Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions.

Benzene was mineralized to CO2 by aquifer-derived microorganisms under strictly anaerobic conditions. The degradation occurred in microcosms containing gasoline-contaminated subsurface sediment from Seal Beach, California, and anaerobic, sulfide-reduced defined mineral medium supplemented with 20 mM sulfate. Benzene, at initial concentrations ranging from 40 to 200 microM, was depleted in all microcosms and more than 90% of 14C-labeled benzene was mineralized to 14CO2.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

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.     

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.     

Chloroethene biodegradation in sediments at 4 degrees C

Microbial reductive dechlorination of [1,2-14C]trichloroethene to [14C]cis-dichloroethene and [14C]vinyl chloride was observed at 4 degrees C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-14C]cis-dichloroethene and [1,2-14C]vinyl chloride to 14CO2 also was observed under these conditions.     

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.     

Microbial oxidation of 1,2-dichloroethane under anoxic conditions with nitrate as electron acceptor in mixed and pure cultures

Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.     

Degradation of 2-sec-butyl-4,6-dinitrophenol (dinoseb) by Clostridium bifermentans KMR-1.

A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6-dinitrophenol (dinoseb) to a level below the limit of detection by high-performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates. KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source. KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2. The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2. During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases. This is the first demonstration of dinoseb degradation by a pure microbial culture.     

Anaerobic Benzene Biodegradation: A Microcosm Survey

Benzene-amended microcosms prepared with saturated soil or sediment from five hydrocarbon-contaminated sites and one pristine site were monitored for a year and a half to determine the rate of benzene biodegradation under a variety of electron-accepting conditions. Sustainable benzene degradation was observed under specific conditions in microcosms from four of the six sites. Significant differences were observed between sites with respect to lag times before the onset of degradation, rates of degradation, sustainability of the activity, and environmental conditions supporting degradation. Benzene degradation was observed under sulfate-reducing, nitrate-reducing, and iron(III)-reducing conditions, but not under methanogenic conditions. The presence of competing substrates such as toluene, xylenes, and ethylbenzene was found to inhibit anaerobic benzene degradation in microcosms where sulfate or possibly nitrate was the electron acceptor for benzene degradation, but not in microcosms from where iron(III) was the electron acceptor. The presence of organic matter, indicated by a high fraction organic carbon (f_oc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest f_oc exhibited the longest lag times before the onset of benzene degradation. The initial extent of hydrocarbon contamination did not appear to correlate with anaerobic benzene-degrading activity.     

Acetogenic Capacities and the Anaerobic Turnover of Carbon in a Kansas Prairie Soil

To assess the anaerobic capacities of a temperate grassland soil, a Kansas prairie soil was incubated anaerobically as either soil-water (1:2) suspensions or as soil microcosms at 78% soil water-holding capacity. Prairie soil formed acetate and CO(inf2) as the two main initial carbonaceous products from the anaerobic turnover of endogenous organic matter. Metabolic capacities of soil suspensions and microcosms were similar. Rates of acetate formation from endogenous organic matter in soil-water suspensions incubated at 40, 30, and 15(deg)C approximated 3.3, 2.4, and 1.1 (mu)g of acetate per g (dry weight) of soil per h, respectively. Supplemental H(inf2) and CO(inf2) were subject to consumption with the apparent concomitant synthesis of acetate in both soil suspensions and soil microcosms. In soil microcosms, rates of H(inf2)-dependent acetogenesis at 30 and 55(deg)C were nearly equivalent. The uptake of supplemental H(inf2) was not coupled to methanogenesis under any condition examined. These anaerobic activities were relatively stable when soils were subjected to either aerobic drying or alternating periods of O(inf2) enrichment. On the basis of the formation of nitrogen (N(inf2)), denitrification was engaged during anaerobic incubation periods; nitrous oxide (N(inf2)O) was also formed under certain conditions. Although extended incubation of soil induced the delayed methanogenic turnover of acetate, acetate was subject to immediate turnover under either O(inf2)- or nitrate-enriched conditions. These studies support the following concepts: (i) obligately anaerobic bacteria such as acetogenic bacteria are stable to periods of aerobiosis and are active in the anaerobic microsites of oxic soils, and (ii) acetate synthesized in anaerobic microsites of oxic terrestrial soils constitutes a trophic link to both aerobic and anaerobic microbial communities.     

Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1.

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.     

