The Choice of PCR Primers Has Great Impact on Assessments of Bacterial Community Diversity and Dynamics in a Wastewater Treatment Plant

Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample

Diversity and dynamics of Archaea in an activated sludge wastewater treatment plant

BACKGROUND: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. RESULTS: The Archaea community was dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. CONCLUSIONS: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge

Hydrodynamic modelling of the microbial water quality in a drinking water source as input for risk reduction management

To mitigate the faecal contamination of drinking water sources and, consequently, to prevent waterborne disease outbreaks, an estimation of the contribution from different sources to the total faecal contamination at the raw water intake of a drinking water treatment plant is needed. The aim of this article was to estimate how much different sources contributed to the faecal contamination at the water intake in a drinking water source, Lake Rådasjön in Sweden. For this purpose, the fate and transport of faecal indicator Escherichia coli within Lake Rådasjön were simulated by a three-dimensional hydrodynamic model. The calibrated hydrodynamic model described the measured data on vertical temperature distribution in the lake well (the Pearson correlation coefficient was 0.99). The data on the E. coli load from the identified contamination sources were gathered and the fate and transport of E. coli released from these sources within the lake were simulated using the developed hydrodynamic model, taking the decay of the E. coli into account. The obtained modelling results were compared to the observed E. coli concentrations at the water intake. The results illustrated that the sources that contributed the most to the faecal contamination at the water intake in Lake Rådasjön were the discharges from the on-site sewers and the main inflow to the lake – the river Mölndalsån. Based on the modelling results recommendations for water producers were formulated. The study demonstrated that this modelling approach is a useful tool for estimating the contribution from different sources to the faecal contamination at the water intake of a drinking water treatment plant and provided decision-support information for the reduction of risks posed to the drinking water source

The IncP-1 plasmid backbone adapts to different host bacterial species and evolves through homologous recombination

Plasmids are important members of the bacterial mobile gene pool, and are among the most important contributors to horizontal gene transfer between bacteria. They typically harbour a wide spectrum of host beneficial traits, such as antibiotic resistance, inserted into their backbones. Although these inserted elements have drawn considerable interest, evolutionary information about the plasmid backbones, which encode plasmid related traits, is sparse. Here we analyse 25 complete backbone genomes from the broad-host-range IncP-1 plasmid family. Phylogenetic analysis reveals seven clades, in which two plasmids that we isolated from a marine biofilm represent a novel clade. We also found that homologous recombination is a prominent feature of the plasmid backbone evolution. Analysis of genomic signatures indicates that the plasmids have adapted to different host bacterial species. Globally circulating IncP-1 plasmids hence contain mosaic structures of segments derived from several parental plasmids that have evolved in, and adapted to, different, phylogenetically very distant host bacterial species

Resistance of aerobic granular sludge microbiomes to periodic loss of biomass

Granular sludge is a biofilm process used for wastewater treatment which is currently being implemented worldwide. It is important to understand how disturbances affect the microbial community and performance of reactors. Here, two acetate-fed replicate reactors were inoculated with acclimatized sludge and the reactor performance, and the granular sludge microbial community succession were studied for 149 days. During this time, the microbial community was challenged by periodically removing half of the reactor biomass, subsequently increasing the food-to-microorganism (F/M) ratio. Diversity analysis together with null models show that overall, the microbial communities were resistant to the disturbances, observing some minor effects on polyphosphate-accumulating and denitrifying microbial communities and their associated reactor functions. Community turnover was driven by drift and random granule loss, and stochasticity was the governing ecological process for community assembly. These results evidence the aerobic granular sludge process as a robust system for wastewater treatment

Effects of storage on mixed-culture biological electrodes

Storage methods are important to preserve the viability and biochemical characteristics of microbial cultures between experiments or during periods when bioreactors are inactive. Most of the research on storage has focused on isolates; however, there is an increasing interest in methods for mixed cultures, which are of relevance in environmental biotechnology. The purpose of this study was to investigate the effect of different storage methods on electrochemically active enrichment cultures. Acetate-oxidizing bioanodes generating a current density of about 5 A m−2 were enriched in a microbial electrolysis cell. The effect of five weeks of storage was evaluated using electrochemical techniques and microbial community analysis. Storage by refrigeration resulted in quicker re-activation than freezing in 10% glycerol, while the bioelectrochemical activity was entirely lost after storage using dehydration. The results showed that the bioelectrochemical activity of bioanodes stored at low temperature could be retained. However, during the re-activation period the bioanodes only recovered 75% of the current density generated before storage and the bacterial communities were different in composition and more diverse after storage than before

Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms

The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors designed for nitrogen removal in wastewater treatment, where suppression of nitrite oxidizers like\ua0Nitrospira\ua0is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence of aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms, treating water from a high-rate activated sludge process, were constantly or intermittently exposed to anaerobic sidestream wastewater, which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among\ua0Nitrospira\ua0in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within\ua0Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing\ua0Nitrosomonas\ua0populations. The different exposure regimens were associated with metagenomic assembled genomes of\ua0Nitrosomonas,\ua0Nitrospira, and\ua0Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment

Hill-based dissimilarity indices and null models for analysis of microbial community assembly

BackgroundHigh-throughput amplicon sequencing of marker genes, such as the 16S rRNA gene in Bacteria and Archaea, provides a wealth of information about the composition of microbial communities. To quantify differences between samples and draw conclusions about factors affecting community assembly, dissimilarity indices are typically used. However, results are subject to several biases, and data interpretation can be challenging. The Jaccard and Bray-Curtis indices, which are often used to quantify taxonomic dissimilarity, are not necessarily the most logical choices. Instead, we argue that Hill-based indices, which make it possible to systematically investigate the impact of relative abundance on dissimilarity, should be used for robust analysis of data. In combination with a null model, mechanisms of microbial community assembly can be analyzed. Here, we also introduce a new software, qdiv, which enables rapid calculations of Hill-based dissimilarity indices in combination with null models.ResultsUsing amplicon sequencing data from two experimental systems, aerobic granular sludge (AGS) reactors and microbial fuel cells (MFC), we show that the choice of dissimilarity index can have considerable impact on results and conclusions. High dissimilarity between replicates because of random sampling effects make incidence-based indices less suited for identifying differences between groups of samples. Determining a consensus table based on count tables generated with different bioinformatic pipelines reduced the number of low-abundant, potentially spurious amplicon sequence variants (ASVs) in the data sets, which led to lower dissimilarity between replicates. Analysis with a combination of Hill-based indices and a null model allowed us to show that different ecological mechanisms acted on different fractions of the microbial communities in the experimental systems.ConclusionsHill-based indices provide a rational framework for analysis of dissimilarity between microbial community samples. In combination with a null model, the effects of deterministic and stochastic community assembly factors on taxa of different relative abundances can be systematically investigated. Calculations of Hill-based dissimilarity indices in combination with a null model can be done in qdiv, which is freely available as a Python package (https://github.com/omvatten/qdiv). In qdiv, a consensus table can also be determined from several count tables generated with different bioinformatic pipelines

Three-dimensional stratification of bacterial biofilm populations in a moving bed biofilm reactor for nitritation-anammox.

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers

The mechanisms of granulation of activated sludge in wastewater treatment, its optimization, and impact on effluent quality

Granular activated sludge has gained increasing interest due to its potential in treating wastewater in a compact and efficient way. It is well-established that activated sludge can form granules under certain environmental conditions such as batch-wise operation with feast-famine feeding, high hydrodynamic shear forces, and short settling time which select for dense microbial aggregates. Aerobic granules with stable structure and functionality have been obtained with a range of different wastewaters seeded with different sources of sludge at different operational conditions, but the microbial communities developed differed substantially. In spite of this, granule instability occurs. In this review, the available literature on the mechanisms involved in granulation and how it affects the effluent quality is assessed with special attention given to the microbial interactions involved. To be able to optimize the process further, more knowledge is needed regarding the influence of microbial communities and their metabolism on granule stability and functionality. Studies performed at conditions similar to full-scale such as fluctuation in organic loading rate, hydrodynamic conditions, temperature, incoming particles, and feed water microorganisms need further investigations