Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal,India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.     

Isolation and Characterization of Arsenate-Reducing Bacteria from Arsenic-Contaminated Sites in New Zealand

Two environmental sites in New Zealand were sampled (e.g., water and sediment) for bacterial isolates that could use either arsenite as an electron donor or arsenate as an electron acceptor under aerobic and anaerobic growth conditions, respectively. These two sites were subjected to widespread arsenic contamination from mine tailings generated from historic gold mining activities or from geothermal effluent. No bacteria were isolated from these sites that could utilize arsenite or arsenate under the respective growth conditions tested, but a number of chemoheterotrophic bacteria were isolated that could grow in the presence of high concentrations of arsenic species. In total, 17 morphologically distinct arsenic-resistant heterotrophic bacteria isolates were enriched from the sediment samples, and analysis of the 16S rRNA gene sequence of these bacteria revealed them to be members of the genera Exiguobacterium, Aeromonas, Bacillus, Pseudomonas, Escherichia, and Acinetobacter. Two isolates, Exiguobacterium sp. WK6 and Aeromonas sp. CA1, were of particular interest because they appeared to gain metabolic energy from arsenate under aerobic growth conditions, as demonstrated by an increase in cellular growth yield and growth rate in the presence of arsenate. Both bacteria were capable of reducing arsenate to arsenite via a non-respiratory mechanism. Strain WK6 was positive for arsB, but the pathway of arsenate reduction for isolate CA1 was via a hitherto unknown mechanism. These isolates were not gaining an energetic advantage from arsenate or arsenite utilization, but were instead detoxifying arsenate to arsenite. As a subsidiary process to arsenate reduction, the external pH of the growth medium increased (i.e., became more alkaline), allowing these bacteria to grow for extended periods of time.     

高效生物表面活性剂产生菌筛选及其性质研究

目的:获得产高效生物表面活性剂的菌株并获得优化培养基。方法:通过从山东文登某加油站附近长期污染富含油质的土壤中逐步采用富集培养基和平板筛选培养基分离筛选菌株并进行优化培养寻找最优生长培养和高产生物表面活性剂的条件。结果:筛选出产表面活性剂的微生物12株,分别命名为BSF1#-BSF12#,从中筛选出1株高效表面活性剂产生菌BSF8#,优化培养结果表明BSF8#的最佳生长pH在7.5左右,最佳碳源为葡萄糖,最佳氮源为蛋白胨,BSF8#培养基中最佳NaCl浓度为2g/L。BSF8#菌株可将发酵液的表面张力由最初的48.29mN/m降到27.79 mN/m,上层乳化状发酵液的排油圈最大直径超过7.5cm,并经红外光谱分析确定其生物表面活性剂为1个糖肽类化合物。结论:BSF8#菌株产生的生物表面活性剂活性突出,有较大的开发潜力。     

Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil

Aims: To screen and identify biosurfactant producers from petroleum‐contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. Methods and Results: We successfully isolated three biosurfactant producers from petroleum‐contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57·5% increase of the growth‐associated biosurfactant production by Acinetobacter sp. YC‐X 2 with an optimized medium: beef extract 3·12 g l^?1; peptone 20·87 g l^?1; NaCl 1·04 g l^?1; and n‐hexadecane 1·86 g l^?1. Biosurfactant produced by Acinetobacter sp. YC‐X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121°C) and high salinities [up to 18% (w/v) Na⁺ and Ca²⁺], which was more sensitive to Ca²⁺ than Na⁺. Conclusions: Two novel biosurfactant producers were isolated from petroleum‐contaminated soil. Biosurfactant from Acinetobacter sp. YC‐X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. Significance and Impact of the Study: The fact, an increasing demand of high‐quality surfactants and the lack of cost‐competitive bioprocesses of biosurfactants for commercial utilization, motivates researchers to develop cost‐effective strategies for biosurfactant production through isolating new biosurfactant producers with special surface‐active properties and optimizing their cultural conditions. Two novel biosurfactant producers in this study will widen our knowledge about this kind of micro‐organism. This work is the first application of RSM designs for cultural optimization of biosurfactant produced by Acinetobacter genus and the first report that biosurfactant may be more sensitive to Ca²⁺ than Na⁺.     

Physical,Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.     

Isolation and Characterization of Methanogenic Bacteria from Landfills

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.     

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).     

Environmental Risk Controllability and Management of VOCs during Remediation of Contaminated Sites

The environmental risk controllability assessment system and its method of controlling volatile organic compounds (VOCs) during remediation of contaminated sites are established in this article based on soil vapor extraction (SVE) technology. According to the properties of VOCs and the technical and operational characteristics of the site remediation process, the environmental risk controllability index system includes environmental risk identification, risk source analysis, and risk assessment. Environmental risk management during site remediation was focused on technical control methods and engineering control technologies. Specifically, acceptance based on risk management was suitable for low-risk levels such as RRI3 and RRI4. Furthermore, control methods for high-level risk (RRI1 or RRI2) could be developed along with transformation and control, combined with the necessary emergency risk plan.     

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.     

