天山1号冰川底部沉积层产β-半乳糖苷酶低温菌株的系统发育分析及生理多样性

[目的]通过对天山1号冰川底部沉积层冻土中细菌的分离和产β-半乳糖苷酶低温菌株的筛选,了解天山冻土微生物的物种多样性,并对产β-半乳糖苷酶低温菌株的系统发育和生理多样性进行分析.[方法]以乳糖为主要碳源,X-Gal为显色剂,分离筛选出产低温β-半乳糖苷酶菌株.对细菌常规生理生化实验、最适生长温度、耐盐性、药物敏感性进行测定.根据16S rRNA基因序列初步确定产β-半乳糖苷酶低温菌种的系统进化地位,并采用BOX-PCR指纹图谱技术对16S rRNA基因高度同源性的菌株进一步区分.[结果]分离到90株可培养低温菌中25株可产β-半乳糖苷酶,其中76％为革兰氏阳性菌.依据生长温度,产酶菌株80％为嗜冷菌,20％为耐冷菌.在系统发育上,产酶菌株隶属于4个类群,其中肠球菌属(Enterococcus)占26％,短波单胞菌属(Brevundimonas)占22％,假单胞菌属(Pseudomonas)占13％.[结论]天山1号冰川底部沉积层冻土中产β-半乳糖苷酶的低温细菌具有比较丰富的物种和生理多样性.     

一株分离自西藏高山草甸土壤的肠球菌的分离鉴定及代谢特征

[目的]了解高海拔低温缺氧条件下土壤环境中微生物生理生化特性及代谢产物[方法]采用Hungate厌氧操作技术从西藏纳木措高山草甸土壤中分离到一株肠球菌C J-1.通过生理生化特征分析和16S rRNA基因序列的系统发育学分析确定该菌株的系统发育学地位.[结果]菌株C J-1为兼性厌氧的革兰氏阳性菌,菌体呈不规则球形,大小为直径约1 μm-1.5 μm,无鞭毛,不运动,成串珠型或成对排列.生长温度范围为10℃-50℃(最适温度为25℃) ;pH范围为5.0-8.5(最适pH为7.0) ;NaC1浓度为0％ -7％(最适NaC1浓度为5％).能够利用葡萄糖、核糖、松三糖以及纤维二糖等多种碳水化合物,发酵纤维二糖的产物是乳酸、乙酸、丙酸、CO2、H2以及少量的丁酸.菌株CJ-1的(G+C)Mo1％为39.2％.与Enterococcus aquimarinus( CCM7283)的相似性达95.9％.菌株可利用纤维二糖,其在沼气发酵过程中充当中间代谢物.[结论]菌株CJ-1为耐低温肠球菌,可以降解纤维二糖等,且适应性较强,在发酵过程中具有重要意义,将其暂定命名为Enterococcus namtsoensis,模式菌株DSM23475T(=ACCC 00521T).     

天山一号冰川底部沉积层产蛋白酶耐低温菌株的筛选及其系统发育

[目的]通过对天山一号冰川底部沉积层耐低温菌的分离和其中产蛋白酶菌株的筛选,了解冰川微生物生理多样性和系统发育多样性,为高效低温蛋白酶生物技术的研发奠定基础.[方法]采用稀浓度的R2A、TSB平板涂布分离可培养细菌,通过脱脂乳选择性培养基筛选产蛋白酶的耐低温菌株.对分离菌株表型特征、生理生化特性、最适生长温度、耐盐性、产酶性能进行了比较,结合16S rRNA基因序列同源性分析确定产蛋白酶菌株的多样性和系统进化地位,通过BOX-PCR指纹技术分析16S rRNA基因序列高相似度的近缘菌株的遗传差异.[结果]从125株分离物中筛选到27株产蛋白酶的耐低温菌株,其中21株为适冷菌,仅6株菌为专性嗜冷菌,革兰氏阴性菌居多,假单胞菌属(Pseudomonas)菌株占40.7％.产酶菌株隶属于5个系统发育类群、9个属,其中γ-Proteobacteria、Actinobacteria、CFB(Cytophaga-Flexibacter-Bacteroides)为优势类群.[结论]天山1号冰川底部沉积层冻土中产蛋白酶的耐低温细菌多样性较丰富,本研究筛选得到的同属近缘种群较多,其产酶性状存在差异,适合开展微生物种群的生物地理学研究.     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

