一个新的产氢细菌的鉴定及产氢特性的研究

利用Hungate滚管技术从福建省漳州垃圾处理厂厌氧消化器的颗粒污泥中分离到一株产氢的细菌L15。菌株L15为严格厌氧的革兰氏阳性杆菌,菌体大小为0.5μm～0.7μm×2.5μm～5.0μm,以侧生鞭毛运动。在孢肉培养基上产生端生的卵圆形芽孢。温度生长范围15℃～45℃（最适温度30℃～37℃）;pH 范围5.0～8.4（最适pH 6.3～6.8）。该菌株不水解明胶和七叶灵,不还原硫酸盐,牛奶变酸但不凝固,发酵多糖和少数的单糖、双糖和寡糖;发酵葡萄糖的最终产物为乙酸、丁酸、H2和CO2。G+C含量为298mol%。16S rDNA序列分析表明,该菌株属于梭菌的簇Ⅰ,与Clostridium paraputrificum较为接近（相似性为97.1%）。通过生理特征和16S rDNA序列的同源性分析,表明菌株L15应是梭菌属簇Ⅰ中的一个新种,命名为Clostridium defluvii。菌株L15保藏在中国普通微生物菌种保藏中心,保藏号为AS1.3489。菌株L15的最佳产氢温度为34℃、pH为7.0。当葡萄糖浓度为0.4%时,氢气产率可达到1.41mol H2/mol 葡萄糖。该菌可利用下列底物产酸产氢,括号内为产氢率(底物浓度1%)：果糖(1.00mol H2/mol)、麦芽糖(2.17mol H2/mol)、蔗糖(1.69mol H2/mol)、菊糖(4.70mol H2/mol)、糖原(5.49mmol H2/g)、淀粉(7.34mmol H2/g)。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp. H-61为原始菌株, 先后经亚硝基胍(NTG)、紫外(UV)诱变, 选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下, 其产氢量为3024 mL/L, 比原始菌株提高了69.89%; 其最大产氢速率为33.19 mmol H2/g DW·h, 比原始菌株(19.74 mmol H2/g DW·h)提高了68.14%。经过多次传代试验, 稳定性良好。其发酵末端产物以乙醇和乙酸为主, 属于典型乙醇型发酵代谢类型。其最适产氢初始pH为6.5, 最适生长温度为36℃, 以蔗糖为最佳碳源。与原始菌株相比, 突变株HCM-23的产氢特性发生了改变, 如生长延滞期延长, 可利用无机氮源等。     

木糖发酵产氢菌的筛选及其生长产氢特性研究

利用改进的Hungate厌氧技术, 从牛粪堆肥中分离出一株能有效利用木糖发酵产氢的中温菌HR-1。通过16S rRNA系统发育树分析表明, 菌株 HR-1 与丙酮丁醇梭菌Clostridium acetobutylicum ATCC 824 相似性最高为96%, 结合生理生化和生长特性分析表明, HR-1是梭菌属Clostridium的一个新种, 命名为Clostridium sp. HR-1。菌株HR-1为单胞生长的规则杆状菌(0.3 mm ~0.6 mm)×(1.4 mm~2.3 mm), 革兰氏染色为阴性, 无荚膜、无鞭毛、表面光滑、无明显凸起, 专性厌氧菌。HR-1可在10°C~45°C, pH 4.0~10.0条件下生长; 37°C和pH 8.0分别为其最适生长条件。发酵PYG的主要发酵产物有氢气、二氧化碳、乙酸、丁酸及少量乙醇。HR-1可以利用有机氮源和无机氮源生长并产氢, 酵母提取物是其最佳产氢氮源。HR-1在木糖浓度为3 g/L和初始pH 6.5条件下, 其比产氢量为1.84 mol-H2/mol-木糖, 最大比产氢速率为10.52 mmol H2/h·g-细胞干重。HR-1可以亦利用葡萄糖、半乳糖、纤维二糖、甘露糖和果糖等碳源生长并发酵产氢, 发酵葡萄糖时比产氢量为2.36 mol-H2/mol-葡萄糖。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp.H-61为原始菌株,先后经亚硝基胍(NTG)、紫外(UV)诱变,选育得到1株高产突变株HCM-23.在葡萄糖浓度为10 g/L的条件下,其产氢量为3024 mL/L,比原始菌株提高了69.89%;其最大产氢速率为33.19 mmol H2/g DW·h,比原始菌株(19.74 mmolH2/g DW·h)提高了68.14%.经过多次传代试验,稳定性良好.其发酵末端产物以乙醇和乙酸为主,属于典型乙醇型发酵代谢类型.其最适产氢初始pH为6.5,最适生长温度为36℃,以蔗糖为最佳碳源.与原始菌株相比,突变株HCM-23的产氢特性发生了改变,如生长延滞期延长,可利用无机氮源等.     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatum Z7产氢效率的因素,采用生理学方法对其进行了研究。结果表明：乙醇型发酵菌A. elongatum Z7的最适产氢温度为37℃, 最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度> 60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

