Bacterial community dynamics in full-scale activated sludge bioreactors: operational and ecological factors driving community assembly and performance

The assembling of bacterial communities in conventional activated sludge (CAS) bioreactors was thought, until recently, to be chaotic and mostly unpredictable. Studies done over the last decade have shown that specific, and often, predictable random and non-random factors could be responsible for that process. These studies have also motivated a "structure-function" paradigm that is yet to be resolved. Thus, elucidating the factors that affect community assembly in the bioreactors is necessary for predicting fluctuations in community structure and function. For this study activated sludge samples were collected during a one-year period from two geographically distant CAS bioreactors of different size. Combining community fingerprinting analysis and operational parameters data with a robust statistical analysis, we aimed to identify relevant links between system performance and bacterial community diversity and dynamics. In addition to revealing a significant β-diversity between the bioreactors' communities, results showed that the largest bioreactor had a less dynamic but more efficient and diverse bacterial community throughout the study. The statistical analysis also suggests that deterministic factors, as opposed to stochastic factors, may have a bigger impact on the community structure in the largest bioreactor. Furthermore, the community seems to rely mainly on mechanisms of resistance and functional redundancy to maintain functional stability. We suggest that the ecological theories behind the Island Biogeography model and the species-area relationship were appropriate to predict the assembly of bacterial communities in these CAS bioreactors. These results are of great importance for engineers and ecologists as they reveal critical aspects of CAS systems that could be applied towards improving bioreactor design and operation.     

Overall functional gene diversity of microbial communities in three full-scale activated sludge bioreactors

Understanding microbial community composition is thought to be crucial for improving process functioning and stabilities of full-scale activated sludge reactors in wastewater treatment plants (WWTPs). However, functional gene compositions of microbial communities within them have not been clearly elucidated. To gain a complete picture of microbial community, in this study, GeoChip 4.2 was used to profile the overall functional genes of three full-scale activated sludge bioreactors, the 16S rRNA gene diversities of which had been unveiled by 454-pyrosequencing in our previous investigation. Triplicate activated sludge samples from each system were analyzed, with the detection of 38,507 to 40,654 functional genes. A high similarity of 77.3–81.2 % shared functional genes was noted among the nine samples, verified by the high 16S rRNA gene similarity with shared operational taxonomic units (OTUs) constituting 66.4–70.0 % of the detected sequences in each system. Correlation analyses showed that the abundances of a wide array of functional genes were associated with system performances. For example, the abundances of carbon degradation genes were strongly correlated to chemical oxygen demand (COD) removal efficiencies (r?=?0.8697, P?<?0.01). Lastly, we found that sludge retention time (SRT), influent total nitrogen concentrations (TN inf), and dissolved oxygen (DO) concentrations were key environmental factors shaping the overall functional genes. Together, the results revealed vast functional gene diversity and some links between the functional gene compositions and microbe-mediated processes.     

活性污泥微生物群落宏组学研究进展

活性污泥是全球最常用的废水生物处理人工生态系统,微生物是驱动其污染净化能力的关键。活性污泥微生物群落所有物种与基因(简称"微生物组")的研究先后经历了"显微镜观察和纯菌培养分离"(1915)、"PCR扩增-测序"(1994)和"高通量测序-宏组学分析"(2006)三个重要阶段的发展变迁。相应地,我们对活性污泥微生物组的认知经历了从最早对微型动物(如钟虫和轮虫)及其他微生物的形貌观察和纯种培养鉴定到今天对整个微生物组的全局多样性认识的飞跃。近13年来,基于高通量测序的宏组学方法被广泛应用于揭示活性污泥微生物群落组成结构和功能,我们现在充分意识到活性污泥微生物组蕴藏着大量不可培养新物种和基因多样性,驱动着各类污染物的降解与转化。目前,特异性分子标记基因的扩增子测序技术已经被广泛应用于揭示城市和工业废水处理活性污泥微生物组和典型功能种群(如硝化细菌和聚磷菌)的时空多样性和群落构建机制,进而为未来实现活性污泥微生物组功能的精准调控奠定理论基础。宏基因组学研究在群落、种群和个体基因组水平全面解析了活性污泥微生物组驱动的碳、氮、磷元素循环过程,以及有机微污染物的生物降解和转化机理。将来活性污泥微生物组学研究需要在"标准化的组学分析方法和绝对定量""高通量培养组学""高通量功能基因组学"和"多组学方法的结合及多种方法并用"4个方面取得实现精准生态基因组学所需的技术突破,以最大限度发掘活性污泥微生物组在污水处理与资源回收领域的生态学与工程学价值。     

