Substrate type determines microbial activity and community composition in bioreactors for nitrate removal by denitrification at low temperature.

High levels of nitrogen originating from blasting operations, for example at mining sites or quarries, risk contaminating water bodies through leaching from waste rock dumps. Woodchip bioreactors can be a simple and cost-effective way of reducing nitrate concentrations in the leachate. In this study we investigated how bottle sedge, barley straw, and pine woodchips used as electron donors for denitrification influenced microbial community composition and nitrate removal in lab-scale bioreactors during 270 days. The reactors were operated to ensure that nitrate was never limiting and to achieve similar nitrate removal (%). Distinct bacterial communities developed due to the different substrates, as determined by sequencing of the 16S rRNA gene. Sedge and straw reactors shared more taxa with each other than with woodchips and throughout the experimental period, sedge and straw were more diverse than woodchips. Cellulose degrading bacteria like Fibrobacteres and Verrucomicrobia were detected in the substrates after 100-150 days of operation. Nitrate removal rates were highest in the sedge and straw reactors. After initial fluctuations, these reactors removed 5.1-6.3 g N m-3 water day-1, which was 3.3-4.4 times more than in the woodchip reactors. This corresponded to 48%, 42%, and 44% nitrate removal for the sedge, straw, and woodchip reactors respectively. The functional communities were characterized by quantitative PCR and denitrification was the major nitrate removing process based on genetic potential and water chemistry, although sedge and straw developed a capacity for ammonification. Gene ratios suggested that denitrification was initially incomplete and terminating with nitrous oxide. An increase in abundances of nitrous oxide reducing capacity in all substrate types towards the end increased the potential for less emissions of the greenhouse gas nitrous oxide.

Spatial impacts of inorganic ligand availability and localized microbial community structure on mitigation of zinc laden mine water in sulfate-reducing bioreactors.

Sulfate-reducing bioreactors (SRBRs) represent a passive, sustainable, and long-term option for mitigating mining influenced water (MIW) during release. Here we investigate spatial zinc precipitation profiles as influenced by substrate differentiation, inorganic ligand availability (inorganic carbon and sulfide), and microbial community structure in pilot-scale SRBR columns fed with sulfate and zinc-rich MIW. Through a combination of aqueous sampling, geochemical digests, electron microscopy and energy-dispersive x-ray spectroscopy, we were able to delineate zones of enhanced zinc removal, identify precipitates of varying stability, and discern the temporal and spatial evolution of zinc, sulfur, and calcium associations. These geochemical insights revealed spatially variable immobilization regimes between SRBR columns that could be further contrasted as a function of labile (alfalfa-dominated) versus recalcitrant (woodchip-dominated) solid-phase substrate content. Both column subsets exhibited initial zinc removal as carbonates; however precipitation in association with labile substrates was more pronounced and dominated by metal-sulfide formation in the upper portions of the down flow columns with micrographs visually suggestive of sphalerite (ZnS). In contrast, a more diffuse and lower mass of zinc precipitation in the presence of gypsum-like precipitates occurred within the more recalcitrant column systems. While removal and sulfide-associated precipitation were spatially variable, whole bacterial community structure (ANOSIM) and diversity estimates were comparatively homogeneous. However, two phyla exhibited a potentially selective relationship with a significant positive correlation between the ratio of Firmicutes to Bacteroidetes and sulfide-bound zinc. Collectively these biogeochemical insights indicate that depths of maximal zinc sulfide precipitation are temporally dynamic, influenced by substrate composition and broaden our understanding of bio-immobilized zinc species, microbial interactions and potential operational and monitoring tools in these types of passive bioreactors.

Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes.

Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.

Draft Genome Sequence of a Novel Desulfobacteraceae Member from a Sulfate-Reducing Bioreactor Metagenome.

Sulfate-reducing bacteria are important players in the global sulfur cycle and of considerable commercial interest. The draft genome sequence of a sulfate-reducing bacterium of the family Desulfobacteraceae, assembled from a sulfate-reducing bioreactor metagenome, indicates that heavy-metal- and acid-resistance traits of this organism may be of importance for its application in acid mine drainage mitigation.

Draft Genome Sequences of Two Novel Acidimicrobiaceae Members from an Acid Mine Drainage Biofilm Metagenome.

