一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌,经过富集培养、分离纯化得到56株细菌,将其接种到基础盐酵母培养基,7d后用紫外分光光度计法初筛得到降解率较高的一株菌,编号为D-1.通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株茵.对菌体降解DDT的特性的研究表明,在培养温度为3℃,底物质量浓度为40 mg/L, pH 7.0,摇床转速为200 r/min的条件下,该菌株对DDT降解10d的降解率为69.0%.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌, 经过富集培养、分离纯化得到56株细菌, 将其接种到基础盐酵母培养基, 7 d后用紫外分光光度计法初筛得到降解率较高的一株菌, 编号为D-1。通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株菌。对菌体降解DDT的特性的研究表明, 在培养温度为30℃, 底物质量浓度为40 mg/L, pH 7.0, 摇床转速为200 r/min的条件下, 该菌株对DDT降解10 d的降解率为69.0%。     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterium

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga. To obtain enzymes that digest the fucoidan, we screened bacteria in the gut contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it. The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52%. The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division Verrucomicrobia. However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division Verrucomicrobia. When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C. okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).     

1株产琼脂酶细菌的分离鉴定及其胞外酶活性测定

从四川省成都市青城山采集土壤,以琼脂作为唯一碳源,筛选到产琼脂酶细菌CMCK136;通过形态观察、生化鉴定、16S r DNA测序及序列分析鉴定其种属;随后测定了菌株CMCK136的胞外酶活性。菌株CMCK136被鉴定为芽胞杆菌属细菌,命名为Bacillus sp.CMCK136。菌株CMCK136的胞外琼脂酶的最适酸碱度为p H 7.0,最适温度为35℃。菌株CMCK136是产琼脂酶细菌家族的新成员,该菌株的发现进一步提示芽胞杆菌属很可能蕴含有尚待开发的琼脂酶资源。     

一种短杆状耐辐射菌的分离与鉴定

从北京地区公园湖岸土壤中分离到一株橙红色杆状耐辐射菌,细胞壁革兰氏染色为阴性,电镜显示菌体大小为06μm～16μm,略大于日本学者报道的Deinobacter grandis菌,过氧化氢酶的含量和分子量不同于D.radiodurans R1菌,分离菌的(G+C)mol%含量为707%, 16S rDNA序列分析表明,分离到的杆状耐辐射菌(RR5332)16S rRNA基因序列与Deinobacter grandis菌高度同源,提示RR5332归于Deinobacter菌属,并可能是该菌属中的一个新种。     

一株海洋细菌的初步鉴定及其产黑色素相关新基因(簇)的分离

对分离自近海沼泽地大米草根际的一株供试海洋细菌MWYL1进行了形态观察、生理特性检测以及16SrDNA序列分析。实验结果表明:该菌株属于海洋单胞菌属(Marinomonas),革兰氏染色呈阴性,直杆状,好氧生长,适于28℃生长。基于16SrDNA序列的Blast分析表明,菌株MWYL1与Marinomonas pontica和Marinomonas dokdonensis的序列相似性分别为97%和95%。通过基因组fosmid文库的构建,直接分离到一个产生黑色素的克隆,进一步亚克隆和测序后获得与黑色素产生相关的功能新基因(簇),并且对其进行了生物信息学的初步分析。     

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,Pseudomonas sp. Strain DO-1

Five microbial strains that removed hydrogen sulfide (H₂S) or methylmercaptan (CH₃SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it’s immobilized cells removed 1 or 10 ppm of H₂S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H₂ S gas without preliminary feeding or exposure to sulfur com-pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.     