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 Cellulolytic Bacteria from Activated Sludge

Cellulolytic aerobic bacteria were isolated from activated sludge systems. Of the media tested for enumeration, only filter paper media gave reliable counts. Five isolates were studied further for characterization. It was found that one strain (DK) belonged to the genus Cellulomonas. The other four strains expressed similarity to the genus Pseudomonas. The different characteristics that were studied, however, do not permit them to be identified with any recognized species. Based on certain characters we believe that they are alcaligenes-like pseudomonads.     

Isolation and Characterization of Bacteria from the Baltic Sea

A bacteriological examination was done on samples of water and sediment from three localities in the Baltic. The highest numbers of bacteria were recovered from areas subjected to pollution. The isolates included members of the family Enterobacteria-ceae, the genus Pseudomonas and strains of Aeromonas hydrophila, Alteromonas putrefaciens and some Gram positive bacteria. It is suggested tentatively that H2S production in the black sediments was caused by Alt. putrefaciens. None of the isolates had an absolute requirement for NaCl, although all of them were salt-tolerant to varying degrees, and most were able to grow aerobically at salinities comparable with those found in seawater. Isolates belonging to the family Enterobacteriaceae were, however, unable to grow anaerobically under comparable conditions. Freshwater strains of several genera of the family Enterobacteriaceae and of Aeromonas hydrophila and Aer. sobria displayed salt tolerance identical with that of the Baltic isolates. One strain each of Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica survived well during three weeks at 17°C in artificial seawater lacking both carbon and nitrogen sources. These results suggest the need for a re-evaluation of the persistence of potentially pathogenic bacteria in the sea.     

TOPSIS-Based Screening Method of Soil Remediation Technology for Contaminated Sites and its Application

A soil remediation technology screening system has not yet been established in China, and the existing supporting conditions for soil remediation technology cannot meet the supervision needs of contaminated sites’ remediation. Considering the soil remediation technology screening methods for contaminated sites in developed countries and the supervision requirements of contaminated sites in China, this article briefly introduces the main characteristics of decision-making methods, and the screening indices, including technical, economic, social, and environmental factors, are suggested. The technique for order performance by similarity to ideal solution (TOPSIS) was used to establish a screening process for soil remediation technology, and the analytic hierarchy process (AHP) method was utilized to obtain the weights of the screening indices. Tthe suggested screening method was then applied in a chromite ore-processing residue site in China, and suitable remediation technology (in-situ chemical reduction-oxidation) was proposed. The research results could provide scientific and technological support for the supervision of contaminated sites in China.     

Isolation and Characterization of Four Gram-Positive Nickel-Tolerant Microorganisms from Contaminated Sediments

Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.     

The Geomicrobiological Role of Sulphate-Reducing Bacteria in Environments Contaminated by Petroleum Products

This paper reviews the geomicrobiological role of sulphate-reducing bacteria (SRB) in environments contaminated with petroleum products and describes the habitats of SRB and their capacity for bioremediation in anaerobic conditions. Moreover, the participation of SRB in biocorrosion and formation of different minerals and sediments is discussed.     

Isolation and Characterization of Polyester-Based Plastics-Degrading Bacteria from Compost Soils

Four potential polyester-degrading bacterial strains were isolated from compost soils in Thailand. These bacteria exhibited strong degradation activity for polyester biodegradable plastics, such as polylactic acid (PLA), polycaprolactone (PCL), poly-(butylene succinate) (PBS) and polybutylene succinate-co-adipate (PBSA) as substrates. The strains, classified according to phenotypic characteristics and 16S rDNA sequence, belonging to the genera Actinomadura, Streptomyces and Laceyella, demonstrated the best polyester- degrading activities. All strains utilized polyesters as a carbon source, and yeast extract with ammonium sulphate was utilized as a nitrogen source for enzyme production. Optimization for polyester-degrading enzyme production by Actinomadura sp. S14, Actinomadura sp. TF1, Streptomyces sp. APL3 and Laceyella sp. TP4 revealed the highest polyester-degrading activity in culture broth when 1% (w/v) PCL (18 U/mL), 0.5% (w/v) PLA (22.3 U/mL), 1% (w/v) PBS (19.4 U/mL) and 0.5% (w/v) PBSA (6.3 U/mL) were used as carbon sources, respectively. All strains exhibited the highest depolymerase activities between pH 6.0–8.0 and temperature 40–60°C. Partial nucleotides of the polyester depolymerase gene from strain S14, TF1 and APL3 were studied. We determined the amino acids making up the depolymerase enzymes had a highly conserved pentapeptide catalytic triad (Gly-His-Ser-Met-Gly), which has been shown to be part of the esterase-lipase superfamily (serine hydrolase).     

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.     

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.     

Isolation and Characterization of Bacteria from the Swedish West Coast

Most of the bacteria isolated from water and sediment samples from a locality off the west coast of Sweden had an absolute requirement for Na+. On the basis of phenotypic characterization and determination of DNA base composition, the strains could be assigned to the genera Beneckea, Alteromonas and Pseudomonas. Apart from a group of sulphide-forming alteromonads, none of the isolates appeared to be identical with organisms described previously.