Evaluating Biodegradation as a Primary and Secondary Treatment for Removing RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) from a Perched Aquifer

Ground water beneath the U.S. Department of Energy (USDOE) Pantex Plant is contaminated with the high explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). The authors evaluated biodegradation as a remedial option by measuring RDX mineralization in Pantex aquifer microcosms spiked with ¹⁴C-labeled RDX (75 g soil, 15 ml of 5 mg RDX/L). Under anaerobic conditions and constant temperature (16°C), cumulative ¹⁴CO₂ production ranged between 52% and 70% after 49 days, with nutrient-amended (C, N, P) microcosms yielding the greatest mineralization (70%). The authors also evaluated biodegradation as a secondary treatment for removing RDX degradates following oxidation by permanganate (KMnO₄) or reduction by dithionite-reduced aquifer solids (i.e., redox barriers). Under this coupled abiotic/biotic scenario, we found that although unconsumed permanganate initially inhibited biodegradation, > 48% of the initial ¹⁴C-RDX was recovered as ¹⁴CO₂ within 77 days. Following exposure to dithionite-reduced solids, RDX transformation products were also readily mineralized (> 47% in 98 days). When we seeded Pantex aquifer material into Ottawa Sand that had no prior exposure to RDX, mineralization increased 100%, indicating that the Pantex aquifer may have an adapted microbial community that could be exploited for remediation purposes. These results indicate that biodegradation effectively transformed and mineralized RDX in Pantex aquifer microcosms. Additionally, biodegradation may be an excellent secondary treatment for RDX degradates produced from in situ treatment with permanganate or redox barriers.     

Anaerobic biodegradation of aliphatic polyesters: poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) and poly(epsilon-caprolactone)

Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate), PHBO, represents a class of PHA copolymers that contain both short-chain-length and medium-chain-length repeat units. Radiolabeled and cold PHBO, containing 90 mol % 3-hydroxybutyrate and 10 mol % 3-hydroxyoctanoate were chemically synthesized using a new difunctional alkoxyzinc initiator. (14)C-PHBO was incubated with samples of anaerobic digester sludge, septage, freshwater sediment, and marine sediment under conditions resembling those in situ. In addition, it was incubated in laboratory-scale landfill reactors. (14)C-PCL (poly-epsilon-caprolactone) was incubated with anaerobic digester sludge and in landfill reactors. Biodegradation was determined by measuring generation of (14)CO(2) and (14)CH(4) resulting from mineralization of the radiolabeled polymers. PHBO was extensively mineralized in digester sludge, septage sediments, and the landfill reactors, with half-lives less than 30 days. PCL was not significantly mineralized in digester sludge over 122 days. In the landfill reactors, PCL mineralization was slow and was preceded by a long lag period (>200 days), suggesting that PCL mineralization is limited by its rate of hydrolysis. The results indicate that PHBO is practically biodegradable in the major anaerobic habitats that it may enter. In contrast, anaerobic biodegradation of PCL is less ubiquitous and much slower.     

Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia.

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions.     

Benzene Oxidation Coupled to Sulfate Reduction

Highly reduced sediments from San Diego Bay, Calif., that were incubated under strictly anaerobic conditions metabolized benzene within 55 days when they were exposed initially to 1 (mu)M benzene. The rate of benzene metabolism increased as benzene was added back to the benzene-adapted sediments. When a [(sup14)C]benzene tracer was included with the benzene added to benzene-adapted sediments, 92% of the added radioactivity was recovered as (sup14)CO(inf2). Molybdate, an inhibitor of sulfate reduction, inhibited benzene uptake and production of (sup14)CO(inf2) from [(sup14)C]benzene. Benzene metabolism stopped when the sediments became sulfate depleted, and benzene uptake resumed when sulfate was added again. The stoichiometry of benzene uptake and sulfate reduction was consistent with the hypothesis that sulfate was the principal electron acceptor for benzene oxidation. Isotope trapping experiments performed with [(sup14)C]benzene revealed that there was no production of such potential extracellular intermediates of benzene oxidation as phenol, benzoate, p-hydroxybenzoate, cyclohexane, catechol, and acetate. The results demonstrate that benzene can be oxidized in the absence of O(inf2), with sulfate serving as the electron acceptor, and suggest that some sulfate reducers are capable of completely oxidizing benzene to carbon dioxide without the production of extracellular intermediates. Although anaerobic benzene oxidation coupled to chelated Fe(III) has been documented previously, the study reported here provides the first example of a natural sediment compound that can serve as an electron acceptor for anaerobic benzene oxidation.     

Microbial mineralization of diisopropanolamine.

