为了探究梭菌属作为高效产氢发酵菌株的产氢性能。该研究通过从浓香型白酒窖泥中筛选出了1株具有产氢能力的菌株WZ-1,经过鉴定后为广西梭菌(Clostridium guangxiense)。在35 ℃厌氧条件下,WZ-1菌株能够以葡萄糖为底物的培养基上进行发酵,产生高含量氢气(58.99%)和丁酸(1 960 mg/L)。为了全面解析其功能特性,对WZ-1菌株进行基因组测序和注释。基因组分析显示,WZ-1基因组总长度为4 406 933 bp,GC含量31.95%,包含4 172条编码序列,3个rRNA基因和52个tRNA基因。通过菌株的KEGG代谢途径分析,揭示了WZ-1的丙酮酸脱羧过程产氢、NADH+H+氧化还原平衡调节产氢、一氧化碳产氢、固氮产氢及其产丁酸途径。研究结果从基因组层面深入解析了WZ-1的产氢产丁酸特性,展现了其在生物制氢、生物制丁酸和促进循环经济等方面具有一定的应用前景,也为开发清洁能源生产技术提供了参考。
To investigate the hydrogen production performance of Clostridium spp. as an efficient hydrogen-producing fermentation strain, a strain named WZ-1, identified as Clostridium guangxiense, was isolated from the pit mud of Nongxiangxing Baijiu due to its hydrogen-producing ability.Under anaerobic conditions at 35 ℃, strain WZ-1 demonstrated the capacity to produce high levels of hydrogen (58.99%) and butyric acid (1 960 mg/L) through fermentation on a medium with glucose as the substrate.To comprehensively analyze its functional properties, the genome of strain WZ-1 was sequenced and annotated.Genomic analysis revealed that the total length of the WZ-1 genome was 4 406 933 bp with a guanine cytosine content of 31.95%, including 4 172 coding sequences, 3 rRNA genes, and 52 tRNA genes.Analysis of KEGG metabolic pathways of the strain revealed hydrogen production during pyruvate decarboxylation, hydrogen production by NADH+H+ redox balance regulation, hydrogen production by carbon monoxide, hydrogen production by nitrogen fixation, and its butyric acid-producing pathway in WZ-1.The results of this study analyzed the hydrogen and butyric acid production characteristics of WZ-1 at the genomic level and indicated that WZ-1 holds promising application prospects in the fields of hydrogen production, butyric acid production, and promoting circular economy initiatives.Furthermore, these findings provide valuable insights for the development of clean energy production technologies.
[1] ABE J O, POPOOLA A P I, AJENIFUJA E, et al.Hydrogen energy, economy and storage:Review and recommendation[J].International Journal of Hydrogen Energy, 2019, 44(29):15072-15086.
[2] 叶思岑. 梭状芽孢杆菌属近缘产氢菌种的产氢与代谢差异研究[D].沈阳:东北大学, 2021.
YE S C.Differences in hydrogen production and metabolism in closely related species of Clostridium[D].Shenyang:Northeastern University, 2021.
[3] WANG J L, YIN Y N.Progress in microbiology for fermentative hydrogen production from organic wastes[J].Critical Reviews in Environmental Science and Technology, 2019, 49(10):825-865.
[4] 姜淑敏, 汪吉霞, 王悦佳, 等.光合细菌产氢研究进展[J].现代农业科技, 2023(19):136-142.
JIANG S M, WANG J X, WANG Y J, et al.Research progress on hydrogen production by photosynthetic bacteria[J].Modern Agricultural Science and Technology, 2023(19):136-142.
[5] ZHAO X, LI D Y, XU S H, et al.Clostridium guangxiense sp.nov.and Clostridium neuense sp.nov., two phylogenetically closely related hydrogen-producing species isolated from lake sediment[J].International Journal of Systematic and Evolutionary Microbiology, 2017, 67(3):710-715.
[6] 杨静, 齐男, 赵鑫, 等.复合有机氮对产氢新菌C.guangxiense产氢的促进作用[J].可再生能源, 2019, 37(3):317-321.
YANG J, QI N, ZHAO X, et al.Promoting of compound organic nitrogen on hydrogen production by the novel species of C.guangxiense[J].Renewable Energy Resources, 2019, 37(3):317-321.
[7] 黄治国, 蒲领平, 罗惠波, 等.浓香型白酒酯化菌协同产酯的工艺优化[J].现代食品科技, 2021, 37(2):64-71;146.
HUANG Z G, PU L P, LUO H B, et al.Optimization of the conditions for synergistic production of esters by esterifying bacteria of luzhou-flavor liquor[J].Modern Food Science and Technology, 2021, 37(2):64-71;146.
[8] 郭燕, 邓杰, 任志强, 等.响应面优化酿酒酵母与窖泥酯化细菌协同发酵产丁酸乙酯和己酸乙酯[J].食品科学, 2021, 42(10):209-217.
