Kerry L. Sublette

Utilization of Microbial Biofilms as Monitors of Bioremediation

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Program’s Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2–13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

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.

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

ABSTRACT 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.

Degradation of munition wastes by Phanerochaete chrysosporium

Abstract“Pink water” is a waste-water stream generated by munitions LAP (loading, assembly, and packing) operations. The major components of this waste water are trinitrotoluene (TNT) and cyclotrimethylene trinitramine (RDX) at concentrations of 120-175 mg/L and 25 mg/L, respectively. Currently, pink water is treated by activated carbon adsorption. Removal efficiencies of >99.5% have been reported. However, this treatment method suffers a serious limitation in that the carbon cannot be safely regenerated. Loaded carbon is disposed of by incineration after a single use.We have demonstrated that TNT, RDX, simulated, and actual pink water can be effectively treated byPhanerochaete chrysosporium immobilized on the disks of a rotating biological contractor (RBC) in both batch and continuous modes. Greater than 90% removal of TNT from a simulated pink water was observed in a continuous RBC with a residence time of about 24 h. The disk area required was about 10,000 ft2/gpm (4091 m2/m3h) feed. RDX was amenable to treatment, but RDX removal rates were somewhat slower. A full-scale treatment system was designed on the basis of laboratory data, and a cost analysis was performed. This analysis has shown that biotreatment of pink water can be a cost-effective alternative to carbon adsorption.

Characterization of a Novel Biocatalyst System for Sulfide Oxidation

It has been demonstrated that an enrichment culture dominated by Thiomicrospira sp. CVO may be cultured on H2S(g) as an energy source under sulfide‐limiting conditions in suspended culture with nitrate as the electron acceptor. Hydrogen sulfide (10,000 ppmv) was completely removed from the feed gas and oxidized to sulfate in <3 s of gas‐liquid contacting time. Maximum loading of the biomass for sulfide oxidation was observed to be 5.8 mmol H2S/h‐g biomass protein, comparable to that reported previously for Thiobacillus denitrificans under similar conditions. However, the enrichment culture was shown to be more tolerant of extremes in pH and elevated temperature than T. denitrificans. Coupled with a reported tolerance of CVO for up to 10% NaCl, these observations suggest that a CVO‐based culture is potentially a more robust biocatalyst system for sulfide oxidation than cultures based on Thiobacilli.

Simultaneous combined microbial removal of sulfur dioxide and nitric oxide from a gas stream

A program is under way at the University of Tulsa to develop a viable process concept whereby a microbial process can impact on the problem of flue gas desulfurization and NOx removal. We have previously reported studies of SO2 reduction byDesulfovibrio desulfuricans and NOx reduction byThiobacillus denitrificans. One potential process concept is the simultaneous combined removal of SO2 and NOx from cooled flue gas by contact with cultures of sulfate-reducing bacteria (SO2→H2S) andT. denitrificans (H2S→SO4-2) as cultures-in-series or in coculture in a single contacting stage. Each of these contacting schemes has been investigated.

Microbial Control of Hydrogen Sulfide Production in a Porous Medium

The ability of a sulfide- and glutaraldehyde-tolerant strain ofThiobacillus denitrificans (strain F) to control sulfide production in an experimental system of cores and formation water from the Redfield, Iowa natural gas storage facility was investigated. A stable, sulfide-producing biofilm was established in two separate core systems, one of which was inoculated with strain F, and the other core system (control) was treated in an identical manner, but was not inoculated with strain F. When formation water with 10 mM acetate and 5 mM nitrate was injected into both core systems, the effluent sulfide concentrations in the control core system ranged from 200–460 μM. In the test core system inoculated with strain F, the effluent sulfide concentrations were lower, ranging from 70–110 μM. In order to determine whether strain F could control sulfide production under optimal conditions for sulfate-reducing bacteria, the electron donor was changed to lactate, and inorganic nutrients (nitrogen and phosphate sources) were added to the formation water. When nutrient-supplemented formation water with 3.1 mM lactate and 10 mM nitrate was used, the effluent sulfide concentrations of the control core system initially increased to about 3800 μM, and then decreased to about 1100 μM after 5 wk. However, in the test core system inoculated with strain F, the effluent sulfide concentrations were much lower, 160–330 μM. Nitrate consumption (5 mM) and high concentrations (107–108 cells/mL) of strain F were detected in the test core system. An accumulation of biomass occurred in the influent lines during 2 mo of continuous operation, but only a small increase in injection pressure was observed. These studies showed that inoculation with strain F was needed for effective control of sulfide production, and that significant plugging or loss of injectivity owing to microbial inoculation did not occur.

