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Diversity and interactions of microbial functional genes under differing environmental conditions: insights from a membrane bioreactor and an oxidation ditch

The effect of environmental conditions on the diversity and interactions of microbial communities has caused tremendous interest in microbial ecology. Here, we found that with identical influents but differing operational parameters (mainly mixed liquor suspended solid (MLSS) concentrations, solid retention time (SRT) and dissolved oxygen (DO) concentrations), two full-scale municipal wastewater treatment systems applying oxidation ditch (OD) and membrane bioreactor (MBR) processes harbored a majority of shared genes (87.2%) but had different overall functional gene structures as revealed by two datasets of 12-day time-series generated by a functional gene array-GeoChip 4.2. Association networks of core carbon, nitrogen and phosphorus cycling genes in each system based on random matrix theory (RMT) showed different topological properties and the MBR nodes showed an indication of higher connectivity. MLSS and DO were shown to be effective in shaping functional gene structures of the systems by statistical analyses. Higher MLSS concentrations resulting in decreased resource availability of the MBR system were thought to promote positive interactions of important functional genes. Together, these findings show the differences of functional potentials of some bioprocesses caused by differing environmental conditions and suggest that higher stress of resource limitation increased positive gene interactions in the MBR system.

Wastewater treatment plants (WWTPs) are the largest applications of bioprocess engineering used for domestic and industrial wastewater treatment, with microbial consortia playing the central role. System-level functions (e.g., biodegradation and mineralization of organic pollutants, and nitrogen and phosphorus cycling) and ecosystem stability are accomplished through the growth, activities and interactions of vastly diverse microbial populations. As a unique artificial microbial ecosystem which is chemically and physically well-defined, WWTPs are considered as a fertile testing ground for a range of fundamental ecological questions 1 .
Environmental heterogeneity, defined as spatial and temporal variation in the physical, chemical and biological environment, is a fundamental property of ecosystems 2 . Its role in shaping microbial community diversity and composition is commonly valued and an intriguing topic. WWTPs are meant to provide proper environmental conditions to maintain the abundances of microbial populations, especially those functionally important groups, within normal levels for system performance and stability. Therefore, numerous studies focus on the relationship between microbial community diversity and environmental conditions mainly based on 16S rRNA genes or specific functional genes. They reveal the differences in microbial diversity and composition resulting from the differentiations of, such as, influent chemical oxygen demand (COD inf) concentrations 3 , dissolved oxygen (DO) concentrations 4 and solid retention time (SRT) 5 . Specifically, it has been suggested that the fixed volume bioreactors operated at high SRTs will be highly saturated with organisms that are capable of efficiently utilizing scarce resources ('K-strategists'), while low SRTs will help enrich for fast-growing organisms that are adapted for Scientific RepoRts | 6:18509 | DOI: 10.1038/srep18509 two systems, involved in each category. In spite of the shared genes, each system possessed some unique genes associated in each category. The genes only present in the MBR system accounted for 3.7% and the value was 9.1% for the OD system.
The two systems displayed similar relative abundance distributions of each category of functional genes. The OD system showed slightly higher overall relative abundances of the genes associated with bacterial phage (p < 0.05, absolute value of Cohen's d = 0.89), nitrogen cycling (p < 0.001, absolute value of Cohen's d = 1.31) and phosphorus utilization (p < 0.05, absolute value of Cohen's d = 0.99) (Fig. 1a).

Diversity of the genes related to key bioprocesses of wastewater treatment.
To clarify the details of the functional potential differentiations of the two systems, we focused on the diversity of three important functional gene categories -carbon cycling, nitrogen cycling and phosphorus utilization, as the removal of carbon, nitrogen and phosphorus pollutants are the main task of MWTPs.
A total of 4,129 genes associated with carbon cycling were detected, with 3,976 genes present in the OD system and 3,735 genes present in the MBR system. Among them, carbon degradation genes were predominant, accounting for 76.6%. These genes played roles in the removal of several polysaccharides such as starch, cellulose and chitin. Carbon degradation genes were slightly more abundant within the MBR system than in the OD system (p < 0.05, Cohen's d = 0.95) (Fig. 1b).
