安徽定远盐矿可培养嗜盐微生物多样性

【背景】嗜盐微生物多生活于高盐环境,具有独特的生理代谢特征,是一类重要的极端环境微生物资源。【目的】为更好地认识我国陆相盐矿的嗜盐微生物多样性组成,更好地开发利用嗜盐微生物资源积累丰富的微生物菌种。【方法】对安徽定远盐矿盐芯样品进行嗜盐微生物的纯培养分离,并对所分离菌株进行基于16SrRNA基因的测序和序列相似性分析,并对所分离菌株进行物种多样性分析。在此基础上,对代表菌株进行菌落形态和耐盐度及酶活测定。【结果】通过纯培养共分离获得了嗜盐微生物264株,其中嗜盐古菌150株,占56.8%;嗜盐细菌114株,占43.2%。嗜盐古菌物种分别来自于Halorubrum、 Halopenitus、 Haloterrigena、 Natrinema、 Natronoarchaeum和Natronomonas等6个属;嗜盐细菌物种分别来自于Pseudomonas、Aliifodinibius、Halobacillus、Halomonas和Halospina等5个属。通过代表菌株的酶活平板检测,发现产胞外蛋白酶菌株1株,酯酶1株,淀粉酶2株;能液化明胶菌株2株。在物种多样性组成方面,发现嗜盐古菌的物种多样性指数高于嗜盐细菌。【结论】本研究对我国安徽定远陆相盐矿的可培养嗜盐微生物多样性进行探究,积累了丰富的嗜盐微生物菌株资源。     

Distribution and diversity of halophilic bacteria in a subsurface salt formation

The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy. The facility will be used for permanent disposal of transuranic wastes. This underground repository has been constructed in the geologically stable Permian age Salado salt formation. Of the wastes to be placed into the facility, 85% will be biodegradable cellulose. A 3-year survey of the bacterial populations existing within the facility was conducted. Bacterial populations were found to be heterogeneously distributed throughout the mine. Populations in some mine areas reached as high as 1.0 × 10⁴ colony-forming units per gram of NaCl. The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity. A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups. The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds. When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation. Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids. The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations. Received: September 29, 1997 / Accepted: January 29, 1998     

Characterization of moderately halotolerant selenate- and tellurite-reducing bacteria isolated from brackish areas in Osaka

Moderately halotolerant selenate- and tellurite-reducing bacteria were characterized for wastewater treatment applications. A selenate-reducing strain 9a was isolated from the biofilm of a leachate treatment plant at a sea-based waste disposal site. A tellurite-reducing strain Taa was isolated from an enrichment culture derived from brackish sediment. Both bacterial strains were Shewanella species. Strain 9a could anaerobically remove 45–70% of 1.0 mM selenate and selenite from water containing up to 3% NaCl within 4 days, while strain Taa could anaerobically and aerobically remove 70–90% of 0.4 mM tellurite from water containing up to 6% NaCl within 3 days. Globular particles of insoluble selenium were observed both outside and inside the cells of strain 9a. The insoluble tellurium formed by strain Taa was globular under microaerobic conditions but nanorod under aerobic conditions. These bacteria will yield a range of useful selenium and tellurium nanomaterials as well as wastewater treatment applications.     

