Assessment of activated sludge microbial community analysis in full-scale biological wastewater treatment plants using patterns of fatty acid isopropyl esters (FAPEs)

This investigation introduces the application of a relatively rapid technique to obtain information about the dynamic nature of microbial communities in activated sludge. The objective has been to consider variability due to measurement errors and protocol changes within the same quantitative framework as the analysis of systematic differences in microbial communities in large-scale aerobic activated sludge secondary wastewater treatment systems. Adjustments to the methodology were considered due to their potential for simplifying and shortening the analysis procedure. All modifications to the protocols used to assay the composition of microbial fatty acids (MFAs) of activated sludge imposed some bias to the chromatographic data. This methodological bias was similar in magnitude to the level of discrimination between activated sludge microbial community structures that were considered as part of the present study. MFA analysis supported the expectations of subtle but systematic community structure differences and shifts in activated sludge based on the current understanding of these wastewater treatment systems. A standardized MFA methodology was shown to be sensitive to minor systematic changes in activated sludge communities due the anticipated underlying factors of selective pressures from the process configuration, history, operational conditions and/or nutrient status. The chemometric approach of fatty acid isopropyl ester analysis of activated sludge can provide a routine tool for meaningful and quantitative information of changes in activated sludge quality in full-scale treatment systems.     

Challenges in determining causation in structure-function studies using molecular biological techniques

The use of molecular biological techniques for determining the levels and types of different microbial populations in bioreactors has led to the emergence of the microbial community ‘structure-function’ paradigm that is often used in research. Typically, lab- or full-scale systems are monitored for the relevant parameters, and these parameters are related to the changes in microbial populations. Research in activated sludge phenomena, such as filamentous bulking, filamentous foaming, nitrogen removal, and phosphorus removal, are replete with many examples of this ‘structure-function’ paradigm, most commonly those that involve 16S rRNA gene-based analysis of the microbial populations. In many cases, such studies assume a causal microbial population (e.g., a species that causes bulking or foaming), or conclude in identifying a causal population. However, assigning cause to specific organisms and populations is problematic in a complex environment such as wastewater bioreactors. The Koch-Henle postulates, the gold standard in evaluating causation of disease, have limitations when applied to systems with mixed microbial communities with complex interactions, particularly if pure cultures are not available. Molecular techniques that allow specific identification and quantification of organisms have been used by researchers to overcome the limitations of culture-based techniques, and at the same time, raised new questions on the applicability of causation postulates in environmental systems. In this paper, various causation criteria improving on the Koch-Henle postulates are presented. Complicating issues in assigning cause in wastewater bioreactors are identified. Approaches for determining cause-effect relationships are illustrated using 16S rDNA-based investigations of filaments that cause bulking and foaming in activated sludge. The hope is that a causation framework that accounts for the assumptions in molecular studies, as applied to wastewater treatment research, will lead to improved experimental design and analysis of data.     

Systematic study of the effect of operating variables on reactor performance and microbial diversity in laboratory-scale activated sludge reactors

Biological treatment processes are “complex systems” where many different kinds of microbes grow and interact in a dynamic manner. Understanding the relationship between microbial diversity and bioreactor performance could facilitate the optimisation of bioreactor design and enable the solution of bioreactor-related problems. However, systematic studies of the effects of operating variables on microbial diversity and reactor performance are rare. In this study, we determined the effects of different operating conditions and system configurations on the performance of laboratory-scale activated sludge reactors and microbial diversity, based on experiments designed using the factorial design approach. We found that the overall system performance and the diversity of the microbial communities in the reactors were affected by changes in the operating parameters. However, the relationship between diversity and performance was sometimes counterintuitive, as increases in system performance were not always associated with increased community diversity. Reactor configuration and addition of soil had the biggest effects on reactor performance, while the effects of organic loading rates and feed composition were less marked. Of all these parameters, reactor configuration was the only one that had a consistent effect on reactor community diversity.     

The inoculum effect on the ammonia-oxidizing bacterial communities in parallel sequential batch reactors

Three identical sequential batch reactors (SBRs) were each inoculated with sludge from a full-scale wastewater treatment plant (WWTP) treating a waste stream of different origin, i.e. a hospital, a meat processing company, and a municipal WWTP. The SBRs were run in parallel for 84 consecutive days to investigate whether the reactors would become more phylogenetically similar or stay separated concerning their functionality and microbial communities. Overall, the nitrification functionality was high throughout the experiment, and the size and structure of the sludge flocs were very similar. The total bacterial and ammonia-oxidizing bacterial (AOB) communities were analyzed by PCR-DGGE. Cluster analysis demonstrated very distinct bacterial communities in the three SBRs, not showing any trend becoming more similar. The carrying capacity, dynamics and functional organization of the communities were assessed by DGGE analysis and based on these patterns the range-weighted richness, moving window analysis, and constructing Pareto-Lorenz evenness distribution curves were calculated. Between the SBRs, highly comparable internal structure and dynamics of the AOB communities were observed, although they had only one AOB DGGE band in common. These observations indicate that community characteristics such as the extent of biodiversity and dynamics are more important indicators of good microbial functionality than the presence of certain specific species.     

Fine-scale bacterial community dynamics and the taxa-time relationship within a full-scale activated sludge bioreactor

In activated sludge bioreactors, aerobic heterotrophic communities efficiently remove organics, nutrients, toxic substances, and pathogens from wastewater, but the dynamics of these communities are as yet poorly understood. A macroecology metric used to quantify community shifts is the taxa-time relationship, a temporal analog of the species-area curve. To determine whether this metric can be applied to full-scale bioreactors, activated sludge samples were collected weekly over a one-year period at a local municipal wastewater treatment plant. Bacterial community dynamics were evaluated by monitoring 16S rRNA genes using Terminal Restriction Fragment Length Polymorphism (T-RFLP), corroborated by clone libraries. Observed taxa richness increased with time according to a power law model, as predicted by macroecological theory, with a power law exponent of w = 0.209. The results reveal strong long-term temporal dynamics during a period of stable performance (BOD removal and nitrification). Community dynamics followed a gradual succession away from initial conditions rather than periodicity around a mean “equilibrium”, with greater within-month then among-month community similarities. Changes in community structure were significantly associated via multivariate statistical analyses with dissolved oxygen, temperature, influent silver, biomass (MLSS), flow rate, and influent nitrite, cadmium and chromium concentrations. Overall, our results suggest patterns of bacterial community dynamics likely regulated in part by operational parameters and provide evidence that the taxa-time relationship may be a fundamental ecological pattern in macro- and microbial systems.     

Tracing biofouling to the structure of the microbial community and its metabolic products: A study of the three-stage MBR process

The biofouling characteristics of a sequential anoxic/aerobic-membrane bioreactor (A/O MBR) were analyzed during the three-stage process (fast-slow-fast transmembrane pressure (TMP) increasing). The results indicated: during the stage 1 (before day 1), the microbial communities in the activated sludge (AS), cake sludge (CS) and biofilm (BF) were similar to each other, and the adsorption of microbes and the metabolic products was the main factor that led to TMP increase; during the stage 2 (between day 1 and day 7), the cake layer begun to form and the TMP continued to rise gradually at a reduced rate compared to stage 1, at this point a characteristic microbial community colonized the CS with microorganisms such as Saprospiraceae and Comamonadaceae thriving on the membrane surface (BF) probably due to greater nutrient availability, and the predominance of these species in the microbial population led to the accumulation of biofouling metabolic products in the CS, which resulted in membrane fouling and the associated rise in TMP; during the final stage (after day 7), the biofilm had matured, and the activity of anaerobes stimulated cake compaction. The statistical analysis showed a correlation between the TMP changing rate and the carbonhydrates of soluble microbial products (SMPc) content in the CS. When the SMPc concentration rose slowly there was a low level of biofouling. However, when the SMPc accumulating rate was greater, it resulted in the more severe biofouling associated with the TMP jump. Furthermore, the correlation coefficient for the TMP increase and protein concentrations of extracellular polymeric substances (EPSp) in the CS was highly significant. The cluster analysis suggested that the AS microbial community remained stable during the three TMP change stages, while the CS and BF community were changed accompanied with the TMP change. The interaction between the microbial communities and the metabolic products lead to the significant correlation between them. The EPSp in conjunction with the SMPc were the main factors that accelerate the membrane fouling. The rapid rise of SMPc triggered a sudden increase in the TMP, while the accumulation of EPSp caused the sustained rise in TMP.     

