Biodegradation of tert-butylphenyl diphenyl phosphate

The biodegradation of tert-butylphenyl diphenyl phosphate (BPDP) was examined in microcosms containing sediment and water from five different ecosystems as part of our studies to elucidate the environmental fate of phosphate ester flame retardants. Biodegradation of [14C]BPDP was monitored in the environmental microcosms by measuring the evolution of 14CO2. Over 37% of BPDP was mineralized after 8 weeks in microcosms from an ecosystem which had chronic exposure to agricultural chemicals. In contrast, only 1.7% of BPDP was degraded to 14CO2 in samples collected from a noncontaminated site. The exposure concentration of BPDP affected the percentage which was degraded to 14CO2 in microcosms from the two most active ecosystems. Mineralization was highest at a concentration of 0.1 mg of BPDP and was inhibited with 10- and 100-fold higher concentrations of BPDP in these microcosms. Indigenous heterotrophic and BPDP-utilizing microbial populations and phosphoesterase enzyme activities were highest in sediments which had the highest biodegradation of BPDP. We observed adaptive increases in both microbial populations and phosphoesterase enzymes in some sediments acclimated to BPDP. Chemical analyses of the residues in the microcosms indicated undegraded BPDP and minor amounts of phenol, tert-butylphenol, diphenyl phosphate, and triphenyl phosphate as biodegradation products. These data suggest that the microbial degradation of BPDP results from at least three catabolic processes and is highest when low concentrations of BPDP are exposed to sediment microorganisms of eutrophic ecosystems which have high phosphotri- and diesterase activities and previous exposure to anthropogenic chemicals.     

Naphthalene biodegradation in environmental microcosms: estimates of degradation rates and characterization of metabolites.

Naphthalene biodegradation was investigated in microcosms containing sediment and water collected from three ecosystems which varied in past exposure to anthropogenic and petrogenic chemicals. Mineralization half-lives for naphthalene in microcosms ranged from 2.4 weeks in sediment chronically exposed to petroleum hydrocarbons to 4.4 weeks in sediment from a pristine environment. Microbiological analysis of sediments indicated that hydrocarbon-utilizing microbial populations also varied among ecosystems and were 5 to 12 times greater in sediment after chronic petrogenic chemical exposure than in sediment from an uncontaminated ecosystem. Sediment from an ecosystem exposed to agricultural chemicals had a mineralization half-life of 3.2 weeks for naphthalene and showed about a 30-fold increase in heterotrophic bacterial populations in comparison to uncontaminated sediments, but only a 2- to 3-fold increase in hydrocarbon-degrading bacteria. Analysis of organic solvent-extractable residues from the microcosms by high-pressure liquid chromatography detected polar metabolites which accounted for 1 to 3% of the total radioactivity. Purification of these residues by thin-layer chromatography and further analysis by gas chromatography-mass spectrometry indicated that cis-1,2-dihydroxy-1,2-dihydronaphthalene, 1-naphthol, salicylic acid, and catechol were metabolites of naphthalene. These results provide useful estimates for the rates of naphthalene mineralization in different natural ecosystems and on the degradative pathway for microbial metabolism of naphthalene in freshwater and estuarine environments.     

Naphthalene biodegradation in environmental microcosms: estimates of degradation rates and characterization of metabolites

Naphthalene biodegradation was investigated in microcosms containing sediment and water collected from three ecosystems which varied in past exposure to anthropogenic and petrogenic chemicals. Mineralization half-lives for naphthalene in microcosms ranged from 2.4 weeks in sediment chronically exposed to petroleum hydrocarbons to 4.4 weeks in sediment from a pristine environment. Microbiological analysis of sediments indicated that hydrocarbon-utilizing microbial populations also varied among ecosystems and were 5 to 12 times greater in sediment after chronic petrogenic chemical exposure than in sediment from an uncontaminated ecosystem. Sediment from an ecosystem exposed to agricultural chemicals had a mineralization half-life of 3.2 weeks for naphthalene and showed about a 30-fold increase in heterotrophic bacterial populations in comparison to uncontaminated sediments, but only a 2- to 3-fold increase in hydrocarbon-degrading bacteria. Analysis of organic solvent-extractable residues from the microcosms by high-pressure liquid chromatography detected polar metabolites which accounted for 1 to 3% of the total radioactivity. Purification of these residues by thin-layer chromatography and further analysis by gas chromatography-mass spectrometry indicated that cis-1,2-dihydroxy-1,2-dihydronaphthalene, 1-naphthol, salicylic acid, and catechol were metabolites of naphthalene. These results provide useful estimates for the rates of naphthalene mineralization in different natural ecosystems and on the degradative pathway for microbial metabolism of naphthalene in freshwater and estuarine environments.     

