Abstract
By using hydrogenase gene-targeted polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), the predominant clostridial hydrogenase that may have contributed to biohydrogen production in an anaerobic semi-solid fermentation system has been monitored. The results revealed that a Clostridium pasteurianum-like hydrogenase gene sequence can be detected by both PCR and RT-PCR and suggested that the bacterial strain possessing this specific hydrogenase gene was dominant in hydrogenase activity and population. Whereas another Clostridium saccharobutylicum-like hydrogenase gene can be detected only by RT-PCR and suggest that the bacterial strain possessing this specific hydrogenase gene may be less dominant in population. In this study, hydrogenase gene-targeted fluorescence in situ hybridization (FISH) and flow cytometry analysis confirmed that only 6.6% of the total eubacterial cells in a hydrogen-producing culture were detected to express the C. saccharobutylicum-like hydrogenase, whereas the eubacteria that expressed the C. pasteurianum-like hydrogenase was 25.6%. A clostridial strain M1 possessing the identical nucleotide sequences of the C. saccharobutylicum-like hydrogenase gene was then isolated and identified as Clostridium butyricum based on 16S rRNA sequence. Comparing to the original inoculum with mixed microflora, either using C. butyricum M1 as the only inoculum or co-culturing with a Bacillus thermoamylovorans isolate will guarantee an effective and even better production of hydrogen from brewery yeast waste.
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References
Amann RI, Binder BJ, Olsen RJ, Chrisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed populations. Appl Environ Microbiol 56:1919–1925
Auling G, Pilz F, Busse HJ, Karrasch S, Streichan M, Schön G (1991) Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic–aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp. Appl Environ Microbiol 57:3585–3592
Cammack R (1999) Hydrogenase sophistication. Nature 397:214–215
Chang JJ, Chen WE, Shih SY, Yu SJ, Lay JJ, Wen FS, Huang CC (2006) Molecular detection of the clostridia in an anaerobic biohydrogen fermentation system by hydrogenase mRNA-targeted reverse transcription-PCR. Appl Microbiol Biotechnol 70:598–604
Chen WM, Tseng ZJ, Lee KS, Chang JS (2005) Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int J Hydrogen Energy 30:1063–1070
Cloete TE, Steyn PL (1988) A combined membrane filter-immunofluorescent technique for the in situ identification and enumeration of Acinetobacter in activated sludge. Water Res 22:961–969
Devereux R, Mundfrom GW (1994) A phylogenetic tree of 16S rRNA sequences from sulfate-reducing bacteria in sandy marine sediment. Appl Environ Microbiol 60:3437–3439
Esteso MA, Estrella CN, Podestá JJ (1996) Evaluation of the absorption on mild steel of hydrogen evolved in glucose fermentation by pure cultures of Clostridium acetobutylicum and Enterobacter. Sens Actuators B Chem 32:27–31
Fang HH, Zhang T, Liu H (2002) Microbial diversity of a mesophilic hydrogen-producing sludge. Appl Microbiol Biotechnol 58:112–118
Gorwa MF, Croux C, Soucaille P (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 178:2668–2675
Hiraishi A (1988) Respiratory quinone profiles as tools for identifying different bacterial populations in activated sludge. J Gen Appl Microbiol 34:39–56
Howgrave-Graham AR, Steyn PL (1988) Application of the fluorescent-antibody technique for the detection of Sphaerotilus natans in activated sludge. Appl Environ Microbiol 54:799–802
Karube I, Urano N, Matsunaga T, Suzuki S (1982) Hydrogen production from glucose by immobilized growing cells of Clostridium butyricum. Eur J Appl Microbiol 16:5–9
Kataoka N, Miya A, Kiriyama K (1997) Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria. Water Sci Technol 36:41–47
Lay JJ (2000) Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 68:269–278
Lay JJ (2001) Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose. Biotechnol Bioeng 74:280–287
Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial population by DGGE analysis of polymerase chain reaction amplified genes encoding for 16S rRNA. Appl Environ Microbiol 62:2676–2680
Nielsen AT, Liu WT, Filipe C, Grady L, Molin S, Stahl DA (1999) Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl Environ Microbiol 65:1251–1258
Palmer CJ, Tsai YL, Paszko-Kolva C, Mayer C, Sangermano LR (1993) Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods. Appl Environ Microbiol 59:3618–3624
Snaidr J, Amann R, Huber I, Ludwig W, Schleifer KH (1997) Phylogenetic analysis and in situ identification of bacteria in activated sludge. Appl Environ Microbiol 63(7):2884–2896
Ueno Y, Otauka S, Morimoto M (1996) Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture. J Ferment Bioeng 82:194–197
Völsch A, Nader WF, Geiss HK, Nebe G, Birr C (1990) Detection and analysis of two serotypes of ammonia-oxidizing bacteria in sewage plants by flow-cytometry. Appl Environ Microbiol 56:2430–2435
Zhang T, Fang HP (2000) Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities. Biotechnol Lett 22:399–405
Acknowledgement
This work was supported by the grants (NSC92-2211-E-005-008, NSC94-2211-E-005-015, and NSC95-ET-7-005-001-ET) from the National Science Council, Taiwan and a grant (no. 93-D0125) from the Bureau of Energy, Ministry of Economic Affairs, Taiwan, Republic of China.
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Jen, C.J., Chou, CH., Hsu, PC. et al. Flow-FISH analysis and isolation of clostridial strains in an anaerobic semi-solid bio-hydrogen producing system by hydrogenase gene target. Appl Microbiol Biotechnol 74, 1126–1134 (2007). https://doi.org/10.1007/s00253-006-0740-8
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DOI: https://doi.org/10.1007/s00253-006-0740-8