Microbial communities in activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor

Microbial communities of activated sludge in an anaerobic/aerobic sequencing batch reactor (SBR) supplied with acetate as sole carbon source were analyzed to identify the microorganisms responsible for enhanced biological phosphorus removal. Various analytical methods were used such as electron microscopy, quinone, slot hybridization, and 16S rRNA gene sequencing analyses. Electron photomicrographs showed that coccus-shaped microorganisms of about 1 microm diameter dominated the microbial communities of the activated sludge in the SBR, which had been operated for more than 18 months. These microorganisms contained polyphosphate granules and glycogen inclusions, which suggests that they are a type of phosphorus-accumulating organism. Quinones, slot hybridization, and 16S rRNA sequencing analyses showed that the members of the Proteobacteria beta subclass were the most abundant species and were affiliated with the Rhodocyclus-like group. Phylogenetic analysis revealed that the two dominating clones of the beta subclass were closely related to the Rhodocyclus-like group. It was concluded that the coccus-shaped organisms related to the Rhodocyclus-like group within the Proteobacteria beta subclass were the most dominant species believed responsible for biological phosphorus removal in SBR operation with acetate.     

Phenotypic fingerprinting of microbial communities in wastewater treatment systems

Community-level characterization of microbial biomass from a municipal activated sludge plant, and two bleached kraft mill effluent (BKME) treatment systems, an oxygenated activated sludge system and an aerated lagoon system, was carried out using the BIOLOG redox-based carbon-substrate utilization assay. Several extraction procedures for separating microbial cells from the dark colloidal samples, including culture enrichment, homogenization, gravel agitation and sonication, were evaluated in order to overcome interference with colour development in the assay. Optimal microbial recovery was achieved by homogenization in the presence of deflocculating agents, sodium pyrophosphate and Tween 80. Principal component analysis was used to differentiate among the microbial communities in the wastewater treatment systems. Biomass obtained from each of the reactors displayed different composite metabolic profiles suggesting a unique indigenous microbial population in each system. Characterization of separate bacterial and protozoal components of one community also showed that each fraction displayed different substrate utilization patterns. Substitution of the already prepared BIOLOG panel substrates with individual chemicals typically associated with BKME allowed for the determination of biodegradation potential in wastewater treatment systems.     

Drivers of microbial community composition in mesophilic and thermophilic temperature-phased anaerobic digestion pre-treatment reactors

Temperature-phased anaerobic digestion (TPAD) is an emerging technology that facilitates improved performance and pathogen destruction in anaerobic sewage sludge digestion by optimising conditions for 1) hydrolytic and acidogenic organisms in a first-stage/pre-treatment reactor and then 2) methogenic populations in a second stage reactor. Pre-treatment reactors are typically operated at 55–65 °C and as such select for thermophilic bacterial communities. However, details of key microbial populations in hydrolytic communities and links to functionality are very limited. In this study, experimental thermophilic pre-treatment (TP) and control mesophilic pre-treatment (MP) reactors were operated as first-stages of TPAD systems treating activated sludge for 340 days. The TP system was operated sequentially at 50, 60 and 65 °C, while the MP rector was held at 35 °C for the entire period. The composition of microbial communities associated with the MP and TP pre-treatment reactors was characterised weekly using terminal-restriction fragment length polymorphism (T-RFLP) supported by clone library sequencing of 16S rRNA gene amplicons. The outcomes of this approach were confirmed using 454 pyrosequencing of gene amplicons and fluorescence in-situ hybridisation (FISH). TP associated bacterial communities were dominated by populations affiliated to the Firmicutes, Thermotogae, Proteobacteria and Chloroflexi. In particular there was a progression from Thermotogae to Lutispora and Coprothermobacter and diversity decreased as temperature and hydrolysis performance increased. While change in the composition of TP associated bacterial communities was attributable to temperature, that of MP associated bacterial communities was related to the composition of the incoming feed. This study determined processes driving the dynamics of key microbial populations that are correlated with an enhanced hydrolytic functionality of the TPAD system.     

