棒状类细菌电击转化中多种条件对转化效率的影响

以质粒PXZl0145电击转化不同棒状类细菌菌株,研究了影响电击转化效率的诸个因素,在含4%甘氨酸的培养基中生长至对数前期的菌体最适用于电击转化,当以同源DNA进行电击转化时,1．μgDNA中转化效率最高可达到8×1O⁶转化子,但用异源DNA时,转化效率要比前者低10²～10³倍。     

Bioaccumulation of metals by Coryneform SL-1

Summary An environmental isolate defined as Coryneform SL-1 has the capacity to remove large quantities of Pb, Ag, Cd, Hg, Ni, and Zn from aqueous solution. The organism effectively removed a mixture of Pb, Zn, Cd, Cu, and Ni from a simulated waste water. In examining the metal resistance of the organism it was found that agar-containing assay systems yield misleadingly high MIC values.     

高温蛋白质降解菌的筛选及其在鸡粪发酵中的应用

目的:本研究通过对高温蛋白质降解菌的富集培养、筛选与分离,旨在为鸡粪资源的合理利用,生产高质量的有机肥料奠定基础。方法:通过在高温条件下对高效降解蛋白质的高温菌种进行富集培养、纯化与分离。结果:分离得到24株高温高效降解蛋白质菌株,经鉴定其分别属于短短芽孢杆菌属(Brevibacillus spp.)、嗜热芽孢杆菌属(Bacillus spp.)、假黄单孢菌(Pseudoxanthomonas spp.)等8种菌属。将制备的复合菌液接种于鸡粪中发现,实验组升温速率明显快于对照组,在发酵后16 h左右到达高温阶段,比空白组提前将近8 h,且加入耐高温蛋白质降解菌的FJ3组,高温期比空白组延长24 h左右,持续时间长达56 h;发酵过程pH值比空白组下降较快,pH值最低到5.5左右;物料的含水率从65%降至20%左右,N、P、K等营养元素所占比例有所增加,且发酵产物质地松散、柔软,带有较浓的酸香味。结论:实验表明添加外源高温降解蛋白质菌株,能够减少鸡粪发酵产生的恶臭,活化养分,提高堆肥效果,对鸡粪便的处理有重要意义。     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Possible Role of Coryneform Bacteria in Age Gelation of Ultrahigh-Temperature-Processed Milk

Two strains of psychrotrophic, gram-positive, proteolytic bacteria isolated from raw milk were identified as Arthrobacter or closely related coryneform species. We inoculated raw-milk samples with the strains (one strain per sample) and compared rates of gelation after ultrahigh-temperature processing with that of ultrahigh-temperature-processed controls. The trial indicated that either organism could play a role in age gelation of ultrahigh-temperature-processed milk.     

棒杆菌宿主中质粒不稳定性的研究

重组质粒pNAR4是由钝齿棒杆菌质粒pNAT65和大肠杆菌质粒pACYCl84构建的穿梭质粒。质粒pNAR4转化不同棒杆菌,在钝齿棒杆菌T6—13和答氨酸棒杆菌l0l47中为结构型不稳定,在钝齿棒杆菌B9中为分离型不稳定。而pNAT65转化谷氨酸棒杆菌10147后,转化于中的质粒分子大小及主要酶切位点与pxz10145相同。DNA杂交实验结果表明．在10147菌中有一种与z10145高度同源的超螺旋DNA组分,而这一组分与pxz10145的来源宿主中的另一小质粒具有相同的分子量。提出了质粒结构不稳定与宿主中存在的pxz10145高度同源的小质粒(超螺旋组分)有关,并提出产生质粒分子结构反复变化原因的假设。     

苦豆子根瘤内生细菌分离及表型多样性分析

对分离自新疆苦豆子(Sophora alopecuroides)根瘤的60株内生细菌进行了108项表型性状的测定,包括唯一碳源利用、唯一氮源利用、对抗生素和染料的抗性、耐盐性、初始pH值生长、生长温度范围及石蕊牛奶反应、氧化酶、过氧化氢酶和脲酶等.结果表明:新疆苦豆子根瘤内生细菌具有较强的抗逆性,具有较强的耐受高温和耐盐、碱能力.77%的菌株能在50℃条件下生长,43%的菌株能耐受6.0%的NaCl,73%的菌株能在pH为10的碱性环境下生长.聚类分析结果表明在Watson距离为0.26时可聚为11个表观群,代表菌株分别属于芽孢杆菌属(Bacillus sp.)和气芽孢杆菌属(Aerobacillus sp.).     

Selection and Characterization of Biofuel-Producing Environmental Bacteria Isolated from Vegetable Oil-Rich Wastes

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.     

Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes.     

