Isolation and Characterization of Coal Solubilizing Aerobic Microorganisms from Salt Range Coal Mines,Pakistan

Microbial solubilization of coal has been considered as a promising technology to convert raw coal into valuable products. In the present study, initially a total of 50 different aerobic bacterial and fungal isolates have been isolated from soil, coal and water samples of Dulmial Coal Mines, Chakwal, Pakistan, but on the basis of solubilization potential, only four isolates were selected for further study. The intensity of biosolubilization was measured by determining the weight loss of the coal pieces, which was observed to be about 25.93% by Pseudomonas sp. AY2, 36.36% by Bacillus sp. AY3 and 50% by Trichoderma sp. AY6, while Phanerochaete sp. AY5 showed maximum coal solubilization potential i.e. 66.67% in 30 days. UV/Vis spectrum revealed an increase in the pattern of absorbance of all treated samples compared to control referring to solubilization. Fourier transform infrared spectroscopy indicated alterations in the structure of treated coal in comparison to control coal suggesting breakdown in the complex structure of coal. The major absorbance bands in infrared spectroscopy for solubilization product were attributed to carbonyl (1,600 cm^?1), hydroxyl (3,450 cm^?1), cyclane (2,925 cm^?1), ether linkage (1,000–1,300 cm^?1), carboxyl (3,300–2,500 cm^?1) and side chains of aromatic ring (1,000–500 cm^?1). The presence of microorganisms and surface erosion of coal residues compared to control samples were observed by scanning electron microscopy, which suggested that isolated microorganisms were able to survive in coal for a longer period of time. Therefore, the present study concluded that microorganisms isolated from coal mines have excellent potential for coal solubilization which is considered as a crucial step in coal methanogenesis allowing them to be used successfully for in situ methane production to meet future energy demands.     

酸性半纤维素降解细菌的筛选与鉴定

从南方酸性水稻土和腐烂秸秆中采集样品,经初筛、复筛获得4株产生明显水解圈且D/d值较大同时传代效果稳定的酸性半纤维素降解细菌。DNS法测定4株细菌半纤维素酶活力,最终结合其产酶速度及酶活大小得到菌株NB8,液体摇瓶发酵72 h其半纤维素酶活达85.12 U/mL。经形态学观察和生理生化指标测定,初步确定该菌为芽胞杆菌属。结合16S rDNA基因序列分析结果,可以初步鉴定NB8为短小芽胞杆菌(Ba-cillus pumilus)。     

一株产纤维素酶细菌的分离鉴定及酶学特性研究

从采集的含腐烂树叶的土壤中,筛选到1株产纤维素酶能力较高的菌株JJ-3,经16S r RNA基因序列分析,鉴定该菌株为产酸克雷伯氏杆菌(Klebsiella oxytoca)。产酶条件及酶学特性研究表明:以滤纸为碳源、蛋白胨为氮源、初始p H为8.0的培养基中发酵3 d更利于纤维素酶的合成;菌株发酵液在中性和碱性条件下均有较高的滤纸酶活力,分别可达118.7 U/m L(p H7.0),167.8 U/m L(p H8.0)和120 U/m L(p H9.0);所产纤维素酶的最适酶反应p H为7.0,最适酶反应温度为40℃,对温度比较敏感,在p H7.0-8.0的范围内具有较好的稳定性,能满足中性和碱性纤维素酶的要求。     

雁门关地区羊致病性大肠杆菌的分离及生物特性分析

为了有效防控羊致病性大肠杆菌病,以雁门关地区养羊企业和规模专业户为研究对象,对临床健康羊和腹泻症状羊进行致病性大肠杆菌的分离、培养、生化试验、血清型鉴定、致病性试验以及耐药性研究.结果显示,从180头份(临床健康羊的小肠150头份,腹泻病羊的肛门棉拭子30头份)材料中分离培养出符合羊大肠杆菌生化特性的72株,其中25株...     

Prevalence of Anaplasma marginale in cattle blood samples collected from two important livestock regions in Punjab (Pakistan) with a note on epidemiology and phylogeny of parasite

Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96–99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.     