硇洲岛潮汐带牡蛎相关可培养细菌多样性

[目的]了解南海硇洲岛潮汐带香港巨牡蛎(Crassostrea hongkongensis)相关可培养细菌的多样性.[方法]应用纯培养法分离样品中的细菌(含放线菌),采用基于16S rRNA基因序列的系统发育分析方法研究这些分离菌株的生物多样性.[结果]用补充0-25％(W/V) NaC1的MA、MH和NA培养基从样品中分离到102株细菌,在形态观察和部分生理生化实验结果的基础上去冗余,选取74个代表性菌株进行基于16S rRNA基因序列的系统发育分析.结果表明,这些菌株归为38个物种,属于4个大的系统发育类群(Gamma-Proteobacteria,Alpha-Proteobacteria,Bacteroidetes,Firmicutes)的16个科、18个属.大多数菌株属于Gamma-Proteobacteria亚门(45株,60.8％),其余依次是Firmicutes门(12株,16.2％)、Bacteroidetes门(11株,14.9％)和Alpha-Proteobacteria亚门(6株,8.1％).大多数菌株与其系统发育关系最密切的有效发表种典型菌株之间(16S rRNA基因序列相似性为94.3％-99.8％)存在一定的遗传差异,其中JSM 111069可能代表Carnobacteriaceae科的潜在新属;菌株JSM 111039和JSM 111085可能分别代表Bacillus属的两个新物种,菌株JSM 111020、JSM 111072和JSM 111090可能分别代表Pseudoalteromonas、Proteus和Idiomarina属的新物种.[结论]南海硇洲岛潮汐带牡蛎中存在较为丰富的原核生物多样性,并潜藏着一定数量的微生物新类群(物种).     

厌氧丁草胺降解菌BAD-20的分离鉴定及降解特性研究

【目的】分离并鉴定能够降解除草剂丁草胺的厌氧微生物菌株,研究其厌氧降解丁草胺的特性和代谢途径,为深入研究丁草胺厌氧降解机制提供依据。【方法】以丁草胺为碳源作为选择压力从水稻田土壤中富集驯化丁草胺降解菌,利用16S rRNA基因系统发育分析结合菌株培养特征对降解菌株进行初步鉴定,利用液相色谱-时间飞行质谱(LC-TOF-MS)检测菌株降解丁草胺的代谢产物。【结果】筛选到一株降解丁草胺的厌氧细菌,命名为BAD-20,初步鉴定为嗜蛋白质菌属(Proteiniphilum),菌株BAD-20降解丁草胺的最适条件为温度30–35℃、pH 7.5–8.0和0–0.5%NaCl,在有氧条件下该菌不能降解丁草胺。最适条件下,菌株BAD-20在10d降解90%的20mg/L丁草胺。菌株BAD-20还能降解甲草胺、乙草胺、丙草胺,降解效率从高到低依次为甲草胺乙草胺丙草胺丁草胺,对这些氯乙酰胺除草剂的降解动力学符合一级动力学方程。鉴定到2个丁草胺降解代谢产物,分别是N-(2,6-二乙基苯基)-N-(丁氧甲基)乙酰胺(DEPBMA)和N-(2,6-二乙基苯基)乙酰胺(DEPA),表明菌株BAD-20降解丁草胺的起始步骤为脱氯,随后脱去N-丁氧甲基。【结论】本研究富集分离到一株降解丁草胺的厌氧细菌嗜蛋白质菌属(Proteiniphilum) BAD-20,为深入研究丁草胺厌氧降解机制及研发含丁草胺废水厌氧生物处理技术提供依据。     

