强化生物除磷系统的功能微生物研究进展

人类活动过程中排放的磷是导致水体富营养化的重要原因之一,因此,采取强化生物除磷(Enhanced biological phosphorus removal,EBPR)技术去除污水中磷,减轻对环境不利影响。由于具有经济、可持续的优点,EBPR系统在污水除磷中得到广泛应用,而体系中微生物群落组成合理、功能完整是EBPR系统高效稳定运行的关键所在。为了深入了解EBPR系统除磷机理和实现高效稳定运行,对系统中微生物群落结构和主要功能微生物进行了大量研究。EBPR系统中除了具有聚磷能力的聚磷菌(Polyphosphate-accumulating organisms,PAOs)外,还包括没有聚磷能力的非聚磷菌(non-PAOs),主要为聚糖菌(Glycogen-accumulating organisms,GAOs)和一些辅助细菌等。目前,发现与聚磷相关的功能微生物种类越来越多,研究最多的PAOs和GAOs分别为Accumulibacter和Defluviicoccus。PAOs和GAOs在不同的环境条件下存在竞争或合作关系,但是PAOs在特定条件下是否能够表现出GAOs的代谢特性这一问题还存在争论。除传统碳源、p H和温度等因素影响生物除磷外,外源污染物(如抗生素和重金属)对EBPR系统中功能微生物也产生影响。为了获得高效PAOs,传统分离方法、蓝白斑筛选法和人工构建工程菌的方法先后得到应用。现代分子生物学技术的发展为EBPR系统中功能微生物研究提供了先进可靠的技术手段,通过高效聚磷菌的构建实现高效除磷是未来提高实际污水中生物除磷效率的一个重要发展方向。     

反硝化聚磷菌的脱氮除磷机制及其在废水处理中的应用

摘要:水体富营养化是当前水污染治理的重点关注对象,利用微生物脱氮除磷开展富营养化水体治理是一种重要的技术。基于反硝化细菌和聚磷菌的脱氮除磷功能,兼具反硝化和聚磷功能的微生物研究及其在污水工艺中的应用越来越广泛。在厌氧和好氧/缺氧环境中,反硝化聚磷菌的脱氮除磷机制有很大差别,且在化学和酶学方面都有所体现。其中,质子驱动力/电子受体理论能够很好地解释反硝化聚磷的化学过程,而反硝化酶系和多聚磷酸盐激酶是酶学过程的主要参与者。当前研究已明确在不同氧含量环境中氮素对磷去除的影响机制,但是否存在磷对除氮作用的影响仍有待进一步研究。在此基础上,本文以氮-磷的偶联过程为切入点,分别从反硝化聚磷的化学过程和酶学机制方面进行简要综述。此外,介绍了反硝化聚磷菌在实验室以及工厂化污水处理中的应用近况,并提出了今后的研究重点与方向,以期为反硝化聚磷菌在环境修复中的进一步开发应用提供理论依据。     

一株高效聚磷解淀粉芽孢杆菌的分离鉴定及其除磷条件优化

[背景]磷是引起水体富营养化的主要营养物质.生物除磷具有成本低、效率高且适用范围广等特点,成为近年来水处理研究领域的热点.目前虽然有一些聚磷菌被筛选获得,但它们的除磷效率不高,其除磷条件尚需优化.聚磷菌的固定化及回收利用也亟待研究.[目的]分离筛选并鉴定高效聚磷菌株,优化除磷环境条件,研究吸附材料对所筛菌株除磷的影响,...     

聚磷菌和聚糖菌的竞争影响因素研究进展

目前, 强化生物除磷工艺(EBPR)以其经济有效而得到广泛的应用, 该工艺关键在于聚磷菌的富集。然而已经发现, 有一类细菌—聚糖菌(GAOs)能够和聚磷菌(PAOs)竞争, 导致除磷效果恶化。关于PAOs-GAOs的竞争, 研究已经很多, 但是其结论有趋同也有矛盾, 有必要对此进行分析讨论。根据近年来国内外的相关报道, 阐述了聚磷菌与聚糖菌的竞争影响因素, 其中碳磷比、碳源种类、温度、pH值是关键因素, 而污泥龄、溶解氧以及水力停留时间等因素对于PAOs和GAOs的竞争也起一定的作用。此外, 在EBPR系统中, 缺氧条件下, 存在反硝化聚磷菌(DPB)和反硝化聚糖菌(DGAO)也会对聚磷菌富集和系统除磷产生影响。最后对EBPR系统未来的发展方向进行了展望。     

