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Comment on “Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge”
International Journal of Hydrogen Energy 31 (2006) 1797 – 1798 www.elsevier.com/locate/ijhydene
Reply
Comment on “Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge” Chen et al. [1] examined the optimal conditions to improve hydrogen-gas production in batch reactions with Clostridium butyricum, varying medium composition, pH, and initial substrate concentration (sucrose). The study compared hydrogen gas, biomass, ethanol, and total volatile fatty acids with different pHs from 5 to 6.5 at a constant initial sucrose concentration of 20 g/L. Total volatile fatty acids were the sum of acetic acid, propionic acid, and butyric acid in this study. The authors reported that the highest yield was 2.78 mol H2 /mol sucrose at pH 5.5, with lower values for pHs 6.0 and 6.5 and no H2 gas at a pH of 5.0. They concluded that the highest hydrogen production was associated with a large generation of total volatile fatty acids and that hydrogen production decreased at pH 6.0 and 6.5 due to biomass synthesis becoming a larger electron sink. However, their conclusions are not reliable, because the measured fermentation products did not close the electron-equivalent mass balance and also were inconsistent among the experiments. The electron-equivalent mass balance (expressed as mg COD) is CODsuc,in = CODsuc,res + CODbio + CODgas + CODinter
(1)
CODsuc,in : mg COD of sucrose initially, CODsuc,res : mg COD of residual sucrose, CODbio : mg COD of biomass in reactor liquid, CODgas : mg COD of H2 evolved, CODinter : mg COD of organic fermentation products in reactor liquid; volatile acids and ethanol were measured and converted to COD of all intermediates by Chen et al. [1]. Table 1 lists the measured mg COD values for all the components in Eq. (1). The notes of Table 1 indicate how we obtained each value. The most important conclusion from Table 1 is that none of the experiments showing hydrogen generation had more than
69% closure of the electron-equivalent balance, and two experiments were missing 62–64% of the electron equivalents. Furthermore, the percentage closures of the electron-equivalent balances varied widely among the experiments, with the experiment showing no hydrogen having 98% closure. The lack of closure and the inconsistency among the closures mean that interpretations about what conditions were optimal and why are unreliable. Further inspection of Table 1 shows that biomass was a relatively small sink for electrons, and it did not change much among the experiments. Therefore, the conclusion that biomass is the reason by pH = 5.5 is “optimal” almost certainly is incorrect. The fact that up to 64% of the electron equivalents were missing in the experiments was due to one or more of three causes. First, an important (perhaps dominant) electron sink was not measured. Possibilities could be CH4 , H2 S, and H2 O from O2 , butanol, propanol, and atypical acids. The first two possibilities should be impossible, since C. butyricum does not do methanogenesis [3], and the sulfate input was equal to only about 10 mg of COD. Air leakage into the experimental system would provide O2 , but the experimental system seems to have been sealed and should have been at positive pressure. Therefore, the most likely missing sinks are organic products not measured. Yu and Fang [4] reported that propanol and butanol increased significantly with increasing substrate concentration, their fractions of total intermediates reaching 32.8% for 20 g COD/L. Zheng and Yu [5] demonstrated a butyrate supplement caused significant buildup of valeric and capric acids, representing 27% of total intermediates. Annous et al. [6], van Ginkel and Sung [7], and Ren et al. [8] reported that alcohols were main intermediates under acidic conditions. Second, the analytical measurements for one or more of the fermentation products could have been systematically low. In general, the analytical methods used are trustworthy if properly carried out. Third, the calculations to convert from the measured concentrations of organic products to COD may be been
1798
Reply / International Journal of Hydrogen Energy 31 (2006) 1797 – 1798
Table 1 Electron mass balance (as mg COD) for the different pH conditions when the initial sucrose concentration contained 34,000 mg COD (20,000 mg COD/L × 1.7 L) pH
Residual sucrose COD
Intermediates CODa
Biomass CODb
H2 CODc
COD Sumd
Missing Electron Equivalentse (%)
6.5 6.0 5.5 5.0
680 680 340 32,980
7330 7720 16,870 450
2460 2270 1860 0
1810 2280 4380 0
12,280 12,950 23,450 33,430
64 62 31 2
a Sum of mg COD of acetic acid, butyric acid, propionic acid, and ethanol, as reported in the original paper as SMP in mgCOD/L × 1.7 L. b Calculated with [mg cell/mg sucrose removed] × mg sucrose removed × [1.42 mg COD/mg cell], assuming that cell formula is
C5 H7 O2 N [2]. c Calculated with [mole H /mole sucrose removed] × mole sucrose removed × [16g COD/mole H ] × [1000 mg/g]. 2 2 d Calculated as the sum of mg COD of residual sucrose, intermediates, biomass, and H from previous four columns. 2 sumd (mg COD) e Calculated with 1 − × 100. initial sucrose (34,000 mg COD)
in error; the primary data and calculations are not shown in the paper. In summary, it is infeasible to conclude anything about optimization of H2 production from experiments whose electron balances are so incomplete and inconsistent. Our discussion underscores the importance of establishing electron balance (and other mass balances) in research addressing biohydrogen production. When electron mass balances are significantly deficient, as is the case in [1], researchers need to identify and correct the problem before interpreting the results. References [1] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int J Hydrogen Energy 2005;30: 1063–70. [2] Rittmann BE, McCarty PL. Environmental biotechnology: principle and applications. New York: McGraw-Hill; 2001. [3] Ferry J. Methanogenesis: ecology, physiology, biochemistry, & genetics. New York: Chapman & Hall Inc.; 1993.