Paraimene tuberculata,a new genus and species of Isopoda (Sphaermomatidae) from Karachi,Pakistan

A new genus,Paraimene is established for a new Isopod species from the intertidal zone of Karachi Coast (Pakistan).Paraimene tuberculata sp. nov. is described and illustrated together with notes on the habitat of the species.     

甘草内生细菌的分离及拮抗菌株鉴定

从乌拉尔甘草健康植株的根茎叶中共分离到内生细菌98株,经初步鉴定芽孢杆菌属(Bacillus sp.)为优势种群,约占30%;从不同生长年份甘草的根、茎、叶组织中分离内生细菌种群密度从5.0×104cfu/g~2.9×107cfu/g鲜重不等。采用平板对峙方法筛选出6株对植物病原菌有明显体外拮抗活性的菌株,通过菌落、菌体形态观察、生理生化反应及16S rDNA序列分析,同时结合Biolog细菌自动鉴定系统验证,鉴定这6株拮抗菌分属萎缩芽孢杆菌(Bacillus atrophaeus)、多粘类芽孢杆菌(Paenibacillus polymyxa)、枯草芽孢杆菌(Bacillus subtilis)、Paenibacillus ehimensis。     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

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).     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Isolation and Characterization of Coumaphos-Metabolizing Bacteria from Cattle Dip

Coumaphos, an organophosphate insecticide, is used for tick control in cattle dipping vats along the U.S.-Mexican border. Recently, several vats (problem vats) have experienced a loss of efficacy because of microbial degradation. Three morphologically distinct bacteria (designated B-1, B-2, and B-3) that metabolized coumaphos were isolated from enrichment cultures that were initiated from problem vat dip material. In general, amino acids, pyrimidines, and acetate supported growth; carbohydrates were not utilized. Only B-2 required growth factors. In resting cell experiments, coumaphos was hydrolyzed to diethylthiophosphoric acid and chlorferon by all three isolates. Chlorferon was subsequently metabolized by B-1 and B-2 to α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Only B-1 produced additional metabolites. Experiments with [benzo ring-labeled U-¹⁴C]coumaphos or chlorferon demonstrated that B-1 was capable of both mineralizing and incorporating into biomass the aromatic portion of the molecule. The majority of label, however, was recovered in the form of soluble products, including α-chloro-β-methyl-2,3,4-trihydroxy-trans-cinnamic acid. Although B-1 had the capacity to use chlorferon as a carbon source at low concentrations (100 μg/ml), visible growth at higher concentrations (1,000 μg/ml) was not observed. The addition of 400 μg of chlorferon per ml to B-1 cells in the mid-log phase of growth resulted in complete inhibition of growth, while the addition of 100 to 200 μg of chlorferon per ml resulted in partial inhibition. The growth of B-2 and B-3 was inhibited by 100 μg of chlorferon per ml. These data suggest that, although B-1 and, to a lesser extent, B-2 and B-3 are responsible for the primary degradation of coumaphos, other organisms in the enrichment culture may play a secondary role in coumaphos degradation by removing inhibitory products of coumaphos metabolism.     

长裙竹荪凝集素的分离纯化与部分生化性质

凝集素是一类与糖专一结合的蛋白质或糖蛋白 ,具有众多的生物学性质[1～ 5] ,在细胞凝集、鉴别人类血型物质和分离纯化某些高分子化合物等都有着非常重要的作用 ,已成为生物化学、细胞学、免疫学及医学等领域中有用的科研材料 ,并被应用于临床诊断、治疗和某些工业生产[1] .自 1888年Ｈ .Ｓｔｉｌｌｍａｒｋ首次从蓖麻籽中发现凝集素以来 ,已分离纯化 10 0多种凝集素 ,约有 60种已成为商品 ,其研究开发日益受到人们的重视 .竹荪是一种著名的食、药兼用菌 ,具有许多药用功效 .由于竹荪含有多种生理活性物质 ,从竹荪子实体或菌丝体中分离到的…     

解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。     

Distribution,species composition and relative abundances of sandflies in North Waziristan Agency,Pakistan

