Valdis Krumins

PCB dechlorination enhancement in Anacostia River sediment microcosms

In situ treatment of PCB contaminated sediments via microbial dechlorination is a promising alternative to dredging, which may be reserved for only the most contaminated areas. Reductive dechlorination of low levels of weathered PCB mixtures typical of urban environments may occur at slow rates. Here, we report that biostimulation and bioaugmentation enhanced dechlorination of low concentration (2.1 mg PCBs/kg dry weight) historical PCBs in microcosms prepared with Anacostia River, Washington, DC, sediment. Treatments included electron donors butyrate, lactate, propionate and acetate (1 mM each); alternate halogenated electron acceptors (haloprimers) tetrachlorobenzene (TeCB, 25 microM), pentachloronitrobenzene (PCNB, 25 microM), or 2,3,4,5,6-PCB (PCB116, 2.0 microM); and/or bioaugmentation with a culture containing Dehalococcoides ethenogenes strain 195 (3 x 10(6)cells/mL). Dechlorination rates were enhanced in microcosms receiving bioaugmentation, PCNB and PCNB plus bioaugmentation, compared to other treatments. Microcosm subcultures generated after 415 days and spiked with PCB116 showed sustained capacity for dechlorination of PCB116 in PCNB, PCNB plus bioaugmentation, and TeCB treatments, relative to other treatments. Analysis of Chloroflexi 16S rRNA genes showed that TeCB and PCNB increased native Dehalococcoides spp. from the Pinellas subgroup; however this increase was correlated to enhanced dechlorination of low concentration weathered PCBs only in PCNB-amended microcosms. D. ethenogenes strain 195 was detected only in bioaugmented microcosms and decreased over 281 days. Bioaugmentation with D. ethenogenes strain 195 increased PCB dechlorination rates initially, but enhanced capacity for dechlorination of a model congener, PCB116, after 415 days occurred only in microcosms with enhanced native Dehalococcoides spp.

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates (“haloprimers”), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.

Methane production from horse manure and stall waste with softwood bedding.

Substantial stall waste is generated from horses on softwood bedding. The methane potential (G(pot)) of horse manure and constructed mixtures of stall waste with softwood bedding was determined at 35°C. G(pot) of 68, 191 and 273 mL/g volatile solids (VS) were estimated for three separate batches of horse manure, indicating variability in the material. Cumulative energy production over 20-40 days ranged from 3.11 ± 0.92 to 8.45 ± 5.42 × 10(5)kJ/metric ton wet weight horse manure alone, and from 1.69 ± 0.39 to 3.91 ± 0.47 × 10(5)kJ/metric ton wet weight horse manure plus softwood stall bedding (mixed at a 1:1 ratio on a VS basis). Softwood bedding was barely degradable and diluted the energy production of the stall waste; however, it did not cause inhibition of methane production from manure. Manually separated used softwood bedding contained substantial methane potential.

Profiling microbial community in a watershed heavily contaminated by an active antimony (Sb) mine in Southwest China.

Located in Southwest China, the Chahe watershed has been severely contaminated by upstream active antimony (Sb) mines. The extremely high concentrations of Sb make the Chahe watershed an excellent model to elucidate the response of indigenous microbial activities within a severe Sb-contaminated environment. In this study, water and surface sediments from six locations in the Chahe watershed with different levels of Sb contamination were analyzed. Illumina sequencing of 16S rRNA amplicons revealed more than 40 phyla from the domain Bacteria and 2 phyla from the domain Archaea. Sequences assigned to the genera Flavobacterium, Sulfuricurvum, Halomonas, Shewanella, Lactobacillus, Acinetobacter, and Geobacter demonstrated high relative abundances in all sequencing libraries. Spearmans rank correlations indicated that a number of microbial phylotypes were positively correlated with different speciation of Sb, suggesting potential roles of these phylotypes in microbial Sb cycling. Canonical correspondence analysis further demonstrated that geochemical parameters, including water temperature, pH, total Fe, sulfate, aqueous Sb, and Eh, significantly structured the overall microbial community in Chahe watershed samples. Our findings offer a direct and reliable reference to the diversity of microbial communities in the presence of extremely high Sb concentrations, and may have potential implications for in situ bioremediation strategies of Sb contaminated sites.

