The effects of pH on carbon material and energy balances in hydrogen-producing Clostridium tyrobutyricum JM1

Bioresource Technology 99 (2008) 8485–8491

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Short Communication

The effects of pH on carbon material and energy balances in hydrogen-producing Clostridium tyrobutyricum JM1 Ji Hye Jo a, Dae Sung Lee b,*, Jong Moon Park a,c,* a

Advanced Environmental Biotechnology Research Center, School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, South Korea b Department of Environmental Engineering, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701, South Korea c Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, South Korea

a r t i c l e

i n f o

Article history: Received 20 December 2007 Received in revised form 30 March 2008 Accepted 31 March 2008 Available online 15 May 2008 Keywords: Clostridium tyrobutyricum Carbon material balance Energy balance Hydrogen production pH effect

a b s t r a c t The effects of pH on hydrogen fermentation of glucose by newly isolated H2-producing bacterium Clostridium tyrobutyricum JM1 were investigated in batch cultivations. The changes of carbon material and energy balances by pH conditions provided useful information for understanding and interpreting the regulatory system of the microorganism, and for optimization of a desired product, in this case, molecular hydrogen. The most probable metabolic pathways of C. tyrobutyricum JM1 were determined through an accurate analysis of stoichiometry and the consistency of the experimental data, checked by high carbon recovery. The carbon material and energy balances were adequately applied to estimate the carbon-flow distribution. They suggested that pH 6.3 was appropriate to maximize hydrogen production with a high concentration of butyrate and balanced activities of NADH. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Hydrogen is recognized as an ideal and renewable energy source producing no green gases, 50% more efficient than gasoline, and 2.75-fold greater than hydrocarbon fuel sources (Mizuno et al., 2000). Anaerobic fermentation involves the acidogenesis phase, during which various end products such as VFAs (volatile fatty acids) and hydrogen gas can be produced depending on the strain and environmental conditions. The community of fermentative bacteria responsible for hydrogen production includes facultative anaerobic bacteria and strict anaerobic bacteria. The representative of the former is Enterobacter sp. and that of the latter is Clostridium sp. (Nath and Das, 2004; Tanisho et al., 1987). Especially, Clostridium tyrobutyricum, one of the saccharolytic clostridia, is a low-G+C gram-positive anaerobe and exhibits special metabolic routes to produce short-chain fatty acids and hydrogen gas from carbohydrates and amino acids (Balow et al., 1992; Dürre, 2005). A part of the carbon source is usually utilized to produce an energy-rich compound, from which ATP is formed via substrate level phosphorylation in the absence of oxygen, whereas the reducing equivalent ðNADHþ 2 Þ is disposed of on the other part of the substrate (Dürre, 2005). Molecular hydrogen is a common end product of * Corresponding authors. Tel.: +82 53 950 7286; fax: +82 53 950 6579 (D.S. Lee); Tel.: +82 54 279 2275; fax: +82 54 279 2699 (J.M. Park). E-mail addresses: [email protected] (D.S. Lee), [email protected] (J.M. Park). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.03.060

clostridial fermentation. The reversible cleavage of hydrogen into two protons and electrons is catalyzed by the enzyme called hydrogenase. Especially, FeFe (iron-only) hydrogenase is known to be a 10 times more active hydrogen producer (Kcat 6 9000 s1) than the NiFe-type present in many bacteria and methanogenic archaea (Dürre, 2005). Besides hydrogenase, enzymes such as pyruvate:ferredoxin oxidoreductase (PFOR) and NADH:ferredoxin oxidoreductase (NFOR) have important roles in hydrogen formation (Hallenbeck, 2005; Jungermann et al., 1971). The pyruvate generated by glycolysis is used to yield acetyl-CoA, from which ATP can be derived, and reduced ferredoxin (Fdred), which drives hydrogen evolution by hydrogenase as follows (Hallenbeck, 2005) Pyruvate þ CoA þ 2Fdox ! Acetyl-CoA þ CO2 þ 2Fdred PFOR