一株甲胺磷分解菌的分离及其特性研究

通过富集驯化和选择性培养,从福建三农集团污水处理池的活性污泥中筛选到一株能以甲胺磷为惟一碳源和氮源的细菌DM-1。研究了该菌形态与部分生理生化特性。DM-1菌在无机盐基础培养基中对甲胺磷的最高耐受浓度为1500mg／L,DM-1菌最适生长条件：起始pH值8．0,培养温度28℃,接种量3．0％,摇床转速150r／min,培养时间72h。最佳碳、氮源分别为D-甘露醇和蛋白胨。     

Isolation and Characterization of a Novel Equol-Producing Bacterium from Human Feces

An equol-producing bacterium was newly isolated from the feces of healthy humans and its morphological and biochemical properties were characterized. The cells were obligate anaerobes. They were non-sporulating, non-motile, gram-positive bacilliform bacteria with a pleomorphic morphology. The strain was catalase-positive, and oxidase-, urease-, and indole-negative. The only other sugar utilized by the strain was glycerin. The strain also degraded gelatin, but not esculin. It was most closely related to Eggerthella hongkongensis HKU10, with 93.3% 16S rDNA nucleotide sequence homology. Based on these features, the isolate was identified as a novel species of the genus Eggerthella. It was named Eggerthella sp. YY7918. Strain YY7918 converted substrates daidzein and dihydrodaidzein into S-equol, but did not convert daidzin, glysitein, genistein, or formononetin into it. An antimicrobial susceptibility assay indicated that strain YY7918 was susceptible to aminoglycoside-, tetracycline-, and new quinolone-antibiotics.     

Enrichment and Physiological Characterization of a Novel Nitrospira-Like Bacterium Obtained from a Marine Sponge

Members of the nitrite-oxidizing genus Nitrospira are most likely responsible for the second step of nitrification, the conversion of nitrite (NO₂⁻) to nitrate (NO₃⁻), within various sponges. We succeeded in obtaining an enrichment culture of Nitrospira derived from the mesohyl of the marine sponge Aplysina aerophoba using a traditional cultivation approach. Electron microscopy gave first evidence of the shape and ultrastructure of this novel marine Nitrospira-like bacterium (culture Aa01). We characterized these bacteria physiologically with regard to optimal incubation conditions, especially the temperature and substrate range in comparison to other Nitrospira cultures. Best growth was obtained at temperatures between 28°C and 30°C in mineral medium with 70% North Sea water and a substrate concentration of 0.5 mM nitrite under microaerophilic conditions. The Nitrospira culture Aa01 is very sensitive against nitrite, because concentrations higher than 1.5 mM resulted in a complete inhibition of growth. Sequence analyses of the 16S rRNA gene revealed that the novel Nitrospira-like bacterium is separated from the sponge-specific subcluster and falls together with an environmental clone from Mediterranean sediments (98.6% similarity). The next taxonomically described species Nitrospira marina is only distantly related, with 94.6% sequence similarity, and therefore the culture Aa01 represents a novel species of nitrite-oxidizing bacteria.Numerous sponges have the capacity to accommodate large amounts of diverse microbes and represent significant sources for bioactive natural compounds (13). Many marine invertebrates excrete ammonium as a metabolic waste product (9), and the excretion of nitrite and nitrate has been taken as primary evidence that nitrifiers are active in these animals (10). By modulation of their pumping, sponges are a suitable habitat not only for aerobic microbes but also for anaerobic microbes. Accordingly, Hoffmann et al. (19) were able to detect major microbial pathways of the nitrogen cycle in the sponge Geodia barretti, including nitrification, the anammox process, and denitrification.Nitrification involves the biological oxidation of ammonia (NH₃) to nitrite (NO₂⁻) and further to nitrate (NO₃⁻) for energy purposes. It is of fundamental importance for the global nitrogen cycle in aquatic and terrestrial habitats. Nitrification is catalyzed by two phylogenetically distinct groups of microorganisms: in the first step, ammonia-oxidizing bacteria and archaea (AOB and AOA) take part in the oxidation of ammonia to nitrite, and in the second step nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate (38).Nitrite has a central position in the nitrogen cycle, connecting aerobic and anaerobic pathways. Nitrite-oxidizing bacteria play a major role in removing nitrite from the environment because it is toxic for living organisms (31). Based on morphological characteristics, NOB have been divided into five genera. This classification also reflects the phylogenetic diversity of NOB, which includes Nitrobacter and Nitrococcus (Alpha- and Gammaproteobacteria), Nitrospina (putative Deltaproteobacteria), and the candidate genus “Candidatus Nitrotoga” (Betaproteobacteria) (2). The genus Nitrospira is more distantly related to the other known NOB because it is part of its own deep-branching bacterial phylum Nitrospirae. Marine species are present in all genera of NOB except in the newly identified genus “Candidatus Nitrotoga.”As all known nitrifying prokaryotes are slow growing and hard to maintain, their enrichment and isolation from environmental samples is difficult. Most physiological studies have been performed with pure cultures of a few “model” nitrifiers, in particular AOB related to the genus Nitrosomonas and NOB of the genus Nitrobacter. For the genus Nitrospira there are only four pure cultures available: the marine species Nitrospira marina (37), Nitrospira moscoviensis (12), “Candidatus Nitrospira bockiana” (25), and Nitrospira calida (E. Lebedeva, personal communication).Sponges of the family Aplysinidae contain large amounts of bacteria embedded within the sponge tissue matrix (15). For example, the biomass of Aplysina aerophoba consists of up to 40% bacteria (36). These sponges are able to differentiate between food bacteria and their own bacterial symbionts (41). Investigations of the diversity of sponge-associated bacteria, including different genetic and also cultivation approaches, have been made with several specimens (15, 16, 39). In terms of nitrification, Hentschel et al. (17) gave first evidence for the presence of nitrite oxidizers, and it has been verified that sponges harbor AOB and AOA (8). Most of the recognized NOB in sponges are Nitrospira-like bacteria (17, 32, 35), although in the beginning, there were further hints to 16S rRNA sequences, which are most closely related to Nitrospina gracilis (17). However, as these sequences were found only once, it could be assumed that Nitrospira is the main nitrite oxidizer in this environment. Nitrospira-like bacteria are deemed to be recalcitrant and fastidious, and they are easily overgrown by other bacteria under suboptimal conditions. Despite these limitations in the laboratory, Nitrospira was determined to be the most important nitrite oxidizer during wastewater treatment (21, 33), in aquaculture biofilters (14) and in freshwater systems (20, 29).Identification of sponge-associated microorganisms has been performed largely with culture-independent methods, which are 16S rRNA gene based (denaturing gradient gel electrophoresis [DGGE], terminal restriction fragment-length polymorphism [TRFLP]) or visual (fluorescence in situ hybridization [FISH], electron microscopy) (8, 11). Nevertheless, the cultivation of microorganisms is still essential for the investigation of their physiological potential and function in the environment. Information about physiological characteristics helps us to understand the metabolism and possible nutritional interactions of nitrifiers with the host sponge (8).This is the first report about cultivation of nitrifying bacteria originating from a marine sponge. We obtained a nitrite-oxidizing enrichment culture of a Nitrospira-like bacterium derived from Aplysina aerophoba, characterized it phylogenetically, and analyzed the most important physiological features.     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterial Strain and Its Fucoidanase