一株油藏嗜热厌氧杆菌的分离、鉴定及代谢产物特征

[目的]了解油藏环境中细菌的生理生化特性及代谢产物.[方法]采用Hungate厌氧操作技术从胜利油田罗801区块油层采出水中分离到一株厌氧杆菌SC-2.采用生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株的系统发育地位,用气相色谱分析其代谢产物.[结果]菌株SC-2为严格厌氧的革兰氏阴性杆菌,菌体大小为0.38 um×1.7um-3.9um,单生、成对或成串生长,产端生芽孢.温度生长范围40℃-75℃(最适温度70℃);pH范围5.5-9.5(最适pH 6.5);NaCl浓度范围0%～5%(最适NaCl浓度0%).能够利用葡萄糖、麦芽糖、甘露糖、木糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、丙酸、H2、CO2及少量的乳酸.菌株SC-2的(G C)mol%含量为30.8%,与Thermoanaerobacter mathranii subsp.mathranii的16S rDNA序列相似性为99.85%.菌株利用葡萄糖产乙酸、乙醇的最佳初始pH为8.0;酵母粉能刺激生长并显著提高发酵葡萄糖的产酸、产醇率;培养基中添加4%(V/V)的乙醇能明显抑制菌体生长.[结论]菌株SC-2是从特殊生境(油层采出水)中分离到的一株嗜热、耐盐的厌氧菌,其发酵葡萄糖产生的代谢产物有利于改善油藏中的微环境.菌株SC-2与T.mathranii subsp.mathranii 11426T的最适pH和最大耐受NaCl浓度有所不同,且二者的(G C)mol%含量差异较大.     

发酵产氢菌株的筛选及影响因素研究

采用双层平板法从污泥池中筛选出一株产氢较高的发酵菌株,经生理生化鉴定表明,分离菌株初步鉴定为消化链球菌属(Peptostreptococcus).研究静态培养条件下葡萄糖、pH、温度及和酵母膏对菌株产氢的影响及不同发酵时间段的产氢情况.结果表明,在葡萄糖浓度20.0 g/L,pH 7.0,温度37 ℃和酵母膏2.0 g/L时,产氢量达21.07 mmol/L,为初始培养条件下的4.14倍.同时,在24～36 h时间段产氢率达到最高,为0.44 mmol/(L·h),并且在60 h时产氢量达到最大累计产氢量的89.2%.     

浮霉菌门严格厌氧产氢细菌(Thermopirellula anaerolimosa)的分离及其生理特性

[目的]厌氧颗粒污泥中含有大量未知微生物资源,利用低浓度底物及添加抗生素的培养基进行厌氧发酵细菌的筛选,并对分离菌株进行生理生化特性研究.[方法]利用系列稀释法及亨盖特厌氧滚管技术从制糖废水厌氧处理反应器的颗粒污泥中分离到一株高温厌氧产氢细菌VM20-7T,通过16S rRNA基因序列同源性确定其系统发育地位.[结果]菌株VM20-7T为高温、严格厌氧、革兰氏阴性梨形细菌,细胞大小为(0.7-2.0)μm×(0.7-2.0) μm,不运动,不产芽胞.其生长温度范围为35℃-50℃(最适温度45℃),pH范围为6.0-8.3(最适pH7.0-7.5),NaCl耐受范围为0％-0.5％(w/v,最适浓度0％).菌株VM20-7T可利用葡萄糖、麦芽糖、核糖等多种糖类为唯一碳源生长,葡萄糖发酵终产物是乙酸和H2.该菌株不利用硝酸盐、硫酸盐等作为电子受体生长.G+C含量为60.9 mol％,16S rRNA基因序列同源性显示菌株属于浮霉菌门,但与已培养菌株的同源性较低,与梨形菌属一红小梨形菌属-芽殖小小梨形菌属(Pirellula-Rhodopirellul -Blastopirellula,PRB)分支的亲缘关系最近,但序列相似性也仅为82.7％-84.3％.[结论]利用低浓度糖类并添加抗生素分离厌氧颗粒污泥中的微生物,获得了浮霉菌门首例严格厌氧细菌VM20-7T.生理生化特性和系统发育分析显示,菌株VM20-7T为浮霉菌目的新属新种,命名为Thermopirellula anaerolimosa.该菌株的菌种保藏号为CGMCC 1.5169T=JCM 17478T=DSM 24165T.     

