Christopher M. DeRito

Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment.

Microorganisms maintain the biosphere by catalyzing biogeochemical processes, including biodegradation of organic chemical pollutants. Yet seldom have the responsible agents and their respective genes been identified. Here we used field-based stable isotopic probing (SIP) to discover a group of bacteria responsible for in situ metabolism of an environmental pollutant, naphthalene. We released 13C-labeled naphthalene in a contaminated study site to trace the flow of pollutant carbon into the naturally occurring microbial community. Using GC/MS, molecular biology, and classical microbiological techniques we documented 13CO2 evolution (2.3% of the dose in 8 h), created a library of 16S rRNA gene clones from 13C labeled sediment DNA, identified a taxonomic cluster (92 of 95 clones) from the microbial community involved in metabolism of the added naphthalene, and isolated a previously undescribed bacterium (strain CJ2) from site sediment whose 16S rRNA gene matched that of the dominant member (48%) of the clone library. Strain CJ2 is a β proteobacterium closely related to Polaromonas vacuolata. Moreover, strain CJ2 hosts the sequence of a naphthalene dioxygenase gene, prevalent in site sediment, detected before only in environmental DNA. This investigative strategy may have general application for elucidating the bases of many biogeochemical processes, hence for advancing knowledge and management of ecological and industrial systems that rely on microorganisms.

Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA

ABSTRACT Our goal was to develop a field soil biodegradation assay using 13C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13CO2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13CO2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF6, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13CO2. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13CO2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community

ABSTRACT The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that 13C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of α-, β-, and γ-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.

Field-based and laboratory stable isotope probing surveys of the identities of both aerobic and anaerobic benzene-metabolizing microorganisms in freshwater sediment

Laboratory incubations of coal-tar waste-contaminated sediment microbial communities under relatively controlled physiological conditions were used to interpret results of a field-based stable isotope probing (SIP) assay. Biodegradation activity of 13C-benzene was examined by GC/MS determination of net 13CO2 production and by GC headspace analysis of benzene loss. Key experimental variables were: the site of the assays (laboratory serum-bottle incubations and in situ field sediments), benzene concentration (10, 36 or 200 p.p.m. in laboratory assays), and physiological conditions (anaerobic with or without sulfate or nitrate additions versus aerobic headspace or the uncontrolled field). In anaerobic laboratory incubations of benzene at 10 p.p.m., greater than 60% of the substrate was eliminated within 15 days. During anaerobic incubations of 200 p.p.m. benzene (70 days), 0.9% benzene mineralization occurred. When benzene (36 p.p.m.) was added to sediment with air in the serum-bottle headspace, 14% of the initial 13C was mineralized to 13CO2 in 2.5 days. In the field experiment (178 microg 13C-benzene dosed to undisturbed sediments), net 13CO2 production reached 0.3% within 8.5 h. After isopycnic separation of 13C (heavy)-labelled DNA from the above biodegradation assays, sequencing of 13C-DNA clone libraries revealed a broad diversity of taxa involved in benzene metabolism and distinctive libraries for each biodegradation treatment. Perhaps most importantly, in the field SIP experiment the clone libraries produced were dominated by Pelomonas (betaproteobacteria) sequences similar to those found in the anaerobic 10 p.p.m. benzene laboratory experiment. These data indicate that the physiological conditions that prevail and govern in situ biodegradation of pollutants in the field may be interpreted by knowing the physiological preferences of potentially active populations.

Investigating the biodegradability of perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is an industrial chemical that has become disseminated globally in aquatic and terrestrial habitats, humans, and wildlife. Understanding PFOAs biodegradability (susceptibility to microbial metabolic attack) is a crucial element in developing an informed strategy for predicting and managing this compounds environmental fate. Reasoning that PFOA might be susceptible to reductive defluorination by anaerobic microbial communities, we embarked on a 2-phase experimental approach examining the potential of five different microbial communities (from a municipal waste-water treatment plant, industrial site sediment, an agricultural soil, and soils from two fire training areas) to alter PFOAs molecular structure. A series of primarily anaerobic incubations (up to 259d in duration) were established with acetate, lactate, ethanol, and/or hydrogen gas as electron donors and PFOA (at concentrations of 100 ppm and 100 ppb) as the electron acceptor. Cometabolism of PFOA during reductive dechlorination of trichloroethene (TCE) and during reduction of nitrate, iron, sulfate, and methanogenesis were also examined. Endpoints of potential PFOA transformation included release of fluoride and detection of potential transformation products by LC/Orbitrap MS and LC/accurate radioisotope counting in a (14)C radiotracer study. The strongest indication of PFOA transformation occurred during its potential cometabolism at the 100 ppb concentration during reductive dechlorination of TCE. Despite an extensive search for transformation products to corroborate potential cometabolism of PFOA, we were unable to document any alteration of PFOAs chemical structure. We conclude that, under conditions examined, PFOA is microbiologically inert, hence environmentally persistent.