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.     

Temporal dynamics of activated sludge bacterial communities in two diversity variant full-scale sewage treatment plants

Bacterial community in activated sludge (AS) is diverse and highly dynamic. Little is known about the mechanism shaping bacterial community composition and dynamics of AS and no study had quantitatively compared the contribution of abiotic environmental factors and biotic associations to the temporal dynamics of AS microbial communities with significantly different diversity. In this study, two full-scale sewage treatment plants (STPs) with distinct operational parameters and influent composition were sampled biweekly over 1 year to reveal the correlating factors to whole and sub-groups of AS bacterial community diversity and dynamics. The results show that the bacterial communities of the two STPs were entirely different and correlated with the influent composition and operating configurations. Bacterial associations represented by cohesion metrics and the environmental factor temperature were the primary correlated factors to the temporal bacterial community dynamics within each STP. The STP with high diversity and evenness could treat influent with higher suspended solid and a shorter sludge retention time, and was less correlated with environmental factors, implying the importance of diversity for AS system.

     

Phage-host associations in a full-scale activated sludge plant during sludge bulking

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of “Candidatus Microthrix parvicella” led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with “Candidatus Accumulibacter phosphatis,” suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.     

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.     

Biodegradability of imidazolium and pyridinium ionic liquids by an activated sludge microbial community

Ionic liquids (ILs) are novel organic salts that have enormous potential for industrial use as green replacements for harmful volatile organic solvents. Varying the cationic components can alter the chemical and physical properties of ILs, including solubility, to suit a variety of industrial processes. However, to complement designer engineering, it is crucial to proactively characterize the biological impacts of new chemicals, in order to fully define them as environmentally friendly. Before introduction of ILs into the environment, we performed an analysis of the biodegradability of six ILs by activated sludge microorganisms collected from the South Bend, Indiana wastewater treatment plant. We examined biodegradability of 1-butyl, 1-hexyl and 1-octyl derivatives of 3-methyl-imidazolium and 3-methyl-pyridinium bromide compounds using the standard Organisation for Economic Cooperation and Development dissolved organic carbon Die-Away Test, changes in total dissolved nitrogen concentrations, and ¹H-nuclear magnetic resonance analysis of initial and final chemical structures. Further, we examined microbial community profiles throughout the incubation period using denaturing gradient gel electrophoresis (DNA-PCR-DGGE). Our results suggest that hexyl and octyl substituted pyridinium-based ILs can be fully mineralized, but that imidazolium-based ILs are only partially mineralized. Butyl substituted ILs with either cation, were not biodegradable. Biodegradation rates also increase with longer alkyl chain length, which may be related to enhanced selection of a microbial community. Finally, DGGE analysis suggests that certain microorganisms are enriched by ILs used as a carbon source. Based on these results, we suggest that further IL design and synthesis include pyridinium cations and longer alkyl substitutions for rapid biodegradability.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.     

