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%。     

Effect of Hydrologic and Geochemical Conditions on Oxygen-Enhanced Bioremediation in a Gasoline-Contaminated Aquifer

Oxygen addition to enhance bioremediation of gasoline-contaminated ground water was performed in two locations of a shallow aquifer in South Carolina characterized by benzene, toluene, and methyl tert-butyl ether (MTBE) at concentrations greater than 1 mg/L, respectively. Oxygen addition with an oxygen-release compound (a proprietary form of magnesium peroxide [MgO₂]) produced markedly different results with respect to dissolved oxygen (DO) generation and contaminant decrease in the two locations. Oxygen-release compound injected at the former underground storage tank (UST) source area did not significantly change measured concentrations of DO, benzene, toluene, or MTBE. Conversely, oxygen-release compound injected 200 m downgradient of the former UST source area rapidly increased DO levels, and benzene, toluene, and MTBE concentrations decreased substantially. The different results can be related to differences in hydrologic and geochemical conditions that characterized the two locations prior to oxygen addition. For example, the contaminated aquifer downgradient of the former UST source area was anoxic, but frequently received oxygen-saturated recharge during rainfall events. As such, the aquifer was characterized by low concentrations of reduced species (such as ferrous iron (Fe²⁺), as well as relatively high numbers of aerobic heterotrophic bacteria (as most probable number [MPN] per milliliter). In contrast, recharge does not occur in the paved, former UST source area. The anoxic aquifer was characterized by higher concentrations of Fe²⁺ that exerted a significant chemical oxygen demand on the oxygen injected, and much lower numbers of aerobic heterotrophic bacteria. The results of this investigation indicate the important role that pre-existing hydrologic, geochemical, and microbiologic conditions have on the outcome of oxygen-based remediation strategies, and suggest that these properties should be evaluated prior to the implementation of oxygen-based remedial strategies.     

津巴布韦烟叶中淀粉酶和蛋白酶产生菌的分离及鉴定

目的:从津巴布韦烟叶中分离产蛋白酶菌和产淀粉酶能力最高的菌株,并对其进行鉴定。方法:采用淀粉富集培养基和酪蛋白富集培养基分别分离津巴布韦烟叶中的产淀粉酶和产蛋白酶菌株,通过生理生化实验和16SrRNA序列分析鉴定分离的菌株。结果:产蛋白酶菌株菌体不透明、表面有褶皱,蛋白酶酶活为52.10±0.13 U/mL;产淀粉酶菌株菌体表面呈黏状,淀粉酶酶活为3.69±0.07 U/mL;产蛋白酶与产淀粉酶的2株菌均与枯草芽孢杆菌的16S rRNA序列有100%的相似性,结合生理生化指标初步鉴定为枯草芽孢杆菌。结论:获得的2株菌在降解烟叶的蛋白质和淀粉过程中可能起重要作用。     

Aerobic Biodegradation of Methyl tert-Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to ¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

PCR–DGGE method for analyzing the bacterial community in a high temperature petroleum reservoir

Different PCR–denaturing gradient gel electrophoresis (DGGE) protocols were employed to investigate bacterial communities in a high temperature and water flooded petroleum reservoir in Dagang oil field, China. Bacterial universal primers sets frequently used in PCR–DGGE were evaluated. Three primers sets P1 (341F-GC and 534R), P2 (341F-GC and 907R) and P3 (1055F and 1406R-GC) showed different DGGE patterns. Good separation and quality of patterns were obtained in DGGE analysis with the set P3. A total of 12 DNA fragments were excised from the DGGE gels and their sequences were determined. Clustering analysis of the DGGE profiles showed that bacteria in this petroleum reservoir belonged to four clusters. These results indicate that the procedure of DGGE analysis with the primer P3 (1055F and 1406R-GC) is suitable for investigating microbial community in petroleum reservoirs.     

Characterization of Thermophilic Proteolytic Spore-Forming Bacteria from a Geothermal Site in Lithuania Based on 16S rDNA RFLP and ITS-PCR Analyses1

Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania. All of the strains were able to hydrolyze collagen and/or casein. Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses. On the basis of RFLP patterns of 16S rDNA and 16S–23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.     

Impacts of Bioremediation Schemes on Bacterial Population in Naphthalene-Contaminated Marine Sediments

Microcosm experiments were conduced in which the surface of marine sediment was contaminated with naphthalene and subjected to either of three different bioremediation schemes, i.e., biostimulation (BS) by supplementing with slow-release nitrogen and phosphorus fertilizers, bioaugmentation (BA) by inoculating with Cycloclasticus sp. E2, an aromatics-degrading bacterium identified to play an important role for aromatic-hydrocarbon degradation in marine environments and combination (CB) of BS and BA. These three schemes were found to be similarly effective for removing naphthalene, while naphthalene disappearance in sediment without any treatment (WT) was slower than those in the treated sediments. Shifts in bacterial populations during and after bioremediation were analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. It was found that the Cycloclasticus rRNA type occurred as the strongest bands in the course of naphthalene degradation. Clustering analysis of DGGE profiles showed that bacterial populations in the WT, BS and CB sediments differed consistently from those in the uncontaminated control, while the profile for the BA sediment was finally included in the cluster for uncontaminated control sediments after a 150-day treatment. The results suggest that bioaugmentation with ecologically competent pollutant-degrading bacteria is an ecologically promising bioremediation scheme.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

