Growth enhancement of effective microorganisms for bioremediation of crude oil contaminated waters

The bioremediation of polluted groundwater, wastewater aeration pond and biopond sites was investigated using bacteria isolated from these sites located at the oil refinery Terengganu Malaysia. Out of 62 isolates, only 16 isolates from groundwater (8) and wastewater aeration pond (3) and biopond (5) were chosen based on growth medium containing 1% (v/v) Tapis crude oil. Only four isolates; Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata showed percentage biodegradation of crude oil more than 50% after 5 days using Mineral Salts Medium (MSM). The effect of physical parameters (temperature, pH and agitation) on growth by all four strains showed a maximum growth in MSM medium with 1% Tapis crude oil at 37 degrees C with pH 7 and agitation of 130 rpm.     

香蕉内生细菌分离、活性评价及数量分布

从香蕉健康植株的根、假茎、叶柄、叶片等组织中分离获得386份内生细菌分离物。通过体外活性评价筛选出对9种常见的植物病原真菌具有一定抗性的内生细菌分离物28份,占总分离物的7.25%,其中对香蕉枯萎病菌抑制率达到80%~100%的分离物有9份。初步测定结果表明,这些分离物的基础生物学特性的分类地位初步归属为不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、短杆菌属(Brevibacterium)、产碱杆菌属(Alcaligenes)和伯克霍尔德菌属(Burkholderia)细菌。9份分离物与病原真菌菌丝体对峙培养镜检结果表明,供试的9种病原真菌菌丝体均出现不同程度的肿胀与断裂。内生细菌在香蕉植株体内的数量分布分析发现,内生细菌在根、假茎、叶柄、叶片等组织中的数量分布呈递减趋势。     

Respiratory bacterial flora from healthy as well as respiratory symptoms' subjects

Respiratory system is the primary settlement place of opportunistic organisms and considered as chief carrier of common respiratory pathogens. The aim of the study was to know the opportunistic organisms present in the healthy subjects as well as subjects that were suffering from respiratory symptoms. The organisms were identified as per standard bacteriological protocol and pathogenicity tests of the identified organisms were performed in mouse model. Antibiotic sensitivity of the identified organisms was performed. The bacterial flora present in the throat swab of apparently healthy as well as subjects suffering from respiratory symptoms were: Staphylococcus spp. (39.44%) of which Coagulase positive Staphylococcus (21.13%) and Coagulase negative Staphylococcus (18.31%), Klebsiella spp. (19.72%), Pseudomonas spp. (15.49%), Proteus spp. (4.23%), E. coli (9.86%) and Bacillus spp. (11.27%). Among the isolates Staphylococcus, Klebsiella and Pseudomonas were the predominant species. Percentages of identified bacteria were higher in respiratory symptoms exhibiting individuals (53.52%) than apparently healthy individuals (46.48%). All coagulase positive Staphylococcus, Klebsiella spp. and Pseudomonas spp. isolated from respiratory symptoms' subjects were found to be pathogenic. The isolated bacteria were resistant to amoxicillin and ampicillin but sensitive to ciprofloxacin and norfloxacin. Isolated Pseudomonas spp. showed multidrugs resistant properties. The study provided information about the pathogenic organisms' present respiratory systems of apparently healthy as well as subjects suffering from respiratory symptoms. The pathogenic natures of the isolated organisms were determined to make aware of scientists as well as clinicians. Antibiotics sensitivity assays would provide information to the clinicians for the selection of appropriate antibiotics to treat their patients.     

Enhancement of Growth and Grain Yield of Rice in Nutrient Deficient Soils by Rice Probiotic Bacteria

Plant associated bacteria are promising alternatives to chemical fertilizers for plant growth and yield improvement in an eco-friendly manner.In this study,rice associated bacteria were isolated and assessed for mineral phosphate solubilization and indole-3-acetic acid(IAA) production activity in vitro.Six promising strains,which were tentatively identified as phylotaxon Pseudochrobactrum sp.(BRRh-1),Burkholderia sp.(BRRh-2),Burkholderia sp.(BRRh-3),Burkholderia sp.(BRRh-4),Pseudomonas aeruginosa(BRRh-5 and BRRh-6) based on their 16 S rRNA gene phylogeny,exhibited significant phosphate solubilizing activity in National Botanical Research Institute phosphate growth medium,and BRRh-4 displayed the highest phosphate solubilizing activity,followed by BRRh-5.The p H of the culture broth declined,resulting in increase of growth rate of bacteria at p H 7,which might be due to organic acid secretion by the strains.In presence of L-tryptophan,five isolates synthesized IAA and the maximum IAA was produced by BRRh-2,followed by BRRh-1.Application of two most efficient phosphate solubilizing isolates BRRh-4 and BRRh-5 by root dipping(colonization) of seedling and spraying at the flowering stage significantly enhanced the growth and grain yield of rice variety BRRI dhan-29.Interestingly,application of both strains with 50% of recommended nitrogen,phosphorus and potassium fertilizers produced equivalent or higher grain yield of rice compared to the control grown with full recommended fertilizer doses,which suggests that these strains may have the potential to be used as bioinoculants for sustainable rice production.     