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

一株高效聚磷细菌的分离及其聚磷特性研究

目的:从太湖沉积物中分离高效聚磷细菌,初步鉴定并研究其聚磷特性。方法:利用合成废水培养基完成菌株分离和纯化,通过单因素实验,研究不同条件对聚磷菌的生长和除磷效率的影响。结果:筛选到一株高效聚磷细菌WK-3并完成了分类鉴定;单因素实验结果表明,其生长的对数期为20~49 h;最适生长和聚磷的碳源为蔗糖,最大除磷率50.1%;最适生长pH为6.0,最适聚磷pH为7.0,除磷率51.2%;最适生长和聚磷的起始磷含量为3μmol/L,除磷率53.9%;最适生长和聚磷的接种量为3%,除磷率34.6%。结论:筛选到聚磷菌株WK-3,初步鉴定为微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila),确定了聚磷的碳源、pH、起始磷量、接种量条件。     

反硝化聚磷菌及其脱氮除磷机理研究进展

水体富营养化是当前水环境保护工作的重点关注问题,微生物修复富营养化水体具有高效、低耗且不产生二次污染等特点,已经成为富营养化水体生态修复的一种重要方式。近年来,对反硝化聚磷菌的研究及其在污水处理工艺中的应用越来越广泛。不同于传统的反硝化细菌联合聚磷菌去除氮磷工艺,反硝化聚磷菌在交替厌氧、缺氧/好氧条件下能同时进行脱氮除磷而被广泛关注与研究。值得注意的是,近几年报道的部分微生物仅在好氧条件下就可进行同时脱氮除磷,但是其脱氮除磷机理仍未理清。基于此,文中总结了目前发现的反硝化聚磷菌和同时硝化反硝化聚磷微生物的种类及特点,并对其脱氮与除磷的关系及其机理进行了系统性分析,对目前反硝化除磷存在的问题进行了梳理,最后对今后的研究方向进行了展望,以期为完善反硝化聚磷菌的脱氮除磷机理及工艺改进提供参考。     

强化生物除磷系统主要微生物及其代谢机理研究进展

强化生物除磷(enhanced biological phosphorus removal,EBPR)工艺在废水除磷处理中应用广泛.主要功能微生物及其代谢机理的研究是有效调控EBPR工艺稳定运行与效能提升的基础.本文选取EBPR系统中最主要的两类微生物(聚磷菌和聚糖菌),从底物吸收机制、糖酵解途径、TCA途径的贡献以及聚磷菌和聚糖菌的代谢相似性等方面对这些微生物的代谢机理进行综述,评价了分子生物学技术在研究EBPR系统微生物学及其代谢机理方面的应用现状,在此基础上对EBPR系统今后的研究方向进行了展望.
     

生物除磷系统中积磷小月菌研究进展

水体中磷元素超标是引起水体富营养化的重要原因,而强化生物除磷(Enhanced biological phosphorus removal,EBPR)是污水除磷最行之有效的方法。聚磷菌(Phosphate accumulating organisms,PAOs)在EBPR中发挥重要作用,本文首先概述了典型PAOs在EBPR的作用和机理:厌氧条件下,典型PAOs分解Poly-P合成聚羟基烷酸(Polyhydroxyalkanoates,PHA);好氧条件下,利用分解PHA产生的能量超量吸收磷合成Poly-P。其次评述了积磷小月菌在EBPR中的作用和机理:积磷小月菌作为PAOs的一种,在PAOs中所占比例较多,且有超强的磷去除能力,研究表明积磷小月菌体内存在PHA,但合成系统与典型PAOs不同;另外,积磷小月菌可直接利用葡萄糖作为碳源,这是典型PAOs不具备的,其超强除磷能力与积磷小月菌有效的磷转运能力和其Poly-P合成代谢能力有关。探讨并总结积磷小月菌在强化生物除磷系统中的作用和机理对进一步研究如何提高积磷小月菌的除磷效果有重要理论意义与应用价值。     

磷酸盐浓度及pH对聚磷菌吸磷能力的影响

在运行良好的A/O生物除磷系统中,研究质子、镁离子和磷酸盐浓度与聚磷菌释磷及超量摄磷的关系及影响。结果表明,正常生物除磷过程中,水中镁离子浓度变化与磷酸盐浓度变化明显正相关;在中性和碱性条件下,外加少量磷酸盐,可以诱导聚磷菌进一步的超量吸磷,达到更好的生物除磷效果;但在好氧段额外分别投加5 mg/L和10 mg/L的磷酸盐,好氧段前期吸磷量由对照的14.4 mg/L分别下降为10.2 mg/L和7.4 mg/L,投加磷抑制了PAOs好氧吸磷速率。在外碳源充足的情况下,对照组及额外投加5 mg/L和10 mg/L磷酸盐的三个系统中PHB最大合成量分别为25.2 mg/g、21.2 mg/g和17.9 mg/g VSS,外加磷的系统PHB合成量明显下降导致好氧段PAO摄磷动力降低是引起系统除磷率下降的直接原因。好氧段外加磷酸盐后镁离子含量水中偏高。说明水体中镁离子与磷酸盐摄入聚磷菌细胞具有同步性。研究表明相对厌氧释磷,PAO好氧摄磷更容易受到影响,从而影响到整个生物除磷效果。     

