Frank Persson

Structure and composition of biofilm communities in a moving bed biofilm reactor for nitritation-anammox at low temperatures.

It is a challenge to apply anaerobic ammonium oxidation (anammox) for nitrogen removal from wastewater at low temperatures. Maintenance of anammox- and aerobic ammonia oxidizing bacteria (AOB) and suppression of nitrite oxidizing bacteria (NOB) are key issues. In this work, a nitritation-anammox moving bed biofilm pilot reactor was operated at 19-10°C for 300 d. Nitrogen removal was decreasing, but stable, at 19-13°C. At 10°C removal became unstable. Quantitative PCR, fluorescence in situ hybridization and gene sequencing showed that no major microbial community changes were observed with decreased temperature. Anammox bacteria dominated the biofilm (0.9-1.2 × 10(14) 16S rRNA copies m(-2)). Most anammox bacteria were similar to Brocadia sp. 40, but another smaller Brocadia population was present near the biofilm-water interface, where also the AOB community (Nitrosomonas) was concentrated in thin layers (1.8-5.3 × 10(12) amoA copies m(-2)). NOB (Nitrobacter, Nitrospira) were always present at low concentrations (<1.3 × 10(11) 16S rRNA copies m(-2)).

The red alga Bonnemaisonia asparagoides regulates epiphytic bacterial abundance and community composition by chemical defence

Ecological research on algal-derived metabolites with antimicrobial activity has recently received increased attention and is no longer only aimed at identifying novel natural compounds with potential use in applied perspectives. Despite this progress, few studies have so far demonstrated ecologically relevant antimicrobial roles of algal metabolites, and even fewer have utilized molecular tools to investigate the effects of these metabolites on the natural community composition of bacteria. In this study, we investigated whether the red alga Bonnemaisonia asparagoides is chemically defended against bacterial colonization of its surface by extracting surface-associated secondary metabolites and testing their antibacterial effects. Furthermore, we compared the associated bacterial abundance and community composition between B. asparagoides and two coexisting macroalgae. Surface extracts tested at natural concentrations had broad-spectrum effects on the growth of ecologically relevant bacteria, and consistent with this antibacterial activity, natural populations of B. asparagoides had significantly lower densities of epibacteria compared with the coexisting algae. Terminal restriction fragment length polymorphism analysis further showed that B. asparagoides harboured surface-associated bacteria with a community composition that was significantly different from those on coexisting macroalgae. Altogether, these findings demonstrate that B. asparagoides produces surface-bound antibacterial compounds with a significant impact on the abundance and composition of the associated bacterial community.

Distribution and activity of ammonia oxidizing bacteria in a large full-scale trickling filter

The biofilm in a full-scale nitrifying trickling filter (NTF) treating municipal wastewater has been investigated with microbiological methods using fluorescence in situ hybridization (FISH) with 16S rRNA oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM) and mathematical modeling using a dynamic multi-species biofilm reactor model. Ammonia oxidizing bacteria (AOB) were found to belong to the genus Nitrosomonas at different depths in the NTF at every sampling occasion, corresponding to different long-term operational conditions for the NTF. Both the measurements and the corresponding simulated predictions showed the same general trend of a decrease with filter depth of the amount of biofilm, the proportion of AOB to all bacteria and the total amount of AOB. The latter decreased by several times from top to bottom of the NTF. Measurements and simulations of potential ammonium oxidizing activity in the biofilm also showed a decreasing activity with depth in the NTF, which generally was operating at close to complete nitrification. However, no difference was observed when the activity was normalized to the amount of biofilm, despite decreasing proportions of AOB to all bacteria with depth in the NTF. This could be explained by diffusion limitations in the biofilm from the upper parts of the NTF according to the biofilm reactor model. The relatively good agreement between the simulations and the measurements shows that the kind of biofilm reactor model used can qualitatively describe an averaged behavior and averaged composition of the biofilm in the reactor.