Monitoring thiocyanate-degrading microbial community in relation to changes in process performance in mixed culture systems near washout

Changes in microbial community structure, associated with changes in process performance, were investigated with respect to the sludge retention time (SRT) in bioreactors treating thiocyanate. Among the seven reactors operated at 0.8-3.0 d SRTs, respectively, the reactor at 2.0 d SRT displayed the maximal thiocyanate removal rate of 240.2mg/L/d. However, the thiocyanate removal efficiency suddenly decreased from 96.1% to 43.1% when the SRT was reduced from 2.0 to 1.8d, corresponding to a 50.1% drop in the removal rate. Microbial communities in the reactors operated at short SRTs, near washout, were analyzed by denaturing gradient gel electrophoresis (DGGE) based on bacterial 16S rRNA genes. All band sequences recovered were assigned to two phyla, Proteobacteria and Bacteriodetes. A Thiobacillus-like microorganism was commonly detected in all the reactors and is suggested to be the main organism responsible for thiocyanate decomposition. Several DGGE band sequences were closely related to the environmental clones detected in environments rich in sulfur and/or nitrogen compounds. Statistical analysis of the DGGE profiles demonstrated that the structure of thiocyanate-degrading communities, as well as the process performance, changed with change in SRT. The microbial community profiles were not always more closely related to those at similar SRT than those at less similar SRT on the non-metric multidimensional scaling (NMDS) map. This was also supported by clustering analysis. These results were contrary to the general notion that the community structures in continuous systems will be controlled by the washout of microbial populations. Our experimental results suggest that the structure of a microbial thiocyanate-degrading community at a given SRT would not be determined only by the washout effect.     

Flow cytometry for fast microbial community fingerprinting

Characterizing the microbial community of water is important in different domains, ranging from food and beverage production to wastewater treatment. Conventional methods, such as heterotrophic plate count, selective plating and molecular techniques, are time consuming and labor intensive. A flow cytometry based approach was developed for a fast and objective comparison of microbial communities based on the distribution of cellular features from single cells within these communities. The method consists of two main parts, firstly the generation of fingerprint data by flow cytometry and secondly a novel statistical pipeline for the analysis of flow cytometric data. The combined method was shown to be useful for the discrimination and classification of different brands of drinking water. It was also successfully applied to detect changes in microbial community composition of drinking water caused by changing environmental factors. Generally, the method can be used as a fast fingerprinting method of microbial communities in aquatic samples and as a tool to detect shifts within these communities.     

Characterization of microbial trophic structures of two anaerobic bioreactors processing sulfate-rich waste streams

A multi-compartment anaerobic bioreactor, designated the anaerobic migrating blanket reactor (AMBR), did not perform well in terms of chemical oxygen demand (COD) removal after an increase in sulfate load, compared to a conventional upflow anaerobic sludge blanket (UASB) reactor. The trophic structures of the bioreactors were characterized by analyzing the electron flows, formation and consumption of fermentation intermediates and terminal product (methane and hydrogen sulfide) formation. Critical performance parameters were linked to operational perturbations such as increase in sulfate load and changes in flow reversal schemes in the AMBR. Both of these manipulations affected the microbial communities, which were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the bacterial and archaeal domains. The less stable AMBR did not produce granular biomass, and in response to increased sulfate concentrations, experienced a reversal in the distribution of hydrogenotrophic methanogens that correlated with a shift in electron flow from butyrate to propionate. As this shift occurred, bacterial populations such as butyrate-producing clostridia, became predominant, thus leading to reactor imbalance. The stable UASB reactor developed and retained granules and maintained a relatively stable archaeal community. Sulfate perturbation led to the selection of a novel bacterial group (Thermotogaceae), which was most likely well adapted to the increasingly sulfidogenic conditions in the bioreactor.     

