Isolation and characterization of halophilic bacteria from Urmia Lake in Iran

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 2535 degrees C, pH 6-9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [ 11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050T, Halovibrio denitrificans HGD 3T and Halospina denitrificans HGD 1-3T, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.     

Isolation and characterization of halophilic bacteria from Urmia Lake in Iran

Urmia Lake is one of the most permanent hypersaline lakes in the world which is threatened by hypersalinity and serious dryness. In spite of its importance no paper has been published regarding bacterial community of this lake. Accordingly, the present study aimed to investigate the halophilic bacteria in the aforementioned lake. In so doing, thirty seven strains were isolated on six different culture media. The isolated strains were characterized using phenotypic and genotypic methods. Growth of the strains occurred at 25–35°C, pH 6–9 and 7 to 20% (w/v) NaCl indicating that most of the isolates were moderately halophiles. Catalase, oxidase and urease activities were found to be positive for the majority of the isolates. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolated bacteria belonged to two major taxa: Gammaproteobacteria (92%, including Salicola [46%], Pseudomonas [13.5%], Marinobacter [11%], Idiomarina [11%], and Halomonas [8%]) and Firmicutes (8%, including Bacillus [5%] and Halobacillus [3%]). In addition, a novel bacterium whose 16S rRNA gene sequence showed almost 98% sequence identity with the taxonomically troubled DSM 3050^T, Halovibrio denitrificans HGD 3^T and Halospina denitriflcans HGD 1–3, each, was isolated. 16S rRNA gene similarity levels along with phenotypic characteristics suggest that some of the isolated strains could be regarded as potential type strain for novel species, on which further studies are recommended.     

Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes

AIMS: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. METHODS AND RESULTS: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates. CONCLUSIONS: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.     

Isolation of bacteria producing polyhydroxyalkanoates (PHA) from municipal sewage sludge

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.     

Molecular adaptation of enzymes, metabolic systems and transport systems in halophilic bacteria

Abstract A review is presented of the special properties and behaviour of enzymes, ribosomes, metabolic systems, protein turnover and active transport systems that are associated with the ability of halophilic archaebacteria and eubacteria to grow in different salt concentrations.     

Isolation and identification of moderately halophilic bacteria producing hydrolytic enzymes from the largest hypersaline playa in Iran

Iran has many hypersaline environments, both the permanent and seasonal ones. One of the seasonal hypersaline lakes in the central desert zone is Aran-Bidgol Lake in which microbial diversity has not been characterized, thus the potential usage of this microbial community in biotechnology remained unknown. In this study, screening the halophilic hydrolytic enzyme-producing bacteria from different areas of this lake led to isolation of 61 gram-positive and 22 gram-negative moderately halophilic bacteria. These bacterial isolates were shown to produce a wide variety of hydrolytic enzymes including DNase, inulinase, amylase, lipase, pectinase, protease, chitinase, pullulanase, cellulase, and xylanase. The most common enzymes were DNase and inulinase in gram-positive bacteria, lipase in gram-negative bacteria, and pullulanase and cellulase in gram-positive cocci. Interestingly, combined hydrolytic activates were observed in some isolates. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the moderately halophilic strains belonged to the genera Halobacillus, Thalassobacillus, Bacillus, Salinicoccus, Idiomarina, Salicola, and Halomonas.     

高产铁载体棉田土壤细菌SS05的筛选与鉴定

【目的】研究从棉田土壤中筛选得到的高产铁载体细菌产铁载体能力、分类地位和抑菌活性。【方法】通过改良蔗糖-天冬氨酸培养基选择性筛选产铁载体细菌,通过分光光度计法测定铁载体活性,通过混菌法测定产铁载体细菌上清液对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,采用形态学、生理生化鉴定及16S rDNA序列系统发育分析对高产铁载体菌株进行鉴定。【结果】从棉田土壤中筛选到162株产铁载体细菌,30株产铁载体能力较强的细菌中21株具有较高产铁载体能力,菌株SS05的铁载体活性单位达到98.3%;在低铁条件下,SS05上清液对F.oxysporum具有显著的抑制作用;SS05与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近。【结论】SS05是高产铁载体菌株,与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近,在低铁培养条件下其上清液对F.oxysporum具有显著的抑制作用。     