A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is widely used for removal of phosphorus from wastewater. In this study, a metagenome (18.2 Gb) was generated using Illumina sequencing from a full-scale EBPR plant to study the community structure and genetic potential. Quantitative fluorescence in situ hybridization (qFISH) was applied as an independent method to evaluate the community structure. The results were in qualitative agreement, but a DNA extraction bias against gram positive bacteria using standard extraction protocols was identified, which would not have been identified without the use of qFISH. The genetic potential for community function showed enrichment of genes involved in phosphate metabolism and biofilm formation, reflecting the selective pressure of the EBPR process. Most contigs in the assembled metagenome had low similarity to genes from currently sequenced genomes, underlining the need for more reference genomes of key EBPR species. Only the genome of ‘Candidatus Accumulibacter'', a genus of phosphorus-removing organisms, was closely enough related to the species present in the metagenome to allow for detailed investigations. Accumulibacter accounted for only 4.8% of all bacteria by qFISH, but the depth of sequencing enabled detailed insight into their microdiversity in the full-scale plant. Only 15% of the reads matching Accumulibacter had a high similarity (>95%) to the sequenced Accumulibacter clade IIA strain UW-1 genome, indicating the presence of some microdiversity. The differences in gene complement between the Accumulibacter clades were limited to genes for extracellular polymeric substances and phage-related genes, suggesting a selective pressure from phages on the Accumulibacter diversity.     

Polyphosphate kinase genes from full-scale activated sludge plants

The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the polyphosphate kinase gene (ppk1) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1, to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G1PAO, G2PAO, and G3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.     

Proton motive force generation from stored polymers for the uptake of acetate under anaerobic conditions

The bacteria facilitating enhanced biological phosphorus removal gain a selective advantage from intracellularly stored polymer-driven substrate uptake under anaerobic conditions during sequential anaerobic : aerobic cycling. Mechanisms for these unusual membrane transport processes were proposed and experimentally validated using selective inhibitors and highly-enriched cultures of a polyphosphate-accumulating organism, Accumulibacter, and a glycogen-accumulating organism, Competibacter. Acetate uptake by both Accumulibacter and Competibacter was driven by a proton motive force (PMF). Stored polymers were used to generate the PMF -Accumulibacter used phosphate efflux through the Pit transporter, while Competibacter generated a PMF by proton efflux through the ATPase and fumarate reductase in the reductive TCA cycle.     

Environmental distribution and population biology of Candidatus Accumulibacter, a primary agent of biological phosphorus removal

Members of the uncultured bacterial genus Candidatus Accumulibacter are capable of intracellular accumulation of inorganic phosphate in activated sludge wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal, but were also recently shown to inhabit freshwater and estuarine sediments. Additionally, metagenomic sequencing of two bioreactor cultures enriched in Candidatus Accumulibacter, but housed on separate continents, revealed the potential for global dispersal of particular Candidatus Accumulibacter strains, which we hypothesize is facilitated by the ability of Candidatus Accumulibacter to persist in environmental habitats. In the current study, we used sequencing of a phylogenetic marker, the ppk1 gene, to characterize Candidatus Accumulibacter populations in diverse environments, at varying distances from WWTPs. We discovered several new lineages of Candidatus Accumulibacter which had not previously been detected in WWTPs, and also uncovered new diversity and structure within previously detected lineages. Habitat characteristics were found to be a key determinant of Candidatus Accumulibacter lineage distribution while, as predicted, geographic distance played little role in limiting dispersal on a regional scale. However, on a local scale, enrichment of particular Candidatus Accumulibacter lineages in WWTP appeared to impact local environmental populations. These results provide evidence of ecological differences among Candidatus Accumulibacter lineages.     