A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory. The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36%. The cells of the strain were rod-shaped (2.0 m long × 1.0 m wide), and each cell was motile by means of one polar flagellum. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae. It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme. The enzyme was purified with 3500-fold purity at 12.0% yield. Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30° to 35°C. The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.     

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H₂, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (α-FeOOH) forms, and above pH 7.2 magnetite (Fe₃O₄) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ~10⁻⁵. To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B₁₂) biosynthesis.     

Isolation and Characterization of a Chlorinated-Pyridinol-Degrading Bacterium

The isolation of a pure culture of bacteria able to use 3,5,6-trichloro-2-pyridinol (TCP) as a sole source of carbon and energy under aerobic conditions was achieved for the first time. The bacterium was identified as a Pseudomonas sp. and designated ATCC 700113. [2,6-(sup14)C]TCP degradation yielded (sup14)CO(inf2), chloride, and unidentified polar metabolites.     

一株生物表面活性剂产生菌的分离及其特性研究

模炼油厂污泥中分离得到1株生物表面活性剂产生菌C-3, 根据其生理生化特性和16S rDNA序列相似性分析, 将其鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。初步研究了其产生物表面活性剂的最适条件, 在以植物油为碳源、30°C、初始pH 8、Ca2+浓度20 mg/L、250 mL三角瓶中装75 mL发酵液的条件下, 最利于菌株的生长和生物表面活性剂产生。它的成分为糖脂类物质, 临界胶束浓度(CMC)为50 mg/L, 具有很好的增溶效果。     

Isolation and Characterization of a Cellulose-utilizing Bacterium

A cellulose-decomposing aerobic and mesophilic bacterium has been isolated from soils of sugar cane fields. The terminal dilution method was adapted to isolate a single clone of cellulolytic organism from closely related contaminants. The cultural and physiological characteristics of the isolate were studied, and the organism was identified as a member of the genus Cellulomonas. The isolate excreted cellulase into the menstruum, and it hydrolyzed various cellulosic materials producing cellobiose as the final breakdown product in the menstruum. When sugar cane bagasse was properly treated with alkali and heat, the organism could decompose up to 90% of the initial substrate within 5 days. Amino acid analysis of the cell crop revealed a high content of lysine, and the essential amino acid pattern compared favorably with that of Food and Agricultural Organization reference protein.     

光合细菌PSB-1菌株的分离鉴定及其生物学特性的研究

光合细菌菌株ＰＳＢ－１,初步定为Ｒｈｏｄｏｐｓｅｕｄｏｍｏｎａｓ ａｃｉｄｏｐｈｉｌａ,系由生活污水中分离获得,革兰氏反应阴性,菌体大小为：长０．２５～０．４５μｍ,宽０．８～２．０μｍ,单个细胞卵圆至球形,出芽生殖,未观察到鞭毛。菌落为玫瑰红色,菌体液体培养物为深红色,菌体中含有丰富的细菌叶绿素ａ和类胡萝卜素,光合内膜结构为片层状,位于质膜之下并与质膜平行。ＰＳＢ－１在黑暗和光照中均能生长,但在黑暗中培养时菌液颜色不变,能利用多种有机碳源和氮源,ＤＮＡ中Ｇ＋Ｃ摩尔分数为６５．７％。     

A Gliding Bacterium Strain Inhibits Adhesion and Motility of Another Gliding Bacterium Strain in a Marine Biofilm

Two species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genus Cytophaga. One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.     

Alkaline Anaerobic Respiration: Isolation and Characterization of a Novel Alkaliphilic and Metal-Reducing Bacterium

Iron-reducing enrichments were obtained from leachate ponds at the U.S. Borax Company in Boron, Calif. Based on partial small-subunit (SSU) rRNA gene sequences (approximately 500 nucleotides), six isolates shared 98.9% nucleotide identity. As a representative, the isolate QYMF was selected for further analysis. QYMF could be grown with Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors, and yeast extract and lactate could serve as electron donors. Growth during iron reduction occurred over the pH range of 7.5 to 11.0 (optimum, pH 9.5), a sodium chloride range of 0 to 80 g/liter (optimum, 20 g/liter), and a temperature range of 4 to 45°C (optimum, approximately 35°C), and iron precipitates were formed. QYMF was a strict anaerobe that could be grown in the presence of borax, and the cells were straight rods that produced endospores. Sodium chloride and yeast extract stimulated growth. Phylogenetic analysis of the SSU rRNA gene indicated that the bacterium was a low-G+C gram-positive microorganism and had 96 and 92% nucleotide identity with Alkaliphilus transvaalensis and Alkaliphilus crotonatoxidans, respectively. The major phospholipid fatty acids were 14:1, 16:1ω7c, and 16:0, which were different from those of other alkaliphiles but similar to those of reported iron-reducing bacteria. The results demonstrated that the isolate might represent a novel metal-reducing alkaliphilic species. The name Alkaliphilus metalliredigens sp. nov. is proposed. The isolation and activity of metal-reducing bacteria from borax-contaminated leachate ponds suggest that bioremediation of metal-contaminated alkaline environments may be feasible and have implications for alkaline anaerobic respiration.     

Isolation and Characterization of Marine Brevibacillus sp. S-1 Collected from South China Sea and a Novel Antitumor Peptide Produced by the Strain

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells.