Biofilm colonization and succession in a full-scale partial nitritation-anammox moving bed biofilm reactor.

BACKGROUND: Partial nitritation-anammox (PNA) is a biological nitrogen removal process commonly used in wastewater treatment plants for the treatment of warm and nitrogen-rich sludge liquor from anaerobic digestion, often referred to as sidestream wastewater. In these systems, biofilms are frequently used to retain biomass with aerobic ammonia-oxidizing bacteria (AOB) and anammox bacteria, which together convert ammonium to nitrogen gas. Little is known about how these biofilm communities develop, and whether knowledge about the assembly of biofilms in natural communities can be applied to PNA biofilms. RESULTS: We followed the start-up of a full-scale PNA moving bed biofilm reactor for 175 days using shotgun metagenomics. Environmental filtering likely restricted initial biofilm colonization, resulting in low phylogenetic diversity, with the initial microbial community comprised mainly of Proteobacteria. Facilitative priority effects allowed further biofilm colonization, with the growth of initial aerobic colonizers promoting the arrival and growth of anaerobic taxa like methanogens and anammox bacteria. Among the early colonizers were known 'oligotrophic' ammonia oxidizers including comammox Nitrospira and Nitrosomonas cluster 6a AOB. Increasing the nitrogen load in the bioreactor allowed colonization by 'copiotrophic' Nitrosomonas cluster 7 AOB and resulted in the exclusion of the initial ammonia- and nitrite oxidizers. CONCLUSIONS: We show that complex dynamic processes occur in PNA microbial communities before a stable bioreactor process is achieved. The results of this study not only contribute to our knowledge about biofilm assembly and PNA bioreactor start-up but could also help guide strategies for the successful implementation of PNA bioreactors. Video Abstract.

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.

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 Nitrospira is 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 Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, 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.

Metagenomic evidence of a novel family of anammox bacteria in a subsea environment.

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.

Correction to: Hill-based dissimilarity indices and null models for analysis of microbial community assembly.

An amendment to this paper has been published and can be accessed via the original article.

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

BACKGROUND: High-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. RESULTS: Using 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. CONCLUSIONS: Hill-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. Video Abstract.

Long-term stability of partial nitritation-anammox for treatment of municipal wastewater in a moving bed biofilm reactor pilot system.

Nitrogen removal from the mainstream of municipal wastewater with partial nitritation-anammox (PNA) would be highly beneficial with regard to the uses of energy and organic carbon. However, the challenges of process instability, low nitrogen removal rates (NRR) and unwanted aerobic nitrite oxidation need to be solved to reach large-scale implementation. Here, we have operated pilot-scale moving bed biofilm reactors (MBBRs) for mainstream treatment, together with sidestream treatment of sludge liquor from anaerobic digestors, for over 900 days to investigate process stability, reactor performance and microbial community structure at realistic conditions. The MBBR biofilm contained stable and high relative abundances of anammox bacteria (10-32%) consisting of two major Brocadia sp. populations, and several populations of aerobic ammonia-oxidising bacteria (AOB) within Nitrosomonas sp. (0.2-3.1%), as assessed by 16S rDNA amplicon sequencing. In addition, nitrite-oxidising bacteria (NOB) consisting of Nitrospira sp. (0.4-0.8%) and Nitrotoga sp. (up to 0.4%) were present. Nitrogen was removed at a peak rate of 0.66 g N m-2 d-1 (0.13 kg N m-3 d-1) with a nitrate production over ammonium consumption of 15% by the NOB, at operation with continuous aeration at 15 °C. However, during most periods with continuous aeration, the NRR was lower (≈ 0.45 g N m-2 d-1), with larger relative nitrate production (≈40%), presumably due to problems to maintain stable residual ammonium concentrations during wet-weather mainstream flows. Changing reactor operation to intermittent aeration decreased the NRR but did not help in suppressing the NOB. The study shows that with MBBRs, stable mainstream PNA can be attained at realistic NRR, but with need for post-treatment of nitrate, since effective NOB suppression was hard to achieve.

Response to starvation and microbial community composition in microbial fuel cells enriched on different electron donors.

In microbial fuel cells (MFCs), microorganisms generate electrical current by oxidizing organic compounds. MFCs operated with different electron donors harbour different microbial communities, and it is unknown how that affects their response to starvation. We analysed the microbial communities in acetate- and glucose-fed MFCs and compared their responses to 10 days starvation periods. Each starvation period resulted in a 4.2 ± 1.4% reduction in electrical current in the acetate-fed MFCs and a 10.8 ± 3.9% reduction in the glucose-fed MFCs. When feed was resumed, the acetate-fed MFCs recovered immediately, whereas the glucose-fed MFCs required 1 day to recover. The acetate-fed bioanodes were dominated by Desulfuromonas spp. converting acetate into electrical current. The glucose-fed bioanodes were dominated by Trichococcus sp., functioning as a fermenter, and a member of Desulfuromonadales, using the fermentation products to generate electrical current. Suspended biomass and biofilm growing on non-conductive regions within the MFCs had different community composition than the bioanodes. However, null models showed that homogenizing dispersal of microorganisms within the MFCs affected the community composition, and in the glucose-fed MFCs, the Trichococcus sp. was abundant in all locations. The different responses to starvation can be explained by the more complex pathway requiring microbial interactions to convert glucose into electrical current.

Combined Deterministic and Stochastic Processes Control Microbial Succession in Replicate Granular Biofilm Reactors.

Granular sludge is an efficient and compact biofilm process for wastewater treatment. However, the ecological factors involved in microbial community assembly during the granular biofilm formation are poorly understood, and little is known about the reproducibility of the process. Here, three replicate bioreactors were used to investigate microbial succession during the formation of granular biofilms. We identified three successional phases. During the initial phase, the successional turnover was high and α-diversity decreased as a result of the selection of taxa adapted to grow on acetate and form aggregates. Despite these dynamic changes, the microbial communities in the replicate reactors were similar. The second successional phase occurred when the settling time was rapidly decreased to selectively retain granules in the reactors. The influence of stochasticity on succession increased and new niches were created as granules emerged, resulting in temporarily increased α-diversity. The third successional phase occurred when the settling time was kept stable and granules dominated the biomass. Turnover was low, and selection resulted in the same abundant taxa in the reactors, but drift, which mostly affected low-abundant community members, caused the community in one reactor to diverge from the other two. Even so, performance was stable and similar between reactors.

Thickness determines microbial community structure and function in nitrifying biofilms via deterministic assembly.

Microbial biofilms are ubiquitous in aquatic environments where they provide important ecosystem functions. A key property believed to influence the community structure and function of biofilms is thickness. However, since biofilm thickness is inextricably linked to external factors such as water flow, temperature, development age and nutrient conditions, its importance is difficult to quantify. Here, we designed an experimental system in a wastewater treatment plant whereby nitrifying biofilms with different thicknesses (50 or 400 µm) were grown in a single reactor, and thus subjected to identical external conditions. The 50 and 400 µm biofilm communities were significantly different. This beta-diversity between biofilms of different thickness was primarily caused by deterministic factors. Turnover (species replacement) contributed more than nestedness (species loss) to the beta-diversity, i.e. the 50 µm communities were not simply a subset of the 400 µm communities. Moreover, the two communities differed in the composition of nitrogen-transforming bacteria and in nitrogen transformation rates. The study illustrates that biofilm thickness alone is a key driver for community composition and ecosystem function, which has implications for biotechnological applications and for our general understanding of biofilm ecology.

A variety of hydrogenotrophic enrichment cultures catalyse cathodic reactions.

Biocathodes where living microorganisms catalyse reduction of CO2 can potentially be used to produce valuable chemicals. Microorganisms harbouring hydrogenases may play a key role for biocathode performance since H2 generated on the electrode surface can act as an electron donor for CO2 reduction. In this study, the possibility of catalysing cathodic reactions by hydrogenotrophic methanogens, acetogens, sulfate-reducers, denitrifiers, and acetotrophic methanogens was investigated. The cultures were enriched from an activated sludge inoculum and performed the expected metabolic functions. All enrichments formed distinct microbial communities depending on their electron donor and electron acceptor. When the cultures were added to an electrochemical cell, linear sweep voltammograms showed a shift in current generation close to the hydrogen evolution potential (-1 V versus SHE) with higher cathodic current produced at a more positive potential. All enrichment cultures except the denitrifiers were also used to inoculate biocathodes of microbial electrolysis cells operated with H+ and bicarbonate as electron acceptors and this resulted in current densities between 0.1-1 A/m2. The microbial community composition of biocathodes inoculated with different enrichment cultures were as different from each other as they were different from their suspended culture inoculum. It was noteworthy that Methanobacterium sp. appeared on all the biocathodes suggesting that it is a key microorganism catalysing biocathode reactions.

Long-term dynamics of the bacterial community in a Swedish full-scale wastewater treatment plant.

The operational efficiency of activated sludge wastewater treatment plants depends to a large extent on the microbial community structure of the activated sludge. The aims of this paper are to describe the composition of the bacterial community in a Swedish full-scale activated sludge wastewater treatment plant, to describe the dynamics of the community and to elucidate possible causes for bacterial community composition changes. The bacterial community composition in the activated sludge was described using 16S rRNA gene libraries and monitored for 15 months by a terminal restriction fragment (T-RF) length polymorphism (T-RFLP) analysis of the 16S rRNA gene. Despite variable environmental conditions, a large fraction of the observed T-RFs were present at all times, making up at least 50% in all samples, possibly representing a relatively stable core fraction of the bacterial community. However, the proportions of the different T-RFs in this fraction as well as the T-RFs in the more variable fraction showed a significant variation over time and temperature. The difference in community composition between summer and winter coincided with observed differences in floc structure. These observations suggest a relationship between floc properties and bacterial community composition, although additional experiments are required to determine causality.

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.

Effect of Start-Up Strategies and Electrode Materials on Carbon Dioxide Reduction on Biocathodes.

The enrichment of CO2-reducing microbial biocathodes is challenging. Previous research has shown that a promising approach could be to first enrich bioanodes and then lower the potential so the electrodes are converted into biocathodes. However, the effect of such a transition on the microbial community on the electrode has not been studied. The goal of this study was thus to compare the start-up of biocathodes from preenriched anodes with direct start-up from bare electrodes and to investigate changes in microbial community composition. The effect of three electrode materials on the long-term performance of the biocathodes was also investigated. In this study, preenrichment of acetate-oxidizing bioanodes did not facilitate the start-up of biocathodes. It took about 170 days for the preenriched electrodes to generate substantial cathodic current, compared to 83 days for the bare electrodes. Graphite foil and carbon felt cathodes produced higher current at the beginning of the experiment than did graphite rods. However, all electrodes produced similar current densities at the end of the over 1-year-long study (2.5 A/m2). Methane was the only product detected during operation of the biocathodes. Acetate was the only product detected after inhibition of the methanogens. Microbial community analysis showed that Geobacter sp. dominated the bioanodes. On the biocathodes, the Geobacter sp. was succeeded by Methanobacterium spp., which made up more than 80% of the population. After inhibition of the methanogens, Acetobacterium sp. became dominant on the electrodes (40% relative abundance). The results suggested that bioelectrochemically generated H2 acted as an electron donor for CO2 reduction.IMPORTANCE In microbial electrochemical systems, living microorganisms function as catalysts for reactions on the anode and/or the cathode. There is a variety of potential applications, ranging from wastewater treatment and biogas generation to production of chemicals. Systems with biocathodes could be used to reduce CO2 to methane, acetate, or other high-value chemicals. The technique can be used to convert solar energy to chemicals. However, enriching biocathodes that are capable of CO2 reduction is more difficult and less studied than enriching bioanodes. The effect of different start-up strategies and electrode materials on the microbial communities that are enriched on biocathodes has not been studied. The purpose of this study was to investigate two different start-up strategies and three different electrode materials for start-up and long-term operation of biocathodes capable of reducing CO2 to valuable biochemicals.

Comparison of the bacterial community composition in the granular and the suspended phase of sequencing batch reactors.

Granulation of activated sludge is an increasingly important area within the field of wastewater treatment. Granulation is usually achieved by high hydraulic selection pressure, which results in the wash-out of slow settling particles. The effect of the harsh wash-out conditions on the granular sludge ecosystem is not yet fully understood, but different bacterial groups may be affected to varying degrees. In this study, we used high-throughput amplicon sequencing to follow the community composition in granular sludge reactors for 12 weeks, both in the granular phase and the suspended phase (effluent). The microbiome of the washed out biomass was similar but not identical to the microbiome of the granular biomass. Certain taxa (e.g. Flavobacterium spp. and Bdellovibrio spp.) had significantly (p < 0.05) higher relative abundance in the granules compared to the effluent. Fluorescence in situ hybridization images indicated that these taxa were mainly located in the interior of granules and therefore protected from erosion. Other taxa (e.g. Meganema sp. and Zooglea sp.) had significantly lower relative abundance in the granules compared to the effluent, and appeared to be mainly located on the surface of granules and therefore subject to erosion. Despite being washed out, these taxa were among the most abundant members of the granular sludge communities and were likely growing fast in the reactors. The ratio between relative abundance in the granular biomass and in the effluent did not predict temporal variation of the taxa in the reactors, but it did appear to predict the spatial location of the taxa in the granules.

Microbial Population Dynamics and Ecosystem Functions of Anoxic/Aerobic Granular Sludge in Sequencing Batch Reactors Operated at Different Organic Loading Rates.

The granular sludge process is an effective, low-footprint alternative to conventional activated sludge wastewater treatment. The architecture of the microbial granules allows the co-existence of different functional groups, e.g., nitrifying and denitrifying communities, which permits compact reactor design. However, little is known about the factors influencing community assembly in granular sludge, such as the effects of reactor operation strategies and influent wastewater composition. Here, we analyze the development of the microbiomes in parallel laboratory-scale anoxic/aerobic granular sludge reactors operated at low (0.9 kg m-3d-1), moderate (1.9 kg m-3d-1) and high (3.7 kg m-3d-1) organic loading rates (OLRs) and the same ammonium loading rate (0.2 kg NH4-N m-3d-1) for 84 days. Complete removal of organic carbon and ammonium was achieved in all three reactors after start-up, while the nitrogen removal (denitrification) efficiency increased with the OLR: 0% at low, 38% at moderate, and 66% at high loading rate. The bacterial communities at different loading rates diverged rapidly after start-up and showed less than 50% similarity after 6 days, and below 40% similarity after 84 days. The three reactor microbiomes were dominated by different genera (mainly Meganema, Thauera, Paracoccus, and Zoogloea), but these genera have similar ecosystem functions of EPS production, denitrification and polyhydroxyalkanoate (PHA) storage. Many less abundant but persistent taxa were also detected within these functional groups. The bacterial communities were functionally redundant irrespective of the loading rate applied. At steady-state reactor operation, the identity of the core community members was rather stable, but their relative abundances changed considerably over time. Furthermore, nitrifying bacteria were low in relative abundance and diversity in all reactors, despite their large contribution to nitrogen turnover. The results suggest that the OLR has considerable impact on the composition of the granular sludge communities, but also that the granule communities can be dynamic even at steady-state reactor operation due to high functional redundancy of several key guilds. Knowledge about microbial diversity with specific functional guilds under different operating conditions can be important for engineers to predict the stability of reactor functions during the start-up and continued reactor operation.

Community structure of partial nitritation-anammox biofilms at decreasing substrate concentrations and low temperature.

Partial nitritation-anammox (PNA) permits energy effective nitrogen removal. Today PNA is used for treatment of concentrated and warm side streams at wastewater treatment plants, but not the more diluted and colder main stream. To implement PNA in the main stream, better knowledge about microbial communities at the typical environmental conditions is necessary. In order to investigate the response of PNA microbial communities to decreasing substrate availability, we have operated a moving bed biofilm reactor (MBBR) at decreasing reactor concentrations (311-27 mg-N l-1 of ammonium) and low temperature (13°C) for 302 days and investigated the biofilm community using high throughput amplicon sequencing; quantitative PCR; and fluorescence in situ hybridization. The anammox bacteria (Ca. Brocadia) constituted a large fraction of the biomass with fewer aerobic ammonia oxidizing bacteria (AOB) and even less nitrite oxidizing bacteria (NOB; Nitrotoga, Nitrospira and Nitrobacter). Still, NOB had considerable impact on the process performance. The anammox bacteria, AOB and NOB all harboured more than one population, indicating some diversity, and the heterotrophic bacterial community was diverse (seven phyla). Despite the downshifts in substrate availability, changes in the relative abundance and composition of anammox bacteria, AOB and NOB were small and also the heterotrophic community showed little changes in composition. This indicates stability of PNA MBBR communities towards decreasing substrate availability and suggests that even heterotrophic bacteria are integral components of these communities.

The inhibitory effects of reject water on nitrifying populations grown at different biofilm thickness.

Suppression of nitrite oxidizing bacteria (NOB) is of vital importance to achieve successful, energy efficient, mainstream anammox processes for wastewater treatment. In this study, biofilm carriers from a fully nitrifying MBBR system, fed with mainstream wastewater, were temporarily exposed to reject water from sludge dewatering, to evaluate this as a possible strategy to inhibit NOB and achieve nitrite production under realistic conditions. Two different carrier types were compared, in which biofilm thickness was maintained at approximately 400 and 50 µm, respectively, and reject treatment was tested at different exposure time and loading rates. Reject exposure almost always resulted in an increased nitrite production in the thinner biofilm, and overall, nitrifiers growing in the thin biofilm were more sensitive than those grown in the thicker biofilm. The effect from reject exposure remained in the systems for four days after returning to mainstream operation, with nitrite production gradually increasing for three days. Increased concentrations of free ammonia correlated with reject exposure and may be the cause of inhibition, although other factors cannot be excluded.

Nonoxidative removal of organics in the activated sludge process.

The activated sludge process is commonly used to treat wastewater by aerobic oxidation of organic pollutants into carbon dioxide and water. However, several nonoxidative mechanisms can also contribute to removal of organics. Sorption onto activated sludge can remove a large fraction of the colloidal and particulate wastewater organics. Intracellular storage of, e.g., polyhydroxyalkanoates (PHA), triacylglycerides (TAG), or wax esters can convert wastewater organics into precursors for high-value products. Recently, several environmental, economic, and technological drivers have stimulated research on nonoxidative removal of organics for wastewater treatment. In this paper, we review these nonoxidative removal mechanisms as well as the existing and emerging process configurations that make use of them for wastewater treatment. Better utilization of nonoxidative processes in activated sludge could reduce the wasteful aerobic oxidation of organic compounds and lead to more resource-efficient wastewater treatment plants.

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.