A total of 2,983 genes associated with nitrogen cycling were detected, consisting of the genes involved in ammonification (12.9%), nitrification (14.2%), denitrification (43.4%), anammox (0.2%) and others (29.3%). The detected 424 nitrification genes consisted of 408 amoA genes and 16 hao genes, of which 55.9% were from putative heterotrophic nitrifiers such as Pseudomonas putida. In addition, 11.3% of the nitrification genes were from the order of Nitrosomonadales, including Nitrosomonas (seven genes), Nitrosospira (15 genes) and Nitrosovibrio (one gene), which are the commonly known ammonia oxidizing bacteria (AOB) in WWTPs. The genes from Nitrosospira were slightly of higher relative abundance within the MBR (p < 0.05, absolute value of Cohen's d = 0.88), with the percentage change of relative abundances being 8.3%. Also, archaeal genes were detected, accounting for 18.4% of the nitrification genes. In the 1295 genes detected associated with denitrification, there were 88.5% from uncultured isolates. The relative abundance of denitrification genes of the OD system was slightly higher than that of the MBR system (p < 0.01, absolute value of Cohen's d = 1.09) (Fig. 1c).
A total of 528 genes associated with phosphorus utilization were detected, including ppk genes (37.3%), ppx genes (57.6%) and phytase genes (5.1%). Polyphosphate kinase (ppk) is responsible for polyphosphate (polyP) synthesis and ppk genes did not show significant differences in relative abundances within the two systems (Fig. 1d). Exopolyphosphatase (ppx) is a highly processive enzyme catalyzing the anaerobic hydrolysis process of terminal residues of long-chain polyP to phosphate (Pi). The relative abundances of ppx genes were significantly higher within the OD system than in the MBR system (p < 0.001, absolute value of Cohen's d = 1.39) (Fig. 1d). Phytase catalyzes the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen 14 . Phytase genes were of slightly higher relative abundance within the MBR system than in the OD system (p < 0.05, Cohen's d = 0.96) (Fig. 1d).
Functional gene structures of the two systems. The nonmetric multidimensional scaling (NMDS) result revealed that the samples from each system grouped (Fig. 2). Moreover, dissimilarity tests indicated that the two systems showed distinct structures of overall functional genes, carbon degradation genes, nitrogen cycling genes and phosphorus cycling genes ( phosphorus removal of the systems did not rely merely on biological processes, phosphorus removal efficiencies were not directly linked to functional gene structures when doing the analysis. In each system, the samples did not show obvious patterns in the NMDS plot and the abundances of each category of functional genes had small variations during the 12 days (Fig. 1), indicating that there was no obvious daily succession of overall functional genes.  Error bars represent the standard deviation of the relative abundances of each category or subcategory of genes in the 12 samples from the same system. Significant differences between the systems indicated by repeated measures analysis of variance (ANOVA) are indicated by asterisk above the bars, '*' (P < 0.05), '**' (P < 0.01), '***' (P < 0.001).
Scientific RepoRts | 6:18509 | DOI: 10.1038/srep18509 structures, and wastewater characteristics and bioreactor temperature and pH were revealed by the mantel test (data not given).

Possible interactions of core genes involved in the cycling of carbon, nitrogen and phosphorus.
The coexistence of a number of genes was noticed within each system. In the OD system, there were 50.9% genes present in all of the 12 daily samples. For the MBR system the value was 47.6%. Detailed information of the coexistent genes in the 12 sampling days of each system is shown in Supplementary Table S4 online. We consider these coexistent genes to be the core functional genes of each system. Possible interactions of these core genes implicated in carbon, nitrogen and phosphorus cycling were revealed by network construction based on a RMT algorithm. Consequently, a network of 1,116 nodes and 1741 links (4.2% negative and 95.8% positive) was   Table 2. Significance tests of the differences of functional genes of the two systems based on nonparametric multivariate analysis of variance (ADONIS), analysis of similarity (ANOSIM) and multiple response permutation procedure (MRPP). obtained for the MBR system and a network of 1,426 nodes and 1,462 links (9.4% negative and 90.6% positive) for the OD system. The two networks exhibited general topological features of ecological networks including scale free, small world and modularity (Supplementary Text S1 online). The topological structures of the two networks differed significantly, including average geodesic distance, average clustering coefficient, modularity and transitivity (Table 3). Average connectivity, average clustering coefficient and transitivity of the MBR network were higher than those of the OD network. The links between the nodes whose connectivity ranked top five in each network were shown in Fig. 4. Only 22.0% (458) of the nodes were shared by the two networks. Significant differences were observed in connectivity (p = 9.51 × 10 −6 ) and clustering coefficients (p = 2.19 × 10 −8 ) of these shared nodes in the two networks, as revealed by paired t tests.