GUO Y, DENG J, REN Z Q, et al.Optimization of the production of ethyl hexanoate and ethyl butyrate by cofermentation of Saccharomyces cerevisiae and esterifying bacteria from pit mud of Chinese Baijiu using response surface methodology[J].Food Science, 2021, 42(10):209-217.
[9] SCHUBERT M, LINDGREEN S, ORLANDO L.AdapterRemoval v2:Rapid adapter trimming, identification, and read merging[J].BMC Research Notes, 2016, 9:88.
[10] LUO R B, LIU B H, XIE Y L, et al.SOAPdenovo2:An empirically improved memory-efficient short-read de novo assembler[J].GigaScience, 2012, 1(1):18.
[11] BANKEVICH A, NURK S, ANTIPOV D, et al.SPAdes:A new genome assembly algorithm and its applications to single-cell sequencing[J].Journal of Computational Biology, 2012, 19(5):455-477.
[12] WALKER B J, ABEEL T, SHEA T, et al.Pilon:An integrated tool for comprehensive microbial variant detection and genome assembly improvement[J].PLoS One, 2014, 9(11):e112963.
[13] BESEMER J, LOMSADZE A, BORODOVSKY M.GeneMarkS:A self-training method for prediction of gene starts in microbial genomes.Implications for finding sequence motifs in regulatory regions[J].Nucleic Acids Research, 2001, 29(12):2607-2618.
[14] KALVARI I, ARGASINSKA J, QUINONES N, et al.Rfam 13.0:Shifting to a genome-centric resource for non-coding RNA families[J].Nucleic Acids Research, 2017, 46:D335-D342.
[15] CHOU K C, SHEN H B.Cell-PLoc:A package of Web servers for predicting subcellular localization of proteins in various organisms[J].Nature Protocols, 2008, 3(2):153-162.
[16] LUO H, LI T, ZHENG J, et al.Isolation, identification, and fermentation medium optimization of a caproic acid-producing Enterococcus casseliflavus strain from pit mud of Chinese strong flavor Baijiu ecosystem[J].Polish Journal of Microbiology, 2022, 71(4):563-575.
[17] CONESA A, GÖTZ S.Blast2GO:A comprehensive suite for functional analysis in plant genomics[J].International Journal of Plant Genomics, 2008, 2008:619832.
[18] ARRIAZA-GALLARDO F J, ZHENG Y C, GEHL M, et al.Fe]-hydrogenase, cofactor biosynthesis and engineering[J].Chembiochem, 2023, 24(20):e202300330.
[19] ŁUKAJTIS R, HOŁOWACZ I, KUCHARSKA K, et al.Hydrogen production from biomass using dark fermentation[J].Renewable and Sustainable Energy Reviews, 2018, 91:665-694.
[20] KNAPPE J, BLASCHKOWSKI H P, GRÖBNER P, et al.Pyruvate formate-lyase of Escherichia coli:The acetyl-enzyme intermediate[J].European Journal of Biochemistry, 1974, 50(1):253-263.
[21] 吴志宇. 纳米水铁矿对梭菌菌株BZ-1发酵产氢的促进机制研究[D].烟台:烟台大学, 2022.
WU Z Y.Promoting mechanism of ferrihydrite nanomaterials on hydrogen fermentation by Clostridium sp.BZ-1[D].Yantai:Yantai University, 2022.
[22] MENON A L, HENDRIX H, HUTCHINS A, et al.The delta-subunit of pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus is a redox-active, iron-sulfur protein:Evidence for an ancestral relationship with 8Fe-type ferredoxins[J].Biochemistry, 1998, 37(37):12838-12846.
[23] BRAGA J K, STANCARI R A, MOTTERAN F, et al.Metals addition for enhanced hydrogen, acetic and butyric acids production from cellulosic substrates by Clostridium butyricum[J].Biomass and Bioenergy, 2021, 150:105679.
[24] 尹梦. 丁酸梭菌的产氢影响因素及其代谢特性的研究[D].上海:上海师范大学, 2020.
YIN M.The influencing factors in hydrogen production and metabolic characteristics of Clostridium butyricum[D].Shanghai:Shanghai Normal University, 2020.
[25] WILCOXEN J, ZHANG B, HILLE R.Reaction of the molybdenum- and copper-containing carbon monoxide dehydrogenase from Oligotropha carboxydovorans with quinones[J].Biochemistry, 2011, 50(11):1910-1916.
[26] RAPSON T D, WOOD C C.Analysis of the ammonia production rates by nitrogenase[J].Catalysts, 2022, 12(8):844.
[27] DAVID BRITT R, RAO G D, TAO L Z.Bioassembly of complex iron-sulfur enzymes:Hydrogenases and nitrogenases[J].Nature Reviews.Chemistry, 2020, 4(10):542-549.
[28] WANG Z C, WATT G D.Large anions induce H2-production from the nitrogenase MoFe proteins of Clostridium Pasteurianum and Azotobacter vinelandii[J].Journal of Inorganic Biochemistry, 2020, 208:111075.