This study was conducted to investigate the diversity of sandflies (Psychodidae: Phlebotominae) and the incidence of leishmaniasis in three villages of North Waziristan Agency, Pakistan. Sandflies were sampled monthly during 2012, at dusk and dawn, in selected indoor habitats including both bedrooms and animal sheds using a knock‐down spray catch method. A total of 3687 sandflies were collected, including 1444 individuals in Drezanda, 1193 in Damdil and 1050 in Dattakhel. This study revealed 14 species of two genera, Phlebotomus (Phlebotomus sergenti, Phlebotomus papatasi, Phlebotomus caucasicus, Phlebotomus kazeruni, Phlebotomus alexandri and Phlebotomus salehi) and Sergentomyia (Sergentomyia dentate, Sergentomyia baghdadis, Sergentomyia babu, Sergentomyia theodori, Sergentomyia sumbarica, Sergentomyia dreyfussitur kestanica, Sergentomyia hogsoni pawlowskyi and Sergentomyia fallax afghanica) (both: Diptera: Psychodidae). Phlebotomus sergenti was the most abundant species (42.1%), followed by S. dentata (17.7%) and S. baghdadis (17.4%). The number of males collected represented about twice that of female flies, and the maximum number was collected in July, followed by August. The determination of the species composition of sandfly populations, seasonal variations, relative abundances and estimations of infection in the vector population may provide information about the dynamics of leishmaniasis transmission that is useful in planning vector control activities.     

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.     

Taxonomy and distribution of agriculturally important plusiinae (Lepidoptera: Noctuidae) from southern Punjab,Pakistan

Taxonomic identification and classifications of insect pest genera Chrysodeixis and Ctenoplusia of the subfamily Plusiinae is very compulsory due to their phytophagous nature and potential to damage the cash as well as cereal crops. Taxonomy plays a key role in proper not only in identification and classification of the pest but also in designing a successful managing strategy. In current study, specimens of Chrysodeixis and Ctenoplusia genera were collected from different geographical areas of south Punjab, Pakistan and their diagnostic features were examined following taxanomic keys. The data of temperature, relative humidity and rainfall were also recorded during the study period. Genitalia was extracted by dissecting of the abdomen and inspected under Stereo microscope. The results revealed two new species, Chryodeixis maxus and Ctenoplusia oleraceaus, from south Punjab region in addition to previously reported species: Chrysodeixis furihatai. Suitable management of the voracious insect pest at appropriate time may help in sustaining the agriculture in Pakistan.     

Isolation and Characterization of Cytotoxic Compounds from Corn

A host-vector system was constructed in Bacillus megaterium strain NK84–0128, an oxetanocin A producer. The replication origin of an endogeneous plasmid, P–4, was used to construct a potential plasmid vector, pSM5, which had a chloramphenicol resistance gene as a selective marker. Plasmid transformation by a protoplast method was used in B. megaterium strain NK84–0128. The maximum transformation frequency attained with the pSM5 plasmid was 2.0 x 10⁴cfu/µg DNA.     

枯草芽胞杆菌L-天冬氨酸a脱羧酶的酶学性质

b-丙氨酸是一种重要的医药化工原料,目前主要依靠化学法进行生产。探寻更为环保和高效的生物生产法是未来研究的一个方向。L-天冬氨酸a脱羧酶 (PanD) 能特异地脱去L-天冬氨酸的a羧基,生成b-丙氨酸。本文比较了3种分别来源于大肠杆菌、谷氨酸棒状杆菌及枯草芽胞杆菌的PanD比酶活 (分别为0.98、7.52和8.4 U/mg)。后两者的最适pH均为6.5,最适反应温度分别为65 ℃及60 ℃。与目前研究最多的来源于大肠杆菌和谷氨酸棒状杆菌的PanD相比,来源于枯草芽胞杆菌的PanD具有更好的活性和热稳定性,具有更强的工业应用潜力。同时,本文对该酶特有的翻译后自剪切及机理性失活现象进行了分析和讨论。     

Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a V_max of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.