Bacterial community composition over a dry winter in meso- and eutrophic Portuguese water bodies

In order to investigate the bacterial diversity in a number of rivers, reservoirs and lakes in northern and central Portugal during the winter of 2004/5 (atypically dry), we applied molecular methodologies, namely denaturing gradient gel electrophoresis with primers targeting fractions of the bacterial 16S rRNA gene. Environmental parameters such as pH, conductivity, inorganic nutrients, total suspended solids and chlorophyll a were determined in order to characterize the trophic status of the studied water bodies. We found water bodies with oligotrophic to hypereutrophic characteristics. Organisms belonging to the Bacteroidetes and Alphaproteobacteria were found at the highest pH environment. Bacteroidetes were also related to high nutrient concentrations. Verrucomicrobia were associated with the most oligotrophic reservoir and low pH values. Actinobacteria were present in all samples from lakes and reservoirs, indicating its preference for lentic water bodies. Cyanobacteria dominance was related to high pH and conductivity levels. In general the conductivity values recorded in winter 2005 were the highest over recent years and chlorophyll a also reached very high levels. The data emphasize an enhanced risk of eutrophication for the studied water bodies, especially in the subsequent months when the temperature rises.     

Molecular Characterization of Prokaryotic Communities Associated with Lonar Crater Basalts

The Lonar crater is an unusually well-preserved meteorite impact structure that is located in one of the largest volcanic provinces on Earth (i.e., the Deccan Traps in India). The diversity of endoliths in Lonar crater basalts or Deccan flood basalts is not known. Here, the phylogenetic diversity of endolithic Bacteria and Archaea inhabiting basalts retrieved from four discrete sampling sites on the Lonar crater walls and the lake-bed was assessed using culture-independent molecular methods. Taxonomic classification of 16S rRNA gene sequences from all four basalt samples revealed similarities as well as dissimilarities in the presence or absence of several prokaryotic taxa. Cluster analysis of Denaturing gradient gel electrophoresis fingerprints and UniFrac analysis of clone library sequences suggested substantial variations in bacterial and archaeal diversity between crater-wall and lake-bed sites. Although sequences affiliated to the bacterial phyla Actinobacteria, Acidobacteria and Chloroflexi were relatively more abundant in crater-wall basalts than in lake-bed basalts; the reverse was observed for sequences related to Proteobacteria, Firmicutes, Cyanobacteria and Bacteroidetes. Archaea in crater-wall and lake-bed basalt libraries were almost completely represented by Thaumarchaeota and Euryarchaeota, respectively. Diversity indices and richness estimates suggested the diversity of endolithic Bacteria to be higher than that of Archaea in the Lonar crater basalts. A substantial number of clone library sequences did not affiliate with extant Bacteria and Archaea. The detection of several putative lineages associated with C, N and S cycling suggests that the Lonar crater basalts are colonized by metabolically diverse prokaryotic communities involved in biogeochemical cycling of major elements.     

一株耐盐细菌的16S rRNA基因序列分析

从舟山群岛滩涂土壤中获得了海水和底泥样品,并从中提取到了一株耐盐细菌,通过用不同盐浓度的培养基培养,挑取单菌落,反复划线纯化,得到了耐盐菌的单菌落。通过菌株基因组DNA的提取、菌株的抗性实验、质粒的提取、16S rRNA的PCR扩增及克隆、16S rRNA的全序列分析等手段,对该菌株的16S rRNA的基因序列进行了研究。     

Phylogenetic analysis of bacterial communities associated with larvae of the Atlantic halibut propose succession from a uniform normal flora

Halibut, the largest of all flatfishes is a valuable species with a great potential for aquaculture. Bacteria play an important role in regulating the health of the early life stages. The present article is the first broad-range molecular analysis of bacterial communities in larvae of the Atlantic halibut (Hippoglossus hippoglossus). DNA was extracted from larvae, water and silo biofilm from hatcheries in Norway, Scotland, Iceland and Canada. Eubacterial 16S rRNA gene fragments were amplified by polymerase chain reaction (PCR) with broad-range primers. Sequences spanning the hyper variable V3 region representing individual bacterial species were separated into community profiles by denaturing gradient gel electrophoresis (DGGE). The profiles revealed simple communities after hatching and bacterial succession following growth. Sequencing and phylogenetic analysis of excised DGGE bands suggested aerobic heterotrophs related to groups of Pseudomonas, Janthinobacterium and possibly Marinomonas to be the primary colonisers of the larvae. After onset of feeding, fermentative species (Vibrio) were detected as well. Comparative analysis of bacterial communities from different geographical regions indicated that larvae of the Atlantic halibut possess a distinct and specific normal flora.     