ð1Þ

NFOR recycles the NAD+ and also produces Fdred, which can in turn drive hydrogen production by hydrogenase (Hallenbeck, 2005; Jungermann et al., 1971) NADH ¢ e ¢ Fd ¢ NFOR

e

hydrogenase

¢ H2

ð2Þ

where Fd is ferredoxin. However, it is known that the NADH oxidation by NFOR is inhibited under standard conditions and the NADH is usually used to drive the more energetically favorable formation of other metabolites such as butyrate or butanol (Hallenbeck, 2005). Therefore, the maximum allowable yield of hydrogen is near 2 or slightly above, which is a major bottleneck of fermentative processes (Hallenbeck, 2005; Nath and Das, 2004). To overcome such a low

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Nomenclature X YATP/G YX/G YX/ATP eq.

biomass concentration, gDW/L1 yield of ATP production on glucose consumption, molATP/molG yield of growth on glucose consumption, C-molDW/ molG yield of growth on ATP production, gDW/molATP equivalent

Greek Symbols in Appendix a molar production of lactate, mollactate/molG (Eq. (3)) b molar production of pyruvate, molpyruvate/molG (Eq. (4))

yield of hydrogen, the knowledge of microbial metabolism and of its behavior is preferentially required. The studies on metabolic pathway provide useful information for understanding and interpreting the regulatory system of the microorganism, for estimating the upper bounds of by-product yields, or for finding the directions for optimization of a desired product (here, hydrogen gas), particularly in fermentations with several by-products (Zeng et al., 1993). Previous research relevant to C. tyrobutyricum focused on butyrate fermentation of xylose using immobilized cells (Zhu and Yang, 2004). Up to now, however, nothing is known concerning the effects of pH on hydrogen production from glucose by C. tyrobutyricum through metabolic engineering methodologies. The pH is one of the most important factors in hydrogen production due to its effects on FeFe-hydrogenase activity, metabolic pathways, and the duration of lag phase (Dabrock et al., 1992; Lay, 2000; Tanisho et al., 1987). Therefore, if the initial pH does not inhibit bacterial growth or residence, a higher initial pH value would delay the beginning of the pH inhibition caused by the metabolic shift from acidogenesis to solventogenesis (Lee et al., 2002). Khanal et al. (2004) also reported that low initial pH values of 4.0-4.5 cause longer lag periods. On the other hand, high initial pH values such as 9.0 decrease lag time, but have a lower yield of hydrogen production (Zhang et al., 2003). It is reported that the effect of pH is due to the change of the ionization state of the components in enzymatic reactions (Fabiano and Perego, 2002). The objective of this work is to estimate and investigate how pH influences an anaerobic metabolism, especially hydrogen production, through the carbon flow distribution in hydrogen-producing C. tyrobutyricum JM1. 2. Methods 2.1. Microorganism Clostridium tyrobutyricum JM1 was isolated from a food waste treating process and used as an inoculum (Jo et al., 2007). The cells were pre-cultured in a Reinforced Clostridial Medium (RCM) at 37 °C under an anaerobic condition. The composition of the RCM was as follows (all % wt/vol): meat extract, 1.0; peptone, 0.5; yeast extract, 0.3; D(+) glucose 0.5; starch, 0.1; sodium chloride, 0.5; sodium acetate, 0.3; and L-cysteinium chloride, 0.05. The cells were harvested at the end of the exponential phase, centrifugated and washed with a PBS (Phosphate Buffered Saline) buffer and used as inocula for the batch experiment. 2.2. Experiment for the determination of carbon-flow distribution The distribution of the carbon-flow in the metabolic pathways at different pHs (6.0, 6.3 and 6.7) was investigated related to hydrogen fermentation. At low pHs (<5.7), lactate and acetate be-

b1 b2 b3 c

molar production of acetate, molacetate/molG (Eq. (10)) molar production of butyrate, molbutyrate/molG (Eq. (17)) molar production of ethanol, molethanol/molG (Eq. (20)) molar production of biomass, C-molDW/molG (Eq. (22))

