Isolation and Characterization of Diuron-degrading Bacteria from Lotic Surface Water

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis (TTGE) of 16 S rDNA gene V1–V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation assays. The TTGE fingerprints revealed that diuron had a strong impact on bacterial community structure and highlighted both diuron-sensitive and diuron-adapted bacterial strains. Two bacterial strains, designated IB78 and IB93 and identified as belonging to Pseudomonas sp. and Stenotrophomonas sp., were isolated and shown to degrade diuron in pure resting cells in a first-order kinetic reaction during the first 24 h of incubation with no 3,4-DCA detected. The percentages of degradation varied from 25% to 60% for IB78 and 20% to 65% for IB93 and for a diuron concentration range from 20 mg/L to 2 mg/L, respectively. It is interesting to note that diuron was less degraded by single isolates than by mixed resting cells, thereby underlining a cumulative effect between these two strains. To the best of our knowledge, this is the first report of diuron-degrading strains isolated from lotic surface water.     

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.     

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.     

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 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 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.     

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 Dehalogenases from 2,2-Dichloropropionate-Degrading Soil Bacteria

Five bacterial strains were isolated from polluted soils capable of degrading 2,2-dichloropropionate. In crude extracts, dehalogenase activity against haloacetates and longer-chained 2-haloalkanoic acids could be detected. Results from activity staining indicated that all bacterial strains expressed a single dehalogenase. In further biochemical characterization, two types of D,L-specific 2-haloalkanoic acid dehalogenases were described, which are different from each other not only in molecular weight and electrophoretic mobility, but also in sensitivity towards thiol reagents. Dehalogenases of these strains have been shown to be inducible and are catalyzing halide hydrolysis with inversion of product configuration. Received: 5 July 1996 / Accepted: 1 August 1996     

Isolation and Characterization of Extremely Thermophilic Bacteria from Hot Springs

In order to investigate the mechanism of microbial growth at elevated temperatures, it was tried to isolate different thermophilic microorganisms from wide origins, such as soils, composts, manure piles and hot spring waters. As the result, 5 strains of extremely thermophilic bacteria, the maximum, the optimum and the minimum temperatures for growth of which were 80, 70~75, and 40°C, respectively, were isolated from Izu-Atagawa hot spring and Beppu hot springs. These bacteria were gram-negative, yellow-pigmented, non-motile and non-sporulating rods of 0.5~0.7 μ in diameter and 2~5 μ in length. They were heterotrophs requiring several amino acids (such as glutamate, aspartate, et al.) and vitamins (such as biotin, folic acid and p-aminobenzoic acid) and grew well at neutral to slight alkali pH. The content of GC pairs of DNAs from the 5 strains was 69~70%, and this seemed to be one of the highest values in bacteria so far known. Among the 5 strains, strain AT–62 was named as Thermus flavus sp. n. AT–62 from its morphological and physiological characteristics. Comparison between Thermus flavus and other extremely thermophilic bacteria as Thermus aquaticus and Flavobacterium thermophilum is described and discussed in reference to classification of extremely thermophilic bacteria.     

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 of an Insertion Sequence from Ralstonia solanacearum Race 1 and Its Potential Use for Strain Characterization and Detection

A new insertion sequence (IS), IS1405, was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS5 family, but the extent of nucleotide sequence identity in 5′ and 3′ noncoding regions between IS1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS1405 and additional five endogenous IS1405s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.     

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.     

氯苯降解菌的筛选鉴定及降解特性研究

本文采集化工厂排污口的污泥样品, 在含有氯苯为唯一碳源的基本培养基中, 先后分离筛选出7株能够降解氯苯的微生物菌株。通过对分离菌株的16S rRNA基因序列进行分析, 发现其中5株细菌分别属于放线菌目的考克氏菌属(KD139)、红球菌属(KD140和KD142)和节杆菌属(KD230和KD232), 1株细菌属于杆菌目的芽胞杆菌d属(KD178), 另外1株细菌属于黄色单孢菌目的寡食单胞菌属(KD237); 同时我们构建了系统进化树, 确定分离菌株的相对进化地位。本文还利用气相色谱方法, 对分离菌株降解氯苯的能力进行了初步分析, 其中寡食单胞菌KD237降解氯苯能力最高, 24 h内氯苯分解率达60.78%。     

Isolation and Characterization of Phenol-Degrading Denitrifying Bacteria

Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of Azoarcus species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus Azoarcus. The results of this study add three new members to the genus Azoarcus, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.     

Isolation and Selection of Potential Probiotic Bacteria from the Pig Gastrointestinal Tract

The present study aimed to isolate bacterial strains from the pig gastrointestinal tract that have antagonistic activity against potential pathogens and are able to produce antimicrobial compounds. That ability would be a first requirement for the strains’ possible use as probiotics. Samples obtained from pig intestinal mucosa and contents were screened for the presence of antagonistic activity against pathogenic indicator strains of Escherichia coli, Salmonella, and Listeria by means of the double-layer technique. Samples displaying the largest inhibitory halos were further studied for the production of inhibitory substances using the agar diffusion and microtitration methods. The three most promising isolates were identified by sequencing of the 16S rRNA gene and showed highest affiliation to Lactobacillus salivarius. Optimal growth conditions and bacteriocin production were recorded in de Man, Rogosa, and Sharpe broth under anaerobic conditions at 37 °C. The antimicrobial substances were found to be sensitive to proteolytic enzymes but showed good stability at pH values below 6. Our findings suggest that these three intestinal strains are able to produce antimicrobial substances capable of inhibiting the growth of potential enteric pathogens and might have potential as probiotic feed additives for the prevention of gastrointestinal diseases.