Influence of Bulking Agents,Fertilizers and Bacteria on the Removal of Diesel from a Newfoundland Soil

Laboratory experiments in culture flasks, containing diesel-contaminated Newfoundland soil samples, were undertaken to compare the influence of fertilizers, microorganisms and bulking agents on bioremediation. In Phase I experiments only one fertilizer (cow manure or poultry manure), one bulking agent (sand or hay), or one inoculum (cold-tolerant indigenous bacteria or exogenous commercial bacteria) was added to a soil sample. In Phase II experiments, Design-Expert® Version 6 design of experiment software determined the combinations of fertilizers, bulking agents and inocula to be mixed with the soil samples to study the interactions among the amendments. The maximum diesel removal at 90 days occurred in the sample with sand (Phase I) and in the sample with cow manure, an inoculum of cold-tolerant indigenous bacteria, and sand (Phase II). Diesel removal at 45 days for the same two samples was 85.4% (Phase I) and 91.9% (Phase II), suggesting the cow manure and/or cold-tolerant bacteria inoclum accelerated the process. The poultry manure, commercial bacteria and hay were less effective than their counterparts. The commercial bacteria were more sensitive to diesel concentration than the indigenous cold-tolerant bacteria. The addition of sand, cow manure, and poultry manure improved diesel removal.     

一株甲烷氧化菌的分离鉴定与特性

甲烷氧化菌是一类能以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物。从若尔盖高原不同地点采集的样品中筛选得到一株名为XN1的甲烷氧化菌,根据此菌株的形态与16SrRNA序列同源性分析,证实该菌株属于Methylomonas属。对该菌株的培养条件进行研究的结果表明,以甲烷与甲醇共同作为碳源,硝酸钾和氯化铵共同为氮源时菌生长最好,最适生长温度为25℃,最适生长pH为6.5,培养基中CuSO4·5H2O和FeSO4·7H2O的浓度以0.03mg/L和0.4mg/L为宜。     

大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定

内生细菌存在于健康植物体内,一些内生细菌具有促生长、抗病和固氮等生物学功能.本项研究采用化学药剂表面灭菌方法从黑龙江省大豆品种合丰25的根、茎、叶和种子中分离到大量内生细菌,其种群数量在根部最多,为3.4×103CFU/g,在叶部次之,为2.8×103CFU/g,在茎部和种子中最少,为2.9×102 CFU/g和1.4×102CFU/g.从121株内生细菌中筛选到31株对大豆根腐病菌Fusarium oxysporum f. sp.soybean具有较强抑制作用的拮抗内生细菌,其中菌株TF28抑菌谱广,抑菌率高,对不同植物的病原菌F. oxysporum的抑菌率为80.2%～96.7%.经形态、生理生化和16S rRNA鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).     

Transposon mutagenesis of coryneform bacteria

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10⁴ mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.     

Characterization of Communities of Heterotrophic Bacteria Associated with Healthy and Diseased Corals in Nha Trang Bay (Vietnam)

A comparative investigation of the heterotrophic microflora of 11 species of healthy corals and of white-band-diseased and yellow-band-diseased corals inhabiting the reefs of Nha Trang Bay (Vietnam), which has been exposed to anthropogenic impact, was performed. Fifty-nine strains of heterotrophic bacteria isolated on Y/K and Endo media were investigated and characterized. All the isolates were identified at the genus level by consideration of the results of analysis of their phenotypic properties, determination of the molar percent of G+C bases in their DNA, and the composition of the fatty acids of lipids. In the composition of the microflora of tissues of healthy corals, γ-proteobacteria prevailed, with halomonads being dominant among them. In addition, the gram-negative bacteria included Pseudomonas and Vibrio spp., members of the Cyto-phaga-Fla-vobacterium-Bacteroides (CFB) phylogenetic cluster, and Moraxella sp. The gram-positive bacteria revealed included Bacillus, Staphylococcus, Halococcus, and Micrococcus spp., and coryneform bacteria. In the composition of the microflora of the tissues of affected corals, bacteria of the family Enterobacteriaceae and of the genera Planococcus and Arthrobacter, which were not revealed in healthy hydrobionts, were found. The anthropogenic impact is not the sole factor determining the infection of corals.     

Isolation and Characterization of Rumex acetosa-Associated Mineral-Weathering Bacteria

A total of 121 bacterial strains were isolated from the rhizosphere soils, root and stem interiors of Rumex acetosa to characterize the microenvironment-related changes in the mineral-weathering effectiveness, weathering mechanisms and populations of the bacteria. Among the 121 bacterial strains, 118 bacterial strains were found to weather biotite. The relative abundance of the highly effective mineral-weathering bacteria was different among the rhizosphere soils, root and stem interiors. Notably, the highest and lowest relative abundances of the highly effective mineral-weathering bacteria were observed in the stem and root interiors, respectively. Furthermore, the relative abundance of the highly acid-producing bacteria was significantly higher in the rhizosphere soils and stem interiors, while the highest and lowest relative abundances of the highly siderophore-producing bacteria were found in the stem interiors and rhizosphere soils, respectively. The mineral-weathering bacteria from the rhizosphere soils, root and stem interiors were affiliated with 11, 7 and 4 genera, respectively. In addition, 25–73% of the bacterial genera were specific to the plant-associated environments. The results showed diverse mineral-weathering bacteria in the plant-associated environments and microenvironment-related changes in weathering effectiveness and pattern and populations of the mineral-weathering bacteria. The results also suggested the different biotite-weathering mechanisms used by the bacteria among the plant-associated environments.     