新疆额尔齐斯河流域冷水鱼肠道耐低温乳酸菌的分离筛选及其遗传差异

[目的]从新疆阿勒泰地区额尔齐斯河流域冷水鱼肠道中分离耐低温的乳酸菌,挖掘乳酸菌的物种和遗传多样性,为低温乳酸菌生物技术研发提供优良菌种.[方法]利用MRS、Elliker两种不同培养基从9种冷水鱼肠道中,分离筛选出耐低温菌,测定细菌最适生长温度并进行常规生理生化实验.根据16S rRNA基因序列初步确定耐低温菌的系统进化地位,并采用BOX、(GTG)5、ERIC三种不同的引物进行rep-PCR,对16S rRNA基因高度同源性的菌株进一步区分.[结果]分离得到78株耐冷菌,最终确定有24株为耐低温乳酸菌,菌株的最适生长温度在15-24℃之间.系统发育分析结果表明:24株耐低温乳酸菌隶属于6个属,其中肉杆菌属(Carnobacterium)3株,乳球菌属(Lactococcus)9株,肠球菌属(Enterococcus)7株,环丝菌属(Brochothrix)1株,魏斯氏菌属(Weissella)2株,链球菌属(Streptococcus)2株.指纹图谱聚类分析树状图显示乳球菌属(Lactococcus)和肠球菌属(Enterococcus)存在种及菌株水平上的遗传差异,前者包括4个种,后者2个种.[结论]新疆额尔齐斯河流域冷水鱼肠道中分离的耐低温乳酸菌以球菌为主,没分离到常见的乳杆菌,需进一步完善分离培养的方法,深入挖掘和研究其物种多样性和遗传多样性.     

一株分离自美国龙虾的麦氏弧菌的鉴定及其低温状态下细胞脂肪酸的变化

[目的]对从美国龙虾(Homarus americanus)中分离的菌株F5-1进行种属鉴定并分析其低温状态下脂肪酸组成变化.[方法]通过VITEK 2 Compact全自动微生物鉴定仪分析菌株的生理生化特征和进行药敏试验;16S rRNA基因序列同源性分析确定该菌株的系统发育学地位;通过气相色谱和质谱联用法(GC-MS)分析菌株的全细胞脂肪酸.[结果]菌株F5-1为革兰氏阴性菌,对弧菌抑制剂O/129敏感,对青霉素有耐药性;生理生化特征与麦氏弧菌(Vibrio metschnikovii)的相似性为96％,16S rRNA序列与麦氏弧菌(GenBank No.HQ658055)的相似性为99％;菌株的主要脂肪酸组成为C12∶0、C14∶0、C16∶0和C16∶1(n-7),不饱和脂肪酸(棕榈油酸)相对含量达34％,低温培养状态下,不饱和脂肪酸(棕榈油酸)相对含量增加至40％.[结论]将美国龙虾中分离的菌株F5-1鉴定为麦氏弧菌,该菌对多种药物敏感,菌细胞脂肪酸组成与来源于俄罗斯海参威某饮用水水库中分离的麦氏弧菌有较大差异.     

一株产1-氨基环丙烷-1-羧酸脱氨酶的氢氧化细菌的分离鉴定及酶活力测定

[目的]以结瘤豆科植物紫花苜蓿根际土壤为研究材料,筛选具有ACC脱氨酶活力的氢氧化细菌,探索氢氧化细菌植物促生作用机制.[方法]利用持续通H2 的气体循环培养体系、矿质盐固体培养基,分离、培养氢氧化细菌,观察菌株形态并测定生理生化特征;16S rDNA序列分析法构建系统发育树;采用薄层层析法筛选ACC脱氨酶阳性菌株,茚三酮显色法测定ACC脱氨酶活力.[结果]分离的37株细菌中有8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌,从中筛选出1株ACC脱氨酶阳性菌株WMQ-7.菌株WMQ-7的形态特征、生理生化特征与恶臭假单胞菌(Pseudomonas putida)的特征基本一致;16s rDNA序列(GenBank登录号为EU807744)在系统发育树中与恶臭假单胞菌同属一个类群,序列同源性99%.鉴定菌株WMQ-7为恶臭假单胞菌,其ACE脱氨酶活力为0.671 U/μg[结论]采用气体循环培养体系分离氢氧化细菌,克服了传统配气法的局限.ACC脱氨酶阳性菌株的筛选,为深入研究氢氧化细菌作为植物根际促生菌的菌株特性和促生机制提供理论依据.     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

Paraliobacillus ryukyuensis gen. nov., sp. nov., a new Gram-positive,slightly halophilic,extremely halotolerant,facultative anaerobe isolated from a decomposing marine alga