There were 200 modules detected within the MBR network and 295 modules detected in the OD network. In each module, various categories or subcategories of functional genes were included. For each network, most of the nodes were evaluated as peripheral nodes, which only have a few links almost always to the nodes within their modules and represent specialists from an ecological perspective. Few of the nodes were defined as module hubs in each system, which were highly connected to several nodes in their own modules. In the MBR network 12 module hubs were detected, consisting of the genes involved in carbon degradation (66.7%), nitrification (8.3%), denitrification (8.3%), nitrogen fixation (8.3%) and dissimilatory N reduction (8.3%). In the OD network, 16 module nodes were detected, consisting of the genes related to carbon degradation (37.5%), carbon fixation (12.5%), phosphorus  Table 3. Major topological properties of the empirical association networks of core genes involved in the cycling of carbon, nitrogen and phosphorus in the MBR and OD systems and their associated random networks. a The number of genes originally used for network construction using the RMT-based approach. b The number of nodes in a network. c The correlation coefficient (r) of the linear relationship in log[P(k)] ~ -γlog(k), where P(k) is the fraction of connectivity k and γ is a constant. d The random networks were generated by rewiring all of the links of a network with the identical numbers of nodes and links to the corresponding empirical network. e Significant difference (P < 0.001) between the networks of OD and MBR. utilization (18.8%), denitrification (12.5%), nitrogen fixation (12.5%) and dissimilarity N reduction (6.3%). Only one connector (highly linked to several modules) associated with nitrogen ammonification was detected within the MBR network, and no connectors were detected within the OD network. From an ecological perspective, module hubs and connectors are generalists. None of the module hubs or connectors of the two networks were the same. The details of the module hubs and connectors of each network are shown in Supplementary Table S5 online. For each network, the relationships between node connectivity and seven environmental variables (DO, pH and temperature of the bioreactors, COD inf, NH 4 + -N inf, TN inf and TP inf) were examined. In the OD network, none of the gene significance (GS) of the environmental variables showed a significant correlation to node connectivity. In the MBR network, the GS of bioreactor pH showed a weak linkage to node connectivity (Supplementary Table S6 online).

Discussion
A high similarity of overall functional genes of the two systems was detected. It is known that the removal of wastewater-borne organic and inorganic pollutants (for example, PO 4 3− and heavy metals) relies heavily on the coexistence of several key microbial groups and the occurrence of particular microbial species 7,15 . The two systems were both designed for the removal of organic carbon, nitrogen and phosphorus pollutants. Several overlapped functional genes may participate in each bioprocess in both systems. Besides, inoculum sludge from the OD system was used for the start-up of the MBR system. Original MBR populations came from the OD community. Also, identical wastewater would facilitate the community similarity, as the bioreactor communities were exempt from the differences in substrate composition and source community, compared to the populations of the systems receiving differing influents. Moreover, identical wastewater and location leaded to similar temperature and pH of the bioreactors, providing the community with similar physical-chemical conditions. Similar results were obtained in the conventional enhanced biological phosphorus removal (EBPR) and MBR plants in Denmark, which showed a majority of shared 16S rRNA genes 10 .
