Bio-Sep Beads Research Articles

Natural Attenuation in Streambed Sediment Receiving Chlorinated Solvents from Underlying Fracture Networks.

Contaminant discharge from fractured bedrock formations remains a remediation challenge. We applied an integrated approach to assess the natural attenuation potential of sediment that forms the transition zone between upwelling groundwater from a chlorinated solvent-contaminated fractured bedrock aquifer and the receiving surface water. In situ measurements demonstrated that reductive dechlorination in the sediment attenuated chlorinated compounds before reaching the water column. Microcosms established with creek sediment or in situ incubated Bio-Sep beads degraded C1-C3 chlorinated solvents to less-chlorinated or innocuous products. Quantitative PCR and 16S rRNA gene amplicon sequencing revealed the abundance and spatial distribution of known dechlorinator biomarker genes within the creek sediment and demonstrated that multiple dechlorinator populations degrading chlorinated C1-C3 alkanes and alkenes co-inhabit the sediment. Phylogenetic classification of bacterial and archaeal sequences indicated a relatively uniform distribution over spatial (300 m horizontally) scale, but Dehalococcoides and Dehalobacter were more abundant in deeper sediment, where 5.7 ± 0.4 × 105 and 5.4 ± 0.9 × 106 16S rRNA gene copies per g of sediment, respectively, were measured. The microbiological and hydrogeological characterization demonstrated that microbial processes at the fractured bedrock-sediment interface were crucial for preventing contaminants reaching the water column, emphasizing the relevance of this critical zone environment for contaminant attenuation.

Microbial attenuation of atrazine in agricultural soils: Biometer assays, bacterial taxonomic diversity, and catabolic genes

The purpose of this study was to examine the potential biomineralization of atrazine and identification of atrazine-degrading bacteria in agricultural soils. Different atrazine application histories of soils impacted the kinetics of biomineralization but not the presence of catabolic genes of two atrazine degradative pathways (Trz and Atz). Biomineralization was based on the measurement of 14CO2 from [U-ring-14C]-atrazine in surface soil (0–7 cm) samples incubated in biometers. Aerobic atrazine biomineralization rate constants (k) varied in the range of 0.004–0.508 d−1 depending on the specific soil sample and glucose amendment. The corresponding k-values for anaerobic biometers ± nitrate, ferrihydrite or sulfate were 0.002–0.360 d−1. Glucose enhancement of atrazine biomineralization was not consistent. Aerobic enrichments from soil samples and in-situ incubated BioSep beads yielded mixed cultures, four of which were characterized by 16S rRNA gene amplification, cloning and sequencing. Twelve pure cultures were isolated from enrichments and they were primarily Arthrobacter spp. (10/12). The presence of eight atrazine catabolic genes representing two degradative pathways was investigated in seven bacterial isolates by PCR amplification and sequencing. Several combinations of atrazine catabolic genes were detected; each contained at least atzBC. A complete set of genes for the Atz pathway was not found among the isolates. Our data indicate that atrazine metabolism involves multiple microorganisms and cooperative pathways diverging from atrazine metabolites.

Molecular analysis of atrazine-degrading bacteria and catabolic genes in the water column and sediment of a created wetland in an agricultural/urban watershed

Abstract Atrazine-mineralizing bacteria were enriched from water and sediment samples from a constructed flow-through wetland that received water from a river with an agricultural watershed. Bio–Sep beads laden with atrazine were submerged in the wetland sediment and water column in an attempt to sorb atrazine-metabolizing microbes on beads. Biometer experiments with in-situ incubated beads did not detect 14CO2 formation from 14C-atrazine. HPLC analysis was used to rank atrazine biodegradation by enrichment cultures from wetland samples. Analysis of the 16S rRNA gene sequences of 60 clones from aerobic enrichment cultures revealed the predominance of the genera Acidovorax (33%), Ralstonia (22%), Azorhizobium (12%), and Xanthobacter (12%) with sequence match ≥0.90 in wetland samples. Similar analysis of 12 clones from anaerobic enrichment cultures revealed the predominance of the genera Hydrogenophaga (42%) and Rhizobium (25%). Pure culture isolates (n = 18) were primarily Arthrobacter spp. (89%) at sequence match ≥0.97. Pure cultures were screened qualitatively for the presence of 8 catabolic genes associated with known pathways of atrazine biodegradation. None of the cultures had all Atz pathway genes and none contained atzD and trzD genes. AtzC was found in all isolates. The data indicate that the diversity of atrazine degraders was comparable to findings in agricultural soil studies, with the notable cluster of Arthrobacter spp. which may be favored by the culture conditions used for isolation.

