H,O. Instead, a complex food-web is operative, in which the product of one functional group is the substrate of the subsequent functional group (Figure I). Electrons from the oxidation of carbohydrates to CO, are accumulated in the hydrocarbon methane, in contrast to aerobic conditions where electrons are accumu-

Publishers World Journal of Minobiology

0 Biotechnology, Vol 12, 1996

497

P. Wesfermann

Biological

polymers

Hydrolytic Oligo and extracellular monomers enzymes

Oligo- and monomers w

Aerobic organisms

Fermentative

I

H,, CO,

Hydrolytic extracellular enzymes

bacteria

Volatile fatty acids

Acetate

Syntrophic bacteria w CO,reducing methanogenic bacteria w CO,,H,O

CH,,H,O

CH,, CO,

Aerobic conditions Figure

1. Major

pathways

in aerobic

Anaerobic conditions and anaerobic

degradation

of organic

lated in H,O. Fermentative bacteria hydrolyze and ferment biological polymers to mainly acetate, H,, and CO,, and to a minor extent short chained volatile fatty acids such as butyrate and propionate. The volatile fatty acids are further oxidized to acetate, H,, and CO, by syntrophic bacteria, and finally metabolized to CH,, CO, and H,O by methanogenie bacteria. In contrast to the fermentative bacteria, the syntrophic bacteria oxidizing the short-chained fatty acids

498

1 Aceticlastic methanogenic bacteria

World Jouml of Microbiology 6 Biotechnology, Vol 12. 1996

matter.

are obligately symbiotic bacteria (McInerney et al. 1979; Boone & Bryant 1980). Under standard conditions, these oxidation processes are endothermic (energy demanding) (Table I). When the H, partial pressure is kept low by methanogenic bacteria, the oxidation processes become energetically possible and the energy yield increases with decreasing H, partial pressure, since the removal of H, results in a displacement of the equilibria to the right

Regulation of anaerobic degradation Table 1. Values for change in Gibbs free energy 0, T = 25°C) of methanogenesis from H,, acetate,

(AGO), enlhalpy and propionale

(AH”), and entropy (AS’) under and butyrate oxidation (given

Reaction

standard conditions in kJ/reaction).

(1 M, 1 atm, pH =

AG"

AH"

AS"

- 175.5 -31

- 240.3 +5

- 0.218 + 0.121

+ 116.4 + 88.2

+ 192.4 + 135.6

+ 0.255 + 0.159

Methanogenesis (1) 4H, + HCO,+ H+ + CH, + 3H,O (2) CH,COO+ H,O + CH, + HCO,Hydrogen (3) CH,CH,COO(4) CH,CH,CH,COOCalculated

production

+ 3H,O + CH,COO+ HCO,+ H+ + 3H, + 3H,O + PCH,COO+ H+ + 2H,

from

energies

of formation

(Stumm

& Morgan

1981; Conrad

&Wetter

1990).

-150

Methanogenesis

-100 Propionate oxidation

Q

-50

4 B $-

0

50

100

I

0

12

I

I

I

I

I

I

I

3

4

5

6

7

8

9

H2 partial pressure, (-log atm) Figure 2. Free energy change of propionate oxidation and methanogenesis from H, as a function of the H, partial pressure. The free energy change is calculated for both 20°C and 37°C by use of the temperature compensated Nernst equation (equation 2). The following concentrations were used for the calculations: Propionate, 0.1 mM; acetate, 1 mM; HCO,-, 10 mM; H+, 1O-4 mM, methane, 0.6 atm. The hatched area indicates the H, partial pressure range where propionate mineralization is possible at 37°C. The striped area indicates the H, partial pressure range where propionate mineralization is possible at 20°C.

(Table I, equations 3 and 4). Under standard conditions methanogenesis is an exothermic (energy yielding) process. Since H, is a substrate for methanogenesis, a reduction in H, partial pressure will lower the energy yield of the methanogenic bacteria (Table 1, equation 1). This implies that there is a certain narrow H, partial pressure range where oxidation of fatty acids with H, as a product, and