产氢细菌Enterbacter sakazakii HP的分离及产氢特性

在自然环境中分离到一株具有高产氢活性的微生物菌株,经细菌鉴定仪及16S rRNA基因序列分析,鉴定该菌株为Enterbacter sakazakii HP。分析了起始pH值、反应温度、碳源、起始糖浓度、起始氧浓度及菌体密度等因素对菌株产氢活性的影响。研究表明,该菌株发酵产氢较适合的条件为:以葡萄糖为产氢底物,起始pH值8.0,菌体密度OD600=0.7,反应温度35℃,糖浓度为0.1mol/L,氧浓度为0%的条件下,此时产氢菌株的最高产氢活性为5.34μmolH2/h.mgdw,氢的得率为1.94molH2/mol葡萄糖。     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。     

高效产氢细菌新种--Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌     

Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80 degrees C, pH 9.8 spring Tsenher, central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4 was moderately thermophilic with optimum growth at 60 degrees C. Maximum temperature for growth was between 70 and 75 degrees C; minimum temperature for growth was between 35 and 30 degrees C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0-5% (w/v) with the optimum at 0.2-0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol. % (td). On the basis of 16S rRNA gene sequence similarity strain T4(T) was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class "Bacilli"). DNA-DNA hybridization data revealed that strain T4T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4(T) (=DSM 19169 = VKM 2407).     

Eubacterium aggreganssp. nov., a new homoacetogenic bacterium from olive mill wastewater treatment digestor

A strictly anaerobic, homoacetogenic, gram-positive, non spore-forming bacterium, designated strain SR12(T) (T = type strain), was isolated from an anaerobic methanogenic digestor fed with olive mill wastewater. Yeast extract was required for growth but could also be used as sole carbon and energy source. Strain SR12(T) utilized a few carbohydrates (glucose, fructose and sucrose), organic compounds (lactate, crotonate, formate and betaine), alcohols (methanol), the methoxyl group of some methoxylated aromatic compounds, and H2 + CO2. The end-products of carbohydrate fermentation were acetate, formate, butyrate, H2 and CO2. End-products from lactate and methoxylated aromatic compounds were acetate and butyrate. Strain SR12(T) was non-motile, formed aggregates, had a G+C content of 55 mol % and grew optimally at 35 degrees C and pH 7.2 on a medium containing glucose. Phylogenetically, strain SR12(T) was related to Eubacterium barkeri, E. callanderi, and E. limosum with E. barkeri as the closest relative (similarity of 98%) with which it bears little phenotypic similarity or DNA homology (60%). On the basis of its phenotypic, genotypic, and phylogenetic characteristics, we propose to designate strain SR12(T) as Eubacterium aggregans sp. nov. The type strain is SR12(T) (= DSM 12183).     

Marinospirillum alkaliphilum sp. nov., a new alkaliphilic helical bacterium from Haoji soda lake in Inner Mongolia Autonomous Region of China

A new helical, alkaliphilic, gram-negative, chemoorganotrophic bacterium designated strain Z4T was isolated from Haoji soda lake in Inner Mongolia Autonomous Region, China. The isolate grows at salinities between 0.2% and 5.0% (w/v) NaCl and pH range 7.0-11.0, with an optimum at 2.0% (w/v) NaCl and pH 9.5. Its growth temperature ranges from 8 degrees to 49 degrees C with an optimum at 37 degrees C. The G+C content of the DNA is 46.8 mol%. The major isoprenoid quinone is ubiquinone 8 (Q-8). Phylogenetic analyses based on 16S rDNA sequence comparison indicates that strain Z4T is a member of the genus Marinospirillum. Phenotypic features and DNA-DNA homology of less than 20% with the described species of Marinospirillum support the view that strain Z4T represents a new species of the genus Marinospirillum. Strain Z4T (= AS 1.2746) is proposed as the type strain of a new species, named Marinospirillum alkaliphilum sp. nov.     

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).     

Isolation, characterization, and identification of Geobacillus thermodenitrificans HRO10, an alpha-amylase and alpha-glucosidase producing thermophile

Thermophilic and amylolytic aerobic bacteria were isolated from soil through a selective enrichment procedure at 60 degrees C with starch as the carbon source. One of the isolates designated as HRO10 produced glucose aside from limit dextrin as the only hydrolysis product from starch and was characterized in detail. The starch-degrading enzymes produced by strain HRO10 were determined to be alpha-amylase and alpha-glucosidase. Whereas the alpha-amylase activity was detected exclusively in the culture supernatant, alpha-glucosidase occurred intracellular, extracellular, or on the surface of the bacteria depending on the growth phase. The optimum temperature and pH required for the growth of strain HRO10 were about 50 degrees C and pH 6.5 to 7.5. The strain used different carbohydrates as the carbon source, but the maximum production of alpha-amylase occurred when 1.0% (w/v) starch or dextrin was used. The use of organic vs. inorganic nitrogen favored the production of alpha-amylase in strain HRO10. The metal ions Li+, Mg2+, and Mn2+ stimulated the production of both enzymes. Identification of strain HRO10 by physiological and molecular methods including sequencing of the 16S rDNA showed that this strain belongs to the species Geobacillus thermodenitrificans. Biochemically, strain HRO10 differs from the type strain DSM 465 only in its ability to hydrolyze starch.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).