Two decades of warming increases diversity of a potentially lignolytic bacterial community.

As Earth's climate warms, the massive stores of carbon found in soil are predicted to become depleted, and leave behind a smaller carbon pool that is less accessible to microbes. At a long-term forest soil-warming experiment in central Massachusetts, soil respiration and bacterial diversity have increased, while fungal biomass and microbially-accessible soil carbon have decreased. Here, we evaluate how warming has affected the microbial community's capability to degrade chemically-complex soil carbon using lignin-amended BioSep beads. We profiled the bacterial and fungal communities using PCR-based methods and completed extracellular enzyme assays as a proxy for potential community function. We found that lignin-amended beads selected for a distinct community containing bacterial taxa closely related to known lignin degraders, as well as members of many genera not previously noted as capable of degrading lignin. Warming tended to drive bacterial community structure more strongly in the lignin beads, while the effect on the fungal community was limited to unamended beads. Of those bacterial operational taxonomic units (OTUs) enriched by the warming treatment, many were enriched uniquely on lignin-amended beads. These taxa may be contributing to enhanced soil respiration under warming despite reduced readily available C availability. In aggregate, these results suggest that there is genetic potential for chemically complex soil carbon degradation that may lead to extended elevated soil respiration with long-term warming.

Using DNA‐Stable Isotope Probing to Identify MTBE‐ and TBA‐Degrading Microorganisms in Contaminated Groundwater

Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate.

Characterization of trapped lignin-degrading microbes in tropical forest soil.

Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition.

Establishment and characterization of atrazine degrading cultures from Nigerian agricultural soil using traditional and Bio-Sep bead enrichment techniques

Traditional and in situ enrichment using porous Bio-Sep beads fortified with atrazine were used to obtain mixed consortia of atrazine-degrading bacteria. Cultures derived from both enrichment techniques showed rapid degradation of atrazine and the composition of consortia varied depending on enrichment type and atrazine concentration present in both the selective media and in atrazine fortified Bio-Sep beads as determined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis also revealed the presence of many bands corresponding to various bacteria both cultured and uncultured. All enrichment cultures possessed trzND genes. The atzBC genes were detected in all bead enrichments while only atzC was present in one soil enrichment. The atrazine chlorohydrolase gene ( atzA) and cyanuric acid amidohydrolase genes (a tzD) were not detected in any of the enrichment cultures. In soil samples, trzN and trzD were the only known atrazine catabolic genes detected. Parallel degradation studies with atrazine potential metabolites showed extensive loss of these compounds from the culture media. The results indicate that in situ enrichment with Bio-Sep beads might be a viable method to cultivate atrazine-degrading bacteria not currently represented in existing culture collections. The triazine chlorohydrolase encoded by trzN commonly found in most Gram-positive atrazine-degrading bacteria, was more prevalent than atzA. Thus, the detection of known atrazine-catabolic genes in soil or bacteria from tropical African contaminated systems is an indication of the likely global distribution of these important s-triazine genes.

Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and trzN Genes in Viral-Community DNA

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.

In Situ Assessment of Biodegradation Potential Using Biotraps Amended with 13C-Labeled Benzene or Toluene

Stable isotope fractionation analysis of an aquifer heavily contaminated with benzene (up to 850 mg L(-1)) and toluene (up to 50 mg L(-1)) at a former hydrogenation plant in Zeitz (Saxonia, Germany) has suggested that significant biodegradation of toluene was occurring. However, clear evidence of benzene biodegradation has been lacking at this site. Determining the fate of benzene is often a determining factor in regulatory approval of a risk-based management strategy. The objective of the work described here was the demonstration of a new tool that can be used to provide proof of biodegradation of benzene or other organics by indigenous microorganisms under actual aquifer conditions. Unique in situ biotraps containing Bio-Sep beads, amended with 13C-labeled or 12C nonlabeled benzene and toluene, were deployed at the Zeitz site for 32 days in an existing groundwater monitoring well and used to collect and enrich microbial biofilms. Lipid biomarkers or remaining substrate was extracted from the beads and analyzed by mass spectrometry and molecular methods. Isotopic analysis of the remaining amounts of 13C-labeled contaminants (about 15-18% of the initial loading) showed no alteration of the 12C/13C ratio during incubation. Therefore, no measurable exchange of labeled compounds in the beads by the nonlabeled compounds in the aquifer materials occurred. Isotopic ratio analysis of microbial lipid fatty acids (as methyl ester derivatives) from labeled benzene- and toluene-amended biotraps showed 13C enrichment in several fatty acids of up to delta (13C) 13400%o, clearly verifying benzene and toluene biodegradation and the transformation of the labeled carbon into biomass by indigenous organisms under aquifer conditions. Fatty acid profiles of total lipid fatty acids and the phospholipid fatty acid fraction and their isotopic composition showed significant differences between benzene- and toluene-amended biotraps, suggesting that different microbial communities were involved in the biodegradation of the two compounds.