Depth-resolved microbial community analyses in two contrasting soil cores contaminated by antimony and arsenic

Investigation of microbial communities of soils contaminated by antimony (Sb) and arsenic (As) is necessary to obtain knowledge for their bioremediation. However, little is known about the depth profiles of microbial community composition and structure in Sb and As contaminated soils. Our previous studies have suggested that historical factors (i.e., soil and sediment) play important roles in governing microbial community structure and composition. Here, we selected two different types of soil (flooded paddy soil versus dry corn field soil) with co-contamination of Sb and As to study interactions between these metalloids, geochemical parameters and the soil microbiota as well as microbial metabolism in response to Sb and As contamination. Comprehensive geochemical analyses and 16S rRNA amplicon sequencing were used to shed light on the interactions of the microbial communities with their environments. A wide diversity of taxonomical groups was present in both soil cores, and many were significantly correlated with geochemical parameters. Canonical correspondence analysis (CCA) and co-occurrence networks further elucidated the impact of geochemical parameters (including Sb and As contamination fractions and sulfate, TOC, Eh, and pH) on vertical distribution of soil microbial communities. Metagenomes predicted from the 16S data using PICRUSt included arsenic metabolism genes such as arsenate reductase (ArsC), arsenite oxidase small subunit (AoxA and AoxB), and arsenite transporter (ArsA and ACR3). In addition, predicted abundances of arsenate reductase (ArsC) and arsenite oxidase (AoxA and AoxB) genes were significantly correlated with Sb contamination fractions, These results suggest potential As biogeochemical cycling in both soil cores and potentially dynamic Sb biogeochemical cycling as well.

Correlating microbial community profiles with geochemical conditions in a watershed heavily contaminated by an antimony tailing pond

Mining activities have introduced various pollutants to surrounding aquatic and terrestrial environments, causing adverse impacts to the environment. Indigenous microbial communities are responsible for the biogeochemical cycling of pollutants in diverse environments, indicating the potential for bioremediation of such pollutants. Antimony (Sb) has been extensively mined in China and Sb contamination in mining areas has been frequently encountered. To date, however, the microbial composition and structure in response to Sb contamination has remained overlooked. Sb and As frequently co-occur in sulfide-rich ores, and co-contamination of Sb and As is observed in some mining areas. We characterized, for the first time, the microbial community profiles and their responses to Sb and As pollution from a watershed heavily contaminated by Sb tailing pond in Southwest China. The indigenous microbial communities were profiled by high-throughput sequencing from 16 sediment samples (535,390 valid reads). The comprehensive geochemical data (specifically, physical-chemical properties and different Sb and As extraction fractions) were obtained from river water and sediments at different depths as well. Canonical correspondence analysis (CCA) demonstrated that a suite of in situ geochemical and physical factors significantly structured the overall microbial community compositions. Further, we found significant correlations between individual phylotypes (bacterial genera) and the geochemical fractions of Sb and As by Spearman rank correlation. A number of taxonomic groups were positively correlated with the Sb and As extractable fractions and various Sb and As species in sediment, suggesting potential roles of these phylotypes in Sb biogeochemical cycling.

Response of Soil Microbial Communities to Elevated Antimony and Arsenic Contamination Indicates the Relationship between the Innate Microbiota and Contaminant Fractions

Mining of sulfide ore deposits containing metalloids, such as antimony and arsenic, has introduced serious soil contamination around the world, posing severe threats to food safety and human health. Hence, it is important to understand the behavior and composition of the microbial communities that control the mobilization or sequestration of these metal(loid)s. Here, we selected two sites in Southwest China with different levels of Sb and As contamination to study interactions among various Sb and As fractions and the soil microbiota, with a focus on the microbial response to metalloid contamination. Comprehensive geochemical analyses and 16S rRNA gene amplicon sequencing demonstrated distinct soil taxonomic inventories depending on Sb and As contamination levels. Stochastic gradient boosting indicated that citric acid extractable Sb(V) and As(V) contributed 5% and 15%, respectively, to influencing the community diversity. Random forest predicted that low concentrations of Sb(V) and As(V) could enhance the community diversity but generally, the Sb and As contamination impairs microbial diversity. Co-occurrence network analysis indicated a strong correlation between the indigenous microbial communities and various Sb and As fractions. A number of taxa were identified as core genera due to their elevated abundances and positive correlation with contaminant fractions (total Sb and As concentrations, bioavailable Sb and As extractable fractions, and Sb and As redox species). Shotgun metagenomics indicated that Sb and As biogeochemical redox reactions may exist in contaminated soils. All these observations suggest the potential for bioremediation of Sb- and As-contaminated soils.

Characterization of the microbial community composition and the distribution of Fe-metabolizing bacteria in a creek contaminated by acid mine drainage.