Diisopropanolamine (DIPA) is a "sweetening agent" used to remove hydrogen sulfide from sour natural gas, and it is a contaminant at some sour gas treatment facilities in western Canada. To investigate the biodegradation of this alkanolamine, 14C-DIPA was used in anaerobic and aerobic mineralization studies. Between 3 and 78% of the radioactivity from this compound was released as 14CO2 in sediment-enrichment cultures incubated under nitrate-reducing conditions. Similarly, 12-78% of the label was converted to 14CO2 in sediment-enrichment cultures incubated under Mn(IV)-reducing conditions. These activities were observed at 8 degrees C, a typical groundwater temperature in western Canada, and at 28 degrees C. In contrast, DIPA-degrading activity was difficult to sustain under Fe(III)-reducing conditions, and < 25% of the radioactive label from 14C-DIPA was liberated as 14CO2. Two mixed cultures and two isolates (both irregular, non-sporeforming, Gram-positive rods) were used to assess aerobic mineralization of 14C-DIPA. The aerobic mixed cultures released 73 and 79% of the radioactive label as 14CO2, whereas the pure cultures liberated only 39 and 47% as 14CO2. Between one-third and one-half of the nitrogen from DIPA was found as ammonium-N in aerobic batch cultures. These results clearly demonstrate that DIPA is mineralized under a variety of incubation conditions.     

Evidence for aceticlastic methanogenesis in the presence of sulfate in a gas condensate-contaminated aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-(14)C]acetate or [U-(14)C]acetate produced higher quantities of CH(4) compared to CO(2) in the presence or absence of sulfate.(14)CH(4) accounted for 70 to 100% of the total labeled gas in the [(14)C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

Potential for Aerobic and Anaerobic Biodegradation of Petroleum Hydrocarbons in Boreal Subsurface

We studied the role of aerobic and anaerobic petroleum hydrocarbon degradation at a boreal, light-weight fuel and lubrication oil contaminated site undergoing natural attenuation. At the site, anoxic conditions prevailed with high concentrations of CH4 (up to 25% v/v) and CO2 (up to 18% v/v) in the soil gas throughout the year. Subsurface samples were obtained mainly from the anoxic parts of the site and they represented both the unsaturated and saturated zone. The samples were incubated in microcosms at near in situ conditions (i.e. in situ temperature 8 degrees C, aerobic and anaerobic conditions, no nutrient amendments) resulting in the removal of mineral oil (as determined by gas chromatography) aerobically as well as anaerobically. In the aerobic microcosms on average 31% and 27% of the initial mineral oil was removed during a 3- and 4-month incubation, respectively. In the anaerobic microcosms, on average 44% and 15% of the initial mineral oil was removed during a 12- and 10-month anaerobic incubation, respectively, and e.g. n-alkanes from C11 to C15 were removed. A methane production rate of up to 2.5 microg CH4 h(-1) g(-1) dwt was recorded in these microcosms. In the aerobic as well as anaerobic microcosms, typically 90% of the mineral oil degraded belonged to the mineral oil fraction that eluted from the gas chromatograph after C10 and before C15, while 10% belonged to the fraction that eluted after C15 and before C40. Our results suggest that anaerobic petroleum hydrocarbon degradation, including n-alkane degradation, under methanogenic conditions plays a significant role in the natural attenuation in boreal conditions.     

Fate of explosives and their metabolites in bioslurrytreatment processes

Microcosm tests simulating bioslurry reactors with 40% soil content, containing high concentrations of TNT and/or RDX, and spiked with either [14C]-TNT or [14C]-RDX were conducted to investigate the fate of explosives and their metabolites in bioslurry treatment processes. RDX is recalcitrant to indigenous microorganisms in soil and activated sludge under aerobic conditions. However, soil indigenous microorganisms alone were able to mineralize 15% of RDX to CO2 under anaerobic condition, and supplementation of municipal anaerobic sludge as an exogenous source of microorganisms significantly enhanced the RDX mineralization to 60%. RDX mineralizing activity of microorganisms in soil and sludge was significantly inhibited by the presence of TNT. TNT mineralization was poor (< 2%) and was not markedly improved by the supplement of aerobic or anaerobic sludge. Partitioning studies of [14C]-TNT in the microcosms revealed that the removal of TNT during the bioslurry process was due mainly to the transformation of TNT and irreversible binding of TNT metabolites onto soil matrix. In the case of RDX under anaerobic conditions, a significant portion (35%) of original radioactivity was also incorporated into the biomass and bound to the soil matrix.