methanogenesis with H, as a substrate is possible (Do&g 1988). Above this range, fatty acid oxidation is not possible due to product inhibition, and below this range methanogenesis is not possible due to substrate limitation (Figure 2). Under aerobic conditions complete oxidation of glucose yields 2826 kJ/mol corresponding to 38 mol ATP/mol glucose. Under anaerobic conditions most of this energy is first conserved in volatile fatty acids and H,, and after complete mineralization, in methane (Figure 3). This has the consequence that only 10% of the potential energy is available to the ferrnentative bacteria while 90% is conserved in the volatile fatty acids. Of this 90% only 4% is available to the methanogenic bacteria while 86% is conserved in methane, which is the energetic background for biogas production. The 2826 kJ released aerobically per mol of glucose are exploited by only one organism while the 390 kJ released per mol glucose under anaerobic conditions (corresponding to 5 ATP) have to be shared between the members of a complex food-web (Thauer et al. 1977; Schink 1988; Schink & Friedrich 1994). Each group of bacteria in the food-web seeks to gain a maximum energy yield. Dependent upon the H, partial pressure, fermentative bacteria obtain from 2 to 4 ATP/mol glucose metabolised (Thauer et al. 1977). If the H, partial pressure is kept low by methanogenic bacteria, fermentative bacteria mainly produce H,, CO,, and acetate from carbohydrates as exemplified in Figure 4. The necessary reduction of protons with electrons from NADH to maintain the electron balance is endothermic under standard conditions at pH 7 (NADH + H+ +NAD+ + H,, AG” = + 18.8 kJ/reaction) but is energetically favourable at H, partial pressures below lop4 atm (Wolin 1982). The production of acetate allows the fermentative bacteria a maximum of 4 ATP/mol glucose with the consequence that only 1 ATP is left to share between syntrophic bacteria and H, utilising aceticlastic methanogenic bacteria per mol glucose mineralized. Therefore, when the size and composition of the bacterial populations are allowed to adjust to the specific environmental conditions, only smaller amounts of volatile fatty acids longer than acetate are formed, primarily from fermentation of lipids and proteins.

WorldJournal

of Microbmlogy & Bmtecknology. Vol 12. 1996

499

P. Westermann

Nernst equation:

125

AG = AG* + R x T x In [B]/[A]

[] A v m l a b l e e n e r g y

Conserved energy 100

(1)

where AG ° is the change in Gibbs free energy under standard conditions, R is the gas constant, T is temperature in °Kelvin, and [13] and [A] are the concentrations of the two compounds A and B.

c-:-::-:-::-:-::-:::.::-:....-..:->i ::::::::::::::::::::::::::::::::::::::::::: ,;.:,:.:.:,:.:,:,:.:,:,:,:,:.:.:,:.:+:

::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::::::::::2:::::::::::::: :::::::::::::::::::::::::::::::::::::::::::

For 4Ha + HCO~- + H +

::::::::::::::::::::::::::::::::::::::::::

75-

::::::::::::::::::::::::::::::::::::::::::: 4.>: :::::::::::::::::::::::::::::., -.:x.: t:i:i:i:i:i:i:i:i:i:i:i:i:!:i:!~!!i:iiii~i~

: .....................................iili~

50-

HzO:

AG = -175.5 + R x T x ln[CH,]/([H2] 4 x [HCO~-] x [H+])

::::::::::::::::::::::::::::::::::::::::::: ................. -:,:,:,:,:,:,:.:.:,:.:.:.:,:.:.:.:.:.:.:. :i:i. :::::::::::::::::::::::::::::::::::::::::::

=

--* C H 4 +

Temperature compensated Nernst equation:

.....:...:...:.:.:+:.:.:.:.:.:+:
[B]). At decreasing temperature, Gibbs free energy, therefore, will become more positive. Figure 2 illustrates the effect of reducing the incubation temperature from 37°C to 20°C on energetic yields from propionate oxidation and methanogenesis from H z and COz. The increased energy yield of methanogenesis with decreasing temperature can be seen from the decrease in the Ha partial pressure where AG ° is zero. The lower energy yield of propionate oxidation with decreasing temperature can similarly be seen from the necessity of a lower H a to make the reaction energetically feasible. The Hz range where propionate oxidation coupled to methanogenesis is possible is thereby displaced to a lower and narrower range. This phenomenon has been confirmed in two investigations, where environmental samples were incubated at

Regulation of anaerobic degradation

H, c 10m4atm

1 Glucose

Figure 4. The Fd-ferredoxin

effect

of H, partial

Table 2. The effect of temperature in slurries of different anaerobic in atm x lo-@).