However, slight differences in the abundances of certain categories and subcategories of functional genes of the two systems were shown. They may result from the common differences in environmental conditions of these two kinds of treatment processes. MBRs were suggested to have a better disinfection capability than CAS systems 16 . Phage removal in a MBR was suggested to be largely achieved by biofilm accumulation on the membrane surface, which physically reduced the membrane pore size, chemically adsorbed phages, and biologically allowed the predation of phages by other microorganisms 17 . Thus, bacterial phages in the MLSS of the membrane tanks (the MBR samples) may be of a low abundance, and they may be less abundant than those of the OD samples. The longer SRT of the MBR system might allow the enrichment of some microbes responsible for the degradation of high-molecular-weight compounds such as polysaccharide and protein 18 . Besides, the higher concentration of MLSS in the MBR system may lead to an increase of the concentration of colloidal material, which presents a new feed source for the microorganisms able to scavenge these compounds. This is verified by the fact that the concentrations of polysaccharides in the supernatant of the membrane tanks of the MBR system (2.4-6.5 mg/l) were higher than those of the aeration tanks of the OD system (1.3-5 mg/l) during April, 2011 to March, 2012 (unpublished data). Thus, the carbon degradation genes may be more abundant within the MBR samples. Possible reasons for the differences in the abundances of nitrogen cycling genes are as follows. The higher abundance of the nitrification genes from Nitrosospira in the MBR system might result from the lower F/M ratio, as Nitrosospira were suggested to be favored under relatively low concentrations of substrates, compared to Nitrosomonas 19 . The higher abundance of denitrification genes within the OD system might be due to the lower DO concentration and the higher F/M ratio. The former may promote denitrification and the latter may allow the denitrifiers to access more readily biodegradable carbon sources. Possible explanations of the differences in the abundances of phosphorus utilization genes are listed below. The higher abundance of ppx genes within the OD system may mainly result from the lower DO concentration, which may benefit the anaerobic hydrolysis process of terminal residues of long-chain polyP to phosphate (Pi). It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesized in response to a nutrient or energy limitation 20 . The higher abundance of phytase genes of the MBR system may be due to a severer situation of the nutrient or energy limitation the MBR community was faced up with. These significant differentiations in the abundances of certain functional gene groups (for example, higher abundance of carbon degradation genes in the MBR system, and higher abundance of denitrification genes in the OD system) revealed specific effects of environmental gradients on the functional potential of specific bioprocesses. They may explain the observed differences of system performance in COD and TN removal to some extent. Differing structures of overall functional genes and specific categories of functional genes of the two systems were also revealed. This may result from the existence of unique genes of each system and abundance differentiations of certain genes. No significant linkages between system functioning and overall functional genes structures were detected, however. The possible reasons might be that the unique functional genes of each system or the genes with differing abundances may not be so tightly linked to removals of COD, TN and NH 4 + -N. In addition to the differentiations of functional gene abundances and structures, tighter interactions/coupling, especially tighter positive (facilitative) interactions within the MBR community was suggested by the higher average connectivity, average clustering coefficient, transitivity and positive interaction percentages of the MBR network. This may result from the lower F/M ratios of the substrates of carbon, nitrogen and phosphorus in the MBR system. For the MBR populations, amelioration of the stress of substrate scarcity by collaborations with the neighbors may be a good choice. For example, the carbon degradation groups could increase the availability of readily degradable organic carbon for denitrifiers. Thus, the organic carbon degradation population and denitrifiers may be more tightly connected in the MBR than in the OD system. Similarly, facilitation could be expected to be the dominant net outcome at moderate levels of stress driven by resource in plants 21 and it was noticed that positive interactions between plant species were dominant at moderate level of rainfall stress 22  within the two networks were detected, indicating that important roles within or between modules of the MBR and OD systems were played by different nodes (genes).