Subsurface cycling of nitrogen and anaerobic aromatic hydrocarbon biodegradation revealed by nucleic acid and metabolic biomarkers.

ABSTRACT Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ. These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).

Stable isotope probing reveals Trichosporon yeast to be active in situ in soil phenol metabolism.

The aim of this study was to extend the results of our previous stable isotope probing (SIP) investigation: we identified a soil fungus involved in phenol biodegradation at an agricultural field site. DNA extracts from our previous study were examined using fungi-specific PCR amplification of the 18S–28S internal transcribed spacer (ITS) region. We prepared an 80-member clone library using PCR-amplified, 13C-labeled DNA derived from field soil that received 12 daily doses of 13C-phenol. Restriction-fragment-length-polymorphism screening and DNA sequencing revealed a dominant clone (41% of the clone library), the ITS sequence of which corresponded to that of the fungal genus Trichosporon. We successfully grew and isolated a white, filamentous fungus from site soil samples after plating soil dilutions on mineral salts agar containing 250 p.p.m. phenol. Restreaking on both yeast extract–peptone–galactose and Sabouraud dextrose agar plates led to further purification of the fungus, the morphological characteristics of which matched those of the genus Trichosporon. The ITS sequence of our isolated fungus was identical to that of a clone from our SIP-based library, confirming it to be Trichosporon multisporum. High-performance liquid chromatography and turbidometeric analyses showed that the culture was able to metabolize and grow on 200 p.p.m. phenol in an aqueous mineral salts medium within 24 h at room temperature. Gas chromatography–mass spectrometry analysis of 13CO2 respiration from laboratory soil incubations demonstrated accelerated phenol mineralization in treatments inoculated with T. multisporum. These findings show that T. multisporum actively degraded phenol in our field-based, soil experiments.

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community

ABSTRACT Investigations of environmental microbial communities are crucial for the discovery of populations capable of degrading hazardous compounds and may lead to improved bioremediation strategies. The goal of this study was to identify microorganisms responsible for aerobic benzene degradation in coal tar-contaminated groundwater. Benzene degradation was monitored in laboratory incubations of well waters using gas chromatography mass spectrometry (GC-MS). Stable isotope probing (SIP) experiments using [13C]benzene enabled us to obtain 13C-labled community DNA. From this, 16S rRNA clone libraries identified Gammaproteobacteria and Betaproteobacteria as the active benzene-metabolizing microbial populations. Subsequent cultivation experiments yielded nine bacterial isolates that grew in the presence of benzene; five were confirmed in laboratory cultures to grow on benzene. The isolated benzene-degrading organisms were genotypically similar (>97% 16S rRNA gene nucleotide identities) to the organisms identified in SIP experiments. One isolate, Variovorax MAK3, was further investigated for the expression of a putative aromatic ring-hydroxylating dioxygenase (RHD) hypothesized to be involved in benzene degradation. Microcosm experiments using Variovorax MAK3 revealed a 10-fold increase in RHD (Vapar_5383) expression, establishing a link between this gene and benzene degradation. Furthermore, the addition of Variovorax MAK3 to microcosms prepared from site waters accelerated community benzene degradation and correspondingly increased RHD gene expression. In microcosms using uninoculated groundwater, quantitative (q)PCR assays (with 16S rRNA and RDH genes) showed that Variovorax was present and responsive to added benzene. These data demonstrate how the convergence of cultivation-dependent and -independent techniques can boost understandings of active populations and functional genes in complex benzene-degrading microbial communities. IMPORTANCE Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

Diagnostic Tools to Assess Mass Removal Processes During Pulsed Air Sparging of a Petroleum Hydrocarbon Source Zone

During remediation of contaminated aquifers, diagnostic tools can help evaluate whether an intended mass removal process was successfully initiated and acted on specific contaminants of concern. In this study, several diagnostic tools were tested in a controlled-release in situ air sparging experiment that focused on the treatment of target hydrocarbons (e.g., benzene, toluene, ethylbenzene, and xylenes). The tools included compound-specific isotope analysis (CSIA), expression of functional genes (mRNA), and metabolites characteristic of aerobic and anaerobic biodegradation. Total and compound-specific mass balances were established and used, along with traditional monitoring parameters, to validate the results from the various tools. CSIA results indicated biodegradation as the main process contributing to benzene and toluene removal. Removal process-specific isotope shifts were detected in groundwater as well as in the system effluent gas. CSIA, metabolite, and mRNA biomarkers consistently indicated that both aerobic and anaerobic biodegradation of benzene and toluene occurred, but that their relative importance evolved over time and were related to the treatment system operation. While the indicators do not allow quantif cation of the mass removed, they are particularly useful to identify if a removal process has been initiated, and to track relative changes in the predominance of in situ contaminant attenuation processes resulting from remediation efforts.