Isolation of Denitrifying Photosynthetic Bacteria

An alkaline proteinase produced by Bacillus sp. was purified and crystallyzed through isopropanol or ammonium sulfate precipitation, decolorization with DEAE-cellulose and gel filtration with Sephadex G-100. The optimum pH for caseinhydrolysis was 11.5 and the activity was completely inactivated after incubation with DFP. The specific activity on Hammersten casein was about 4,500 units/mg enzyme protein. The enzyme also hydrolyzed synthetic ester substrate such as Ac-Tyr-OEt, Ac-Phe-OEt or Bz-Met-OMe. The isoelectric point was pH 10.7, and the molecular weight was estimated to be about 17,500 by the sedimentation equilibrium method and 16,000 by gel filtration method. Some physicochemical properties and the amino acid composition of the enzyme were investigated, indicating that the enzyme is distinguishable from alkaline proteinase of Bacillus species so far reported.     

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 Phenol-Degrading Soil Bacterium Xanthobacter flavus

A soil bacterium isolated from a contaminated site degraded phenol when provided as the sole carbon and energy source in the medium. The bacterium was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1000 mg L^?1 concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism efficiently utilized phenol and could degrade it completely within 120 h when initial concentration was less than 600 mg L^?1. Degradation of phenol was through ortho pathway, enzyme assay through cell-free extract exhibited the presence of catechol 1,2-dioxygenase. The specific activity was 0.146 μ mol min^?1 mg^?1 protein. However, higher concentrations of phenol in the medium had a negative effect on the growth of the bacterium. Hence this ability of Xanthobacter flavus can be effectively used for bioremediation studies of phenol-contaminated sites.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

好氧反硝化菌的分离鉴定及特性研究

从土壤中分离到1株能以硝酸钠为氮源进行好氧反硝化作用的细菌,命名为Rhodococcussp.DN,分离菌株革兰氏染色为阳性,球状或杆状,菌落颜色为橙红色。该细菌能以乙酰胺为惟一碳源和氮源,能进行氨化和硝化作用并产生亚硝酸。部分长度的16S rDNA序列分析表明,所分离的细菌与Rhodococcus ruber的16S rDNA序列具有99%相似性。     

Isolation and Characterization of High-Strength Phenol-Degrading Novel Bacterium of the Pantoea Genus

A new indigenous soil bacterium Pantoea strain NII-153 utilizing phenol as a sole carbon source was isolated and characterized. Phylogenetic analysis suggested its classification to the Enterobacteriaceae family, with 95.0% gene sequence similarity to Pantoea ananatis ATCC 33244. Biodegradation rates of phenol by NII-153 were found to be more effective at 64 h with initial concentration of 600 mg L^{? 1} of phenol and this is the first report of such activity in Pantoea species. Strain NII-153 has showed high tolerance to phenol concentration (900 mg L^{? 1}). Therefore, strain NII-153 could be used for biotreatment of high-strength phenol-containing industrial effluents and for bioremediation of phenol-contaminated soils.     

Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.     

Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots

An analysis of the molecular diversity of N₂ fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N₂ fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

一株轻度嗜盐反硝化细菌的分离鉴定和反硝化特性初探

从处理高盐度废水的成熟活性污泥中分离筛选得到1株轻度嗜盐反硝化细菌GYL, 通过对该菌株的形态观察、生理生化实验以及16S rDNA序列分析, 确定该菌株为盐单胞菌(Halomonas sp.)。该菌株能在盐度为10%的培养液中生长, 最适盐度为2%~7%, 最适pH为7.5~8.5, 最佳碳源为蔗糖, 在25°C~30°C的温度范围内脱氮效率达到80%以上。对该菌株的异养硝化能力进行了测定, 其对氨氮的去除率可达98.3%, 说明该菌株可实现同步硝化反硝化, 即该菌可以独立完成生物脱氮的全部过程。     

A Medium for Detecting Phenol-Degrading Bacteria

S ummary . A rapid test for the identification of phenol-degrading bacteria was devised using a medium containing phenol as the sole source of carbon and energy. Degradation of phenol in such a medium was assessed by growth and change in pH value. The results obtained are in agreement with those obtained by methods requiring chemical analysis of phenol in spent culture fluid.     

一株苯酚降解菌的分离与鉴定

目的：筛选能高效降解苯酚的微生物,并进行初步鉴定。方法：从某焦化厂排水沟采集污泥,通过逐步驯化筛选苯酚降解菌株;利用形态观察、生理生化检测、16SrDNA序列分析进行初步鉴定。结果：筛选获得1株苯酚降解菌JDM-2—1,该菌能够以苯酚为惟一碳源,耐酚能力高达2200mg／L,在30℃和pH7．0条件下,42h内能将800mg／L的苯酚彻底降解;初步鉴定其为球形芽孢杆菌（Bacillus sphaericus）。结论：菌株JDM-2-1是一株高效降解苯酚的球形芽孢杆菌。     

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%。     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

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.     

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.     

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 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.     

Bromate Reduction by Denitrifying Bacteria

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25 deg C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO(inf3)(sup-) to Br(sup-) but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO(inf3)(sup-), and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br(sup-), indicating that intermediates, e.g., BrO(inf2)(sup-), did not accumulate during bromate reduction.