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.     

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%。     

Molecular Detection,Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.Many scientists have used the rRNA approach (29, 30) to detect microbial populations and to describe the structures of microbial communities in various environments without isolating the component microorganisms. These studies have shown that most 16S ribosomal DNA (rDNA) sequences directly amplified from environmental samples are different from the sequences of comparable laboratory strains. Workers have concluded from such observations that many bacteria that are predominant in the natural environment have not been isolated in the laboratory yet and that the microbial diversity in the natural environment is much greater than the diversity of the bacteria that have been isolated (2, 7, 13, 25, 35, 36, 39, 40).Currently, one important aspect of microbial ecology studies is functional dissection of microbial communities based on structural information obtained by the approach mentioned above. An analysis of a population shift accompanied by a change in the function of a community yields information useful for identifying functionally dominant populations (2, 3, 42), although information concerning the function (activity) of each population can never be obtained by this kind of approach. Hence, workers have emphasized that pure-culture experiments are indispensable for detailed analysis of the functions of each population and that isolation of the functionally dominant populations in a microbial community is quite important.Phenol and its derivatives are some of the major hazardous compounds in industrial wastewater (1, 31, 43), and for this reason biodegradation of phenol has attracted keen attention (34, 46). However, since most studies of phenol biodegradation have been carried out under laboratory conditions with arbitrarily selected phenol-degrading bacteria, phenol biodegradation in the environment is not well understood yet. In the present study, to better understand phenol degradation in activated sludge, we isolated and characterized the phenol-degrading bacteria that were identified by the rRNA approach to be the dominant population in phenol-digesting activated sludge. Physiological and genetic differences between the dominant phenol-degrading bacteria isolated in this study and representative phenol-degrading bacteria characterized previously in several laboratories are discussed below.     

Isolation and Characterization of New Cyclohexylacetic Acid-Degrading Bacteria

Six cyclohexylacetic acid-degrading strains were isolated from soil samples in Japan and identified as members of the genera Cupriavidus (strain KUA-1), Rhodococcus, and Dietzia by 16S rRNA gene sequence analysis. For the first time members of these genera were shown to be capable of degrading cyclohexylacetic acid. A selected strain, KUA-1, which is the first reported Gram-negative organism capable of growth on cyclohexylacetic acid, was identified as a Cupriavidus metallidurans, based on morphologic and physiologic characteristics and its 16S rRNA gene sequence. Metabolite analysis by HPLC-MS indicated that 1-cyclohexenylacetic acid is an intermediate of cyclohexaneacetic acid metabolism in strain KUA-1.     

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。     

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.     

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.     

Autolytic Activity and Butanol Tolerance of Clostridium acetobutylicum

The effects of acetone and butanol on the growth of vegetative cells and the stability of swollen-phase bright-stationary-phase cells (clostridial forms) of Clostridium acetobutylicum P262 and an autolytic deficient mutant (lyt-1) were investigated. There was little difference in the sensitivity of strain P262 and the lyt-1 mutant vegetative cells and clostridial forms to acetone. The stability of the different morphological stages was unaffected by acetone concentrations far in excess of those encountered in factory fermentations. Butanol concentrations between 7 and 16 g/liter, which are within the range obtained in industrial fermentations, increased the degeneration of strain P262 clostridial forms but had no effect on the stability of lyt-1 clostridial forms which never underwent autolysis. Vegetative cells of the lyt-1 mutant were able to grow in higher concentrations of butanol than strain P262 vegetative cells. It was concluded that there is a relationship between butanol tolerance and autolytic activity.     

大蒜内生细菌的分离及拮抗菌筛选与鉴定

利用常规分离方法对大蒜鳞茎进行内生细菌的分离,采用对峙法和平板涂布法对分离的内生菌进行拮抗试验研究,并对菌株DSP6进行16S rDNA全序列鉴定。结果表明:分离得到19株内生细菌,其中10株菌对2种以上植物病原真菌有不同程度的抑制作用,占分离菌总数的52.6%,DSN7对番茄早疫病的抑菌圈半径最大,为13mm;17株菌对5种病原细菌中至少1种有抑制作用,占分离菌总数的89.5%,其中菌株DSP3对大肠杆菌的抑菌圈半径最大,达到10 mm;菌株DSP6对供试的9种病原菌有较强的抑菌作用,且抑菌圈平均半径最大,为6.88mm;16S rDNA全序列鉴定显示,菌株DSP6与芽孢杆菌属Bacillus axarquiensis相似性为100%,表明菌株DSP6为Bacillus axarquiensis。     

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 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.     

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.     

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.     

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 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.