Microbial mineralization of VC and DCE under different terminal electron accepting conditions

Production of 14CO2 from [1,2-14C] dichloroethene (DCE) or [1,2-14C] vinyl chloride (VC) was quantified in aquifer and stream-bed sediment microcosms to evaluate the potential for microbial mineralization as a pathway for DCE and VC biodegradation under aerobic, Fe(III)-reducing, SO4-reducing, and methanogenic conditions. Mineralization of [1,2-14C] DCE and [1,2-14C] VC to 14CO2 decreased under increasingly reducing conditions, but significant mineralization was observed for both sediments even under anaerobic conditions. VC mineralization decreased in the order of aerobic > Fe(III)-reducing > SO4-reducing > methanogenic conditions. For both sediments, VC mineralization was greater than DCE mineralization under all electron-accepting conditions examined. For both sediments, DCE mineralization was at least two times greater under aerobic conditions than under anaerobic conditions. Although significant microbial mineralization of DCE was observed under anaerobic conditions, recovery of 14CO2 did not differ substantially between anaerobic treatments.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate.

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.     

Aerobic biodegradation of methyl tert-butyl ether by aquifer bacteria from leaking underground storage tank sites.

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-(14)C]MTBE was mineralized to (14)CO(2). Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems.

Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation.     

Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems

Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation.     

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.     

Laboratory Evaluations of Factors Affecting Biodegradation of Sulfolane and Diisopropanolamine

Sulfolane and diisopropanolamine (DIPA) are used in the Sulfinol® process to remove hydrogen sulfide from sour natural gas. This process has been used in western Canada since the early 1960s, and contamination of groundwater has occurred from surface spills and from seepage from landfills and unlined process water storage ponds. Aquifer sediments from contaminated and uncontaminated areas, and muds in a wetland downgradient from the contaminated plume, were collected from a gas plant. Vigorously agitated shake-flask cultures and gently agitated 2.5-L microcosms consisting of contaminated sediment, mud and groundwater, or wetland water were used to study the biodegradation of sulfolane and DIPA. The aerobic shake-flask method showed that all five of these materials contained microbial communities that biodegraded both compounds. Microorganisms in all samples, except the uncontaminated aquifer sediment, degraded both compounds in the aerobic 2.5-L microcosms. In general, the biodegradation occurred more rapidly in the shake-flask cultures. The addition of P greatly enhanced the degradation of sulfolane and DIPA, whereas the addition of N yielded little stimulation.     

Kinetic and microbial community analysis of methyl ethyl ketone biodegradation in aquifer sediments

Methyl ethyl ketone (MEK) is a common groundwater contaminant often present with more toxic compounds of primary interest. Because of this, few studies have been performed to determine the effect of microbial community structure on MEK biodegradation rates in aquifer sediments. Here, microcosms were prepared with aquifer sediments containing MEK following a massive spill event and compared to laboratory-spiked sediments, with MEK biodegradation rates quantified under mixed aerobic/anaerobic conditions. Biodegradation was achieved in MEK-contaminated site sediment microcosms at about half of the solubility (356 mg/L) with largely Firmicutes population under iron-reducing conditions. MEK was biodegraded at a higher rate [4.0 ± 0.74 mg/(L days)] in previously exposed site samples compared to previously uncontaminated sediments [0.51 ± 0.14 mg/(L days)]. Amplicon sequencing and denaturing gradient gel electrophoresis of 16S rRNA genes were combined to understand the relationship between contamination levels, biodegradation, and community structure across the plume. More heavily contaminated sediments collected from an MEK-contaminated field site had the most similar communities than less contaminated sediments from the same site despite differences in sediment texture. The more diverse microbial community observed in the laboratory-spiked sediments reduced MEK concentration 47 % over 92 days. Results of this study suggest lower rates of MEK biodegradation in iron-reducing aquifer sediments than previously reported for methanogenic conditions and biodegradation rates comparable to previously reported nitrate- and sulfate-reducing conditions.     

RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Biodegradation in Aquifer Sediments under Manganese-Reducing Conditions

A shallow, RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-contaminated aquifer at Naval Submarine Base Bangor has been characterized as predominantly manganese-reducing, anoxic with local pockets of oxic conditions. The potential contribution of microbial RDX degradation to localized decreases observed in aquifer RDX concentrations was assessed in sediment microcosms amended with [U-¹⁴C] RDX. Greater than 85% mineralization of ¹⁴C-RDX to ¹⁴CO₂ was observed in aquifer sediment microcosms under native, manganese-reducing, anoxic conditions. Significant increases in the mineralization of ¹⁴C-RDX to ¹⁴CO₂ were observed in anoxic microcosms under NO₃-amended or Mn(IV)-amended conditions. No evidence of ¹⁴C-RDX biodegradation was observed under oxic conditions. These results indicate that microbial degradation of RDX may contribute to natural attenuation of RDX in manganese-reducing aquifer systems.     

Effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities.

The adaptation of a mixed aquatic microbial community to phenol was examined in microcosms receiving phenol as a sole carbon source. Extended exposure (adaptation) to phenol resulted in adaptation of the microbial community to the structurally related aromatic compounds m-cresol, m-aminophenol, and p-chlorophenol. The increased biodegradation potential of the phenol-adapted microbial community was accompanied by a concurrent increase in the number of microorganisms able to degrade the three test compounds. Thus, adaptation to the three test chemicals was likely a growth-related result of extended exposure to phenol. The results indicate that adaptation to a single chemical may increase the assimilative capacity of an aquatic environment for other related chemicals even in the absence of adaptation-inducing levels of those materials.     

Microbial communities of the discharge zone of oil- and gas-bearing fluids in low-mineral Lake Baikal

At the site of natural ingress of oil, microbial diversity in the Central Baikal bottom sediments differing in the chemical composition of pore waters was studied by molecular biological techniques. The sediments saturated with oil and methane were found to contain members of 10 bacterial and 2 archaeal phyla. The oxidized sediment layer contained methanotrophic bacteria belonging to the Alphaproteobacteria, which had a specific structure of the pmoA gene and clustered together with uncultured methanotrophs from cold ecosystems. The upper sediment layer also contained oil-oxidizing bacteria and the alkB genes most closely related to those of Rhodococcus. The microbial community of reduced sediments exhibited lower diversity and was represented mostly by the organisms involved in hydrocarbon biodegradation.     

Biodegradation of Trichloroethylene and Its Anaerobic Daughter Products in Freshwater Wetland Sediments

The wide range of redox conditions and diversity of microbial populations in organic-rich wetland sediments could enhance biodegradation of chlorinated solvents. To evaluate potential biodegradation rates of trichloroethylene (TCE) and its anaerobic daughter products (cis-1,2-dichloroethylene; trans-1,2-dichloroethylene; and vinyl chloride), laboratory microcosms were prepared under methanogenic, sulfate-reducing, and aerobic conditions using sediment and groundwater from a freshwater wetland that is a discharge area for a TCE contaminant plume. Under methanogenic conditions, biodegradation rates of TCE were extremely rapid at 0.30 to 0.37 d^-1 (half-life of about 2 days). Although the TCE biodegradation rate was slower under sulfate-reducing conditions (0.032 d^-1) than under methanogenic conditions, the rate was still two orders of magnitude higher than those reported in the literature for microcosms constructed with sandy aquifer sediments. In the aerobic microcosm experiments, biodegradation occurred only if methane consumption occurred, indicating that methanotrophs were involved. Comparison of laboratory-measured rates indicates that production of the 1,2-dichloroethylene isomers and vinyl chloride by anaerobic TCE biodegradation could be balanced by their consumption through aerobic degradation where methanotrophs are active in wetland sediment. TCE degradation rates estimated using field data (0.009 to 0.016 d^-1) agree with the laboratory-measured rates within a factor of 3 to 22, supporting the feasibility of natural attenuation as a remediation method for contaminated groundwater discharging in this wetland and other similar environments.     

Aerobic Biodegradation of Methyl tert-Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to ¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer.

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microscale decoupling of sediment oxygen consumption and microbial biomass in an oligotrophic lake

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