红树林内生细菌的分离及拮抗菌筛选

红树林内生细菌及拮抗菌分离筛选结果表明:各品种红树体内均有大量的内生细菌,不同红树品种及部位内生细菌的数量不同,其中以红海榄体内的含量最高,达4．225×104cfu／g(fw),其它依次为木榄、桐花树、秋茄和白骨壤等;不同部位以茎组织体内内生细菌的含量最多,达1．649×104cfu/g(fw),其次为根和叶。获得的内生细菌中约有43．53％的内生细菌菌株对枯萎病菌(Fusarium oxysporum)、炭疽病菌(Colletotrichum sp．)等病原真菌及番茄青枯病菌(Ralstonia solanaceance)具有较强拮抗作用。对番茄生长测定发现,13株菌株中有9株(69．23％)表现为促进生长效果,4株(30．77％)表现为抑制生长作用。经初步鉴定,上述拮抗细菌均为芽孢杆菌(Bacillus sp．)。     

广西山口红树林内生放线菌的分离、筛选及初步鉴定

从广西山口国家红树林生态自然保护区采集8种真红树植物和5种半红树植物的根、茎、叶及胚轴等组织样品共计41份。经过表面消毒后粉碎,分别以几丁质、腐殖酸、木聚糖等作为主要营养及能量来源设计了8种分离培养基,从植物组织中分离得到118株放线菌。使用6种新药筛选模型对分离菌株进行了生理活性筛选,77株放线菌的发酵液样品在一个或多个药物筛选模型中显示为阳性,初筛阳性率为65.3%。对77株筛选阳性菌株进行的初步分类研究结果显示,其中44株属于链霉菌属,25株为小单孢属的菌株,3株属于糖丝菌属,3株为诺卡氏属菌株,1株是拟诺卡氏属菌株,1株是伦茨氏菌属的菌株。对一株在抗多重耐药检定菌HH22和降血脂模型上都显示出良好活性的菌株I07A-01824进行表型和基因型分析,确定I07A-01824为黄白链霉菌(Streptomyces albidoflavus)菌株。该研究显示,红树植物内生环境不仅仅孕育了丰富多样的内生放线菌,而且红树植物内生放线菌也是新型天然生理活性次级代谢产物的有效来源。     

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。     

微波处理对嗜碱和嗜盐海洋放线菌分离效果的影响

【目的】研究微波处理对于分离嗜碱和嗜盐海洋放线菌的效果。【方法】用微波处理7份海泥样品,梯度稀释后涂布于3种分离培养基,分离具有嗜碱和嗜盐特性的海洋放线菌。【结果】微波处理后的7份样品中,4份样品中嗜碱海洋稀有放线菌和3份样品的嗜盐海洋稀有放线菌数量极显著提高;7份样品中的嗜碱、嗜盐海洋小单孢菌属、游动放线菌属、诺卡氏菌属等稀有放线菌数量均有显著增加,不同样品中新分离到链孢菌属、小双孢菌属、链孢囊菌属及其他未鉴定的海洋稀有放线菌,分离到属的数量提高了1-4个。【结论】微波处理不仅显著提高嗜碱和嗜盐海洋放线菌的分离数量,而且明显增加了海洋稀有放线菌的分离种类。     

艾丁湖可培养嗜盐菌多样性及功能酶、抗菌活性筛选

【目的】探究艾丁湖可培养嗜盐菌的多样性、功能酶活性以及抗菌活性,进一步了解其次级代谢产物情况,为新型生物活性物质的发掘提供依据。【方法】选用以20种糖及糖的衍生物作为唯一碳源的寡营养培养基从艾丁湖5个样点中分离得到298株嗜盐菌,根据形态特征去重复后,选取62株菌运用16S r RNA基因系统发育分析的方法研究样品中嗜盐菌的多样性;从不同类群选取22株代表菌株,采用点接法进行3种功能酶的筛选,运用平板对峙法检测代表菌株对12种病原菌的抗菌活性。【结果】从5%、10%和15%3个盐浓度中分别分得221、54和23株嗜盐菌,获得的嗜盐菌分布在9个科18个属;其中放线菌分布于4个属,细菌分布于14个属;Nocardiopsis和Pontibacillus属为艾丁湖可培养嗜盐菌的优势类群,分别占17.7%和16.1%;有15株嗜盐菌相似性低于98.5%,可能为潜在新种。所选取的22株代表菌株中,分别有68.2%、22.7%和72.7%的实验菌株具有蛋白酶、淀粉酶和酯酶活性;45.5%的代表菌株对12种病原菌表现出了抗至少1种病原菌的活性,其中一株Nocardiopsis属放线菌能抗9种病原菌,表现出了广谱的抗菌活性。【结论】新疆艾丁湖土样中嗜盐菌的多样性较丰富,而且具有较好的生物活性,为后续进一步研究其次级代谢产物提供了依据。     

Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Culture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.     

