Zohre Kurt

Microbial Community Degradation of Widely Used Quaternary Ammonium Disinfectants

ABSTRACT Benzalkonium chlorides (BACs) are disinfectants widely used in a variety of clinical and environmental settings to prevent microbial infections, and they are frequently detected in nontarget environments, such as aquatic and engineered biological systems, even at toxic levels. Therefore, microbial degradation of BACs has important ramifications for alleviating disinfectant toxicity in nontarget environments as well as compromising disinfectant efficacy in target environments. However, how natural microbial communities respond to BAC exposure and what genes underlie BAC biodegradation remain elusive. Our previous metagenomic analysis of a river sediment microbial community revealed that BAC exposure selected for a low-diversity community, dominated by several members of the Pseudomonas genus that quickly degraded BACs. To elucidate the genetic determinants of BAC degradation, we conducted time-series metatranscriptomic analysis of this microbial community during a complete feeding cycle with BACs as the sole carbon and energy source under aerobic conditions. Metatranscriptomic profiles revealed a candidate gene for BAC dealkylation, the first step in BAC biodegradation that results in a product 500 times less toxic. Subsequent biochemical assays and isolate characterization verified that the putative amine oxidase gene product was functionally capable of initiating BAC degradation. Our analysis also revealed cooperative interactions among community members to alleviate BAC toxicity, such as the further degradation of BAC dealkylation by-products by organisms not encoding amine oxidase. Collectively, our results advance the understanding of BAC aerobic biodegradation and provide genetic biomarkers to assess the critical first step of this process in nontarget environments.

Biodegradation of Chlorobenzene, 1,2-Dichlorobenzene, and 1,4-Dichlorobenzene in the Vadose Zone

Much of the microbial activity in nature takes place at interfaces, which are often associated with redox discontinuities. One example is the oxic/anoxic interface where polluted groundwater interacts with the overlying vadose zone. We tested whether microbes in the vadose zone can use synthetic chemicals as electron donors and thus protect the overlying air and buildings from groundwater pollutants. Samples from the vadose zone of a site contaminated with chlorobenzene (CB), 1,2-dichlorobenzene (12DCB), and 1,4-dichlorobenzene (14DCB) were packed in a multiport column to simulate the interface of the vadose zone with an underlying groundwater plume. A mixture of CB, 12DCB, and 14DCB in anoxic water was pumped continuously through the bottom of column to an outlet below the first sampling port to create an oxic/anoxic interface and a capillary fringe. Removal to below the detection limits by rapid biodegradation with rates of 21 ± 1 mg of CB • m(-2) • d(-1), 3.7 ± 0.5 mg of 12DCB • m(-2) • d(-1), and 7.4 ± 0.7 mg of 1.4 DCB • m(-2) • d(-1) indicated that natural attenuation in the capillary fringe can prevent the migration of CB, 12DCB, and 14DCB vapors. Enumeration of bacteria capable of degrading chlorobenzenes suggested that most of the biodegradation takes place within the first 10 cm above the saturated zone. Biodegradation also increased the upward flux of contaminants and thus enhanced their elimination from the underlying water. The results revealed a substantial biodegradation capacity for chlorinated aromatic compounds at the oxic/anoxic interface and illustrate the role of microbes in creating steep redox gradients.

Biodegradation of Chlorobenzene and Nitrobenzene at Interfaces between Sediment and Water

Plumes of contaminated groundwater often pass through an oxic/anoxic interface when they discharge into surface water bodies. We tested the hypothesis that contaminants recalcitrant under anaerobic conditions but degradable under aerobic conditions can be biodegraded at the interface resulting in the protection of the overlying water. Flow-through columns containing sediment and water were used to evaluate degradation of synthetic organic compounds at the thin organic layer at the sediment/water interface. Sediment samples collected from several sites contaminated with nitrobenzene (NB) or chlorobenzene (CB) were tested for their biodegradation capacities in the columns. The biodegradation capacities of sediment in the columns were 2-4.2 g CB·m(-2)·d(-1) and 6.5 g NB·m(2)·d(-1). Bacteria able to carry out rapid and complete biodegradation of CB or NB were detected in the sediments prior to the experiments, which suggested the presence of an active microbial community at the contaminated sites. The results revealed robust biodegradation of toxic compounds migrating across the sediment/water interface and indicate that the biodegradation capacities were sufficient to eliminate transport of the contaminants to the overlying water in the field.

Isotope fractionation associated with the biodegradation of 2- and 4-nitrophenols via monooxygenation pathways

Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding (13)C- and (15)N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1-8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ(15)N vs δ(13)C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary (15)N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.

Modeling aerobic biodegradation in the capillary fringe.

Vapor intrusion from volatile subsurface contaminants can be mitigated by aerobic biodegradation. Laboratory column studies with contaminant sources of chlorobenzene and a mixture of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene showed that contaminants were rapidly degraded in thin reactive zones with high biomass and low substrate concentrations in the vicinity of the capillary fringe. Such behavior was well characterized by a model that includes oxygen-, substrate-, and biomass-dependent biodegradation kinetics along with diffusive transport processes. An analytical solution was derived to provide theoretical support for the simplification of reaction kinetics and the approximation of reactive zone location and mass flux relationships at steady state. Results demonstrate the potential of aerobic natural attenuation in the capillary fringe for preventing contaminant migration in the unsaturated zone. The solution indicates that increasing contaminant mass flux into the column creates a thinner reactive zone and pushes it toward the oxygen boundary, resulting in a shorter distance to the oxygen source and a larger oxygen mass flux that balances the contaminant mass flux. As a consequence, the aerobic biodegradation can reduce high contaminant concentrations to low levels within the capillary fringe and unsaturated zone. The results are consistent with the observations of thin reactive layers at the interface in unsaturated zones. The model considers biomass while including biodegradation in the capillary fringe and unsaturated zone and clearly demonstrates that microbial communities capable of using the contaminants as electron donors may lead to instantaneous degradation kinetics in the capillary fringe and unsaturated zone.