Effect of carbonic anhydrase-containing endophytic bacteria on growth and physiological attributes of wheat under water-deficit conditions

Drought is one of the major limitations to agricultural productivity, suppressing plant growth and yield of food crops throughout the world particularly in arid and semiarid regions. Drought-tolerant carbonic anhydrase (CA; EC 4.2.1.1)-containing endophytic bacteria may improve plant growth under stressed conditions. In the present study, effect of drought-tolerant CA-containing endophytic bacteria on growth and physiology of wheat under water-deficit conditions was studied. One hundred and fifty isolates were isolated from wheat plants and screened for their ability to tolerate polyethylene glycol (PEG) 6000-induced water-deficit stress (?0.31 to ?3.20 MPa). Fifty isolates exhibiting intrinsic ability to tolerate stress were further screened for CA activity. Ten drought-tolerant isolates with higher CA activity were evaluated for improving wheat growth under water-deficit conditions (?0.04, ?1.09, ?1.23 MPa). Results showed that PEG-mediated water-deficit stress significantly reduced growth of wheat. However, inoculation with isolates WR2, WS11 and WL19 significantly enhanced seedling growth by improving maximum root length, shoot length, root and shoot dry weight under non-stressed as well as stressed conditions. These isolates were identified by 16S rRNA as Bacillus marisflavi (WR2) Bacillus thuringiensis (WS11) and Bacillus subtilis (WL19). Isolate WL19 also improved chlorophyll content, photosynthetic rate, CA activity and relative water content compared to uninoculated control plants. Overall, our findings suggest that endophytic bacterial isolates WR2, WS11 and WL19 with CA activity can enhance photosynthesis and biomass of wheat seedlings under water-deficit conditions.

Abbreviations: CA: Carbonic anhydrase; PEG: Polyethylene glycol; CO₂: Carbon dioxide; HCO^3–: Bicarbonate; TSA: Tryptic Soy Agar; LB: Luria Bertani; A: CO₂ assimilation rate; E: Transpiration rate; g_s: Stomatal conductance; C_i: Substomatal CO₂ concentration; RWC: Relative water content; EL: Electrolyte leakage     

Pectic enzyme production by two strains of pseudomonas fluorescens associated with the pinkeye disease of potato tubers

A bacterium identified asPseudomonas fluorescens was isolated from pinkeye lesions of infected Kennebec potato tubers. Two isolates which rapidly macerated tuber slices were selected for further study. Supernatants from macerated potato tuber slices inoculated previously with either isolate 13 or 31 ofP. fluorescens contained pectic enzyme activity. Trace amounts of pectin methylesterase activity were observed in the supernatants. Optimum viscosity reduction of sodium polypectate (NaPP) or pectin substrates by the enzyme complexes occurred at pH 8.6 and 7.2 respectively. Paper chromatography of hydrolysis products indicated that substrates were hydrolyzed terminally as well as randomly when the reaction mixtures were buffered from pH 7.1 to 7.5 NaPP was degraded more rapidly than pectin by both enzyme complexes. Reducing group data indicated the greatest hydrolysis of both substrates occurred near pH 8.6 and that both enzyme complexes hydrolyzed NaPP preferentially. Trans-eliminase activity was found in both enzyme complexes. Optimum activity occurred between pH 8.0 and 9.0 using a NaPP substrate and between pH 7.0 and 8.0 using pectin. The addition of calcium to the reaction mixture increased hydrolysis suggesting the importance of calcium as a co-factor for trans-eliminase activity. Both enzyme complexes macerated potato tuber slices rapidly. Data suggested a relationship between viscosity reduction of NaPP, transelimination of NaPP, and loss of coherence of tuber tissue slices by enzyme complexes of both isolates.     