The CCA unveiled that MLSS and DO were main factors shaping the functional gene structures of the two systems, which was verified by the mantel test. As is discussed above, the higher MLSS of the MBR system, which resulted in lower F/M ratios, may favor K-strategists like Nitrosospira, select against denitrifers and promote phytase genes in the MBR system. Also, it resulted in more polysaccharides of the supernatant, which may contribute to the higher abundance of carbon degradation genes of the MBR system. The higher aeration rates of the MBR system may select against denitrifiers and ppx genes. Due to the numerical correlation between SRT and MLSS concentrations in doing the CCA, SRT was not evaluated as a redundant variable when with the presence of MLSS and excluded from the CCA plot. On the other hand, the significant correlation between SRT and the functional gene structures in the mantel test indicates that SRT may exert some effects on the functional gene structures. Specifically, as is discussed above, the longer SRT of the MBR system might help promote some organisms able to degrade polysaccharides, for example Chloroflexi 5,23 . It may be an explanation of the higher abundance of carbon degradation genes. Short SRTs were shown to have impacts on microbial communities of wastewater treatment plants in previous research. A system operated at a SRT of 3d was indicated to harbor no Nitrospira sp. and Nitrosomonas sp. 5 . Another system operated with SRTs of 10 d, 3 d and 5 d was shown to have more diverse microbial populations when with a higher SRT 24 . However, differing SRTs of the two systems did not lead to significant differences of nitrification gene abundances (Fig. 1c). The possible reason might be that the SRTs of the two systems (20.5 d and 16.2 d) could both allow the nitrifiers to thrive. Besides the effects of operational parameters on the functional gene structures, a low percentage of shared nodes, differing topological structures and different topological roles of the nodes in the two networks indicated that operational parameter differentiations also altered possible microbial interactions of the two systems. The higher MLSS of the MBR system increased the resource limitation stress and may, thus, increase positive interactions between the functional genes associated with carbon, nitrogen and phosphorus cycling. The possible impacts of treatment processes and environmental variables on the diversity and interactions of the functional genes in the two systems are summarized in Supplementary table S1 online.
Wastewater characteristics, and bioreactor temperature and pH, which were vulnerable to influent variations, played relatively small roles in shaping the overall functional gene structures. In addition, no significant or only few weak links between these variables and the topology of each network revealed their inability in affecting the possible microbial interactions. The reason might be that the two systems were with small wastewater characteristics fluctuations and basically stable temperature and pH during the sampling days. Besides, some unmeasured influential variables could also possibly contribute to significant changes in functional gene diversity and affect possible interactions of core functional genes in each system.
When long-term sampling is implemented, larger gradients in other parameters, for example, bioreactor temperature and pH, are covered. The main environmental variables that affect microbial ecology might be different. A long-term investigation on a single WWTP using high-throughput sequencing indicated the importance of temperature and salinity in driving the seasonal dynamics of the genera with significantly changed abundances during an over 4-year period 25 . Another study indicated a lack of strong correlations between environmental factors and several persistent operational taxonomy units (OTUs) in a WWTP. Environmental conditions (mainly SRT and inorganic nitrogen) partially explained the phylogenetic variances and indirectly influenced bacterial assembly 8 . These studies suggest that the effective variables shaping microbial community structures may be case-specific. However, few long-term investigations on the communities of MBRs and activated sludge systems have been reported. Such research did not discuss which variables were predominant in leading to the observed differentiations of microbial diversity either 11 . Long time-series sampling (for example, monthly, seasonal and yearly) of the communities in MBRs and activated sludge systems are suggested to be conducted by further studies to enable profound discussions on the correlation between microbial ecology and environmental gradients, with big environmental gradients in both physical-chemical and operational parameters covered.
In sum, in this study we showed that the differences in operational parameters (MLSS, SRT and DO) of two parallelly operated MBR and OD systems indeed affected not only the functional potentials of certain bioprocesses but the microbial interactions. These findings give a useful insight of the effect of differing environmental conditions on microbial ecology. WWTPs are well controlled microbial ecological systems, but we only know a tip of the iceberg of them. Further studies on these systems targeting at answering key ecological questions and understanding the systems for a better design and operation of them are of great significant.

Methods
Location Description and Sampling. The two full-scale wastewater treatment facilities are located in a WWTP in Wuxi, Jiangsu Province of China. One of them is an orbal OD, and the other is a MBR coupled with anaerobic-anoxic-oxic process (A 2 O-MBR). They treat identical wastewater (domestic : industrial = 0.6 : 0.4) at the same scale (50,000 m 3 /d) and the MBR sludge was originally inoculated with the OD sludge. The removal of organic carbon and nitrogen pollutants of the systems were achieved through biological processes, and the removal of phosphorus of them were through both biological processes and chemical precipitation. They had been operated with good treatment efficiency and stability at least for one year and three months before sampling 26,27 .
