Philip L. Bond

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

ABSTRACT Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) andPropionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related toRhodocyclus and Propionibacter.

Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site.

ABSTRACT An unusually thick (∼1 cm) slime developed on a slump of finely disseminated pyrite ore within an extreme acid mine drainage site at Iron Mountain, near Redding, Calif. The slime was studied over the period of 1 year. The subaerial form of the slime distinguished it from more typical submerged streamers. Phylogenetic analysis of 16S rRNA genes revealed a diversity of sequences that were mostly novel. Nearest relatives to the majority of sequences came from iron-oxidizing acidophiles, and it appears that iron oxidation is the predominant metabolic characteristic of the organisms in the slime. The most abundant of the 16S rRNA genes detected were from organisms related toLeptospirillum species. The dominant sequence (71% of clones) may represent a new genus. Sequences within theArchaea of the Thermoplasmales lineage were detected. Most of these were only distantly related to known microorganisms. Also, sequences affiliating withAcidimicrobium were detected. Some of these were closely related to “Ferromicrobium acidophilus,” and others were affiliated with a lineage only represented by environmental clones. Unexpectedly, sequences that affiliated within the delta subdivision of the Proteobacteria were detected. The predominant metabolic feature of bacteria of this subdivision is anaerobic sulfate or metal reduction. Thus, microenvironments of low redox potential possibly exist in the predominantly oxidizing environments of the slime. These results expand our knowledge of the biodiversity of acid mine drainage environments and extend our understanding of the ecology of extremely acidic systems.

Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems

ABSTRACT This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ∼0.5 to ∼1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “Ferroplasma” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength.Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated.Sulfobacillus spp. were detected as a prominent inhabitant in warmer (∼43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.

Cathodic oxygen reduction catalyzed by bacteria in microbial fuel cells.

Microbial fuel cells (MFCs) have the potential to combine wastewater treatment efficiency with energetic efficiency. One of the major impediments to MFC implementation is the operation of the cathode compartment, as it employs environmentally unfriendly catalysts such as platinum. As recently shown, bacteria can facilitate sustainable and cost-effective cathode catalysis for nitrate and also oxygen. Here we describe a carbon cathode open to the air, on which attached bacteria catalyzed oxygen reduction. The bacteria present were able to reduce oxygen as the ultimate electron acceptor using electrons provided by the solid-phase cathode. Current densities of up to 2.2 A m−2 cathode projected surface were obtained (0.303±0.017 W m−2, 15 W m−3 total reactor volume). The cathodic microbial community was dominated by Sphingobacterium, Acinetobacter and Acidovorax sp., according to 16S rRNA gene clone library analysis. Isolates of Sphingobacterium sp. and Acinetobacter sp. were obtained using H2/O2 mixtures. Some of the pure culture isolates obtained from the cathode showed an increase in the power output of up to three-fold compared to a non-inoculated control, that is, from 0.015±0.001 to 0.049±0.025 W m−2 cathode projected surface. The strong decrease in activation losses indicates that bacteria function as true catalysts for oxygen reduction. Owing to the high overpotential for non-catalyzed reduction, oxygen is only to a limited extent competitive toward the electron donor, that is, the cathode. Further research to refine the operational parameters and increase the current density by modifying the electrode surface and elucidating the bacterial metabolism is warranted.

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.

ABSTRACT Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum YT by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and YT group as a single species. “F. acidarmanus” Fer1T groups separately, and we propose the new species “F. acidarmanus” Fer1T sp. nov.

Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating microorganisms to achieve phosphate removal from wastewater. We used high-resolution community proteomics to identify key metabolic pathways in ‘Candidatus Accumulibacter phosphatis’ (A. phosphatis)-mediated EBPR and to evaluate the contributions of co-existing strains within the dominant population. Overall, 702 proteins from the A. phosphatis population were identified. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Strong similarity in protein profiles under anaerobic and aerobic conditions was uncovered (only 3% of A. phosphatis-associated proteins exhibited statistically significant abundance differences). By comprehensive genome-wide alignment of 13 930 orthologous proteins, we uncovered substantial differences in protein abundance for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the A. phosphatis population. These findings suggest an essential role for genetic diversity in maintaining the stable performance of EBPR systems and, hence, demonstrate the power of integrated cultivation-independent genomics and proteomics for the analysis of complex biotechnological systems.

Metaproteomics provides functional insight into activated sludge wastewater treatment

Background Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR). Methodology/Principal Findings A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism “Candidatus Accumulibacter phosphatis”. When EBPR failed, the sludge was dominated by tetrad-forming α-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from “Candidatus Accumulibacter phosphatis” and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid β oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. Conclusions/Significance Importantly, this study provides direct evidence linking the metabolic activities of “Accumulibacter” to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.

Metabolically Active Eukaryotic Communities in Extremely Acidic Mine Drainage

ABSTRACT Acid mine drainage (AMD) microbial communities contain microbial eukaryotes (both fungi and protists) that confer a biofilm structure and impact the abundance of bacteria and archaea and the community composition via grazing and other mechanisms. Since prokaryotes impact iron oxidation rates and thus regulate AMD generation rates, it is important to analyze the fungal and protistan populations. We utilized 18S rRNA and beta-tubulin gene phylogenies and fluorescent rRNA-specific probes to characterize the eukaryotic diversity and distribution in extremely acidic (pHs 0.8 to 1.38), warm (30 to 50°C), metal-rich (up to 269 mM Fe2+, 16.8 mM Zn, 8.5 mM As, and 4.1 mM Cu) AMD solutions from the Richmond Mine at Iron Mountain, Calif. A Rhodophyta (red algae) lineage and organisms from the Vahlkampfiidae family were identified. The fungal 18S rRNA and tubulin gene sequences formed two distinct phylogenetic groups associated with the classes Dothideomycetes and Eurotiomycetes. Three fungal isolates that were closely related to the Dothideomycetes clones were obtained. We suggest the name “Acidomyces richmondensis” for these isolates. Since these ascomycete fungi were morphologically indistinguishable, rRNA-specific oligonucleotide probes were designed to target the Dothideomycetes and Eurotiomycetes via fluorescent in situ hybridization (FISH). FISH analyses indicated that Eurotiomycetes are generally more abundant than Dothideomycetes in all of the seven locations studied within the Richmond Mine system. This is the first study to combine the culture-independent detection of fungi with in situ detection and a demonstration of activity in an acidic environment. The results expand our understanding of the subsurface AMD microbial community structure.

Geochemical and biological aspects of sulfide mineral dissolution: lessons from Iron Mountain, California

Abstract The oxidative dissolution of sulfide minerals leading to acid mine drainage (AMD) involves a complex interplay between microorganisms, solutions, and mineral surfaces. Consequently, models that link molecular level reactions and the microbial communities that mediate them to field scale processes are few. Here we provide a mini-review of laboratory and field-based studies concerning the chemical, microbial, and kinetic aspects of sulfide mineral dissolution and generation of AMD at the Richmond ore body at Iron Mountain, California.

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.