玉米茎腐病病原禾谷镰孢拮抗菌筛选及分子鉴定

禾谷镰孢引起的玉米茎腐病会导致玉米产量锐减,筛选高效的拮抗菌株对防治玉米茎腐病尤为重要。采用稀释培养法从土壤中分离得到810株细菌,通过平板对峙法和16S rDNA分子测序方法进行拮抗菌的筛选和分子鉴定,共分离出禾谷镰孢拮抗菌23株,占分离细菌总数的2.84%,抑菌圈直径10.12～27.56 mm。拮抗细菌主要分布在厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和放线菌门(Actinobacteria)3个门及芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)、不动杆菌属(Acinetobacter)和节杆菌属(Arthrobacter)5个属中,其中,芽孢杆菌属细菌数量最多且拮抗能力较强,暹罗芽孢杆菌(Bacillus siamensis)的拮抗活性最强,具有较大的生防潜力。实验分离的23株菌株对禾谷镰孢有较好的拮抗活性,在玉米茎腐病防控中具有潜在的应用价值。     

Acclimated biomass that degrades Sulfonated Naphthalene Formaldehyde Condensate

A number of aerobic species were isolated from textile industry activated sludge wastewater. The bacterial consortium was acclimated during seven days before testing its capacity of Sulfonated Naphthalene-Formaldehyde Condensate (SNFC) recalcitrant compound degradation. SNFC's degradation was evaluated by using different techniques including: vapour pressure osmometry, spectroscopy UV-Visible and Chemical Oxygen Demand (COD). The degradation of SNFC by acclimated bacterial consortium was determined by monitoring the decrease of absorbance and of COD at wavelength 288 nm. We were able to deduce that biodegradation of SNFC involves two steps: cleavage of CH2 bridges and the degradation of the aromatic nuclei. The bacteria species community that was able to degrade SNFC consisted of aerobic Gram-negative rods belonging to the Pseudomonadaceae family. The strains were identified as Bukholderia cepacia, Brevundimonas vesicularis, Pseudomonas stutzeri, Ralostonia picketti, Shewanella putrefaciens, Sphingomonas paucimobilis and Agrobacterium radiobacter.     

白叶枯病菌拮抗菌筛选及水稻叶围微生物互作研究初报

 1998年7月～10月, 于田间汕优63水稻叶片上分离采集微生物, 并进行叶围微生物鉴定,以筛选白叶枯病拮抗菌。结果表明:除极少量分布的真菌和细菌外, 主要种群包括真菌类群Alternaria和 Fusarium属; 细菌类群Flavobacterium, Xanthomonas, Pseudomonas和Bacillus属。对白叶枯病菌（Xanthomonas oryzae pv. oryzae）具有明显拮抗作用的微生物为Bacillus属,而且Bacillus属细菌对其余大部分叶围微生物具有程度不同的拮抗作用,可以初步断定其产生的拮抗物质具有一定的广谱性。     

红树共附生微生物分离与抗白色念珠菌活性评价

对湛江市廉江高桥、东简北港2个红树林区红树的根、叶、花、果不同部位27份样品进行共附生微生物的分离及抗白色念珠菌活性的评价。采用改良M1培养基、改良马丁培养基和改良高氏一号培养基进行细菌、真菌和放线菌的分离,用美蓝酶标仪法对所筛菌株进行抗白色念珠菌活性评价。利用SPSS13.0软件对数据进行单因素方差分析和多重比较。从27份样品中共分离出384株红树林共附生微生物,其中细菌170株,真菌160株,放线菌54株,细菌和真菌为优势菌群,两者占分离总菌株数的86.0%;从384株分离菌中检测出64株具有抗白色念珠菌活性,占分离菌的16.7%;统计分析显示,除2个地区的抗白色念珠菌活性数无显著差异外,2个地区的红树共附生微生物的组成、不同类型共附生微生物的数量、不同类型共附生微生物的抗白色念珠菌活性、红树不同部位的共附生微生物数量与活性菌株数量均存在一定的差异。     

盐碱地大豆根内促生拮抗细菌DCX的分离及鉴定

根系内生细菌资源的开发与利用是绿色农业发展的研究重点，而盐碱地大豆根内生细菌的分离与鉴定鲜有报道。本文以大庆盐碱地大豆根系为材料，分离纯化大豆内生细菌，采用平板对峙法和比色法测定内生细菌的抑菌能力和分泌IAA能力，选取抑菌谱广且分泌IAA能力强的株菌，通过检测形态学特征、生理生化性质、结构基因和功能基因序列的系统发育分析，明确细菌的分类地位。结果表明从大庆盐碱地大豆根内分离获得的一株内生细菌DCX，对10种植物病原真菌均具有拮抗作用，其中，对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为50.00%、78.12%、56.25%、43.75%，分泌IAA的能力为2.56±0.41μg·mL-1（n=6）。经形态学、生理生化试验及系统进化分析鉴定，DCX菌株属于解淀粉芽孢杆菌（Bacillus amyloliquefaciens），该菌株在pH11、NaCl浓度 10%的NA培养基中生长良好，可为盐碱地的作物病害防治提供资源。     

