Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

Bacterial communities in full-scale wastewater treatment systems

Bacterial metabolism determines the effectiveness of biological treatment of wastewater. Therefore, it is important to define the relations between the species structure and the performance of full-scale installations. Although there is much laboratory data on microbial consortia, our understanding of dependencies between the microbial structure and operational parameters of full-scale wastewater treatment plants (WWTP) is limited. This mini-review presents the types of microbial consortia in WWTP. Information is given on extracellular polymeric substances production as factor that is key for formation of spatial structures of microorganisms. Additionally, we discuss data on microbial groups including nitrifiers, denitrifiers, Anammox bacteria, and phosphate- and glycogen-accumulating bacteria in full-scale aerobic systems that was obtained with the use of molecular techniques, including high-throughput sequencing, to shed light on dependencies between the microbial ecology of biomass and the overall efficiency and functional stability of wastewater treatment systems. Sludge bulking in WWTPs is addressed, as well as the microbial composition of consortia involved in antibiotic and micropollutant removal.     

Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in china

To determine if there is a core microbial community in the microbial populations of different wastewater treatment plants (WWTPs) and to investigate the effects of wastewater characteristics, operational parameters, and geographic locations on microbial communities, activated sludge samples were collected from 14 wastewater treatment systems located in 4 cities in China. High-throughput pyrosequencing was used to examine the 16S rRNA genes of bacteria in the wastewater treatment systems. Our results showed that there were 60 genera of bacterial populations commonly shared by all 14 samples, including Ferruginibacter, Prosthecobacter, Zoogloea, Subdivision 3 genera incertae sedis, Gp4, Gp6, etc., indicating that there is a core microbial community in the microbial populations of WWTPs at different geographic locations. The canonical correspondence analysis (CCA) results showed that the bacterial community variance correlated most strongly with water temperature, conductivity, pH, and dissolved oxygen (DO) content. Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 25.7% of the variance of bacterial communities independently, followed by operational parameters (23.9%) and geographic location (14.7%). Results of this study provided insights into the bacterial community structure and diversity in geographically distributed WWTPs and discerned the relationships between bacterial community and environmental variables in WWTPs.     

Microbial Community Profiles in Wastewaters from Onsite Wastewater Treatment Systems Technology

The aim of the study was to determine the potential of community-level physiological profiles (CLPPs) methodology as an assay for characterization of the metabolic diversity of wastewater samples and to link the metabolic diversity patterns to efficiency of select onsite biological wastewater facilities. Metabolic fingerprints obtained from the selected samples were used to understand functional diversity implied by the carbon substrate shifts. Three different biological facilities of onsite wastewater treatment were evaluated: fixed bed reactor (technology A), trickling filter/biofilter system (technology B), and aerated filter system (the fluidized bed reactor, technology C). High similarities of the microbial community functional structures were found among the samples from the three onsite wastewater treatment plants (WWTPs), as shown by the diversity indices. Principal components analysis (PCA) showed that the diversity and CLPPs of microbial communities depended on the working efficiency of the wastewater treatment technologies. This study provided an overall picture of microbial community functional structures of investigated samples in WWTPs and discerned the linkages between microbial communities and technologies of onsite WWTPs used. The results obtained confirmed that metabolic profiles could be used to monitor treatment processes as valuable biological indicators of onsite wastewater treatment technologies efficiency. This is the first step toward understanding relations of technology types with microbial community patterns in raw and treated wastewaters.     

Metagenomic analysis reveals the prevalence and persistence of antibiotic- and heavy metal-resistance genes in wastewater treatment plant

The increased antibiotic resistance among microorganisms has resulted into growing interest for investigating the wastewater treatment plants (WWTPs) as they are reported to be the major source in the dissemination of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) in the environment. In this study, we investigated the prevalence and persistence of ARGs and HMRGs as well as bacterial diversity and mobile genetic elements (MGEs) in influent and effluent at the WWTP in Gwangju, South Korea, using high-throughput sequencing based metagenomic approach. A good number of broad-spectrum of resistance genes (both ARG and HMRG) were prevalent and likely persistent, although large portion of them were successfully removed at the wastewater treatment process. The relative abundance of ARGs and MGEs was higher in effluent as compared to that of influent. Our results suggest that the resistance genes with high abundance and bacteria harbouring ARGs and MGEs are likely to persist more through the treatment process. On analyzing the microbial community, the phylum Proteobacteria, especially potentially pathogenic species belonging to the genus Acinetobacter, dominated in WWTP. Overall, our study demonstrates that many ARGs and HMRGs may persist the treatment processes in WWTPs and their association to MGEs may contribute to the dissemination of resistance genes among microorganisms in the environment.     

