119 Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate H.-J. Seitz 1, B. Schink 2 and R. Conrad 1 I Fakultiitfiir Biologie, Unioersiti~'t Konstanz, Konstanz, F.R.G. and 2 Lehrstuhl Mikrobiologie I, Universit&'t Tiibingen, Tiibingen, F.R.G. Key words: Hydrogen partial pressure; Proton reduction; Methanogenesis; Syntrophic coculture; Thermodynamics 1. SUMMARY 2. INTRODUCTION Anaerobic mineralization of organic matter to CO 2 and methane requires a complex food web of Pure cultures of Desulfovibrio vulgaris or Pelo- microorganisms [1,2]. Molecular hydrogen is a key bacter acetylenicus do not grow with lactate or intermediate in these degradation processes, and ethanol, respectively, under obligately proton- re- influences H z-producing as well as hydrogen-con- ducing conditions. However, a small part of these suming bacteria. It has been shown that a mini- substrates was oxidized and molecular hydrogen mum H 2 partial pressure is necessary for net H z was produced up to 4.2 and 3.2 kPa, respectively. oxidation in pure cultures of hydrogen-utilizing During growth in syntrophic methanogenic cocul- bacteria [3,4]. On the other hand, it is well estab- tures with Methanospirillum hungatei as partner, lished by pure culture studies that increased H z maximum hydrogen partial pressures were signifi- partial pressures shift the electron flow in ferment- cantly lower (0.7 to 2.5 kPa) than in the corre- ing bacteria towards reduced organic compounds sponding pure cultures. Calculation of Gibbs free [5-7] or even completely inhibit oxidation of cer- energies for the prevailing culture conditions tain substrates which can only be oxidized via showed that H 2 partial pressures were kept in a proton reduction [8-10]. range at which both, H z-producing and H z-con- H z concentrations permissive for anaerobic de- suming reactions, were thermodynamically per- gradation of various substrates have been esti- missive in pure as well as in syntrophic mixed mated from theoretical calculations [11-14] but cultures. have not yet been determined by measurements in defined cultures. We therefore measured H 2 metabolism of physiologically well-characterized fermenting bacteria under obligately proton-re- ducing conditions in pure culture as well as in Correspondence to: H.-J. Seitz, Fakult~t fiir Biologie, Uni- coculture with a Hz-utilizing methanogenic versitat Konstanz, Postfach 5560, D-7750 Konstanz, F.R.G. bacterium. The data were used to calculate the 120 Gibbs free energies of the H 2 transformation at CO 2 and pH using published equations [19]. Al- various growth phases. Our results show that H 2 cohols and volatile fatty acids were assayed by partial pressures are being kept in a range at standard gas chromatography procedures with which both, H2-producing and H2-consuming re- injector and detector temperatures being 130 or actions were thermodynamically permissive. 170°C, oven temperature being 100 or 130°C, respectively. For analysis of volatile fatty acids, samples were acidified with formic acid to a final 3. MATERIALS AND METHODS concentration of 0.5 mol formic acid/1 [16]. Lactate was determined enzymatically using lactate 3.1. Organisms and cultivation dehydrogenase (Boehringer, Mannheim) according Desulfovibrio vulgar& strain Marburg, DSM to [20]. Sulfide was determined by the methylene 2119, was kindly provided by Prof. Thauer, Mar- blue method [21]. The concentration of HS- was burg, F.R.G. Pelobacter acetylenicus strain calculated from total sulfide and the actual pH, WoAcyl, DSM 2348, and Methanospirillum assuming a pKa of 7.0. Sulfate was measured hungatei strain Mlh, were taken from the culture photometrically after precipitation with barium collection of our laboratory. chloride [22, modified after 23]. t Cells were grown at an initial pH of 7.0-7.2 in The standard Gibbs free energies (AG ° ) of the mineral medium described [15], except that H2-producing and Hz-utilizing reactions were Na2S was increased to 1.5 mmol/1. Acetate (2 calculated from the tabulated Gibbs free energies mmol/1) was added as additional carbon source if of formation (AG O of the individual reactants hydrogen was the sole electron donor. Prior to and products [24]. H 2 and CH 4 were assumed as inoculation, media were reduced with a few crystals gaseous compounds; all other compounds as dis- of sodium-dithionite (less than 0.1 mmol/1). solved. The Gibbs free energy (AG) of a reaction Precultures of hydrogen-consuming bacteria under non-standard conditions was calculated were grown in shaken 120 ml serum bottles under from its standard Gibbs free energy (AG °' ) and an atmosphere of H2/CO 2 (80/20 v/v). P. the actual partial pressures or concentrations of acetylenicus was grown on acetoin (10 mmol/1) in the reactants and products involved, the actual 50 ml screw cap bottles without a gas