Microbial treatment of sulfide-laden water

Abstract Water containing up to 25 mM soluble sulfide has been successfully treated microbially in an upflow bubble column. Sulfides were completely oxidized to sulfate by a sulfide-tolerant strain of the chemoautotroph, Thiobacillus denitrificans , flocculated by co-culture with floc-forming heterotrophs. The sulfide-active floc was shown to be stable for 9 months of continuous operation with no external organic carbon required to support the growth of the heterotrophs. The floc exhibited excellent settling properties throughout the experiment.

Evaluation of a microbial method to reduce hydrogen sulfide levels in a porous rock biofilm

SummaryThe efficacy of nitrate addition, with and without inoculation with a sulfide-resistant strain ofThiobacillus denitrificans (strain F), in reducing sulfide levels in an experimental system using cores and subsurface formation water from a gas storage facility was examined. The addition of nitrate (40 mM) alone to the formation water injected into core systems operated at hydraulic retention times of 3.2 and 16.7 h resulted in lower effluent sulfide concentrations, from an influent concentration of about 170–190 μM to an effluent concentration of 110 and 3 μM, respectively. A reduction in effluent nitrate concentrations in both core systems indicated the presence of indigenous nitrate-using populations. After strain F was inoculated into the core system operated at the shorter retention time, the effluent sulfide concentration decreased from 110 to 16–25 μM. The effluent sulfate concentration increased, and the effluent nitrate concentration decreased concomitant with the presence of high concentrations of denitrifying thiobacilli in the inoculated core system. The denitrifying thiobacilli detected after inoculation were presumed to be strain F since these organisms were not detected in this core system before inoculation, or in any of the samples from the uninoculated core system. These data suggest that the efficacy of the nitrate treatment may depend on the residence times of the liquids in the core system, and that inoculation with strain F was required to reduce sulfide levels to <20 μM in the core system operated at a short hydraulic retention time.

Multi-Species Ecotoxicity Assessment of Petroleum-Contaminated Soil

In 1992, a study was begun to compare the effect of landfarming vs. natural attenuation on the restoration of soil that had been contaminated with crude oil. Each of three lysimeters was filled with a sandy loam topsoil, and crude oil was applied to two of the lysimeters. One of the contaminated lysimeters was tilled, watered, and received a one-time application of fertilizer (N, P, K). No amendments were added to the second contaminated lysimeter, and the third was left uncontaminated. The lysimeters were monitored for 6 months and then left unattended. In 1995 and again in 1997 we sampled these lysimeters to evaluate the long-term effects of contamination and bioremediation. In 1995 we found marked effects on soil chemistry, bacterial, fungal, nematode, and plant populations and a higher rate of bioremediation in the fertilized-contaminated lysimeter (Lawlor et al., 1997). Data from 1997 and previously unreported data from 1995 are the subject of the current report. In 1997, low densities of hydrocarbon-degrading bacteria were found in all the lysimeters and little loss of TPH from the two contaminated lysimeters, suggesting a decreased rate of bioremediation. Nevertheless, there were increases in diversity and number of functional groups of bacteria, nematodes, and native plant species. However, molecular analyses revealed marked differences remained in the composition of dominant eubacterial species, and tests of soybeans indicated field conditions remained unsuitable for these plants.