Diversity and population structure of sewage‐derived microorganisms in wastewater treatment plant influent

The release of untreated sewage introduces non‐indigenous microbial populations of uncertain composition into surface waters. We used massively parallel 454 pyrosequencing of hypervariable regions in rRNA genes to profile microbial communities from eight untreated sewage influent samples of two wastewater treatment plants (WWTPs) in metropolitan Milwaukee. The sewage profiles included a discernible human faecal signature made up of several taxonomic groups including multiple Bifidobacteriaceae, Coriobacteriaceae, Bacteroidaceae, Lachnospiraceae and Ruminococcaceae genera. The faecal signature made up a small fraction of the taxa present in sewage but the relative abundance of these sequence tags mirrored the population structures of human faecal samples. These genera were much more prevalent in the sewage influent than standard indicators species. High‐abundance sequences from taxonomic groups within the Beta‐ and Gammaproteobacteria dominated the sewage samples but occurred at very low levels in faecal and surface water samples, suggesting that these organisms proliferate within the sewer system. Samples from Jones Island (JI – servicing residential plus a combined sewer system) and South Shore (SS – servicing a residential area) WWTPs had very consistent community profiles, with greater similarity between WWTPs on a given collection day than the same plant collected on different days. Rainfall increased influent flows at SS and JI WWTPs, and this corresponded to greater diversity in the community at both plants. Overall, the sewer system appears to be a defined environment with both infiltration of rainwater and stormwater inputs modulating community composition. Microbial sewage communities represent a combination of inputs from human faecal microbes and enrichment of specific microbes from the environment to form a unique population structure.     

污水处理过程中微生物群落多样性及其对环境因子响应的研究进展

污水生物处理系统的性能和稳定性与微生物群落结构和动态密切相关。通过深入了解活性污泥中微生物群落结构及其影响因素,有助于提高污水厂污染物的去除效果。在不同污水活性污泥处理系统中细菌群落主要以变形菌、绿弯菌、放线菌、厚壁菌和拟杆菌为功能菌群;活性污泥中寄居的大多数真菌来自于子囊菌门,还有少量担子菌门;古菌以产甲烷菌为主;而病毒中分布最广的噬菌体和致病性病毒是最主要的关注点。本文通过对相关文献分析及总结,综述了进水组成、不同处理工艺、参数(理化参数和运行参数)、地理位置和气候条件等环境因子对活性污泥中细菌、真菌、古菌以及病毒群落组成的影响,尽可能全面地介绍污水厂微生物群落多样性及其对环境因子的响应。同时,对未来研究方向进行探讨,以期能够为活性污泥中功能微生物的应用及调控提供理论和应用基础。     

京津冀区域市政污水厂活性污泥种群结构的多样性及差异

【背景】活性污泥中微生物的种群结构影响着污水生物处理的高效性及稳定性,是有效保证污水处理效果的关键。【目的】研究活性污泥中细菌的群落结构组成及多样性,并分析相应菌群的主要功能,旨在更好地发挥细菌的净化作用、保持污水处理过程的稳定及提高污水的处理效率。【方法】以京津冀区域内典型市政污水厂活性污泥为研究对象,通过IlluminaMiSeq高通量测序及实时定量PCR技术,对5个污水厂活性污泥的微生物种群结构特征进行了详细解析,研究不同工艺参数下活性污泥中优势种群及脱氮菌群丰度的差异。【结果】5个污水厂活性污泥种群结构具有一定差异,其中Hengshui (HS)厂污泥的群落结构受温度的影响最大,而Shahe (SH)、Daoxianghu (DXH)、Nangong(NG)厂活性污泥群落结构则受总氮、总磷与氨氮的共同影响,氨氮对SH厂活性污泥种群结构影响最大。DXH、NG和HS厂污泥中优势菌均为Anaerolineaceae,而SH和Hejian (HJ)厂的优势菌则为Saprospiraceae与Lactobacillus。活性污泥中反硝化菌丰度最高的为HJ厂,丰度最低的为HS厂,反硝化功能基因nirS比nirK分布更为广泛。【结论】对于不同污水厂,影响其活性污泥群落结构组成的环境因素也是不同的,并且特殊的进水水质也会对污泥菌群组成和生物多样性产生影响。     