基于宏基因组学分析安徽地区畜禽粪便微生物耐药性特征

为深入了解在食用动物生产过程中滥用抗生素,迫使肠道共生细菌成为微生物耐药性（antimicrobial resistance,AMR）重要宿主之一的现象,本研究利用宏基因组学针对食用动物的肠道细菌AMR进行分析。

利用宏基因组学技术对安徽地区猪和鸡粪便中的AMR及可移动遗传元件进行定量和表征,并且在核心抗性基因亚型水平上研究猪和鸡共生细菌群落的耐药性特征。

从抗性基因（antimicrobial resistance gene,ARG）的丰度和多样性来看,鸡肠道共生菌中ARG的多样性和检出率高于猪,而猪肠道共生菌中ARG总丰度高于鸡。大环内酯―林可酰胺―链菌素类抗性基因和四环素抗性基因是所有样本中含量最高的2种ARG,研究也检测到了世界范围内流行的ARG,包括optrA、qnrS、lsaE和tem等,同时,一些可移动遗传元件,包括噬菌体、质粒和插入序列也显示出与ARG存在一定相关性,这揭示了ARG的水平转移潜力。此外,食物链动物的抗性组和微生物组之间在统计学上也被证明存在正相关性。宿主溯源分析表明,在鸡样本中的拟杆菌和大肠埃希菌被检测出含有大量ARG,而在猪的样本中被检测出含有丰富ARG的为瘤胃球菌和罗氏菌。

本研究通过对猪和鸡共生细菌群落中AMR的调查统计,证实肠道微生物群是ARG的重要宿主及传播途径之一,为评价食用动物安全性及后续泛耐药基因数据库的构建提供科学依据,对公共健康具有重要意义。

     

Bioenergetic models for acetate and phosphate transport in bacteria important in enhanced biological phosphorus removal

Most of our understanding of the physiology of microorganisms is the result of investigations in pure culture. However, in order to understand complex environmental processes, there is a need to investigate mixed microbial communities. This is true for enhanced biological phosphorus removal (EBPR), an environmental process that results in the enrichment of the polyphosphate-accumulating organism Accumulibacter spp. and the glycogen non-polyphosphate accumulating organism Defluviicoccus spp. We investigated acetate and inorganic phosphate (P(i)) uptake in enrichments of Accumulibacter spp. and acetate uptake in enrichments of Defluviicoccus spp. For both enrichments, anaerobic acetate uptake assays in the presence of the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the membrane potential (Delta psi) uncoupler valinomycin, indicated that acetate is likely to be taken up by a permease-mediated process driven by the Delta psi. Further investigation with the sodium ionophore monensin suggested that anaerobic acetate uptake by Defluviicoccus spp. may in part be dependent on a sodium potential. Results of this study also suggest that Accumulibacter spp. generate a proton motive force (pmf or Delta p) for anaerobic acetate uptake by efflux of protons in symport with P(i) through an inorganic phosphate transport (Pit) system. In contrast, we suggest that the anaerobic Delta p in Defluviicoccus spp. is generated by an efflux of protons across the cell membrane by the fumarate respiratory system, or by extrusion of sodium ions via decarboxylation of methylmalonyl-CoA. Aerobic P(i) uptake by the Accumulibacter spp. enrichment was strongly inhibited in the presence of an ATPase inhibitor, suggesting that the phosphate-specific transport (Pst) system is important even under relatively high concentrations of P(i). Acetate permease activity in these microorganisms may play an important role in the competition for acetate in the often acetate-limited EBPR process. Activity of a high-velocity Pst system in Accumulibacter spp. may further explain its ability to compete strongly in EBPR.     

Evidence for bacteriophage activity causing community and performance changes in a phosphorus-removal activated sludge

Bacteria are known to play important roles in biogeochemical cycles and biotechnology processes, but little is known about the influence of bacteriophage on these processes. A major impediment to the study of host-bacteriophage interactions is that the bacteria and their bacteriophage are often not available in a pure culture. In this study, we detected an unexpected decline in the phosphorus-removal performance of a granular laboratory-scale wastewater treatment reactor. Investigations by FISH, transmission electron microscopy and proteomics led us to hypothesize that a bacteriophage infection of the uncultured Candidatus 'Accumulibacter phosphatis' was responsible for the decline in performance. Further experiments demonstrated that the addition of a putative bacteriophage-rich supernatant, obtained from the previous failed reactor to phosphorus-removal reactors, caused a decrease in the abundance of Accumulibacter in both granular and floccular activated sludges. This coincided with increases in bacteriophage-like particles and declining phosphorus-removal performance. The granular sludge did not recover after the attack, but the floccular sludge regained Accumulibacter numbers and phosphorus-removal performance. These findings suggest that bacteriophage may play a significant role in determining the structure and function of bacterial communities in activated sludges.