腐殖土活性污泥技术的除污效能及除臭效果

阐述了腐殖土活性污泥工艺的基本原理，从微生物菌群结构、除污效能、控制恶臭气体及改善污泥沉降和脱水性能等方面介绍了该工艺的特性，同时说明了该技术的典型工艺流程及设计参数。实践表明，腐殖土活性污泥工艺可强化对氮、磷的去除，使污泥脱水性能大大改善，实现污水处理流程的无臭化。     

TISTD反应器中的菌群结构及生态变化

以两相一体式污泥浓缩消化反应器(Two-phase Integrated Sludge Thickening and Digestion reactor,TISTD)为对象,研究了反应器稳定运行期间菌群结构及反应器负荷改变时其内部生态结构的变化.在以30％的投配率稳定运行时,从产酸相和产甲烷相分别取样、培养和分离出20...     

Microbial community structure and dynamics in a pilot-scale submerged membrane bioreactor aerobically treating domestic wastewater under real operation conditions

A pilot scale submerged ultra-filtration membrane bioreactor (MBR) was used for the aerobic treatment of domestic wastewater over 9 months of year 2006 (28th March to 21st December). The MBR was installed at a municipal wastewater facility (EMASAGRA, Granada, Spain) and was fed with real wastewater. The experimental work was divided in 4 stages run under different sets of operation conditions. Operation parameters (total and volatile suspended solids, dissolved oxygen concentration) and environmental variables (temperature, pH, COD and BOD₅ of influent water) were daily monitored. In all the experiments conducted, the MBR generated an effluent of optimal quality complying with the requirements of the European Law (91/271/CEE 1991). A cultivation-independent approach (polymerase chain reaction-temperature gradient gel electrophoresis, PCR-TGGE) was used to analyze changes in the structure of the bacterial communities in the sludge. Cluster analysis of TGGE profiles demonstrated significant differences in community structure related to variations of the operation parameters and environmental factors. Canonical correspondence analysis (CCA) suggested that temperature, hydraulic retention time and concentration of volatile suspended solids were the factors mostly influencing community structure. 23 prominent TGGE bands were successfully reamplified and sequenced, allowing gaining insight into the identities of predominantly present bacterial populations in the sludge. Retrieved partial 16S-rRNA gene sequences were mostly related to the α-Proteobacteria, β-Proteobacteria and γ-Proteobacteria classes. The community established in the MBR in each of the four stages of operation significantly differed in species composition and the sludge generated displayed dissimilar rates of mineralization, but these differences did not influence the performance of the bioreactor (quality of the permeate). These data indicate that the flexibility of the bacterial community in the sludge and its ability to get adapted to environmental changes play an important role for the stable performance of MBRs.     