Subscripts ATP adenosine triphosphate ADP adenosine diphosphate DW dry weight G glucose

came the major fermentation products instead of butyrate and H2 gas (Zhu and Yang, 2004). To evaluate and quantify accurately the effects of pH on the carbon material and energy balances, experiments were performed three times in a sterilized bioreactor of 3 L working volume filled with a CGM (Clostridial Growth Medium). The medium was composed of the following materials (all % wt/vol): peptone, 0.4; sodium chloride, 0.3; potassium monophosphate, 0.1; potassium diphosphate 0.1; magnesium chloride, 0.03; ferrous sulfate heptahydrate, 0.03; and L-cysteinium chloride, 0.05. The 5 ml of the concentrated cells were inoculated and incubated in the reactor controlled with three pH conditions. Oxygen within the headspace of the reactor was removed by flushing with nitrogen gas for 10 min. The anaerobic reactor was agitated at 150 rpm with an initial glucose concentration of 111.2 mM and temperature of 37 °C. The pH was maintained by automatic titration with 5 N NaOH and 5N HCl solutions. The experimental data showing the final concentrations of the metabolites (acetate, butyrate, lactate, ethanol, carbon dioxide, and hydrogen), and biomass were utilized to determine the carbon material and energy balances. The glucose conversion stoichiometric equation is depicted in the Appendix. The balance equations were based on the consumption of 1 mol glucose and determined by analyses of all the measurable metabolites, substrate and cell mass during glucose fermentation. As shown in the scheme of glucose catabolism, it was assumed that (i) two molecules of ATP were generated from glucose to pyruvate via an Embden–Meyerhof–Parnas (EMP) pathway, (ii) two extra ATP molecules per glucose equivalent were formed by AK (acetate kinase), (iii) two NAD+ molecules per glucose eq. were reduced to NADHþ 2 by GAPDH (glyceraldehyde-3-phosphate dehydrogenase, not shown in Fig. 1) during EMP pathway (Desvaux et al., 2000), (iv) Two NAD+ per glucose eq. were produced by BHBD (b-hydroxylbutyryl-CoA dehydrogenase) and BCD (butyryl-CoA dehydrogenase), (v) two extra NAD+ were formed by LDH (lactate dehydrogenase) (vi) four NAD+ molecules were generated by ALDH (acetaldehyde dehydrogenase) and ADH (alcohol dehydrogenase), (vii) the carbon fraction for microbial growth was estimated assuming the dry cell composition reported by Zeng et al. (1993) (Fig. 1). In most cases, ammonium is used as either the sole nitrogen source or in combination with another nitrogen source, yielding the following general expression for the degree of reduction k of a compound with the elemental composition CHaObNc: k = 4 + a  2b  3c (Stephanopoulos et al., 1998). 2.3. Analytical methods The biogas and liquid suspension from the anaerobic reactor was constantly sampled for analyses. The total volume of evolved gases was measured using a water displacement method. The biogas composition was analyzed using gas chromatography (model 6890N, Agilent Inc.) using a pulsed discharge detector. The biogas

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Fig. 1. The primary metabolic network map of C. tyrobutyricum JM1 showing the experimental end products yields at three pH conditions (6.0, 6.3 and 6.7). All fluxes were given in C-mol carbon transferred and refer to the consumption of 100 C-mol glucose. The enzymes involved by their abbreviations are: HYDA, hydrogenase; PTA, phosphotransacetylase; AK, acetate kinase; THL, thiolase; BHBD, b-hydroxybutyryl-CoA dehydrogenase; CRO, crotonase; BCD, butyryl-CoA dehydrogenase; PTB, phosphotransbutyrylase; BK, butyrate kinase; LDH, L-lactate dehydrogenase; ALDH, acetaldehyde dehydrogenase; and ADH, alcohol dehydrogenase.

consisted of H2 and CO2. Collected effluents were centrifuged at 8000g for 10 min, and then the supernatants were acidified by formate followed by filtration through a membrane of 0.2-lm pore

size before analysis. The concentrations of ethanol and volatile fatty acids (VFAs) excluding lactate were analyzed using gas chromatography equipped with a flame ionization detector (FID) and a