phase. D. temperature and H+-concentration [25]. vulgar& was cultivated similarly on lactate (40 mmol/1) and limiting amounts of sulfate (5 mmol/1). Experiments with pure cultures and 4. RESULTS defined cocultures were carried out basically as recently described [4]. Purity controls were per- 4.1. H2-metabolism in pure cultures formed microscopically at the beginning and the Pure cultures of Desulfovibrio vulgaris or Pelo- end of each experiment. bacter acetylenicus were unable to grow by fer- mentation of lactate or ethanol, respectively, 3.2. Analytical procedures according to the following equations [24]: Methane was measured with a Perkin Elmer gas chromatograph with flame ionization detector (1) Ethanol + H20---) acetate- + H + + 2 H 2 [16]. H 2 partial pressures above 100 Pa were (AG °' = + 9.6 kJ/mol ethanol) analyzed in a gas chromatograph (Carlo Erba) (2) Lactate- + 2 H20---) with a thermal conductivity detector. H 2 partial pressures below 100 Pa were measured using a H 2 acetate- + H + + HCO 3 + 2 H 2 analyzer based on the HgO-Hg vapour conversion (AG °'= -4.2 kJ/mol lactate) technique [17,18] with a lower detection limit of 0.2 mPa H z. CO 2 was determined with an infrared However, both strains were able to produce analyzer (UNOR, Maihak, Hamburg, F.R.G.). Bi- hydrogen which accumulated in the culture carbonate concentrations were calculated from headspace to partial pressures of up to 3.2 kPa 121 Table 1 5OOO Gibbs free energies of H2-producing and H2-consuming reac- fo tions in pure cultures of fermentative and methanogenic o bacteria / Organism Substrate Maximum or AG /o minimum H2 (kJ/mol) "0 30OO partial pressure fit. (Pa) o Desulfovibrio lactate 4 200 - 34.6/lactate -r / 2000 o vulgaris (-sulfate) Pelobacter / acetylenicus ethanol 3200 - 9.5/ethanol 10120 Methanospirillum hungatei hydrogen 2.5 - 26.0/methane 0 ~ i i .~0 IO0 with ethanol and up to 4.2 kPa with lactate as Time [ h] electron donor (Fig. 1). With both strains, hydro- Fig. 1. Production of hydrogen from ethanol (e) or lactate (o) gen evolution ceased before the H 2 partial pres- by pure cultures of Pelobacter acetylenicus and Desulfooibrio sures reached values at which fermentation of oulgaris. The data are mean values of duplicate experiments. lactate or of ethanol would become endergonic The maximum theoretical H 2 partial pressure would be 24000 (Table 1). Pa, if the total amount of organic electron donor added (10 mmol/l) would have been fermented according to the equa- In cell suspensions of Methanospirillum hungatei tions given in RESULTS. H 2 was oxidized according to the following equa- 0,10 - 100 ¢- t~ 4000 0 0 E ~" o -8o ~, E 3000 O -60 0 .12 D 0,~. 2ooo LLI C "6 (.) - ~0 E .t o E 0 X -6 0 a 1000 - 20 £-9 -1- 2 ~ o < "6" 0 0 2 /, 6 o 2 ~ 6 Time [ d ] Time [d] Fig. 2. Fermentation of ethanol by a homogeneously mixed coculture of Pelobacter acetylenicus and Methanospirillum hungatei: (a) time course of substrate degradation and product formation, (b) change of Gibbs free energies during syntrophic growth. 122 Table 2 5. DISCUSSION Gibbs free energies of Hz-producing and H2-consuming reac- tions in obligately syntrophic cocultures of fermentative and In the absence of external electron acceptors, methanogenic bacteria Desulfovibrio vulgaris and Pelobacter acetylenicus Coculture Sub- H 2 partial AG behaved like typical obligately syntrophic H+-re - strate pressure (k J/tool) ducing bacteria. Thus, growth coupled to oxida- (Pa) tion of organic substrates like lactate or ethanol maxi- mini- was only possible with concomitant hydrogen re- mum mum moval. This reaction can be carried out by hydro- P. acetylenicus - 13.7/ethanol gen-oxidizing bacteria in syntrophic coculture + ethanol 2 500 2.8 [10,26-30]. Recently, effective chemical mecha- M. hungatei - 31.3/methane nisms have also been demonstrated [31]. The D. vulgaris - 34.3/lactate + lactate 1800 2.2 necessity for H 2 removal can easily be explained M. hungatei - 25.7/methane by the fact that the H2-producing reactions are endergonic or only poorly exergonic at standard conditions and pH 7.0, i.e., at a hydrogen partial pressure of approximately 100 kPa. During growth of these strains in syntrophic coculture with M. tion [24]: hungatei, H z partial pressures were about 40 to HCO~- + H + + 4 H 2---~ CH 4 + 3 H20 150 times lower (0.7-2.5 kPa) and thus always kept in a range at which hydrogen producers as × (AG °'= -135.6 kJ/mol methane) well as hydrogen consumers could obtain suffi- cient energy for growth (Fig. 2). This corresponds H 2 oxidation stopped at H 2 partial pressures at well with data based on H 2 measurements in which Ha-dependent production of methane was various methanogenic environments [25]. still exergonic (Table 1). While in homogeneously mixed cocultures H 2 partial pressures were always lower than the maxi- 4.2.