Selection and evaluation of potential biocontrol rhizobacteria from a raised bog environment

Understanding the interactions of bacteria with plants and the role of microorganisms in the lifecycle of plants is becoming more and more important due to the social claim for a clean agriculture. Thus the significance of studying plant associated bacteria is increasing. The main aim of our study was to isolate and characterize bacteria associated with bryophytes from a natural raised bog ecosystem. Characterization of the isolates by their beneficial properties was realized with microbiological and molecular tools. Bacterial strains were identified taxonomically, isolates belonging to the genera Pseudomonas, Bacillus, Serratia, Stenotrophomonas, Delftia, Cedecea, Lysinibacillus and Viridibacillus have been found. Pseudomonas, Serratia and Bacillus were the dominant bacterial genera associated with bryophytes. In vitro study of antagonism showed that a high number of the isolates inhibited the growth of fungal and bacterial plant pathogens – Fusarium oxysporum, Alternaria alternata, Erwinia carotovora – or produced secondary metabolic substances with biocontrol properties. Bacterial strains identified as Serratia fonticola BB17 and Pseudomonas fluorescens BE8 proved to be the most efficient against plant pathogens, with biocontrol effectiveness up to 48.28% and 55.17% respectively, suggesting the potential use of these strains in biotechnological applications.     

油菜菌核病拮抗细菌的筛选和高效菌株的鉴定

从油菜根际和叶围分离得到320个细菌分离物,在马铃薯葡萄糖琼脂培养基上的拮抗实验中,18个分离细菌表现对油菜菌核病菌不同程度的拮抗作用,其中Y1菌株对油菜菌核病菌菌丝的生长具有明显的抑制作用.对Y1进行油菜离体叶片、温室盆栽和田间小区接种实验,该菌均表现对油菜菌核病明显的防病效果;在温室盆栽试验和田间小区试验中,防病效果达到92%.经过鉴定,Y1菌株为枯草芽孢杆菌.     

大豆镰刀菌病害生防菌的分离鉴定及防治效果验证

为解决镰刀菌病害对大豆种植业造成的损失,避免化学药剂对农产品质量和人体健康的风险,本研究分离针对大豆镰刀菌病害的生防菌,通过分子生物学和生物信息学手段鉴定生防菌类型,并通过温室试验验证生防菌对大豆尖孢镰刀菌病害的防治效果。研究共分离得到144株菌,其中1种Y8菌株对尖孢镰刀菌大豆专化型有较强的防治效果,这种菌经鉴定为假单胞菌属微生物。试验结果显示:Y8菌株能够有效保护大豆免受尖孢镰刀菌的侵染,使大豆的病情指数降低42.23%。相对于对照组,Y8菌株使大豆的生物量提高45.9%,使根生物量提高73.3%,大豆籽产量提高35.4%,叶绿素含量提高22.7%。这可能是由于Y8菌株成功的在大豆根际定植,抑制尖孢镰刀菌的生长,并且其次生代谢物能促进大豆的生长。本研究发现的假单胞菌属微生物可为大豆尖孢镰刀菌病害的防治提供一种有效的解决途径。     

Genetic approach for the fast discovery of phenazine producing bacteria

A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.     

中国长江三角洲地区及日本水稻种子细菌多样性研究

1997～1999年,分别从中国长江三角洲稻区的276份和日本稻区30份水稻种子样本中分离出3 200余个及303个非致病菌株。经细菌学、菌落形态及致病性测定后,对386个格兰氏阴性菌及30个阳性菌进行Biolog鉴定,并用3个水稻病原菌进行了拮抗性筛选。从中国的水稻种子中测得假单胞杆菌属的10个种及5个非假单胞杆菌属中的14个种;从日本的水稻种子中测得假单胞杆菌属的6个种及5个非假单胞杆菌属中的10个种。这5个非假单胞菌属中的14个种尚未有从中国稻谷上分离出来的记录。从中国的14个细菌种及日本的10个细菌种中测得拮抗菌,其中占拮抗细菌种总数64%的种来自假单胞杆菌属和芽孢杆菌属。     