墓室酥碱砖壁画及其环境的真菌多样性分析

【背景】酥碱是威胁古代墓室砖壁画保存的主要病害之一,水分和盐分共同作用下壁画产生酥碱病害,而其中真菌类群、来源及耐盐性鲜有报道。【目的】比较敦煌汉墓、晋墓和嘉峪关五号墓内砖壁画酥碱样品及其赋存环境空气中可培养真菌的群落组成、多样性及耐盐性,为砖壁画的防护提供理论依据。【方法】运用扫描电镜观察酥碱样品微观形貌,并结合能谱、X射线衍射进行成分分析;对样品培养、分离、纯化获得真菌纯菌株,运用形态和分子生物学技术鉴定种属;利用梯度含盐培养基研究菌株的耐盐性。【结果】墓室酥碱样品及环境空气中青霉属(Pencillium)、曲霉属(Aspergillus)和枝孢属(Cladosporium)丰度较高,为优势属;其他菌属包括镰孢菌属(Fusarium)和毛壳菌属(Chaetomium)为稀有属。三座墓室砖壁画酥碱中均存在烟曲霉(A.fumigatus)、花斑曲霉(A.versicolor)以及黄灰青霉(P.aurantiogriseum);酥碱样品与墓室内外空气中分离获得的真菌类群间有一定相似性。多数菌株可在含15%NaCl的培养基上生长,一株产黄青霉(P. chrysogenum)甚至可在30%盐浓度条件下生长。【结论】青霉属和曲霉属为墓室共有优势菌,多数菌株有较强的耐盐性。     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

Fatty acids, unusual glycophospholipids and DNA analyses of thermophilic bacteria isolated from hot springs

The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography–mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-(O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15–C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus.     

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7–8 and at temperature close to 35°C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40–45°C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.     

Production,characterization and bioactivities of biosurfactants from newly isolated strictly halophilic bacteria

Eight distinct halophilic bacteria isolated from extreme saline soil samples of Khewra Salt Mines, Pakistan, were investigated for biosurfactant production. Isolates were identified by physiological, phenotypic and genetic characterization. Using 16S rDNA sequence analysis, the strains MB590, MB591, MB593, MB594, MB595 and MB596 were identified as Halomonas elongata, MB588 as Halobacillus karajiensis, and MB589 as Alkalibacillus almallahensis. Preliminary screening of biosurfactant production in halophilic bacteria was done by multiple screening assays. All biosurfactants showed significant emulsification properties and remarkably low surface tension values (up to 16.5 dynes/cm). Structural characterization of partially purified biosurfactants using Gas chromatography–mass spectrometry (GC–MS) and Fourier-transform infrared spectroscopy (FTIR) techniques indicated different fatty acids, glycolipid derivatives and a novel antimicrobial peptide furanomycin. These biosurfactants exhibited strong bioactivities against bacterial/fungal pathogens i.e. Klebsiella pneumoniae (86.5 %), Staphylococcus aureus (97.75 %), Bacillus subtilis (97 %), Enterococcus faecalis (97.6 %), E. coli (54.5 %), Aspergillus niger (87.75 %), Aspergillus fumigatus (93.1 %), Aspergillus flavus (80.4 %), and Fusarium solani (89.05 %). Additionally, biosurfactants also showed 85 % free radical scavenging activity indicating their antioxidant potential. The present study revealed the potential of halophilic bacterial biosurfactants as effective antimicrobial agents against various pathogens, and their possible applications in the biomedical field.     

Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast

A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth (G_t, 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 °C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease.     

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.     

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.     

Identification of lactic acid bacteria isolated from human vaginal secretions

A total of 57 lactic acid bacteria were isolated from the vaginal secretions of 259 patients. Of these strains, 37 were isolated from patients attending pre-natal clinics and the remaining strains from patients attending post-natal clinics. The strains were identified by using simple physiological and biochemical tests and their phenotypic relatedness determined by numerical analysis of total soluble cell protein patterns. The genotypic relatedness of representative strains selected from each of the protein profile clusters was determined by numerical analysis of the DNA banding patterns obtained from RAPD-PCR. The majority of lactobacilli isolated belonged to the species Lactobacillus pentosus, Lactobacillus fermentum and Enterococcus faecium. A few strains of Lactobacillus plantarum and Weissella viridescens were also isolated. One strain, TV 1029, grouped into the same protein profile cluster as E. faecium, but revealed a DNA banding pattern closer related to Enterococcus faecalis. This is the first report of W. viridescens associated with the human vagina. This revised version was published online in June 2006 with corrections to the Cover Date.     

