Release of poly- and perfluoroalkyl substances from finished biosolids in soil mesocosms.

Finished biosolids were collected and characterized from seven municipal water resource recovery facilities. Poly- and perfluoroalkyl substances (PFAS) for the 54 quantified in the biosolids ranged from 323 ± 14.1 to 1100 ± 43.8 µg/kg (dry weight basis). For all biosolids, greater than 75% of the PFAS fluorine mass was associated with precursors. Di-substituted polyfluorinated phosphate esters (diPAPs) were the most abundant PFAS identified in the biosolids. The total oxidizable precursor assay on biosolids extracts generally failed to quantify the amount of precursors present, in large part due to the fact that diPAPS were not fully transformed during the TOP assay. Outdoor biosolids column leaching experiments intended to simulate biosolids land application showed sustained PFAS leaching over the 6-month study duration. Perfluoroalkyl acid (PFAA) concentrations in leachate, when detected, typically ranged in the 10 s to 100 s of ng/L; no diPAPs were detected in the leachate. The PFAA leaching from the biosolids exceeded the PFAA mass initially present in the biosolids (typically by greater than an order of magnitude), but the cumulative PFAA mass leached did not exceed the molar equivalents that could be explained by transformation of quantified precursors. These results highlight the importance of PFAA precursors initially present in biosolids and their contribution to long term leaching of PFAAs from land-applied biosolids.

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.

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.