pressure

on hydrogen environments

Environment

15°C

Alder swampa Lake sedimenta Paddy soilb a Westermann,

2.96 2.47 19.7-59.2 1994; b Chin

on product

8. Conrad,

partial (partial

formation

pressures pressures

30% 14.8 7.40 69.0-88.8

1995

different temperatures and the concentration of H, was measured (Westermann 1994; Chin & Conrad 1995) (Table 2). The concept of a zero-sum community has been introduced by Dolfing (1992). In this model, the increased energy available to one organism following the increase in concentration of its substrate is counterbalanced by a decrease in energy available to the producer of this substrate. The decrease in energy of syntrophic fatty acid oxidation with decreasing temperature is hence compensated by a simultaneous decrease in the H, partial pressure. The consequence is, that the energy yield of the complete mineralization of propionate remains fairly constant with changing temperature despite the fluctuations in the energy yield of the part processes.

and ATP yield

of fermentative

bacteria.

The Effect of Temperature Anaerobic Degradation

AcCoA-acetyl

coenzyme

on Kinetics

A;

of

Most studies of temperature effects on activity (growth or metabolism) in axenic bacterial cultures are carried out under optimal growth conditions with temperature as the only variable. Under these conditions growth and metabolism can be compared to a chemical reaction and the relationship to temperature can, within a certain range, be approximated by the Arrhenius equation. This implies that activity increases with increasing temperature. Since optimal substrate conditions rarely are found in nature, microbial activity occurs at substrate concentrations far below saturation with the result that both temperature and substrate availability limit growth and metabolism. Kinetic studies of combined effects of substrate and temperature limitation can either be carried out by measurements of growth (Monod kinetics) or by measurements of substrate uptake (Michaelis-Menten kinetics). Both types of studies have their advantages and disadvantages. Measurements of growth is obvious, since increase in bacterial number should be the consequence of substrate uptake. However, compounds other than the carbon/energy source most commonly studied (minerals, vitamins, co-factors etc.) have to be taken up simultaneously by the bacteria to support growth. Laboratory kinetic studies in defined media, where

World Journal of Mmbmlogy

b Biotechnology, Vol 12, 1996

501

P. Westermann the concentration of substrate is varied at different temperatures, might, therefore, result in multiple substrate limitation (Law & Button 1977) or a shift in limiting steps with concentration (transport limitation at low concentrations, limitation by reaction with intracellular enzymes at medium concentrations, and limitation by enzyme or ribosomal activity at high concentrations (Button 1993)). Furthermore, at permanently low substrate concentrations as experienced in sediments and wetlands, growth rates are probably rather low. Substrate scavenging for endogenous metabolism, such as maintenance energy for active transport, replacement of leaked constituents or macromolecule replacement might take up a significant part of the energy metabolism. Up to 50% of the ATP produced during fermentative catabolism is used for purposes other than biosynthesis and is not included in growth measurements (Thauer et al. 1977). The maintenance energy has been shown to vary among anaerobic bacteria (Russell & Baldwin 1979), and might further change several-fold with changing temperature (Verduyn 1991). Interpretations of growth in relation to substrate concentrations and temperature should therefore be carried out cautiously. To my knowledge, no kinetic studies of temperature effects on growth of anaerobic bacteria at low substrate concentrations have been carried out. Chemostat studies of aerobic bacteria have shown both increasing and decreasing affinity (K,) with decreasing temperature and therefore, are not conclusive (Herbert & Bell 1977; Nedwell & Rutter 1994). Most studies of temperature effects on anaerobic bacteria are ecologically related and focus on substrate consumption or product formation (e.g. methane emission in relation to climate change or biogas production) rather than growth. This is also a consequence of the low energy yield from anaerobic metabolism of organic matter, where a relatively large amount of substrate has to be converted to increase the cell number measurably. Finally anaerobic environments are typically opaque (sediment, soil, animal waste) and sure measurements of cellular growth is difficult compared to investigations of bacteria in open waters. To my knowledge only one study has so far been published on direct kinetic measurements of temperature effects on substrate kinetics of anaerobic bacteria (Westermann et al. 1989). In this study, concentrations of acetate and H, were shown to affect the temperature sensitivity of a commonly occurring methanogen, Mefhanosarcina barkeri by increasing the affinity (decreasing K,) for substrate uptake and methane production. This response implies a decrease in the apparent activation energy and Q,, and hence a decrease in the temperature dependency as a function of decreasing substrate concentration. A few kinetic studies have been conducted on aerobic bacteria. Laudelout & van Tichelen (1960) and Knowles et al. (1965) demonstrated significant increases in saturation constants of nitrifying bacteria with temperature increases, and Hug & Hunter (19i’4a, 1974b)