Bacteria belonging to the family Acidimicrobiaceae are frequently encountered in heavy metal-contaminated acidic environments. However, their phylogenetic and metabolic diversity is poorly resolved. We present draft genome sequences of two novel and phylogenetically distinct Acidimicrobiaceae members assembled from an acid mine drainage biofilm metagenome.

Organoheterotrophic Bacterial Abundance Associates with Zinc Removal in Lignocellulose-Based Sulfate-Reducing Systems.

Syntrophic relationships between fermentative and sulfate-reducing bacteria are essential to lignocellulose-based systems applied to the passive remediation of mining-influenced waters. In this study, seven pilot-scale sulfate-reducing bioreactor columns containing varying ratios of alfalfa hay, pine woodchips, and sawdust were analyzed over ∼500 days to investigate the influence of substrate composition on zinc removal and microbial community structure. Columns amended with >10% alfalfa removed significantly more sulfate and zinc than did wood-based columns. Enumeration of sulfate reducers by functional signatures (dsrA) and their putative identification from 16S rRNA genes did not reveal significant correlations with zinc removal, suggesting limitations in this directed approach. In contrast, a strong indicator of zinc removal was discerned in comparing the relative abundance of core microorganisms shared by all reactors (>80% of total community), many of which had little direct involvement in metal or sulfate respiration. The relative abundance of Desulfosporosinus, the dominant putative sulfate reducer within these reactors, correlated to representatives of this core microbiome. A subset of these clades, including Treponema, Weissella, and Anaerolinea, was associated with alfalfa and zinc removal, and the inverse was found for a second subset whose abundance was associated with wood-based columns, including Ruminococcus, Dysgonomonas, and Azospira. The construction of a putative metabolic flowchart delineated syntrophic interactions supporting sulfate reduction and suggests that the production of and competition for secondary fermentation byproducts, such as lactate scavenging, influence bacterial community composition and reactor efficacy.

Polygold-FISH for signal amplification of metallo-labeled microbial cells.

An improved in situ hybridization approach (Polygold-FISH) using biotinylated probes targeting multiple locations of the 16 S ribosomal subunit, followed by fluoronanogold-streptavidin labeling and autometallographic enhancement of nanogold particles was developed as a means of signal amplification of metallo-labeled cells, without the need for Catalyzed Reporter Deposition (CARD). Bacterial cells were readily detected based on their gold-particle signal using scanning-electron microscopy and energy-dispersive X-ray spectroscopy when contrasted with controls or cells hybridized with a single probe. Polygold-FISH presents an alternative to CARD-FISH, circumventing the need for aggressive oxidants, which is useful when products of microbial respiration such as those relevant at the microbe-mineral interface could be altered during processing for visualization.

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.

New methods for analysis of spatial distribution and coaggregation of microbial populations in complex biofilms.

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.

Effects of environmental conditions on the nitrifying population dynamics in a pilot wastewater treatment plant.

The effect of environmental conditions, especially ammonium concentration, on community composition and nitrification activity of nitrifying bacterial biofilms in a pilot wastewater treatment plant was examined. A decreasing ammonium gradient was created when four aerated tanks with suspended carrier material were serially fed with wastewater. Community composition was analysed using fluorescence in situ hybridization (FISH) probes as well as partial 16S rRNA and amoA gene analysis using polymerase chain reaction-denaturating gradient gel electrophoresis (PCR-DGGE) and sequencing. Fluorescence in situ hybridization probes identified at least five ammonia-oxidizing bacterial (AOB) and two nitrite-oxidizing bacterial (NOB) populations. A change in nitrifying community was detected in the tanks, indicating that ammonium was an important structuring factor. Further, we found support for different autoecology within the Nitrosomonas oligotropha lineage, as at least one population within this lineage increased in relative abundance with ammonium concentration while another population decreased. Absolute numbers of AOB and NOB growing in biofilms on the carriers were determined and the cell specific nitrification rates calculated seemed strongly correlated to ammonium concentration. Oxygen could also be limiting in the biofilms of the first tank with high ammonium concentrations. The response of the nitrifying community to increased ammonium concentrations differed between the tanks, indicating that activity correlates with community structure.