拮抗大豆疫霉菌植物内生细菌的筛选与鉴定

大豆疫霉根腐病是世界范围内危害严重的毁灭性大豆病害,筛选内生菌进行生物防治是未来该病害防治的重要发展方向。以大豆根系为材料筛选得到67株内生细菌,采用平板对峙生长实验,发现其中18株对大豆疫霉菌的菌丝生长具有抑制作用,其中以A2、A4、A7和B8效果最佳,抑制率分别达到72.8%、55.5%、76.0%和87.1%。利用无菌发酵滤液与大豆疫霉孢子共培养发现,A2和A4对孢子萌发具有明显抑制效果,抑制率达75.4%和85.8%。盆栽实验表明,A2和A4无菌发酵滤液能使经大豆疫霉孢子侵染后的大豆种子萌发率由63.1%分别提高到80.0%和80.3%,健康幼苗率由31.4%提高到45.7%和50.9%。根据16S rDNA序列系统发育分析,初步确定A2为假单胞菌(Pseudomonas sp.),A4、A7和B8皆为芽孢杆菌(Bacillus sp.)。     

Epidemiology and antibacterial susceptibility patterns of bloodstream infections, 2001-2004: an experience with BACTEC 9240 in Southern Iran

This study was conducted to determine the prevalence of bacteria recovered from bloodstream samples by Bactec 9240 at our hospital wards and to evaluate their antibacterial susceptibility patterns. During January 2001 through December 2004, 9407 referred blood samples in Bactec bottles from admitted patients at three main wards, neonates, pediatrics and adults at Nemazee Hospital, affiliated to Shiraz University of Medical Sciences in Shiraz were processed. Positive cultures were purified and identified according to standard methods. Sensitivity of bacteria to different antibiotics was determined by Kirby-Bauer disk diffusion method. Staphylococcus aureus 132(25%), Escherichia coli 64(12.1%) and Pseudomonas aeruginosa 52(9.8%) were the most pathogenic bacteria which were recovered from the blood samples. Pathogenic microorganisms were isolated from blood samples of 305 (57.8%) at pediatrics, from 181 (34.2%) at adults and from 42 (8%) at neonates wards. The highest antibiotics activities against gram positive isolates observed for vancomycin (98.4%), chloramphenicol (86.4%) and ciprofloxacin (77.4%), while in gram negative bacteria imipenem (96.1%), ciprofloxacin (83%) and amikacin (77.9%), were effective antibiotics. Frequency of isolated bacteria at pediatrics compared to adults and neonates wards were approximately two and seven folds high, respectively which indicates special attention should be paid to pediatrics patients both in prevention and treatment aspects. Vancomycin and imipenim are the effective antibiotics and could cover majority of gram positive and negative bacteria. Therefore, combined administrations of these antibiotics seems mandatory for empirical therapy.     

Molecular identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in nasal swabs samples from horses suffering respiratory infections in Iran

The objective of this study was to evaluate the existence of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus as probable agents associated with naturally occurring infection of the equine upper respiratory disease in Mashhad area. Nasal swabs samples from thirty horses with upper respiratory tract infections were collected. The bacteria isolated and identified were Streptococcus equi subsp. equi (1 isolate), Streptococcus equi subsp. zooepidemicus (25 isolates), Pasteurella sp. (11 isolates), Staphylococcus sp. (17 isolates), Bacillus sp. (4 isolates), Pseudomonas sp. (4 isolates), Proteus sp. (1 isolate), Neisseria sp. (1 isolate) and E. coli (1 isolate). All 25 isolates of Streptococcus equi subsp. zooepidemicus and the isolate of Streptococcus equi subsp. equi were characterized by biochemical tests and molecular techniques. For molecular identification of the subspecies S. equi and S. zooepidemicus two genomic region SeM and sodA were amplified. This study is the first report of molecular identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Iran.     

传统豆制品白干与薄百叶中优势腐败菌的分离与初步鉴定

以传统豆制品白干和薄百叶为材料,对贮藏过程中主要腐败微生物进行分离鉴定。利用纯培养的方法共筛选出菌落形态差别比较明显的菌株19株。对细菌菌株进行形态观察、革兰氏染色和生理生化鉴定,同时对细菌和酵母菌纯培养物提取DNA,分别进行16S rDNA和26S rDNA PCR扩增,PCR产物经测序后与NCBI中已知序列进行比对和鉴定,确定各细菌和酵母菌菌株的种属;霉菌纯培养物染色后观察孢子繁殖形态,最终确定这些菌株的种属。结果表明:白干和薄百叶中共有的优势腐败菌是溶酪葡萄球菌,枯草芽孢杆菌,约翰逊不动杆菌,戊糖片球菌;季也蒙毕赤酵母,皮状丝孢酵母;圆弧青霉和黄绿青霉。此外,白干中还有诞沫假丝酵母;薄百叶中有浅黄假单胞菌,芸苔丝孢酵母。