A slightly halophilic, extremely halotolerant, alkaliphilic, and facultatively anaerobic rod bacterium was isolated from a decomposing marine alga collected in Okinawa, Japan. The isolate, designated O15-7(T), was Gram-positive, endospore-forming, catalase-positive, menaquinone-7-possessing bacterium that is motile by peritrichous flagella. The isolate was an inhabitant of marine environments; the optimum NaCl concentration for growth was 0.75-3.0% (w/v) with a range of 0-22.0%, and the optimum pH was 7.0-8.5 with a range of 5.5-9.5. Catalase was produced in aerobic cultivation but not in anaerobic cultivation. Carbohydrate, sugar alcohol or a related carbon compound was required for growth. In aerobic cultivation, the isolate produced pyruvate, acetate and CO(2) from glucose, and in anaerobic cultivation, it produced lactate, formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 for the last three products. No gas was produced anaerobically. Lactate yield per consumed glucose was markedly affected by the pH of the fermentation medium: 51% at pH 6.5 and 8% at pH 9.0. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Phylogenetically, the isolate occupied an independent lineage within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1 with the highest 16S rRNA gene sequence similarity of 95.2% to the genus Gracilibacillus. For this isolate, Paraliobacillus ryukyuensis gen. nov., sp. nov. was proposed. The type strain, O15-7(T) (G+C535.6 mol%), has been deposited in the DSMZ, IAM, NBRC, and NRIC (DSM 15140(T)=IAM 15001(T)=NBRC 10001(T)=NRIC 0520(T)).     

Anaerofustis stercorihominis gen. nov., sp. nov., from human feces

Phenotypic and phylogenetic studies were performed on an unidentified Gram-positive, strictly anaerobic, non-spore-forming, rod-shaped bacterium isolated from human feces. The organism was catalase-negative, resistant to 20% bile, produced acetic and butyric acids as end products of glucose metabolism, and possessed a G+C content of approximately 70 mol%. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of the Clostridium sub-phylum of the Gram-positive bacteria, and formed a loose association with rRNA cluster XV. Sequence divergence values of 12% or greater were observed between the unidentified bacterium and all other recognized species within this and related rRNA clusters. Treeing analysis showed the unknown anaerobe formed a deep line branching near to the base of rRNA cluster XV and phylogenetically represents a hitherto unknown taxon, distinct from Acetobacterium, Eubacterium sensu stricto, Pseudoramibacter and other related organisms. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from feces be classified in a new genus Anaerofustis, as Anaerofustis stercorihominis sp. nov. The type strain of Anaerofustis stercorihominis is ATCC BAA-858(T)=CCUG 47767(T).     

Clostridium bartlettii sp. nov., isolated from human faeces

Seven obligately anaerobic, Gram-positive, rod-shaped, spore-forming organisms isolated from human faecal specimens were characterized using phenotypic and molecular taxonomic methods. Strains of the unidentified bacterium used carbohydrates as fermentable substrates, producing acetic acid, isovaleric acid and phenylacetic acid (PAA) as the major products of glucose metabolism, and possessed a G +C content of approximately 29.8 mol%. Comparative 16S rRNA gene sequencing showed that the 7 strains were genetically highly related to each other (displaying >99.5% sequence similarity) and represent a previously unknown sub-line within the Clostridium Cluster XI. The closest described species to the novel bacterium is Clostridium glycolicum, although a 16S rRNA sequence divergence of 4% demonstrates that they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and C. glycolicum (30.6% similarity between the proposed type strain of the novel species, WAL 16138, and C. glycolicum ATCC 14880(T)). Based on morphologic, phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium be classified as C. bartlettii sp. nov. The type strain of C. bartlettii is WAL 16138(T) (= ATCCBAA-827(T)=CCUG48940(T)).     

Cetobacterium ceti gen. nov., sp. nov., a new Gram-negative obligate anaerobe from sea mammals

Phenotypic and phylogenetic studies were performed on a Gram-negative obligately anaerobic rod-shaped bacterium isolated from two sea mammals. 16S rRNA gene sequence analysis demonstrated the bacterium represents a hitherto unknown line of descent peripherally associated to the fusobacteria and low G + C relatives. Based on the result of the phylogenetic analysis and phenotypic criteria, it is proposed that the bacterium should be assigned to a new genus, Cetobacterium ceti gen. nov., sp. nov. The type strain of Cetobacterium ceti sp. nov. is NCFB 3026.     