Evaluation of support matrices for immobilization of anaerobic consortia for efficient carbon cycling in waste regeneration

Efficient metabolism of fatty acids during anaerobic waste digestion requires development of consortia that include “fatty acid consuming H 2 producing bacteria” and methanogenic bacteria. The objective of this research was to optimize methanogenesis from fatty acids by evaluating a variety of support matrices for use in maintaining efficient syntrophic–methanogenic consortia. Tested matrices included clays (montmorillonite and bentonite), glass beads (106 and 425–600 μm), microcarriers (cytopore, cytodex, cytoline, and cultispher; conventionally employed for cultivation of mammalian cell lines), BioSep beads (powdered activated carbon), and membranes (hydrophilic; nylon, polysulfone, and hydrophobic; teflon, polypropylene). Data obtained from headspace methane (CH 4) analyses as an indicator of anaerobic carbon cycling efficiency indicated that material surface properties were important in maintenance and functioning of the anaerobic consortia. Cytoline yielded significantly higher CH 4 than other matrices as early as in the first week of incubation. 16S rRNA gene sequence analysis from crushed cytoline matrix showed the presence of Syntrophomonas spp. (butyrate oxidizing syntrophs) and Syntrophobacter spp. (propionate oxidizing syntrophs), with Methanosaeta spp. (acetate utilizing methanogen), and Methanospirillum spp. (hydrogen utilizing methanogen) cells. It is likely that the more hydrophobic surfaces provided a suitable surface for adherence of cells of syntrophic–methanogenic consortia. Cytoline also appeared to protect entrapped consortia from air, resulting in rapid methanogenesis after aerial exposure. Our study suggests that support matrices can be used in anaerobic digestors, pre-seeded with immobilized or entrapped consortia on support matrices, and may be of value as inoculant-adsorbents to rapidly initiate or recover proper system functioning following perturbation.

Biotreatment of refinery spent-sulfidic caustic using an enrichment culture immobilized in a novel support matrix.

Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H2S) and mercaptans from various hydrocarbon streams. The resulting sulfide-laden waste stream is called spent-sulfidic caustic. An aerobic enrichment culture was previously developed using a gas mixture of H2S and methyl-mercaptan (MeSH) as the sole energy source. This culture has now been immobilized in a novel support matrix, DuPont BIO-SEP beads, and is used to bio-treat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor. Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H2S and < 2 ppmv of MeSH produced in the bioreactor outlet gas. Excessive buildup of sulfate (> 12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough. Therefore, bioreactor performance was limited by the steady-state sulfate concentration. Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.

Biodesulfurization of flue gases and other sulfate/sulfite waste streams using immobilized mixed sulfate-reducing bacteria.

Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere that cause acid rain. Microbial processes for reducing SO2 to hydrogen sulfide (H2S) have previously been demonstrated by utilizing mixed cultures of sulfate-reducing bacteria (SRB) with municipal sewage digest as the carbon and energy source. To maximize the productivity of the bioreactor for SO2 reduction in this study, various immobilized cell bioreactors were investigated: a stirred tank with SRB flocs and columnar reactors with cells immobilized in either potassium-carrageenan gel matrix or polymeric porous BIO-SEP beads. The maximum volumetric productivity for SO2 reduction in the continuous stirred-tank reactor (CSTR) with SRB flocs was 2.1 mmol of SO2/(h.L). The potassium-carrageenan gell matrix used for cell immobilization was not durable at feed sulfite concentrations greater than 2000 mg/L (1.7 mmol/(h.L)). A columnar reactor with mixed SRB cells that had been allowed to grow into highly stable BIO-SEP polymeric beads exhibited the highest sulfite conversion rates, in the range 16.5 mmol/(h.L) (with 100% conversion) to 20 mmol/(h.L) (with 95% conversion). The average specific activity for sulfite reduction in the column, in terms of dry weight of SRB biomass, was 9.5 mmol of sulfite/(h.g). In addition to flue gas desulfurization, potential applications of this microbial process include the treatment of sulfate/sulfite-laden wastewater from the pulp and paper, petroleum, mining, and chemical industries.