Bacterial community composition and diversity of a full-scale integrated fixed-film activated sludge system as investigated by pyrosequencing

The integrated fixed-film activated sludge (IFAS) system is a variation of the activated sludge wastewater treatment process, in which hybrid suspended and attached biomass is used to treat wastewater. Although the function and performance of the IFAS system are well studied, little is known about its microbial community structure. In this study, the composition and diversity of the bacterial community of suspended and attached biomass samples were investigated in a full-scale IFAS system using a highthroughput pyrosequencing technology. Distinct bacterial community compositions were examined for each sample and appeared to be important for its features different from conventional activated sludge processes. The abundant bacterial groups were Betaproteobacteria (59.3%), Gammaproteobacteria (8.1%), Bacteroidetes (5.2%), Alphaproteobacteria (3.9%), and Actinobacteria (3.2%) in the suspended sample, whereas Actinobacteria (14.6%), Firmicutes (13.6%), Bacteroidetes (11.6%), Betaproteobacteria (9.9%), Gammaproteobacteria (9.25%), and Alphaproteobacteria (7.4%) were major bacterial groups in the attached sample. Regarding the diversity, totals of 3,034 and 1,451 operational taxonomic units were identified at the 3% cutoff for the suspended and attached samples, respectively. Rank abundance and community analyses demonstrated that most of the diversity was originated from rare species in the samples. Taken together, the information obtained in this study will be a base for further studies relating to the microbial community structure and function of the IFAS system.     

Nitrifying bacterial community structure of a full-scale integrated fixed-film activated sludge process as investigated by pyrosequencing

Nitrifying bacterial community structures of suspended and attached biomasses in a full-scale integrated fixed-film activated sludge process were investigated by analyzing 16S rRNA gene sequences obtained from pyrosequencing. The suspended biomass had a higher number of ammoniaoxidizing bacterial sequences (0.8% of total sequences) than the attached biomass (0.07%), although most of the sequences were within the Nitrosomonas oligotropha lineage in both biomasses. Nitrospira-like nitrite-oxidizing bacterial sequences were retrieved in the suspended biomass (0.06%), not in the attached biomass, whereas the existence of Nitrobacter-like sequences was not evident. The suspended biomass had higher nitrification activity (1.13 mg N/TSS/h) than the attached biomass (0.07 mg N/TSS/h). Overall, the results made it possible to conclude the importance of the suspended biomass, rather than the attached biomass, in nitrification in the wastewater treatment process studied.     

碳源对活性污泥微生物细胞膜特性和群落结构影响

【目的】揭示碳源对活性污泥微生物细胞膜特性(磷脂脂肪酸组成和流动性)以及群落结构的影响规律。【方法】采用原子力显微镜、磷脂脂肪酸(PLFA)、荧光漂白恢复(FRAP)和Mi Seq分析技术,考察以葡萄糖、乙酸钠、蛋白胨、葡萄糖?蛋白胨(1?1)和乙酸钠?蛋白胨(1?1)为基质的序批式活性污泥(SBR)反应器中,活性污泥微生物表面粘附力、细胞膜PLFA组成和流动性及群落结构的差异。【结果】含有蛋白胨为碳源时相比于葡萄糖和乙酸钠为单一碳源,细胞膜磷脂流动性增加,且18?1ω9c、15?0iso和17?0iso的含量提高了53.1%-354.7%、135.6%-407.9%和88.1%-264.3%;同时其微生物表面粘附力和群落多样性均增大。优势菌群对碳源的响应呈现出不一致的规律:葡萄糖和乙酸钠为单一碳源时其优势菌门分别为放线菌门和变形菌门,Nakamurella和Flavobacterium分别为其优势菌属,群落多样性指数分别为3.65和4.25;蛋白胨为碳源时促进了Candidatus Saccharibacteria门的累积,群落多样性指数在4.96-5.09。【结论】主成分分析(PCA)表明,含有蛋白胨为碳源时微生物细胞膜PLFA组成具有相似性;冗余分析(RDA)表明,不同碳源驯化出不同的微生物群落,进而也对细胞膜磷脂组成产生了影响。     

Long-term microbial community dynamics at two full-scale biotrickling filters treating pig house exhaust air