Failure of the ammonia oxidation process in two pharmaceutical wastewater treatment plants is linked to shifts in the bacterial communities

AIMS: To investigate whether two different wastewater treatment plants (WWTPs) -- treating the same pharmaceutical influent -- select for a different bacterial and/or ammonia oxidizing bacterial (AOB) community. METHODS AND RESULTS: Molecular fingerprinting demonstrated that each WWTP had its own total bacterial and AOB community structure, but Nitrosomonas eutropha and N. europea were dominant in both WWTP A and B. The DNA and RNA analysis of the AOB communities revealed different patterns; so the most abundant species may not necessarily be the most active ones. Nitritation failures, monitored by chemical parameter analysis, were reflected as AOB community shifts and visualized by denaturing gradient gel electrophoresis (DGGE)-based moving window analysis. CONCLUSIONS: This research demonstrated the link between functional performance (nitritation parameters) and the presence and activity of a specific microbial ecology (AOB). Clustering and moving window analysis based on DGGE showed to be valuable to monitor community shifts in both WWTPs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of specific community shifts together with functional parameter analysis has potential as a tool for relating functional instability (such as operational failures) to specific-bacterial community shifts.     

Pandemic pharmaceutical dosing effects on wastewater treatment: no adaptation of activated sludge bacteria to degrade the antiviral drug oseltamivir (Tamiflu®) and loss of nutrient removal performance

The 2009-2010 influenza pandemic saw many people treated with antivirals and antibiotics. High proportions of both classes of drugs are excreted and enter wastewater treatment plants (WWTPs) in biologically active forms. To date, there has been no study into the potential for influenza pandemic-scale pharmaceutical use to disrupt WWTP function. Furthermore, there is currently little indication as to whether WWTP microbial consortia can degrade antiviral neuraminidase inhibitors when exposed to pandemic-scale doses. In this study, we exposed an aerobic granular sludge sequencing batch reactor, operated for enhanced biological phosphorus removal (EBPR), to a simulated influenza-pandemic dosing of antibiotics and antivirals for 8 weeks. We monitored the removal of the active form of Tamiflu(?), oseltamivir carboxylate (OC), bacterial community structure, granule structure and changes in EBPR and nitrification performance. There was little removal of OC by sludge and no evidence that the activated sludge community adapted to degrade OC. There was evidence of changes to the bacterial community structure and disruption to EBPR and nitrification during and after high-OC dosing. This work highlights the potential for the antiviral contamination of receiving waters and indicates the risk of destabilizing WWTP microbial consortia as a result of high concentrations of bioactive pharmaceuticals during an influenza pandemic.     

我国南北区域城市污水处理系统内真菌群落的差异

【背景】微生物是污水处理系统内污染物去除的主体,真菌作为其中不可或缺的一部分,在去除有毒化合物、提高生物转化等方面发挥着重要作用,应引起人们的关注。【目的】明确我国南北区域城市污水处理系统内真菌群落的多样性、组成差异及其影响因素。【方法】采集我国北方23个污水处理厂的90份活性污泥样品和南方37个污水处理厂的121份活性污泥样品,提取上述样本的总DNA,并应用Illumina MiSeq平台进行ITS高通量测序,结合多种数量生态学分析方法对数据进行分析。【结果】我国南北区域城市污水处理系统内真菌群落结构存在显著差异,真菌群落多样性南方显著高于北方;我国南北区域真菌群落的组成差异显著,南方群落以Sordariomycetes和Glomeromycetes为优势菌纲,以Ophiocordycep和Alternaria为优势菌属,北方群落以Tremellomycetes和Saccharomycetes为优势菌纲,以Trichosporon和Saccharomyces为优势菌属,其中Tremellomycetes和Sordariomycetes是常见的病原菌,对下游生物或者人类的健康具有潜在威胁,Trichosporon属的丝状真菌若大量异常增殖会引发污泥膨胀现象,影响系统的稳定运行;地理纬度、年平均气温、进水NH_4~+浓度、进水总氮浓度是导致我国南北区域真菌群落存在差异的重要影响因子。【结论】我国南北区域真菌群落多样性、组成存在显著差异,地理因素、所在城市的气候因素和进水污染物浓度对真菌群落结构影响显著。针对南北区域,应合理调控相应区域内的重要功能菌群,以调控污水处理厂的高效稳定运行,同时密切关注其中的条件致病菌和易引发污泥膨胀的菌群,建立面向种群的优化控制系统以管控风险。     

