焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因多样性

研究了某焦化废水处理厂接触氧化池中降酚菌群的苯酚羟化酶大亚基基因(thelargestsubunitofthemulti-componentphenolhydroxylase,LmPH)的多样性。通过温度梯度凝胶电泳(temperaturegradientgelelectrophoresis,TGGE)对比分析了氧化池4个区段(O1—O4)中降酚菌群LmPH的组成。它们的TGGE图谱完全一样,相似性为100%,表明该处理池中不同区段的降酚菌群的功能基因组成是高度相似的。以O4段的菌群为代表建立LmPH基因克隆文库,从中挑选了49个克隆测序。依据LmPH基因的DNA序列所推测的氨基酸序列完全相同的归为一类的原则,49个克隆被分为16种类型,其中优势LmPH基因主要有5种类型(多于4个克隆),而另外11种类型都只有1个克隆。与已知基因同源性超过90%的有7种类型,低于80%的有2种类型。基于氨基酸序列的系统进化树分析表明,LmPH文库中绝大部分的类型都属于低亲和常数(low-Ks)的LmPH,占所有克隆的92%。只有一个类型属于高亲和常数(high-Ks)的。因此,处理焦化废水的工业装置中不仅具有丰富多样的苯酚羟化酶基因类型,而且以编码低亲和常数的占优势地位,而过去报道的通过富集培养分离得到的降酚菌则多带有高亲和常数的酶。这提示我们传统的富集培养方法并不能筛选到生态环境中的真正优势功能菌。     

Group-specific monitoring of phenol hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K(s) (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K(s), and high-K(s) groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter(-1)); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

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.     

Comparing activated sludge and aerobic granules as microbial inocula for phenol biodegradation

Activated sludge and acetate-fed granules were used as microbial inocula to start up two sequencing batch reactors (R1, R2) for phenol biodegradation. The reactors were operated in 4-h cycles at a phenol loading of 1.8 kg m^–3 day^–1. The biomass in R1 failed to remove phenol and completely washed out after 4 days. R2 experienced initial difficulty in removing phenol, but the biomass acclimated quickly and effluent phenol concentrations declined to 0.3 mg l^–1 from day 3. The acetate-fed granules were covered with bacterial rods, but filamentous bacteria with sheaths, presumably to shield against toxicity, quickly emerged as the dominant morphotype upon phenol exposure. Bacterial adaptation to phenol also took the form of modifications in enzyme activity and increased production of extracellular polymers. 16S rRNA gene fingerprints revealed a slight decrease in bacterial diversity from day 0 to day 3 in R1, prior to process failure. In R2, a clear shift in community structure was observed as the seed evolved into phenol-degrading granules without losing species-richness. The results highlight the effectiveness of granules over activated sludge as seed for reactors treating toxic wastewaters.     

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH⁺ phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.     

倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究

【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与GenBank中相应酶的序列一致性分别介于92%?100%和87%?100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有LmPH (Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。     

Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K_s (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K_s, and high-K_s groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter⁻¹); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

采用苯酚羟化酶基因特异引物检测苯酚降解菌

根据苯酚羟化酶基因高度保守序列设计了一对该基因的特异PCR引物。采用该特异引物从苯酚降解菌醋酸钙不动杆菌 (Acinetobactercalcoaceticus)PHEA 2的总DNA中扩增到唯一一条大小为 684bp的片段。该DNA片段与已知的A .calcoaceticusNCIB82 50的苯酚羟化酶基因具有高度的同源性 ,其核苷酸序列的同源性为 84% ,推导的氨基酸序列的同源性为 98%。对苯酚和非苯酚降解菌株的PCR扩增结果表明 :所有苯酚降解菌均能扩增出 684bp的特征片段 ,而非苯酚降解菌则无PCR条带。对炼焦废水中的细菌群落进行PCR扩增和生化特性检测表明 :显示 684bp特征片段的菌株均具有苯酚降解特性。上述结果表明 ,利用苯酚羟化酶基因的特异引物可对环境中的苯酚降解菌株进行准确快速的PCR检测。     

TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性

应用TGGE指纹图谱技术对两个曝气池细菌种群的动态变化及多样性进行了研究。每3d进行1次,共8个监测时期中同一曝气池活性污泥的16SrDNAV3-PCRTGGE指纹图谱基本一致,图谱间的相似性系数(Cs)为100％。同一曝气池不同位点活性污泥的TGGE指纹图谱也完全一致。功能不同曝气池活性污泥TGGE指纹图谱存在差异,Cs为83．3％。对TJ1活性污泥TGGE图谱中9条主要条带回收、扩增、克隆建库,每个条带选4个转化子进行序列分析,结果显示TGGE条带是由序列不同的片段组成。32个序列在97％的相似性下分成16个分类操作单元(OTU),14个OTU与GenBank中已登录的细菌种群的同源性≥97％,2个OTU的同源性为95％和94％。与10个OTU同源性较高的细菌类群是在活性污泥或污染环境分离或发现的,与8个OTU相似的细菌类群目前尚无法分离培养。     

ERIC-PCR-based strain-specific detection of phenol-degrading bacteria in activated sludge of wastewater treatment systems

Aims:  To evaluate the use of Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)-derived probes and primers to specifically detect bacterial strains in an activated sludge microbial community.
Methods and Results:  ERIC-PCR was performed on two phenol-degrading bacterial strains, Arthrobacter nicotianae P1-7 and Klebsiella sp. P8-14. Their amplicons were DIG labelled for use as probes and then hybridized with ERIC-PCR fingerprints. The results showed the distinct band patterns for both bacterial strains. Strain-specific PCR primers were designed based on the sequences of ERIC-PCR bands. The DNA of each of these strains was successfully detected from its mixture with activated sludge DNA, either by using their respective ERIC-PCR-based probes for hybridization or by using species-specific primers for amplification, with higher sensitivity by latter method.
Conclusions:  Two phenol-degrading bacterial strains were identified from a mixture of activated sludge by using ERIC-PCR-based methods.
Significance and Impact of the Study:  The study demonstrated that the bacteria, which have important functions in complex wastewater treatment microbial communities, could be specifically detected by using ERIC-PCR fingerprint-based hybridization or amplification.     

Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies

Molecular ecological approaches have detected diverse microorganisms that occur in response to pollution and bioremediation; however, most of these organisms have not been isolated, and their physiological traits are poorly understood. One important objective in current bioremediation studies would therefore be an assessment of the physiology and functions of the diverse microbial population at a polluted site. Among the parameters relating to the diversity of the microbial catabolic potential, e.g., substrate specificity, inducer specificity, number of catabolic routes and kinetics of catabolic enzymes, our studies have focused on the kinetic diversity of phenol-degrading bacteria. In one example, a kinetic analysis allowed functionally important phenol-degrading bacteria to be identified in activated sludge; this information could be used to improve the performance of phenol-degrading activated sludge. In an analysis of phenol-degrading bacteria present in trichloroethylene (TCE)-contaminated aquifer soil, the kinetic data could be linked to group-specific monitoring of their phenol-hydroxylase genes. The results have suggested that one group of phenol-degrading bacteria can effectively contribute to TCE bioremediation, while other groups work poorly. Based on this information, we have succeeded in developing a high-performance TCE-degrading bioreactor. We suggest that a careful analysis of the diversity of microbial catabolic potential, particularly of the kinetic traits, may facilitate the development of new bioremediation strategies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Population Dynamics of Phenol-Degrading Bacteria in Activated Sludge Determined by gyrB-Targeted Quantitative PCR

A method for quantifying bacterial populations introduced into an activated-sludge microbial community is described. The method involves extraction of DNA from activated sludge, appropriate dilution of the extracted DNA with DNA extracted from nonintroduced activated sludge, PCR amplification of a gyrB gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. The adequacy of the method was examined by analyzing the population dynamics of two phenol-degrading bacteria, Pseudomonas putida BH and Comamonas sp. strain E6, that had been introduced into phenol-digesting activated sludge. The density of each of the two populations determined by the PCR method immediately after the introduction was consistent with the density estimated from a plate count of the inoculum. This quantitative PCR method revealed different population dynamics for the two strains in the activated sludge under different phenol-loading conditions. The behavior of both of these strains in the activated sludge reflected the growth kinetics of the strains determined in laboratory axenic cultures.     