A small watershed heavily contaminated by long-term acid mine drainage (AMD) from an upstream abandoned coal mine was selected to study the microbial community developed in such extreme system. The watershed consists of AMD-contaminated creek, adjacent contaminated soils, and a small cascade aeration unit constructed downstream, which provide an excellent contaminated site to study the microbial response in diverse extreme AMD-polluted environments. The results showed that the innate microbial communities were dominated by acidophilic bacteria, especially acidophilic Fe-metabolizing bacteria, suggesting that Fe and pH are the primary environmental factors in governing the indigenous microbial communities. The distribution of Fe-metabolizing bacteria showed distinct site-specific patterns. A pronounced shift from diverse communities in the upstream to Proteobacteria-dominated communities in the downstream was observed in the ecosystem. This location-specific trend was more apparent at genus level. In the upstream samples (sampling sites just below the coal mining adit), a number of Fe(II)-oxidizing bacteria such as Alicyclobacillus spp., Metallibacterium spp., and Acidithrix spp. were dominant, while Halomonas spp. were the major Fe(II)-oxidizing bacteria observed in downstream samples. Additionally, Acidiphilium, an Fe(III)-reducing bacterium, was enriched in the upstream samples, while Shewanella spp. were the dominant Fe(III)-reducing bacteria in downstream samples. Further investigation using linear discriminant analysis (LDA) effect size (LEfSe), principal coordinate analysis (PCoA), and unweighted pair group method with arithmetic mean (UPGMA) clustering confirmed the difference of microbial communities between upstream and downstream samples. Canonical correspondence analysis (CCA) and Spearman’s rank correlation indicate that total organic carbon (TOC) content is the primary environmental parameter in structuring the indigenous microbial communities, suggesting that the microbial communities are shaped by three major environmental parameters (i.e., Fe, pH, and TOC). These findings were beneficial to a better understanding of natural attenuation of AMD.

Microbial diversity and community structure in an antimony-rich tailings dump.

To assess the impact of antimony (Sb) on microbial community structure, 12 samples were taken from an Sb tailings pile in Guizhou Province, Southwest China. All 12 samples exhibited elevated Sb concentrations, but the mobile and bioaccessible fractions were small in comparison to total Sb concentrations. Besides the geochemical analyses, microbial communities inhabiting the tailing samples were characterized to investigate the interplay between the microorganisms and environmental factors in mine tailings. In all samples, Proteobacteria and Actinobacteria were the most dominant phyla. At the genus level, Thiobacillus, Limnobacter, Nocardioides, Lysobacter, Phormidium, and Kaistobacter demonstrated relatively high abundances. The two most abundant genera, Thiobacillus and Limnobacter, are characterized as sulfur-oxidizing bacteria and thiosulfate-oxidizing bacteria, respectively, while the genus Lysobacter contains arsenic (As)-resistant bacteria. Canonical correspondence analysis (CCA) indicates that TOC and the sulfate to sulfide ratio strongly shaped the microbial communities, suggesting the influence of the environmental factors in the indigenous microbial communities.

Remediation of antimony-rich mine waters: Assessment of antimony removal and shifts in the microbial community of an onsite field-scale bioreactor.

An on-site field-scale bioreactor for passive treatment of antimony (Sb) contamination was installed downstream of an active Sb mine in Southwest China, and operated for one year (including a six month monitoring period). This bioreactor consisted of five treatment units, including one pre-aerobic cell, two aerobic cells, and two microaerobic cells. With the aerobic cells inoculated with indigenous mine water microflora, the bioreactor removed more than 90% of total soluble Sb and 80% of soluble antimonite (Sb(III)). An increase in pH and decrease of oxidation-reduction potential (Eh) was also observed along the flow direction. High-throughput sequencing of the small subunit ribosomal RNA (SSU rRNA) gene variable (V4) region revealed that taxonomically diverse microbial communities developed in the bioreactor. Metal (loid)-oxidizing bacteria including Ferrovum, Thiomonas, Gallionella, and Leptospirillum, were highly enriched in the bioreactor cells where the highest total Sb and Sb(III) removal occurred. Canonical correspondence analysis (CCA) indicated that a suite of in situ physicochemical parameters including pH and Eh were substantially correlated with the overall microbial communities. Based on an UPGMA (Unweighted Pair Group Method with Arithmetic Mean) tree and PCoA (Principal Coordinates Analysis), the microbial composition of each cell was distinct, indicating these in situ physicochemical parameters had an effect in shaping the indigenous microbial communities. Overall, this study was the first to employ a field-scale bioreactor to treat Sb-rich mine water onsite and, moreover, the findings suggest the feasibility of the bioreactor in removing elevated Sb from mine waters.