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HP-INNOWax capillary column, with helium as a carrier gas at 2.2 mL/min. Both the injector and detector were kept at 250 °C, while the oven was held at 60 °C for 1 min, heated to 220 °C at the heating rate of 10 °C/min and maintained at 220 °C. Glucose, pyruvate and lactate were analyzed using high performance liquid chromatography (Model 1100, Agilent Inc.) with a refractive index detector (RID) and an Aminex HPX-87H column (300 mm  7.8 mm), and using 8 mN H2SO4 as the mobile phase. Cell growth was analyzed spectrophotometrically at 600 nm and calibrated against a dry cell weight measurement (1OD600 = 0.83 gDW/L). For the analysis of the dry cell weight, 20 mL samples were centrifuged for 5 min at 8000g, washed with a PBS buffer, and dried at 105 °C to a constant weight. The carbon fraction consumed for growth can be estimated assuming the elemental composition of the biomass (CH1.75O0.5N0.25) and neglecting the amount of CO2 produced in biomass synthesis g/mol (Converti and Perego, 2002; Zeng et al., 1993).

Table 1 Carbona material balance of the batch anaerobic fermentation of 1 mol glucose by C. tyrobutyricum JM1, carried out at three pH conditions (values are given in C-mol)

3. Results and discussion

6.3

6.7

6.0

6.0

6.0

Products Acetate Butyrate Lactate Ethanol Biomass CO2b Total products Error (%)

0.674 2.150 0.109 0.000 1.968 0.966 5.867 2.2

0.426 2.752 0.055 0.002 1.245 1.513 5.993 0.1

0.752 2.433 0.000 0.000 2.018 0.732 5.935 1.0

Product (%)c Acetate Butyrate Lactate Ethanol Biomass CO2

11.49 36.65 1.86 0.00 33.54 16.46

7.11 45.92 0.92 0.03 20.77 25.25

12.67 40.99 0.00 0.00 34.00 12.34

b c

Values are the averages of three different experiments. CO2 in the liquid phase was ignored. Carbon distribution (%) from total products-carbon.

Table 2 Energy balance of the batch anaerobic fermentation of 1 mol glucose by C. tyrobutyricum JM1, carried out at different pH conditions pH

6.0

6.3

6.7

Reactants Glucose

24

24

24

Products Acetate Butyrate Lactate Ethanol Biomass Hydrogen Total products Error (%)

2.696 10.75 0.436 0.000 7.872 1.785 23.54 1.9

1.704 13.760 0.219 0.013 4.981 3.241 23.918 0.3

3.008 12.165 0.000 0.000 8.072 1.009 24.25 1.0

lactate can be fermented to butyrate, H2 and CO2 in the presence of acetate. The subsequent conversion of lactate to butyrate is energetically favorable as the following equations:

Lactate þ 0:4Acetate þ 0:7Hþ ! 0:7Butyrate þ 0:6H2 þ CO2 þ 0:4H2 O Lactate þ Acetate þ H ! Butyrate þ 0:8H2 þ 1:4CO2 þ 0:6H2 O

6.0

Reactants Glucose

a

Glucose concentration plays an important role on the yield and production rate of hydrogen (Fabiano and Perego, 2002). A low initial glucose concentration results in the low rate of the fermentation steps, and the fermentation time increases as the initial substrate concentration increases. Temperature is also known to affect the maximum specific growth, substrate utilization rate and the metabolic pathway of the microorganisms, resulting in a shift of by-product compositions (Lay, 2000; Van Ginkel et al., 2001). Therefore, both initial glucose concentration and temperature in all experiments were controlled at 111.2 mM and 37 °C, respectively. Carbon material and energy balances were used to check the consistency of the data and identify the most probable metabolic pathways by the pH conditions in glucose fermentation. All fluxes were given in C-mol carbon transferred and refer to the consumption of 100 C-mol glucose as outlined in Fig. 1. The metabolites including acetate, butyrate, lactate, and ethanol can be simultaneously produced depending on the pH values. The carbon material and energy balance data obtained from H2 fermentation are summarized in Tables 1 and 2. The balances are satisfactory as the error of the carbon recovery compared to the consumption of glucose (C-mol) was less than 2.2% and 1.9%, respectively. In Table 1, the carbon balance calculated by taking into account the conversion of glucose into biomass and metabolites is in the range of

þ

pH

0;