Biodegradation of cis-dichloroethene and vinyl chloride in the capillary fringe.

Volatile chlorinated compounds are major pollutants in groundwater, and they pose a risk of vapor intrusion into buildings. Vapor intrusion can be prevented by natural attenuation in the vadose zone if biodegradation mechanisms can be established. In this study, we tested the hypothesis that bacteria can use cis-dichloroethene (cis-DCE) or vinyl chloride (VC) as an electron donor in the vadose zone. Anoxic water containing cis-DCE or VC was pumped continuously beneath laboratory columns that represented the vadose zone. Columns were inoculated with Polaromonas sp. strain JS666, which grows aerobically on cis-DCE, or with Mycobacterium sp. JS60 and Nocardiodes sp. JS614 that grow on VC. Complete biodegradation with fluxes of 84 ± 15 μmol of cis-DCE · m(-2) · hr(-1) and 218 ± 25 μmole VC·m(-2) · h(-1) within the 23 cm column indicated that microbial activities can prevent the migration of cis-DCE and VC vapors. Oxygen and volatile compound profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place in the first 10 cm above the saturated zone within the capillary fringe. The results revealed that cis-DCE and VC can be biodegraded readily at the oxic/anoxic interfaces in the vadose zone if appropriate microbes are present.

Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution

Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), which is a molecule secreted by Streptomyces scabies, the plant pathogen responsible for potato scab. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis in vitro, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but is readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expand our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates.

Natural Attenuation of Nonvolatile Contaminants in the Capillary Fringe

When anoxic polluted groundwater encounters the overlying vadose zone an oxic/anoxic interface is created, often near the capillary fringe. Biodegradation of volatile contaminants in the capillary fringe can prevent vapor migration. In contrast, the biodegradation of nonvolatile contaminants in the vadose zone has received comparatively little attention. Nonvolatile compounds do not cause vapor intrusion, but they still move with the groundwater and are major contaminants. Aniline (AN) and diphenylamine (DPA) are examples of toxic nonvolatile contaminants found often at dye and munitions manufacturing sites. In this study, we tested the hypothesis that bacteria can aerobically biodegrade AN and DPA in the capillary fringe and decrease the contaminant concentrations in the anoxic plume beneath the vadose zone. Laboratory multiport columns that represented the unsaturated zone were used to evaluate degradation of AN or DPA in contaminated water. The biodegradation fluxes of the contaminants were estimated to be 113 ± 26 mg AN·m(-2)·h(-1) and 76 ± 18 mg DPA·m(-2)·h(-1) in the presence of bacteria known to degrade AN and DPA. Oxygen and contaminant profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place within the lower part of the capillary fringe. The results indicate that bacteria capable of contaminant biodegradation in the capillary fringe can create a sink for nonvolatile contaminants.

Resveratrol as a Growth Substrate for Bacteria from the Rhizosphere

ABSTRACT Resveratrol is among the best-known secondary plant metabolites because of its antioxidant, anti-inflammatory, and anticancer properties. It also is an important allelopathic chemical widely credited with the protection of plants from pathogens. The ecological role of resveratrol in natural habitats is difficult to establish rigorously, because it does not seem to accumulate outside plant tissue. It is likely that bacterial degradation plays a key role in determining the persistence, and thus the ecological role, of resveratrol in soil. Here, we report the isolation of an Acinetobacter species that can use resveratrol as a sole carbon source from the rhizosphere of peanut plants. Both molecular and biochemical techniques indicate that the pathway starts with the conversion of resveratrol to 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde. The aldehydes are oxidized to substituted benzoates that subsequently enter central metabolism. The gene that encodes the enzyme responsible for the oxidative cleavage of resveratrol was cloned and expressed in Escherichia coli to establish its function. Its physiological role in the resveratrol catabolic pathway was established by knockouts and by the reverse transcription-quantitative PCR (RT-qPCR) demonstration of expression during growth on resveratrol. The results establish the presence and capabilities of resveratrol-degrading bacteria in the rhizosphere of the peanut plants and set the stage for studies to evaluate the role of the bacteria in plant allelopathy. IMPORTANCE In addition to its antioxidant properties, resveratrol is representative of a broad array of allelopathic chemicals produced by plants to inhibit competitors, herbivores, and pathogens. The bacterial degradation of such chemicals in the rhizosphere would reduce the effects of the chemicals. Therefore, it is important to understand the activity and ecological role of bacteria that biodegrade resveratrol near the plants that produce it. This study describes the isolation from the peanut rhizosphere of bacteria that can grow on resveratrol. The characterization of the initial steps in the biodegradation process sets the stage for the investigation of the evolution of the catabolic pathways responsible for the biodegradation of resveratrol and its homologs.