Acetobacterium tundrae sp. nov., a new psychrophilic acetogenic bacterium from tundra soil

A new psychrophilic, anaerobic, acetogenic bacterium from the tundra wetland soil of Polar Ural is described. The organism fermented H2/CO2, formate, methanol, and several sugars to acetate as the sole end-product. The temperature range for growth was 1-30 degrees C with an optimum at 20 degrees C. The bacterium showed no growth at 32 degrees C. Cells were gram-positive, oval-shaped, flagellated rods 0.7-1.l x 1.1-4.0 microm in size when grown at 1-20 degrees C. At 25-30 degrees C, the cell size increased up to 2-3 x 10-15 microm due to a defect in cell division. The DNA G+C content of the organism was 39.2 mol%. Based upon 16S rDNA analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium as a new species, for which the name Acetobacterium tundrae sp. nov. is proposed. The type strain is Z-4493 (=DSM 9173T).     

Caldicoprobacter algeriensis sp. nov. a New Thermophilic Anaerobic, Xylanolytic Bacterium Isolated from an Algerian Hot Spring

A thermophilic anaerobic bacterium (strain TH7C1(T)) was isolated from the hydrothermal hot spring of Guelma in the northeast of Algeria. Strain TH7C1(T) stained Gram-positive, was a non-motile rod appearing singly, in pairs, or as long chains (0.7-1 × 2-6 μm(2)). Spores were never observed. It grew at temperatures between 55 and 75°C (optimum 65°C) and at pH between 6.2 and 8.3 (optimum 6.9). It did not require NaCl for growth, but tolerated it up to 5 g l(-1). Strain TH7C1(T) is an obligatory heterotroph fermenting sugars including glucose, galactose, lactose, raffinose, fructose, ribose, xylose, arabinose, maltose, mannitol, cellobiose, mannose, melibiose, saccharose, but also xylan, and pyruvate. Fermentation of sugars only occurred in the presence of yeast extract (0.1%). The end-products from glucose fermentation were acetate, lactate, ethanol, CO(2), and H(2). Nitrate, nitrite, thiosulfate, elemental sulfur, sulfate, and sulfite were not used as electron acceptors. The G+C content of the genomic DNA was 44.7 mol% (HPLC techniques). Phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene sequence indicated that strain TH7C1(T) was affiliated to Firmicutes, order Clostridiales, family Caldicoprobacteraceae, with Caldicoprobacter oshimai (98.5%) being its closest relative. Based on phenotypic, phylogenetic, and genetic characteristics, strain TH7C1(T) is proposed as a novel species of genus Caldicoprobacter, Caldicoprobacter algeriensis, sp. nov. (strain TH7C1(T) = DSM 22661(T) = JCM 16184(T)).     

Atopostipes suicloacale gen. nov., sp. nov., isolated from an underground swine manure storage pit

Phenotypic and molecular genetic studies were performed on an unknown facultative anaerobic, catalase-negative, non-spore-forming, rod-shaped bacterium isolated from a pig manure storage pit. The unknown bacterium was nutritionally fastidious with growth enhanced by the addition of rumen fluid and was phenotypically initially identified as an Eubacterium species. Comparative 16S rRNA gene sequencing studies, however, revealed that the unknown bacterium was phylogenetically distant from Eubacterium limosum (the type species of the genus Eubacterium) and related organisms. Phylogenetically, the unknown species displayed a close association with an uncultured organism from human subgingival plaque and formed an unknown sub-line within a cluster of organisms which includes Alloioccoccus otitis, Alkalibacterium olivoapovliticus, Allofustis seminis, Dolosigranulum pigrum, and related organisms, within the low mol% G+C Gram-positive bacteria. Sequence divergence values of >8% with all known taxonomically recognised taxa, however, clearly indicates the novel bacterium represents a hitherto unknown genus. Based on both phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium from pig manure be classified in a new genus and species, as Atopostipes suicloacale gen. nov., sp. nov. The type strain of Atopostipes suicloacale is PPC79(T)=NRRL 23919(T)=DSM 15692(T).     