河南叶县岩盐可培养中度嗜盐菌的多样性

【背景】嗜盐微生物因为独特的生理和代谢特征而对高盐环境有着良好的适应能力,在环境污染治理、酶制剂等领域具有很高的应用和研究价值,是一类重要的极端环境微生物资源。【目的】为了更好地认识我国岩盐微生物的多样性,开发和利用嗜盐微生物资源,积累丰富的微生物菌种资源。【方法】在5%和10%的盐度下,使用Alkaline oligotrophic medium (AOM)、Neutral haloarchaeal medium (NHM)、Diluted modified marine agar (dmMA)和ISP3 medium (ISP3)四种培养基,分离和纯化河南叶县岩盐矿的卤水和盐土中的嗜盐菌,使用细菌通用引物27F和1492R扩增和测序纯化菌株的16SrRNA基因,使用Ez BioCloud和NCBI上的BLAST比对进行分子鉴定,使用MEGA5.0进行遗传进化分析。【结果】从河南叶县岩盐卤水和盐土中一共分离和纯化到78株细菌,菌株16S rRNA基因序列显示它们来自3个门:厚壁菌门(Firmicutes)的Bacillus 26株、Halobacillus 30株、Oceanobac...     

Exploration,antifungal and antiaflatoxigenic activity of halophilic bacteria communities from saline soils of Howze-Soltan playa in Iran

In the present study, halophilic bacteria communities were explored in saline soils of Howze-Soltan playa in Iran with special attention to their biological activity against an aflatoxigenic Aspergillus parasiticus NRRL 2999. Halophilic bacteria were isolated from a total of 20 saline soils using specific culture media and identified by 16S rRNA sequencing in neighbor-joining tree analysis. Antifungal and antiaflatoxigenic activities of the bacteria were screened by a nor-mutant A. parasiticus NRRL 2999 using visual agar plate assay and confirmed by high-performance liquid chromatography. Among a total of 177 halophilic bacteria belonging to 11 genera, 121 isolates (68.3%) inhibited A. parasiticus growth and/or aflatoxin production. The most potent inhibitory bacteria of the genera Bacillus, Paenibacillus and Staphylococcus were distributed in three main phylogenetic clusters as evidenced by 16S rRNA sequence analysis. A. parasiticus growth was inhibited by 0.7–92.7%, while AFB₁ and AFG₁ productions were suppressed by 15.1–98.9 and 57.0–99.6%, respectively. Taken together, halophilic bacteria identified in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for managing toxigenic fungi growth and subsequent aflatoxin contamination of food and feed in practice.

     

一株来自蚯蚓的产纤溶酶蜡状芽孢杆菌Bacillus cereus的筛选及鉴定

对来自4个不同省份的5条蚯蚓的肠道及体表细菌进行分离,共获得122株细菌。通过脱脂奶粉平板法初筛,纤维蛋白平板法复筛,以透明圈为筛选标记,共筛选出产纤溶酶菌株12株,其中菌株SC-3-W-3的纤溶酶活力较高,达到了538.64 U/mL(相当于尿激酶的活力单位)。通过对其形态、培养、生理生化特征进行研究,发现其与蜡状芽孢杆菌Bacillus cereus Frankland的特征很相符。进一步对SC-3-W-3的16S rDNA序列及系统发育分析表明,该菌株与蜡状芽孢杆菌的同源性高达100%。综合生理生化及16S rDNA序列比对结果,将SC-3-W-3菌株鉴定为蜡状芽孢杆菌。     

Bioprospecting potentials of moderately halophilic bacteria and the isolation of squalene producers from Kuwait sabkha

International Microbiology - Sabkhas in Kuwait are unique hypersaline marine environments under-explored for bacterial community composition and bioprospecting. The 16S rRNA sequence analysis of 46...     

黑麦草根际产铁载体细菌HMGY6B的筛选鉴定及对病原菌的拮抗作用

【目的】从石灰性土壤中分离筛选出铁载体合成能力强、抗病效果好的菌株。【方法】采用刃天青(Chrome azural S,CAS)平板检测法,定性、定量筛选产铁载体能力较强的菌株,通过菌株形态、生理生化特征、16S r RNA基因序列相似性和系统发育分析,鉴定细菌类型,然后采用平板对峙法研究菌株与病原菌的拮抗作用。【结果】从多年生黑麦草根际土壤中分离得到一株产铁载体能力很强的菌株HMGY6B,经鉴定,该菌属假单胞菌属Pseudomonas菌株,其产生的儿茶酚型铁载体对黄瓜灰霉病(Botrytis cinerea)有显著的拮抗作用,在低铁条件下(0.16、2、5、10μmol/L FeCl_3)对黄瓜灰霉病的生长抑制率高达91.2%,但在富铁条件下(50μmol/L FeCl_3)降为30.2%,100μmol/L FeCl_3抑制率仅为5.5%。【结论】菌株HMGY6B可用于今后复合型抗病生物菌肥的开发研制。     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。