Removal of Geosmin and MIB by Biofiltration - an Investigation Discriminating Between Adsorption and Biodegradation

Geosmin and 2-methylisoborneol (MIB) are two substances causing earthy/musty odours that are difficult to remove by conventional chemical drinking water treatment. In this study removal of geosmin and MIB by biofiltration of untreated surface water was investigated using granular activated carbon (GAC) and crushed expanded clay (EC) as filter media. Biofiltration through both GAC and EC removed geosmin and MIB present at low (20 ng l−1) concentrations by at least 97 % at an empty bed contact time of 30 minutes and a temperature of 15°C. At lower temperature (6-12°C) and simultaneously lower biomass concentrations, removal efficiency was similar in the GAC but considerably lower in the EC biofilter, pointing to a second mechanism different from biodegradation. Consequently, microbial activity was suppressed with azide to enable discrimination between biodegradation and adsorption. During azide dosage, the GAC biofilters still removed geosmin and MIB nearly unaffectedly. In the EC biofilter, however, removal of both odorants ceased completely. Methylene blue adsorption confirmed that the GAC, even after almost four years of operation receiving surface water, had capacity to remove geosmin and MIB by adsorption. Since odour episodes commonly occur during the warm season when microbiological activity is high, EC constitutes a viable option as carrier medium for direct biological filtration of surface water. The additional GAC adsorption capacity however adds robustness to the removal process.

Performance of direct biofiltration of surface water for reduction of biodegradable organic matter and biofilm formation potential.

Direct biofiltration of surface water may be considered as pre-treatment for membrane filtration, in order to reduce fouling. The biofiltration process was investigated with regard to biodegradable organic matter and biofilm formation, covering the annual variations under moderately cold climate conditions. Granular activated carbon (GAC) and two types of crushed expanded clay (EC) were compared as filter media. To assess the biological properties of the biofilters, viable biomass and respiratory activity was examined. Biofiltration removed assimilable organic carbon (AOC) and biodegradable dissolved organic carbon (BDOC) by about 30% and also reduced the bacterial concentration in the water phase. Also, biofilm formation in the treated water was reduced by 80-90% during summer and winter conditions. The reductions in the investigated parameters were similar in the biofilters with GAC and EC. Likewise, a similar amount and development of biomass was found in the GAC and EC biofilters of comparable grain size with a pronounced stratification from top to bottom of the filter bed. The specific respiratory activity of the biofilter biomass was dependent on raw water temperature. Even though slight correlations between BDOC removal and temperature or respiratory activity were observed, AOC and BDOC removals were mainly dependent on the feed water concentrations of these compounds. The results indicate that direct biofiltration of surface water, by reducing AOC, BDOC and biofilm formation in the water, may be an advantageous pre-treatment for membrane filtration processes.

New Methods for Analysis of Spatial Distribution and Coaggregation of Microbial Populations in Complex Biofilms

ABSTRACT In biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.

Ecological role of a seaweed secondary metabolite for a colonizing bacterial community.

Bacteria associated with seaweeds can both harm and benefit their hosts. Many seaweed species are known to produce compounds that inhibit growth of bacterial isolates, but the ecological role of seaweed metabolites for the associated bacterial community structure is not well understood. In this study the response of a colonizing bacterial community to the secondary metabolite (1,1,3,3-tetrabromo-2-heptanone) from the red alga Bonnemaisonia hamifera was investigated by using field panels coated with the metabolite at a range of concentrations covering those measured at the algal surface. The seaweed metabolite has previously been shown to have antibacterial effects. The metabolite significantly affected the natural fouling community by (i) altering the composition, (ii) altering the diversity by increasing the evenness and (iii) decreasing the density, as measured by terminal restriction fragment length polymorphism in conjunction with clone libraries of the 16S rRNA genes and by bacterial enumeration. No single major bacterial taxon (phylum, class) was particularly affected by the metabolite. Instead changes in community composition were observed at a more detailed phylogenetic level. This indicates a broad specificity of the seaweed metabolite against bacterial colonization, which is supported by the observation that the bacterial density was significantly affected at a lower concentration (0.02 μg cm−2) than the composition (1–2.5 μg cm−2) and the evenness (5 μg cm−2) of the bacterial communities. Altogether, the results emphasize the role of secondary metabolites for control of the density and structure of seaweed-associated bacterial communities.

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.

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.