酸马奶中乳酸菌的鉴定及生物学特性的研究

[目的]对从酸马奶中分离出来的10株乳酸菌进行鉴定和生理生化特性研究,为工业生产筛选特性优良的菌种.[方法]通过形态学观察、生理生化特性、分子生物学特性及其对致病菌抑制作用的研究对其进行鉴定,并筛选特性优良菌株.[结果]10株乳酸菌分别为2株Lactobacillus plantarum、2株Enterococcus villorum、2株Enterococcus dispar、3株Enterococcus durans和1株Enterococcus raffinosus;其对Staphylococcus aureus、Escherichia coli和Enteritidis bacillus有不同程度的抑制作用.[结论]菌株HZ24、HZ25具有良好的生物学特性和益生功能,可以应用到食品发酵工业生产中.     

高效降解草酸乳酸菌的筛选

尿路结石70%~80%主要由草酸钙结晶构成。人体内的草酸一般通过肠道内微生物降解,经由粪便排出或在泌尿道吸收由尿液排出。本研究对市场上商品化的发酵乳制品、饮料和药品中的乳酸菌进行分离,得到37株菌,包括嗜热链球菌、保加利亚乳杆菌、干酪乳杆菌、鼠李糖乳杆菌、植物乳杆菌、嗜酸乳杆菌、动物双歧杆菌、长双歧杆菌、粪肠球菌、屎肠球菌和乳球菌,并检测这些菌株降解草酸的能力。结果提示,乳酸菌在体外能够有效的降低培养物中的草酸浓度,并筛选出了具有高效降解草酸能力的乳酸菌菌株。有望成为尿石症预防的新措施。     

Probiotic characteristics of lactic acid bacteria isolated from kimchi

Aims: The present work was aimed at identifying strains of lactic acid bacteria (LAB) from kimchi, with properties suitable for use as starter cultures in yogurt fermentation. Methods and Results: A total of 2344 LAB strains were obtained from two different sources, one group consisted of commercial LAB strains from kimchi, and the second group consisted of those strains isolated from various types of kimchi. The LAB strains from both groups were screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that KFRI342 of the four selected strains displayed the greatest ability to reduce the growth of the cancer cells, SNU‐C4. The in vivo efficacy of strains in quinone reductase induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Strain KFRI342 was identified as Lactobacillus acidophilus by 16S rRNA sequence analysis, showed protection against tumour initiation and imparted immunostimulation as well as protection against DNA damage. Conclusions: Strain KFRI342, which showed probiotic characteristics reducing cancer cell growth, could be a suitable starter culture for yogurt fermentation because of its strong acid production and high acid tolerance. Significance and Impact of the Study: This is the first report to describe a bacterium, isolated from kimchi, Lact. acidophilus KFRI342 which has the probiotic characteristics and the acid tolerance needed for its use as a starter culture in yogurt fermentation.     

一平浪盐矿古老岩盐沉积中可培养细菌的系统发育多样性研究

运用纯培养法和基于16S rRNA基因序列的系统发育分析对云南省一平浪盐矿古老岩盐沉积中可培养细菌的多样性进行了研究。用补充0.5~3.5mol/L NaCl的MBA和ISP2琼脂培养基从卤水、岩盐和盐土样品中分离到38株细菌,用细菌通用引物进行16S rRNA基因扩增和序列测定,用相关软件进行序列相似性搜索、比对和系统发育分析。结果表明,38个分离菌株可分为31个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)、17个科、24个属。多数菌株属于Proteobacteria门(18株,47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)和Firmicutes门(13株,34.2%)。这些分离菌株中,至少有3个菌株可能代表3个不同属的3个新物种:Y3、Y15和Y25分别代表Idiomarina属、Salinicoccus属和Saccharospirillum属的新物种;而菌株Y21有可能代表Staphylococcaceae科的一个新属。从以上结果可以看出,一平浪盐矿古老岩盐沉积中存在较为丰富的微生物物种多样性和系统发育多样性,并且潜藏着新的微生物资源。     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。