In this study, the microbial community structure of two full-scale biotrickling filters treating exhaust air from a pig housing facility were evaluated using 16S metabarcoding. The effect of inoculation with activated sludge of a nearby domestic waste water treatment plant was investigated, which is a cheap procedure and easy to apply in practice. The study was performed at a three-stage and a two-stage full-scale biotrickling filter; of which, only the latter was inoculated. Both biotrickling filters evolved towards a rather similar community over time, which differed from the one in the activated sludge used for inoculation. However, the bacterial population at both biotrickling filters showed small differences on the family level. A large population of heterotrophic bacteria, including denitrifying bacteria, was present in both biotrickling filters. In the non-inoculated biotrickling filter, nitrite-oxidizing bacteria (NOB) could not be detected, which corresponded with the incomplete nitrification leading to high nitrite accumulation observed in this system. Inoculation with the wide spectrum inoculum activated sludge had in this study a positive effect on the biotrickling filter performance (higher ammonia removal and lower nitrous oxide production). It could thus be beneficial to inoculate biotrickling filters in order to enrich NOB at the start-up, making it easier to keep the free nitrous acid concentration low enough to not be inhibited by it.     

Systematic analysis of biochemical performance and the microbial community of an activated sludge process using ozone-treated sludge for sludge reduction

Two lab-scale bioreactors (reactors 1 and 2) were employed to examine the changes in biological performance and the microbial community of an activated sludge process fed with ozonated sludge for sludge reduction. During the 122 d operation, the microbial activities and community in the two reactors were evaluated. The results indicated that, when compared with the conventional reactor (reactor 1), the reactor that was fed with the ozonated sludge (reactor 2) showed good removal of COD, TN and cell debris, without formation of any excess sludge. In addition, the protease activity and intracellular ATP concentration of reactor 2 were increased when compared to reactor 1, indicating that reactor 2 had a better ability to digest proteins and cell debris. DGGE analysis revealed that the bacterial communities in the two reactors were different, and that the dissimilarity of the bacterial population was nearly 40%. Reactor 2 also contained more protozoa and metazoa, which could graze on the ozone-treated sludge debris directly.     

Relationship between microbial activity and microbial community structure in six full-scale anaerobic digesters

High activity levels and balanced anaerobic microbial communities are necessary to attain proper anaerobic digestion performance. Therefore, this work was focused on the kinetic performance and the microbial community structure of six full-scale anaerobic digesters and one lab-scale co-digester. Hydrolytic (0.6–3.5 g COD g^?1 VSS d^?1) and methanogenic (0.01–0.84 g COD g^?1 VSS d^?1) activities depended on the type of biomass, whereas no significant differences were observed among the acidogenic activities (1.5–2.2 g COD g^?1 VSS d^?1). In most cases, the higher the hydrolytic and the methanogenic activity, the higher the Bacteroidetes and Archaea percentages, respectively, in the biomasses. Hydrogenotrophic methanogenic activity was always higher than acetoclastic methanogenic activity, and the highest values were achieved in those biomasses with lower percentages of Methanosaeta. In sum, the combination of molecular tools with activity tests seems to be essential for a better characterization of anaerobic biomasses.     

Hydraulics of laboratory and full-scale upflow anaerobic sludge blanket (UASB) reactors

Laboratory-scale upflow anaerobic sludge blanket (UASB) reactors are often used as test platforms to evaluate full-scale applications. However, for a given volume specific hydraulic loading rate and geometry, the gas and liquid flows increase proportionally with the cube root of volume. In this communication, we demonstrate that a laboratory-scale reactor had plug-flow hydraulics, while a full-scale reactor had mixed flow hydraulics. The laboratory-scale reactor could be modeled using an existing biochemical model, and parameters identified, but because of computational speed with plug-flow hydraulics, mixed systems are instead recommended for parameter identification studies. Because of the scaling issues identified, operational data should not be directly projected from laboratory-scale results to the full-scale design.