Microbial biomass, activity and community composition in constructed wetlands

The aim of the current article is to give an overview about microbial communities and their functioning but also about factors affecting microbial activity in the three most common types (surface flow and two types of sub-surface flow) of constructed wetlands. The paper reviews the community composition and structural diversity of the microbial biomass, analyzing different aspects of microbial activity with respect to wastewater properties, specific wetland type, and environmental parameters. A brief introduction about the application of different novel molecular techniques for the assessment of microbial communities in constructed wetlands is also given. Microbially mediated processes in constructed wetlands are mainly dependent on hydraulic conditions, wastewater properties, including substrate and nutrient quality and availability, filter material or soil type, plants, and different environmental factors. Microbial biomass is within similar ranges in both horizontal and vertical subsurface flow and surface flow constructed wetlands. Stratification of the biomass but also a stratified structural pattern of the bacterial community can be seen in subsurface flow systems. Microbial biomass C/N ratio is higher in horizontal flow systems compared to vertical flow systems, indicating the structural differences in microbial communities between those two constructed wetland types. The total activity of the microbial community is in the same range, but heterotrophic growth is higher in the subsurface (vertical flow) system compared to the surface flow systems. Available species-specific data about microbial communities in different types of wetlands is scarce and therefore it is impossible make any general conclusions about the dynamics of microbial community structure in wetlands, its relationship to removal processes and operational parameters.     

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.     

Characterizing the bacterial communities in retail stores in the United States

The microorganisms present in retail environments have not been studied in detail despite the fact that these environments represent a potentially important location for exposure. In this study, HVAC filter dust samples in 13 US retail stores were collected and analyzed via pyrosequencing to characterize the indoor bacterial communities and to explore potential relationships between these communities and building and environmental parameters. Although retail stores contained a diverse bacterial community of 788 unique genera, over half of the nearly 118K sequences were attributed to the Proteobacteria phylum. Streptophyta, Bacillus, Corynebacterium, Pseudomonas, and Acinetobacter were the most prevalent genera detected. The recovered indoor airborne microbial community was statistically associated with both human oral and skin microbiota, indicating occupants are important contributors, despite a relatively low occupant density per unit volume in retail stores. Bacteria generally associated with outdoor environments were present in the indoor communities with no obvious association with air exchange rate, even when considering relative abundance. No significant association was observed between the indoor bacterial community recovered and store location, store type, or season. However, predictive functional gene profiling showed significant associations between the indoor community and season. The microbiome recovered from multiple samples collected months apart from the same building varied significantly indicating that caution is warranted when trying to characterize the bacterial community with a single sampling event.     

Bacteriophages--potential for application in wastewater treatment processes

Bacteriophages are viruses that infect and lyse bacteria. Interest in the ability of phages to control bacterial populations has extended from medical applications into the fields of agriculture, aquaculture and the food industry. Here, the potential application of phage techniques in wastewater treatment systems to improve effluent and sludge emissions into the environment is discussed. Phage-mediated bacterial mortality has the potential to influence treatment performance by controlling the abundance of key functional groups. Phage treatments have the potential to control environmental wastewater process problems such as: foaming in activated sludge plants; sludge dewaterability and digestibility; pathogenic bacteria; and to reduce competition between nuisance bacteria and functionally important microbial populations. Successful application of phage therapy to wastewater treatment does though require a fuller understanding of wastewater microbial community dynamics and interactions. Strategies to counter host specificity and host cell resistance must also be developed, as should safety considerations regarding pathogen emergence through transduction.     

Sludge population optimisation: a new dimension for the control of biological wastewater treatment systems

The activated sludge comprises a complex microbiological community. The structure (what types of microorganisms are present) and function (what can the organisms do and at what rates) of this community are determined by external physico-chemical features and by the influent to the sewage treatment plant. The external features we can manipulate but rarely the influent. Conventional control and operational strategies optimise activated sludge processes more as a chemical system than as a biological one. While optimising the process in a short time period, these strategies may deteriorate the long-term performance of the process due to their potentially adverse impact on the microbial properties. Through briefly reviewing the evidence available in the literature that plant design and operation affect both the structure and function of the microbial community in activated sludge, we propose to add sludge population optimisation as a new dimension to the control of biological wastewater treatment systems. We stress that optimising the microbial community structure and property should be an explicit aim for the design and operation of a treatment plant. The major limitations to sludge population optimisation revolve around inadequate microbiological data, specifically community structure, function and kinetic data. However, molecular microbiological methods that strive to provide that data are being developed rapidly. The combination of these methods with the conventional approaches for kinetic study is briefly discussed. The most pressing research questions pertaining to sludge population optimisation are outlined.