[4] Yu HQ, Fang HHP. Anaerobic acidification of a synthetic wastewater in batch reactors at 55 ◦ C. Wat Sci Technol 2002;46:153–7. [5] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic culture. J Environ Manage 2005;74:65–70. [6] Annous BA, Shieh JS, Shen GJ, Jain MK, Zeikus JG. Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH. Appl Microbiol Biotechnol 1996;45: 804–10. [7] van Ginkel S, Sung S. Biohydrogen production as a function of pH and substrate concentration. Environ Sci Technol 2001;35:4726–30. [8] Ren NQ, Wang BZ, Huang JC. Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol Bioeng 1997;54:428–33.
Hyung Sool Lee, Jinwook Chung, Bruce E. Rittmann Center for Environmental Biotechnology The Biodesign Institute at Arizona State University, 1001 S. McAllister Ave. Tempe, AZ 85287-5701, USA E-mail address: [email protected]
Reply
Comment on “Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge” Chen et al. [1] examined the optimal conditions to improve hydrogen-gas production in batch reactions with Clostridium butyricum, varying medium composition, pH, and initial substrate concentration (sucrose). The study compared hydrogen gas, biomass, ethanol, and total volatile fatty acids with different pHs from 5 to 6.5 at a constant initial sucrose concentration of 20 g/L. Total volatile fatty acids were the sum of acetic acid, propionic acid, and butyric acid in this study. The authors reported that the highest yield was 2.78 mol H2 /mol sucrose at pH 5.5, with lower values for pHs 6.0 and 6.5 and no H2 gas at a pH of 5.0. They concluded that the highest hydrogen production was associated with a large generation of total volatile fatty acids and that hydrogen production decreased at pH 6.0 and 6.5 due to biomass synthesis becoming a larger electron sink. However, their conclusions are not reliable, because the measured fermentation products did not close the electron-equivalent mass balance and also were inconsistent among the experiments. The electron-equivalent mass balance (expressed as mg COD) is CODsuc,in = CODsuc,res + CODbio + CODgas + CODinter
(1)
CODsuc,in : mg COD of sucrose initially, CODsuc,res : mg COD of residual sucrose, CODbio : mg COD of biomass in reactor liquid, CODgas : mg COD of H2 evolved, CODinter : mg COD of organic fermentation products in reactor liquid; volatile acids and ethanol were measured and converted to COD of all intermediates by Chen et al. [1]. Table 1 lists the measured mg COD values for all the components in Eq. (1). The notes of Table 1 indicate how we obtained each value. The most important conclusion from Table 1 is that none of the experiments showing hydrogen generation had more than
69% closure of the electron-equivalent balance, and two experiments were missing 62–64% of the electron equivalents. Furthermore, the percentage closures of the electron-equivalent balances varied widely among the experiments, with the experiment showing no hydrogen having 98% closure. The lack of closure and the inconsistency among the closures mean that interpretations about what conditions were optimal and why are unreliable. Further inspection of Table 1 shows that biomass was a relatively small sink for electrons, and it did not change much among the experiments. Therefore, the conclusion that biomass is the reason by pH = 5.5 is “optimal” almost certainly is incorrect. The fact that up to 64% of the electron equivalents were missing in the experiments was due to one or more of three causes. First, an important (perhaps dominant) electron sink was not measured. Possibilities could be CH4 , H2 S, and H2 O from O2 , butanol, propanol, and atypical acids. The first two possibilities should be impossible, since C. butyricum does not do methanogenesis [3], and the sulfate input was equal to only about 10 mg of COD. Air leakage