Effects of plant and influent C:N:P ratio on microbial diversity in pilot-scale constructed wetlands

Microbial processes within the rhizosphere of constructed wetlands are crucial to wastewater treatment, but the relation between microbial community diversity in rhizosphere, plant growth and water quality are unclear at present. The effects of plant growth, water C:N:P ratio and their interaction on microbial diversity in the rhizosphere were studied in synthetic wastewater in planted and unplanted wetlands during three different seasons. The physiological profile of microbial community-level in each wetland was assessed using substrate utilization patterns gathered via BIOLOG? ECO plates. Plant had a significant effect on AWCD parameter, since the planted wetlands usually had a higher the total microbial activity than the unplanted over the study period. The Shannon, Simpson and McIntosh indices in the planted wetlands were apparently higher than those in the unplanted wetlands under any C:N:P ratio influent condition especially in summer. It was also shown that the unplanted wetlands have a greater shift of the interstitial microbial community than the planted at different seasons, since plant rhizospheres produce a more ecologically stable system in order to resist against shifts in microbial community composition in response to C:N:P ratio change in wastewater. Principal component analysis and clustering analysis indicated that influent C:N:P ratio would induce similar microbial species in the planted wetlands and detach them from the unplanted wetlands.     

Effects of substrate composition on the structure of microbial communities in wastewater using fluorescence in situ hybridisation

The microbial composition in a pulp and paper wastewater aerated lagoon system was analysed using fluorescence in situ hybridisation (FISH) to gain further understanding of the effect of substrate composition on microbial diversity for improved management of wastewater treatment systems. Few experiments have been conducted to tease apart the factors influencing the composition and abundance of certain groups within these wastewaters. Specific probes were used to investigate and enumerate the different bacterial groups present at particular stages through the treatment system over an extended period. Community composition and abundance of specific groups differed through the system however temporal stability was retained despite significant variability in the wastewater. Middle stream wastewater samples were enriched to explore the impact of different carbon/nitrogen/phosphorus (C:N:P) ratios on community composition and provide functionality to groups of micro-organisms within the microbial consortia. Nitrogen and phosphorus conditions did not impact community composition of methanol-fed cultures, which exhibited a dominance of Betaproteobacteria (>75%), namely Methylotrophic bacteria. This was confirmed through 16S rRNA gene sequencing and specific FISH probing, reflecting population observations at the beginning of the treatment system. We conclude that the nutrient and carbon combinations used in the enrichments created an interactive effect, altering the community composition and mimicking the main substrate load in the different stages of the treatment system. Finally, pulp and paper wastewater microbial composition was highly variable across the treatment system but was stable within the time sampled, with the enrichments emulating the substrate loads in the full scale system.     

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.     

Comparison of bacterial diversity in full scale anammox bioreactors operated under different conditions

Bacterial community structure of full‐scale anammox bioreactor is still mainly unknown. It has never been analyzed whether different anammox bioreactor configurations might result in the development of different bacterial community structures among these systems. In this work, the bacterial community structure of six full‐scale autotrophic nitrogen removal bioreactors located in The Netherlands and China operating under three different technologies and with different influent wastewater characteristics was studied by the means of pyrotag sequencing evaluation of the bacterial assemblage yielded a great diversity in all systems. The most represented phyla were the Bacteroidetes and the Proteobacteria, followed by the Planctomycetes. 14 OTUs were shared by all bioreactors, but none of them belonged to the Brocadiales order. Statistical analysis at OTU level showed that differences in the microbial communities were high, and that the main driver of the bacterial assemblage composition was different for the distinct phyla identified in the six bioreactors, depending on bioreactor technology or influent wastewater characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1464–1472, 2015     

Microbial community composition and dynamics of moving bed biofilm reactor systems treating municipal sewage