Clostridium bolteae sp. nov., isolated from human sources

Seven obligately anaerobic, gram-positive, rod-shaped, spore-forming organisms isolated from human sources were characterized using phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the strains were genetically highly related to each other (displaying >99% sequence similarity) and represent a previously unknown sub-line within the Clostridium coccoides rRNA group of organisms. Strains of the unidentified bacterium used carbohydrate as fermentable substrates, producing acetic acid and lactic acid as the major products of glucose metabolism. The closest described species to the novel bacterium corresponded to Clostridium clostridioforme, although a 16S rRNA sequence divergence of 3% demonstrated they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and Clostridium clostridioforme. Based on phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium, be classified as Clostridium bolteae sp. nov. The type strain of Clostridium bolteae is WAL 16351T (= ATCC(T) = BAA-613T, CCUG(T) = 46953T).     

Paenibacillus xylaniclasticus sp. nov., a xylanolytic-cellulolytic bacterium isolated from sludge in an anaerobic digester

A mesophilic, facultative, anaerobic, xylanolytic-cellulolytic bacterium, TW1(T), was isolated from sludge in an anaerobic digester fed with pineapple waste. Cells stained Gram-positive, were spore-forming, and had the morphology of straight to slightly curved rods. Growth was observed in the temperature range of 30 to 50°C (optimum 37°C) and the pH range of 6.0 to 7.5 (optimum pH 7.0) under aerobic and anaerobic conditions. The strain contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The predominant isoprenoid quinone was menaquinone with seven isoprene units (MK-7). Anteiso-C(15:0), iso-C(16:0), anteiso-C(17:0), and C(16:0) were the predominant cellular fatty acids. The G+C content of the DNA was 49.5 mol%. A phylogenetic analysis based on 16S rRNA showed that strain TW1(T) belonged within the genus Paenibacillus and was closely related to Paenibacillus cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) with 97.7, 97.5, and 97.3% sequence similarity, respectively. The DNA-DNA hybridization values between the isolate and type strains of P. cellulosilyticus LMG 22232(T), P. curdlanolyticus KCTC 3759(T), and P. kobensis KCTC 3761(T) were found to be 18.6, 18.3, and 18.0%, respectively. The protein and xylanase patterns of strain TW1(T) were quite different from those of the type strains of closely related Paenibacillus species. On the basis of DNA-DNA relatedness and phenotypic analyses, phylogenetic data and the enzymatic pattern presented in this study, strain TW1(T) should be classified as a novel species of the genus Paenibacillus, for which the name Paenibacillus xylaniclasticus sp. nov. is proposed. The type strain is TW1(T) (=NBRC 106381(T) =KCTC 13719(T) =TISTR 1914(T)).     

Clostridium tunisiense sp. nov., a new proteolytic, sulfur-reducing bacterium isolated from an olive mill wastewater contaminated by phosphogypse

A new sporulated fermentative bacterium designated strain E1(T) (T=type strain), was isolated from an anaerobic mud of an olive mill wastewater basin contaminated by phosphogypse produced by a Tunisian factory. Strain E1(T) was a motile Gram-positive slightly curved rod with spherical terminal spore swelling the cell. It grew between 18 degrees C and 43 degrees C with an optimum at 37 degrees C and pH 7.8 (range 5.5-8.7), without NaCl (range 0-3%). Strain E1(T) was a chemoorganotrophic anaerobic bacterium fermenting only proteins and amino acids. Yeast extract was required for growth. Elemental sulfur was used as terminal electron acceptor. The G+C content of the DNA was 32.6 mol%. The closest phylogenetical relatives of strain E1(T) were Clostridium thiosulfatireducens and C. subterminale (97.3% similarity for partial rRNA gene sequences). DNA-DNA hybridization values between strain E1(T) and both species were 17% and 20.8%, respectively. On the basis of differences in genotypic and phenotypic characteristics, strain E1(T) (DSM 15206(T), CIP 107666(T)) is proposed as the type strain of a new species, C. tunisiense sp. nov. GenBank accession number for the 16S rRNA gene sequence of strain E1(T) is AY187622.