into the experimental system would provide O2 , but the experimental system seems to have been sealed and should have been at positive pressure. Therefore, the most likely missing sinks are organic products not measured. Yu and Fang [4] reported that propanol and butanol increased significantly with increasing substrate concentration, their fractions of total intermediates reaching 32.8% for 20 g COD/L. Zheng and Yu [5] demonstrated a butyrate supplement caused significant buildup of valeric and capric acids, representing 27% of total intermediates. Annous et al. [6], van Ginkel and Sung [7], and Ren et al. [8] reported that alcohols were main intermediates under acidic conditions. Second, the analytical measurements for one or more of the fermentation products could have been systematically low. In general, the analytical methods used are trustworthy if properly carried out. Third, the calculations to convert from the measured concentrations of organic products to COD may be been
1798
Reply / International Journal of Hydrogen Energy 31 (2006) 1797 – 1798
Table 1 Electron mass balance (as mg COD) for the different pH conditions when the initial sucrose concentration contained 34,000 mg COD (20,000 mg COD/L × 1.7 L) pH
Residual sucrose COD
Intermediates CODa
Biomass CODb
H2 CODc
COD Sumd
Missing Electron Equivalentse (%)
6.5 6.0 5.5 5.0
680 680 340 32,980
7330 7720 16,870 450
2460 2270 1860 0
1810 2280 4380 0
12,280 12,950 23,450 33,430
64 62 31 2
a Sum of mg COD of acetic acid, butyric acid, propionic acid, and ethanol, as reported in the original paper as SMP in mgCOD/L × 1.7 L. b Calculated with [mg cell/mg sucrose removed] × mg sucrose removed × [1.42 mg COD/mg cell], assuming that cell formula is
C5 H7 O2 N [2]. c Calculated with [mole H /mole sucrose removed] × mole sucrose removed × [16g COD/mole H ] × [1000 mg/g]. 2 2 d Calculated as the sum of mg COD of residual sucrose, intermediates, biomass, and H from previous four columns. 2 sumd (mg COD) e Calculated with 1 − × 100. initial sucrose (34,000 mg COD)
in error; the primary data and calculations are not shown in the paper. In summary, it is infeasible to conclude anything about optimization of H2 production from experiments whose electron balances are so incomplete and inconsistent. Our discussion underscores the importance of establishing electron balance (and other mass balances) in research addressing biohydrogen production. When electron mass balances are significantly deficient, as is the case in [1], researchers need to identify and correct the problem before interpreting the results. References [1] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int J Hydrogen Energy 2005;30: 1063–70. [2] Rittmann BE, McCarty PL. Environmental biotechnology: principle and applications. New York: McGraw-Hill; 2001. [3] Ferry J. Methanogenesis: ecology, physiology, biochemistry, & genetics. New York: Chapman & Hall Inc.; 1993.
[4] Yu HQ, Fang HHP. Anaerobic acidification of a synthetic wastewater in batch reactors at 55 ◦ C. Wat Sci Technol 2002;46:153–7. [5] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological hydrogen production with mixed anaerobic culture. J Environ Manage 2005;74:65–70. [6] Annous BA, Shieh JS, Shen GJ, Jain MK, Zeikus JG. Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH. Appl Microbiol Biotechnol 1996;45: 804–10. [7] van Ginkel S, Sung S. Biohydrogen production as a function of pH and substrate concentration. Environ Sci Technol 2001;35:4726–30. [8] Ren NQ, Wang BZ, Huang JC. Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol Bioeng 1997;54:428–33.
Hyung Sool Lee, Jinwook Chung, Bruce E. Rittmann Center for Environmental Biotechnology The Biodesign Institute at Arizona State University, 1001 S. McAllister Ave. Tempe, AZ 85287-5701, USA E-mail address: [email protected]