Moving bed biofilm reactor (MBBR) systems are increasingly used for municipal and industrial wastewater treatment, yet in contrast to activated sludge (AS) systems, little is known about their constituent microbial communities. This study investigated the community composition of two municipal MBBR wastewater treatment plants (WWTPs) in Wellington, New Zealand. Monthly samples comprising biofilm and suspended biomass were collected over a 12-month period. Bacterial and archaeal community composition was determined using a full-cycle community approach, including analysis of 16S rRNA gene libraries, fluorescence in situ hybridization (FISH) and automated ribosomal intergenic spacer analysis (ARISA). Differences in microbial community structure and abundance were observed between the two WWTPs and between biofilm and suspended biomass. Biofilms from both plants were dominated by Clostridia and sulfate-reducing members of the Deltaproteobacteria (SRBs). FISH analyses indicated morphological differences in the Deltaproteobacteria detected at the two plants and also revealed distinctive clustering between SRBs and members of the Methanosarcinales, which were the only Archaea detected and were present in low abundance (<5%). Biovolume estimates of the SRBs were higher in biofilm samples from one of the WWTPs which receives both domestic and industrial waste and is influenced by seawater infiltration. The suspended communities from both plants were diverse and dominated by aerobic members of the Gammaproteobacteria and Betaproteobacteria. This study represents the first detailed analysis of microbial communities in full-scale MBBR systems and indicates that this process selects for distinctive biofilm and planktonic communities, both of which differ from those found in conventional AS systems.     

Diversity and assembly patterns of activated sludge microbial communities: A review

Understanding diversity and assembly patterns of microbial communities in activated sludge (AS) is pivotal for addressing fundamental ecological questions and wastewater treatment engineering. Recent applications of molecular methods especially high-throughput sequencing (HTS) have led to the explosion of information about AS community diversity, including the identification of uncultured taxa, and characterization of low-abundance but environmentally important populations such as antibiotic resistant bacteria and pathogens. Those progresses have facilitated the leverage of ecological theories in describing AS community assembly. The lognormal species abundance curve has been applied to estimate AS microbial richness. Taxa-area and taxa-time relationships (TAR and TTR) have been observed for AS microbial communities. Core AS microbial communities have been identified. Meanwhile, the roles of both deterministic and stochastic processes in shaping AS community structures have been examined. Nonetheless, it remains challenging to define tempo-spatial scales for reliable identification of community turnover, and find tight links between AS microbial structure and wastewater treatment plant (WWTP) functions. To solve those issues, we expect that future research will focus on identifying active functional populations in AS using omics- methods integrated with stable-isotope probing (SIP) with the development of bioinformatics tools. Developing mathematic models to understand AS community structures and utilize information on AS community to predict the performance of WWTPs will also be vital for advancing knowledge of AS microbial ecology and environmental engineering.     

人工湿地处理造纸废水后细菌群落结构变化

人工湿地通过模拟自然湿地的生态系统,能够实现污染物的去除,是一种高效经济的污水处理手段,但是对人工湿地处理后污水中细菌群落的研究较少。采集了造纸厂排污口和人工湿地出水口的水样,检测了溶解氧、pH值和盐度等水质指标,对主要污染物的含量进行了测定和分析,并利用DGGE技术对细菌群落的变化进行了研究。结果表明:(1)造纸污水经人工湿地处理后水质有明显提高;(2)人工湿地处理后细菌群落结构发生变化,优势菌由γ变形菌和衣原体变为α变形菌,而且微生物多样性指数Shannon-Wiener's降低;(3)人工湿地处理前污水中存在着大量致病微生物和降解微生物,处理后以环境友好的固氮菌和少量致病菌为主。研究结果揭示了人工湿地不仅可以去除造纸废水中的污染物,改善水质而且可以大大减少向环境中排放的致病微生物,防止由致病微生物引起的生态灾难的发生,为将来人工湿地应用于工业污水处理和微生物生态安全评估提供有效可靠的依据。     

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.

     

Differences in vegetation composition and plant species identity lead to only minor changes in soil-borne microbial communities in a former arable field

To examine the relationship between plant species composition and microbial community diversity and structure, we carried out a molecular analysis of microbial community structure and diversity in two field experiments. In the first experiment, we examined bacterial community structure in bulk and rhizosphere soils in fields exposed to different plant diversity treatments, via a 16S rRNA gene clone library approach. Clear differences were observed between bacterial communities of the bulk soil and the rhizosphere, with the latter containing lower bacterial diversity. The second experiment focused on the influence of 12 different native grassland plant species on bacterial community size and structure in the rhizosphere, as well as the structure of Acidobacteria and Verrucomicrobia community structures. In general, bacterial and phylum-specific quantitative PCR and PCR-denaturing gradient gel electrophoresis revealed only weak influences of plant species on rhizosphere communities. Thus, although plants did exert an influence on microbial species composition and diversity, these interactions were not specific and selective enough to lead to major impacts of vegetation composition and plant species on below-ground microbial communities.