DG0; ¼ 183:9 kJ=mol

DG ¼ 59:4 kJ=mol

97.8–99.9%. Moreover, the fractional carbon distributions in various metabolites were occupied with these ranges at different pHs: butyrate (36.65–45.92%), acetate (7.11–12.67%), lactate (0.00–1.86%), biomass (20.77–34.00) and CO2 (12.34–25.25%). In the case of a metabolite such as ethanol, the carbon distribution was below 0.03% regardless of the pH conditions. The majority of fractions of the metabolites produced were occupied with butyrate, which could explain why C. tyrobutyricum is a good producer of butyrate, i.e., the butyric clostridia. Butyrate is known to be a result of an anaerobic metabolism of Clostridium species bacteria such as Clostridium butyricum because anaerobic bacteria can not use the Tricarboxylic acid (TCA) cycle as a complete pathway (Gaudy and Gaudy, 1981). Moreover, Balow et al. (1992) reported that

ð3Þ ð4Þ

It is well known that a number of microorganisms such as C. acetobutylicum and Butyribacterium methylotrophicum as well as C. tyrobutyricum are related to the above reactions (Hashsham et al., 2000). Like the preceding equations, the predominant fermentation end product of glucose catabolism at each pH condition was butyrate. A maximum concentration of butyrate was obtained at pH 6.3. A metabolic pathway shift from lactate and acetate to butyrate production was observed from pH 6.0 to 6.3. However, the carbon fraction for butyrate decreased from 2.8 to 2.4 C-mol at pH 6.7 (Table 1). This phenomenon could likely be due to a product inhibition phenomenon. It was known that higher butyrate concentrations induced lower yields and productivities of butyrate due to butyrate inhibition (Zhu and Yang, 2004).

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Glucose þ 2H2 O ! 2Acetate þ 2CO2 þ 4H2 Glucose ! Butyrate þ 2CO2 þ 2H2

DG0; ¼ 182:4 kJ

0;

DG ¼ 257:1 kJ

ð5Þ ð6Þ

The above reactions indicate that acetate and butyrate can be formed during H2 fermentation (Hallenbeck, 2005). Therefore, the decrease of butyrate mainly resulted in the increase of acetate at pH 6.7. While H2 production was maximized with the highest concentration of butyrate at pH 6.3, the biomass formation was the lowest (Tables 1 and 2). It was coincident with the result that the highest biomass yield (0.46 mmol/mmol glucose) had the lowest hydrogen and butyrate yield (1.47 and 0.37 mmol/mmol glucose, respectively) (Lin et al., 2007). It is known that butyrate is an inhibitor to microbial growth (Berrios-Rivera et al., 2000; Zhu and Yang, 2004). The carbon portion to lactate increased from pH 6.7 to 6.0, although it was a negligible amount compared to the butyrate and acetate. Lower pH favored lactate production with higher activities of lactate dehydrogenase (LDH) (Dabrock et al., 1992). Appendix and Table 3 show that detailed metabolic reactions and their stoichiometric coefficients, respectively. The ultimate molar yields of products per mol of consumed glucose are presented in the table. From pH 6.0 to 6.3, butyrate production (b2) increased from 0.511 to 0.688 mol butyrate/mol glucose, while the percentage of glucose addressed to the growth (c) decreased from 0.078 to 0.051 C-molDW/mol glucose. Therefore, hydrogen production increased with increase of butyrate concentration and decrease of cell mass at pH 6.3. However, butyrate molar yield decreased at pH 6.7, whereas the portions of cell mass increased by reduced inhibition of butyrate. The experimental values were used to calculate the molar yield of ATP (YATP/G). ATP is generated in the fermentative processes when butyrate and acetate are the main metabolites as shown in Fig. 1. In acid fermentation, Mosey (1983) assumed only two moles of ATP were produced, however, Gaudy and Gaudy (1981) elucidated the possibility for an additional 1 mol of ATP for each mole of butyrate formed. As indicated in Table 3, pH condition scarcely influenced the molar production of ATP (YATP/G) in the range of 2.2–2.5 mol/mol glucose and the yields were very low values similar to those of Enterobacter aerogenes, which is known to be the characteristics of anaerobic fermentation (Converti and Perego, 2002). Under steady–state conditions, the ATP production must be necessarily balanced with the ATP consumption, resulting in a no net accumulation of ATP (Converti and Perego, 2002). Thus, the average value of ATP for the synthesis of biomass precursors and their subsequent polymerization to biomass (i.e., YX/ATP) can be estimated and calculated from the values of YATP/G and YX/G (Table 3). The average value of YX/ATP under three pH conditions (0.17 C-molDW/molATP) was lower than that of the aerobic conditions by Aerobacter aerogenes (0.35–1.0 C-molDW/molATP), which