Phylogenetic Diversity of Aerobic Saprotrophic Bacteria Isolated from the Daqing Oil Field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G+C content of DNA and gram-negative bacteria of the -subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Template DNA ratio can affect detection by genus-specific PCR-denaturing gradient gel electrophoresis of bacteria present at low abundance in mixed populations

The detection of Helicobacter species by genus-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was compared with that by species-specific PCR in murine intestinal samples. Results suggest that, in samples containing multiple Helicobacter species, genus-specific PCR-DGGE may fail to detect all Helicobacter species present and that this relates to the initial template DNA ratio.     

Coexistence of nitrifiers, denitrifiers and Anammox bacteria in a sequencing batch biofilm reactor as revealed by PCR-DGGE

Aims:  The bacterial diversity in a sequencing batch biofilm reactor (SBBR) treating landfill leachate was studied to explain the mechanism of nitrogen removal.
Methods and Results:  The total microbial DNA was extracted from samples collected from landfill leachate and biofilm of the reactor with the removal efficiencies of NH₄⁺-N higher than 97% and that of chemical oxygen demand (determined by K₂Cr₂O₇, COD_Cr) higher than 86%. Denaturing gradient gel electrophoresis (DGGE) fingerprints based on total community 16S rRNA genes were analyzed with statistical methods, and excised DNA bands were sequenced. The results of phylogenetic analyses revealed high diversity within the SBBR biofilm community, and DGGE banding patterns showed that the community structure in the biofilm remained stable during the running period.
Conclusions:  A coexistence of nitrifiers, including ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, denitrifiers, including aerobic or anaerobic denitrifying bacteria and Anammox bacteria were detected, which might be the real matter of high removal efficiencies of NH₄⁺-N and COD_Cr in the reactor.
Significance and Impact of the Study:  The findings in this study indicated that PCR-DGGE analysis could be used for microbial community detection as prior method, and the SBBR technique could provide preferable growing environment for bacteria with N removal function.     

Evaluation of 16S rDNA- and gyrB-DGGE for typing members of the genus Aeromonas

The DNA sequence of the gyrB gene is a suitable phylogenetic marker for bacterial systematics. In this study, the diversity of Aeromonas spp. present in environmental samples was assessed by a PCR combined with DGGE approach. PCR primers targeting the gyrB gene of aeromonads were designed and the resulting amplicons were analyzed by DGGE. The gyrB-DGGE analysis was evaluated with Aeromonas isolates and reference strains allowing discrimination of the majority of strains. The gyrB-DGGE analysis is a powerful tool to monitor the presence and evaluate the diversity of aeromonads in complex samples, as is the case of water from a wastewater treatment plant.     

环己酮降解细菌的筛选和系统发育分析

通过以环己酮为唯一碳源的选择培养基富集培养和细菌环己酮降解能力的测定,从巴陵石化公司环己酮生产车间排水口的污泥样品中分离到12株降解环己酮性能强的细菌菌株。根据形态观察、部分生理生化试验和16SrRNA基因序列的比对分析,初步确定这些菌株代表8个物种,属于3个大的系统发育类群/门(Actinobacteria,Proteobacteria,Bacteroidetes)的5个科、7个属;大多数菌株与其系统发育关系最密切的典型菌株之间存在一定的遗传差异。结果表明降解环己酮性能强的细菌具有较丰富的系统发育多样性。     

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.     

DGGE and 16S rDNA analysis reveals a highly diverse and rapidly colonising bacterial community on different substrates in the rumen of goats

In the rumen, plant particles are colonised and degraded by the rumen micro-organisms. Although numerous important findings about fibre-associated bacterial community were obtained using traditional or molecular techniques, little information is available on the dynamics of bacteria associated with feed particles during incubation in the rumen. In the present study, ryegrass leaf, ryegrass stem and rice straw, representing different carbohydrate compositions, were used as substrates and placed in the rumen of goats by using nylon bags, and PCR/DGGE (denaturing gradient gel electrophoresis) with subsequent sequence analysis were used to monitor the dynamics of and identify bacteria associated with the substrates during 24 h of incubation. DGGE results showed that substrate samples collected from 10 min to 6 h had similar DGGE patterns, with up to 24 predominant bands to each sample, including 14 common bands to all samples, suggesting a rapid and stable colonisation by a highly diverse bacterial community. Substrate samples collected at 12 and 24 h showed similar DGGE patterns but had great difference in DGGE patterns from those collected at 10 min to 6 h, suggesting an apparent shift in bacterial community. Sequence analysis indicated that most substrate-associated bacteria were closely related to fibrolytic bacteria. In conclusion, a highly diverse and similar rumen bacterial community could immediately colonise to different substrates and remained stable during the initial 6 h of incubation, but experienced a marked change after 12 h of incubation. Italian ryegrass leaf, Italian ryegrass stem and rice straw were colonised with a similar bacterial community.