502

WorldJournal of Microbmlogy & Biotechnology, Vol 12, 1996

showed increased affinity for both ammonia-lyase and urocanase of intact cells and cell extracts of Pseudomonas pufida with decreasing temperature. Several studies have focused on product formation in ecosystems as a function of temperature. In these studies, Q,, has been measured and related to substrate concentration. All studies have so far confirmed a positive correlation between substrate concentration and temperature dependency (Conrad et al. 1987, Westermann 1993, Whiting & Chanton 1993). The decrease in activation energy which is a consequence of a decrease in Q,, parallels the results obtained with Mefhanosarcina barkeri pointing towards a reduced temperature dependency when the substrate concentration is reduced to subsaturating levels and hence towards values observed in balanced ecosystems.

The Effect of Temperature Degradation Pathways

on Anaerobic

Only a few studies have been carried out on changes in degradation pathways caused by temperature changes. Kotsyurbenko et al. (1993) studied temperature effects at elevated substrate concentrations in a freshwater sediment. At the artificially high substrate concentrations, methanogenesis from H, was insignificant at low temperatures compared to the energetically less favourable homoacetogenesis from H, and CO,. This implied that virtually all methane was derived from acetate. Chin & Conrad (1995) similarly showed a low temperature inhibition of methanogenesis from H, at in situ substrate concentrations. The resulting elevated H, partial pressure inhibited syntrophic degradation of fatty acids, and a sudden decrease in temperature resulted in accumulation of volatile fatty acids. Fermentation of polysaccharides was then taken over by homoacetogenic bacteria producing only acetate, and aceticlastic methanogenesis was the dominating terminal process, shortcutting syntrophic bacteria.

Conclusion Microorganisms are poikilotherrnic, since maintenance of a constant cell temperature in an environment of changing temperature is impossible due to the high surface to volume ratio. During evolution, microorganisms have developed an array of mechanisms to cope with impacts of the environment (e.g. changing water activity, food availability, toxic compounds etc.). It is, therefore, puzzling that temperature is still a major regulator of microbial activity taking into account that temperature variations have always occurred on earth during the history of life. Interestingly, temperature mediated decrease in activity has been circumvented by many poikilothermic marine animals (e.g. fish and inverte-

Regulafion

brates) living in the cold polar seas. This temperature compensation is ascribed to an increase in affinity with decreasing temperature of most investigated marine animal enzymes (Hochachka & Somero 1984). The studies presented in this review indicate, however, that a certain compensation for changing temperature is possible among anaerobic bacteria but that the overall activity is directly proportional to temperature although Q,, values close to I are observed in well balanced ecosystems and axenic methanogenic cultures growing at low substrate concentrations.

of anaerobic

degradation

Kotsyurbenko, O.R., Nozhevnikova, A.N. & Zavarzin, G.A. 1993 Methanogenic degradation of organic matter by anaerobic bacteria at low temperature. Chemosphere 27, 1745-1761.

Laudelout, H. & van Tichelen, L. 1960 Kinetics of the nitrite oxidation by Nitrobacter winogrudskyi. ]ournal of Bacteriology 79, 39-42. Law, A.T. & Button, D.K. 1977 Multiple-carbon-source-limited growth kinetics of a marine coryneform bacterium. Iournal of Bacteriology 129, 115-123. McInemey, M.J., Bryant, M.P. & Pfennig, N. 1979 Anaerobic bacterium that degrades fatty acids in syntrophic association with methanogens. Archives of Microbiology 122, 129-135.

References Boone, D.R. & Bryant, M.P. 1980 Propionate degrading bacterium Syntrophobucter wolinii sp. nov. gen. nov., from methanogenic ecosystems. Applied and Environmental Microbiology 40, 626-

632. Boone, D.R., Johnson, R.L. & Liu, Y. 1989 Diffusion of the interspecies electron carriers H, and formate in methanogenic ecosystems and its implication in the measurement of K, for H, or formate uptake. Applied and Environmental Microbiology 55,1735-1741.