could be explained that the formation of biomass consumed more ATP under anaerobiosis than in the presence of oxygen (Converti and Perego, 2002; Converti et al., 2003). Moreover, the value of Yx/ATP (C-molDW/molATP) at pH 6.3 was lower than those of the remaining pH conditions, which indicated more energy should be required to shift the metabolisms from metabolite production related to hydrogen to biomass synthesis. Payot et al. (1998) demonstrated that the formation of final products such as butyrate and acetate corresponded to an accumulation of intracellular NADHþ 2. The pathways to the increased formation of reducing equivalents could also regulate the enhanced production of hydrogen by NFOR and [FeFe]-hydrogenase. Therefore, pH 6.3 was of great advantage to the production of hydrogen, with the concomitant formation of butyrate in glucose metabolism by C. tyrobutyricum JM1. 4. Conclusion The shifts in the flows and fluxes of carbon by the pH conditions were determined and estimated through carbon material and energy balances. They could provide qualitative information and critical guidance for effective metabolic design or optimization, in this case, the maximization of hydrogen production by understanding the regulatory metabolism of C. tyrobutyricum JM1. The condition of pH 6.3 was an advantage to the production of hydrogen and butyrate yielding a low biomass with low energetic growth efficiencies. The consistency and accuracy of the experimental data in carbon material and energy balances were proved by the high carbon recoveries. As a further study, the pathway or gene will be knocked-out or over-expressed based on the consequences for enhancing hydrogen productivity. Acknowledgements This work was financially supported by the Korea Science and Engineering Foundation through the Advanced Environmental Biotechnology Research Center (R11-2003-006) at Pohang University of Science and Technology. Appendix. Clostridium tyrobutyricum JM1 metabolism Lactate production aGlucose þ 2aNADþ þ 2aADP þ 2aPi ! 2aPyruvate þ 2aNADHþ 2 þ 2aATP þ 2aH2 O 2aPyruvate þ

2aNADHþ 2

ð1Þ þ

! 2aLactate þ 2aNAD

ð2Þ

Net (Eqs. (1) and (2)): Table 3 Stochiometric coefficients and products molar yields (mol/mol glucose) of glucose fermentations by C. tyrobutyricum JM1 at different pH conditions pH

6.0

6.3

6.7

Coefficient a b b1 b2 b3 c

0.017 0.671 0.160 0.511 0.000 0.078

0.009 0.794 0.106 0.688 0.0005 0.051

0.000 0.703 0.166 0.537 0.000 0.074

Products yields YATP/G YX/G YX/ATP

2.208 0.468 0.212

2.509 0.310 0.124

2.275 0.446 0.196

See Appendix for calculating stochiometric coefficients and molar yields.

aGlucose þ 2aADP þ 2aPi ! 2aLactate þ 2aATP þ 2aH2 O

ð3Þ

Pyruvate–ferredoxin oxidoreductase (b = b1 + b2 + b3) bGlucose þ 2bNADþ þ 2bADP þ 2bPi ! 2bPyruvate þ 2bNADHþ 2 þ 2bATP þ 2bH2 O

ð4Þ

2bPyruvate þ 2bCoA þ 2bFd ! 2bAcetyl-CoA þ 2bCO2 þ 2bFdH2

ð5Þ

Net (Eqs. (4) and (5)): bGlucose þ 2bNADþ þ 2bADP þ 2b Pi þ 2bCoA þ 2bFd ! 2bFdH2 þ 2bNADHþ 2 þ 2bATP þ 2b H2 O þ 2bCO2 þ 2bAcetyl-CoA

ð6Þ

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Anaerobic metabolism of Clostridium tyrobutyricum JM1