Genetic Characterization of Epilithic Cyanobacteria and Their Associated Bacteria

Epilithic phototrophic biofilms develop inside Roman Necropolis and Catacombs on rock surfaces exposed to artificial light sources and are composed by a microbial consortium dominated by cyanobacteria. In this work, six non-axenic cultures of Leptolyngbya sp. strains isolated from biofilms from different Roman hypogea and maintained in cultures from 11 to 20 years were analysed along with their associated bacteria isolated in culture. The employment of PCR-fingerprinting techniques, using HIP1 and ERIC derived primers, allowed the clustering in three groups of the six Leptolyngbya strains and the typing of their isolated bacteria. The bacterial fingerprinting patterns were in agreement with the 16S rRNA gene sequencing and showed the presence in Leptolyngbya isolates of Pseudomonas, Stenotrophomonas, Agrobacterium and Bacillus representatives that were detected also in biofilms sampled from catacombs.     

Metal Distribution and Characterization of Cultivable Lead-Resistant Bacteria in Shooting Range Soils

Shooting ranges represent about 10% of polluted sites in Switzerland. This pollution, mainly consisting of lead (Pb) and antimony (Sb), can spread into the local groundwater, wildlife and plants. In this study, shooting range stop butt soils (elevated soil mount behind the target) of three sites (Versoix, Cartigny and Nyon, Western Switzerland) were investigated for metal contamination and culturable lead-resistant bacteria in contaminated soils. The inductively coupled plasma mass spectrometry analysis of surface stop butt soils (0–5 cm) indicated high metal concentrations especially for Pb and Sb, e.g., the Pb concentration ranging from 61,135 to 395,651, 23,821 to 201,268 and 120 to 27,517 mg kg^?1 in the soil from the sites of Versoix, Cartigny and Nyon, respectively. Molecular analysis of 16S rRNA gene demonstrates the presence of bacteria from diverse classes: Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli that exhibited high Pb minimum inhibitory concentration value from 1,036 to 2,694 mg kg^?1. The isolated strains could be the subject of further studies to evaluate their role in bioremediation of lead-contaminated soil.     

Bioremediation of Agricultural Wastes through Vermicomposting

The post-harvest residues of some local crops, e.g., wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting. The crop residues were amended with animal dung, and three types of vermibeds were prepared: (i) millet straw (S. vulgare + P. typhoides in equal quantity) + sheep manure (1: 2 ratio) (MS); (ii) pulse bran (V. radiata) + wheat straw (T. aestivum) + cow dung (1: 1: 2 ratio) (PWC); and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). Vermicomposting resulted in a significant increase in total N (97.3% to 155%), available P (67.5% to 123.5%), exchangeable K (38.3% to 112.9%), and exchangeable Ca (23.3% to 53.2%), and decrease in organic C content (20.4% to 29.0%) in the different vermibeds. The earthworm showed the higher biomass gain, growth rate (mg wt. worm^?1 day^?1) and cocoon numbers in the CSM vermibed. The quality of crop residues was directly related to the rate of organic matter mineralization during the vermicomposting. This study suggests that agriculture wastes could be converted into some value-added products, e.g., vermicompost and worm biomass through vermicomposting technology. The higher concentrations of plant nutrients in end products indicate a potential for using agriculture wastes in sustainable crop production.     

甘草内生细菌多样性研究

以分离培养的方法对内蒙古鄂尔多斯市甘草基地野生及栽培甘草内生细菌的多样性进行了初步研究.结果表明,野生及栽培甘草植株内存在大量种群丰富的内生细菌.经ERIC-PCR指纹图谱分析,共分离到120株内生细菌,野生及栽培甘草均表现出根和叶部位的内生细菌数量多于茎部.对其中82株进行16S rDNA片段测序分析,结果表明这些内生细菌分别与GenBank中α、β、γ-Proteobacteria、Firmicutes、Actinobacteria五类细菌中的19个已知属相似性达到97%～100%.内生细菌的主要优势种群为芽孢杆菌属(Bacillus sp.)、假单胞菌属(Pseudomonas sp.)、泛菌属(Pantoea sp.)和沙雷氏菌属(Serratia sp.).