Buhr, H.O. & Andrews, J.F. 1977 The thermophilic anaerobic digestion process. Water Research 11, 129-143. Button, D.K. 1993 Nutrient-limited microbial growth kinetics: overview and recent advances. Antonie uun Leeuwenhoek 63,

225-235. Chin, K.-J. & Conrad, R. 1995 Intermediary metabolism in methanogenie paddy soil and the influence of temperature. FEMS Microbiology Ecology 18, 85-102. Conrad, R., Schiitz, H. & Babbel, M. 1987 Temperature limitations of hydrogen turnover and methanogenesis in anoxic paddy soil. FEMS Microbiology Ecology 45, 281-289. Conrad, R. & Wetter, B. 1990 Influence of temperature on energetics of hydrogen metabolism in homoacetogenic, methanogenic, and other anaerobic bacteria. Archives of Microbiology

155,94-98. Do&g, J. 1988 Acetogenesis. In Biology of unuerobic microorgunisms, ed Zehnder, A.J.B. pp. 417-468. New York: Wiley Interscience. Dolfing, J. 1992 The energetic consequences of hydrogen gradients in methanogenic ecosystems. FEMS Microbiology Ecology 101,183-187.

Herbert, R.A. & Bell, obligately psychrophilic

C.R.

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Hochachka, P.W. & Somero, G.N. 1984 Biochemical adaptation. Princeton: Princeton University Press. Hug, D.H. & Hunter, J.K. 1974a Effect of temperature on urocanase from a psychrophile, Psetrdomonus putidu. Biochemistry 13, 1427-1431.

Hug, D.H. & Hunter, J.K. 1974b Effect of temperature on histidine ammonia-lyase from a psychrophile, Pseudomonas putidu. ]oournu2 of Bacteriology 119, 92-97. Knowles, G., Downing, A.L. & Barrett, M.J. 1965 Determination of kinetic constants for nitrifying bacteria in mixed culture, with the aid of an electronic computer. ]otlrnu/ of General Microbiology 38, 263-278.

Nedwell, D.B. & Rutter, M. 1994 Influence of temperature on growth rate and competition between two psychrotolerant antarctic bacteria: Low temperature diminishes affinity for substrate uptake. Applied and Environmental Microbiology 60, 19841992.

Russell, J.B. & Baldwin, R.L. 1979 Comparison of maintenance energy expenditures and growth yields among several rumen bacteria grown on continuous culture. Applied and Environmental Microbiology 37, 537-543. Schink, B. 1988 Principles and limits of anaerobic degradation: Environmental and technological aspects. In Biology of anaerobic microorganisms, ed Zehnder, A.J.B. pp. 771-846. New York: Wiley Interscience. Schink, B. & Friedrich, M. 1994 Energetics of syntrophic fatty acid oxidation. FEMS Microbiology Reviews 15, 85-94. Stumm, W. & Morgan, J.J. 1981 Aquatic Chemistry 2nd. edition New York: Wiley Interscience. Thauer, R.K., Jungermann, K. & Decker, K. 1977 Energy conservation in chemoautotrophic anaerobic bacteria. Bacteriological Reviews 41, 100-180. Verduyn, C. 1991 Physiology of yeasts in relation to biomass yields. Antonie vun Leeuwenhoek 60, 325-353. Watson, R.T., Rodhe, H., Oeschger, H. & Siegenthaler, U. 1990 Greenhouse gases and aerosols. In Climate Change. The IPPC Scientific Assessment, eds Houghton, J.T., Jenkins, G.J. & Ephraums, J.J. pp. l-41. Cambridge: Cambridge University Press. Westermann, P. 1993 Temperature regulation of methanogenesis in wetlands. Chemosphere 26, 321-328. Westermann, P. 1994 The effect of incubation temperature on steady-state concentrations of hydrogen and volatile fatty acids during anaerobic degradation in slurries from wetland sediments. FEMS Microbiology Ecology 13, 295-230 Westermann, P. & Ahring, B.K. 1987 Dynamics of methane production, sulfate reduction and denitrification in a permanently waterlogged alder swamp. Applied and Environmental Microbiology 53, 2554-2559. Westermann, P., Ahring, B.K & Mah, R.A. 1989 Temperature compensation in Methunosurcinu burkeri by modulation of hydrogen and acetate affinity. Applied and Environmental Microbiology

55,1262-1266. Whiting, control

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794-795. Wolin, M.J. 1982 Hydrogen transfer in microbial communities. In Microbial interactions and communities, eds Bull, A.T. & Slater, J.H. pp. 323-357. London: Academic Press.

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