Hydrogen production (Ferredoxin: hydrogenase activity) 2bFdH2 ! 2bFd þ 2bH2

ð7Þ

Net (Eqs. (3), (21)–(23)), where: a + b + c = 1 and b = b1 + b2 + b3: Glucose þ ð2a þ 2b þ 2b1 þ b2 Þ ADP þ ð2a þ 2b þ 2b1 þ b2 ÞPi

Acetate production

þ 1:5cNH3

2b1 Acetyl-CoA þ 2b1 Pi ! 2b1 Acetylphosphate þ 2b1 CoA

ð8Þ

2b1 Acetylphosphate þ 2b1 ADP ! 2b1 Acetate þ 2b1 ATP

ð9Þ

Net (Eqs. (8) and (9)):

! 2aLactate þ 2b1 Acetate þ b2 Butyrate þ 2b3 Ethanol þ 2bCO2 þ ð4b  2b2  4b3  0:873cÞH2 þ ð2a þ 2b þ 2b1 þ b2 ÞATP þ ð2a þ 2b þ b2 þ 3cÞH2 O þ 6cCH1:75 O0:5 N0:25

2b1 Acetyl-CoA þ 2b1 Pi þ 2b1 ADP ! 2b1 Acetate þ 2b1 CoA þ 2b1 ATP

ð24Þ

ð10Þ References

Butyrate production 2b2 Acetyl-CoA ! b2 Acetoacetyl-CoA þ b2 CoA

ð11Þ

b2 Acetoacetyl-CoA þ b2 NADHþ 2 ! b2 3-Hydroxybutyryl-CoA þ b2 NADþ

ð12Þ

b2 3-Hydroxybutyryl-CoA ! b2 Crotonyl-CoA þ b2 H2 O

ð13Þ

þ b2 Crotonyl-CoA þ b2 NADHþ 2 ! b2 Butyryl-CoA þ b2 NAD

ð14Þ

b2 Butyryl-CoA þ b2 Pi ! b2 Butyrylphosphate þ b2 CoA

ð15Þ

b2 Butyrylphosphate þ b2 ADP ! b2 Butyrate þ b2

ð16Þ

ATP Net (Eqs. (11)–(16)): 2b2 Acetyl-CoA þ 2b2 NADHþ 2 þ b2 Pi þ b2 ADP ! b2 Butyrate þ 2b2 CoA þ 2b2 NADþ þ b2 ATP þ b2 H2 O

ð17Þ

Ethanol production 2b3 Acetyl-CoA þ 2b3 NADHþ 2 ! 2b3 Acetaldehyde þ 2b3 NADþ þ 2b3 CoA þ 2b3 Acetaldehyde þ 2b3 NADHþ 2 ! 2b3 Ethanol þ 2b3 NAD

ð18Þ ð19Þ

Net (Eqs. (18) and (19)): þ 2b3 Acetyl-CoA þ 4b3 NADHþ 2 ! 2b3 Ethanol þ 4b3 NAD

þ 2b3 CoA

ð20Þ

Net (Eqs. (6), (7), (10), (17) and (20)): bGlucose þ ð2b  2b2  4b3 ÞNADþ þ ð2b þ 2b1 þ b2 ÞADP þ ð2b þ 2b1 þ b2 Pi ! 2b1 Acetate þ b2 Butyrate þ 2b3 Ethanol þ 2bCO2 þ 2bH2 þ ð2b  2b2  4b3 ÞNADHþ 2 þ ð2b þ 2b1 þ b2 ÞATP þ ð2b þ b2 ÞH2 O

ð21Þ

Cell growth cGlucose þ 1:5cNH3 þ 0:873cNADHþ 2 ! 6cCH1:75 O0:5 N0:25 þ 3cH2 O þ 0:873cNADþ

ð22Þ

Formation of H2 from NADHþ 2 The reducing equivalents generated on all the metabolic pathways are used to produce molecular hydrogen by hydrogenase (Chen et al., 2006) ð2b  2b2  4b3  0:873cÞNADHþ 2 ! ð2b  2b2  4b3  0:873cÞ NADþ þ ð2b  2b2  4b3  0:873cÞH2

ð23Þ

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