MLSS samples were taken from the the aerobic zones of the OD system and from the membrane tanks of the A 2 O-MBR system once a day for 12 concecutive days during April 10 to 21, 2011. On each day, 50 ml MLSS was collected at each site. Each sample was dispensed into a 50 mL sterile Eppendorf tube and centrifuged at 14,000 g for 10 min. The pellets were stored at −80° C for analysis. Daily measurement was done to decide the pollutant concentrations of 24-h composite samples of influents and effluents, and temperatures, pH, and DO concentrations Scientific RepoRts | 6:18509 | DOI: 10.1038/srep18509 of the aeration zones of each system. The approximate values of the MLSS concentrations and SRT were provided by the plant staff.
DNA Extraction and Purification. Microbial genomic DNA was extracted from the pellets of activated sludge samples through a combination of freezing and sodium dodecyl sulfate (SDS) for cell lysis as previously described 28 . The extracted products were then purified employing the Wizard ® SV Genomic DNA Purification Kit (Promega, Madison WI). DNA Labeling and Hybridization. 1 μ g DNA was labeled, purified and dried as previously described 29 . All labeled DNA was resuspended in 10 μ L hybridization solution as previously described 30 and was hybridized with GeoChip 4.2 on a MAUI hybridization station (BioMicro, Salt Lake City, UT, USA) at 42° C with 40% formamide for 16 h. Microarrays were scanned by a ScanArray 5000 Microarray Analysis System (PerkinElmer, Wellesley, MA, USA) at 100% laser power. Data Analysis. Signal intensities of the spots were measured with ImaGene 6.0 (Biodiscovery Inc., El Segundo, CA, USA). Data pretreatment was performed online (ieg.ou.edu). The percentages of thermophile probes (negative controls) in each sample were made less than 5% by the removal of low quality spots. The genes detected only in three or fewer samples out of 12 samples from the same system were removed to avoid potential noise. For each sample, intensities of the genes were then transformed to the natural logarithmic form and divided by the mean signal intensity.
Detailed information of the detected genes and their signal intensities is available at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/, accession number GSE 67307).
To unveil the dissimilarities of microbial communities, three non-parametric multivariate statistical tests including non-parametric multivariate analysis of variance (ADONIS), analysis of similarity (ANOSIM) and multiple response permutation procedure (MRPP) and a NMDS analysis were performed. To test whether the diversity indices and the abundances of each functional gene category and certain subcategories or phylogenetic groups in the two systems differed, repeated measures analysis of variance (ANOVA) was performed. Cohen's d was applied to assess the magnitude of treatment effects and a conventional rule is to consider a Cohen's d of 0.8 as large, suggesting that 79% of the control group would have a score below the subject in the experimental group 31,32 . To evaluate the effects of environmental factors on overall functional community structures, a CCA and a mantel test were performed. In CCA, the VIF for each environmental attribute was used to identify multicollinearity between explanatory variables. The factor with a VIF value over 20 was considered to be a redundant constraint and removed. All the above statistical analyses were done with R code (http://www.r-project.org/).
To unveil possible microbial interactions of the genes associated with carbon, nitrogen and phosphorus cycling, two microbial association networks were constructed based on RMT using a comprehensive Molecular Ecological Network Analysis Pipeline (MENAP) (http://ieg2.ou.edu/MENA/) 13 . A similarity matrix of "Pearson correlation in time-series (allow 1 time point lagging)" was applied. For each system, only those genes detected in all the 12-week samples (majority rule) were kept for network construction. This filtering step removed poorly represented functional genes and reduced network complexity 33 . For each network, 100 corresponding random networks were generated, with the same network size and average number of links. The Z-test was used to test the differences of the indices between the constructed networks and random networks. To characterize the modularity property, each network was separated into modules by the fast greedy modularity optimization. The topological roles of different nodes were divided into the following four subcategories by within-module connectivity (z i ) and among module connectivity (P i ): (i) peripheral nodes; (ii) connectors; (iii) module hubs; and (iv) network hubs 34 . For comparison between the network indices of different systems, the Student t-test was employed using the standard deviations derived from corresponding random networks. Paired t-test was done to make comparisons between the topological structures of the shared nodes of the two networks. The relationships between microbial network topology and environmental characteristics were examined in an indirect way by measuring the correlation between the GS and the connectivity of nodes 12 . Cytoscape_3.2.1 was used for network visualization of the nodes, the connectivity of which ranked top five.