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Microbial community structure and trichloroethylene degradation in groundwater

Trichloroethylene (TCE) is a prevalent contaminant of groundwater that can be cometabolically degraded by indigenous microbes. Groundwater contaminated with TCE from a US Department of Energy site in Ohio was used to characterize the site-specific impact of phenol on the indigenous bacterial community for use as a possible remedial strategy. Incubations of 14C-TCE-spiked groundwater amended with phenol showed increased TCE mineralization compared with unamended groundwater. Community structure was determined using DNA directly extracted from groundwater samples. This DNA was then analyzed by amplified ribosomal DNA restriction analysis. Unique restriction fragment length polymorphisms defined operational taxonomic units that were sequenced to determine phylogeny. DNA sequence data indicated that known TCE-degrading bacteria including relatives of Variovorax and Burkholderia were present in site water. Diversity of the amplified microbial rDNA clone library was lower in phenol-amended communities than in unamended groundwater (i.e., having Shannon-Weaver diversity indices of 2.0 and 2.2, respectively). Microbial activity was higher in phenol-amended ground water as determined by measuring the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride. Thus phenol amendments to groundwater correlated with increased TCE mineralization, a decrease in diversity of the amplified microbial rDNA clone library, and increased microbial activity.     

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four?weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four?weeks, almost 7.07?×?10⁷ more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.     

一株高效降酚菌的质粒特性及柠檬酸细菌表达的研究

在焦化废水处理厂的活性污泥中筛选的降酚效果较好的菌株H,研究其质粒特性并提取其降解质粒将其转入柠檬酸细菌进行表达。结果表明H菌株具有质粒,且质粒片段较大最大的超过10 kb,最小的2 kb左右。通过SDS和原生质体再生法分别对H菌进行质粒消除,结果发现质粒去除的菌株降酚能力也随之消失。说明H菌株的质粒上有控制酚降解基因存在;提取H菌的质粒将其转入柠檬酸细菌进行表达,获得了转化子。转化子具有较好的降酚效果12 h可达77.34%,但转化子的降解速率较小。另证明了转化子内含有与H菌株相同特性的质粒得到具有降酚能力的柠檬酸细菌表达体系。     

RAPD引物数量对施式巴鲵遗传多样性评估结果的影响

应用RAPD技术对神农架施式巴鲵居群内24个个体进行了遗传多样性分析,探讨引物数量对遗传多样性分析结果 的影响,结果表明:遗传多样性分析准确度与引物数量有关,引物数增加到30左右时,PPB(Percentageofpolymorphicbands)、 Shannon信息指数(Shannon′sinformationindex)、Nei′s遗传多样性指数(Nei′sgenediversity)变化趋于稳定。标准误分析表明, 引物数增加到25左右时,再增加引物,标准误变化很小。若要客观反映出个体之间的遗传变异关系,引物数应在25以上,多 态位点数应在250以上。     

Functional and compositional comparison of two activated sludge communities remediating coking effluent

The success of engineered microbiological systems is evident in the global application of activated sludge communities to remediate coking effluent. However, there is a lack of understanding of the microbiology underlying treatment efficiency and stability. In this study, two functionally distinct activated sludge pools, treating the same effluent and operating under the same conditions, were examined to establish a relationship between overall diversity and/or functional diversity with respect to process stability. Molecular profiling, sequencing and RNA-based stable isotope probing were used to examine the bacterial diversity, general composition and functional composition of the most abundant members of the two communities. The inferior process stability in one of the pools could not be explained by reduced total bacterial diversity or evenness. RNA-based stable isotope probing revealed that both pools harboured an abundant phenol-degrading Acidovorax species, and that the pool of inferior stability accommodated an additional closely related phenol-degrading Acidovorax species at high abundance. These results are discussed in the context of deterministic and stochastic models of microbial community assembly.