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)     

Assessment of the microbial potential for nitrate-enhanced bioremediation of a JP-4 fuel-contaminated aquifer

A site that was contaminated with JP-4 jet fuel was characterized microbiologically to assess the feasibility of nitrate-enhanced bioremediation. The results of microcosm studies indicated that the mean pseudo zero-order rate constants for alkylbenzene biodegradation and NO₃ ⁻-N removal were 1.2 and 2.4 mg L⁻¹ per day, respectively. Several alkylbenzenes were removed to a greater extent in samples contaminated with residual JP-4 than in unexposed samples and samples downgradient of the spill; benzene was recalcitrant in all samples. Numbers of total heterotrophs, JP-4-degraders, oligotrophs, total denitrifiers, denitrifiers growing in the presence of JP-4, estimates of cell number by analysis of phospholipid fatty acids, direct counts and aerobic and anaerobic protozoa were determined; however, numbers of microorganisms were not reliable predictors of alkylbenzene biodegradation activity. The presence of aerobic and anaerobic protozoa suggests that protozoa may be active under a variety of different electron acceptor conditions. The results of the characterization study indicated that the site was amenable to nitrate-enhanced bioremediation. Received 12 March 1996/ Accepted in revised form 17 September 1996     

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.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

Anaerobic biodegradation of diesel fuel-contaminated wastewater in a fluidized bed reactor

Diesel fuel spills have a major impact on the quality of groundwater. In this work, the performance of an Anaerobic Fluidized Bed Reactor (AFBR) treating synthetic wastewater is experimentally evaluated. The wastewater comprises tap water containing 100, 200 and 300 mg/L of diesel fuel and nutrients. Granular, inert, activated carbon particles are employed to provide support for biomass inside the reactor where diesel fuel is the sole source of carbon for anaerobic microorganisms. For different rates of organic loading, the AFBR performance is evaluated in terms of the removal of diesel fuel as well as chemical oxygen demand (COD) from wastewater. For the aforementioned diesel fuel concentrations and a wastewater flow rate of 1,200 L/day, the COD removal ranges between 61.9 and 84.1%. The concentration of diesel fuel in the effluent is less than 50 mg/L, and meets the Level II groundwater standards of the MUST guidelines of Alberta.     

Intrinsic potential for immediate biodegradation of toluene in a pristine,energy-limited aquifer

Pristine and energy-limited aquifers are considered to have a low resistance and resilience towards organic pollution. An experiment in an indoor aquifer system revealed an unexpected high intrinsic potential for the attenuation of a short-term toluene contamination. A 30 h pulse of 486 mg of toluene, used as a model contaminant, and deuterated water (D₂O) through an initially pristine, oxic, and organic carbon poor sandy aquifer revealed an immediate aerobic toluene degradation potential. Based on contaminant and tracer break-through curves, as well as mass balance analyses and reactive transport modelling, a contaminant removal of 40 % over a transport distance of only 4.2 m in less than one week of travel time was obtained. The mean first-order degradation rate constant was λ = 0.178 day^?1, corresponding to a half-life time constant T_1/2 of 3.87 days. Toluene-specific stable carbon isotope analysis independently proved that the contaminant mass removal can be attributed to microbial biodegradation. Since average doubling times of indigenous bacterial communities were in the range of months to years, the aerobic biodegradation potential observed is assumed to be present and active in the pristine, energy-limited groundwater ecosystems at any time. Follow-up experiments and field studies will help to quantify the immediate natural attenuation potential of aquifers for selected priority contaminants and will try to identify the key-degraders within the autochthonous microbial communities.     

Aerobic biodegradation of 4-methylquinoline by a soil bacterium.

Methylquinolines and related N-heterocyclic aromatic compounds are common contaminants associated with the use of hydrocarbons in both coal gasification and wood treatment processes. These compounds have been found in groundwater, and many are known mutagens. A stable, five-member bacterial consortium able to degrade 4-methylquinoline was established by selective enrichment using soil collected from an abandoned coal gasification site. The consortium was maintained for 5 years by serial transfer in a medium containing 4-methylquinoline. A gram-negative soil bacterium, strain Lep1, was isolated from the consortium and shown to utilize 4-methylquinoline as a source of carbon and energy during growth in liquid medium. A time course experiment demonstrated that both the isolate Lep1 and the consortium containing Lep1 were able to degrade 4-methylquinoline under aerobic conditions. Complete degradation of 4-methylquinoline by either strain Lep1 alone or the consortium was characterized by the production and eventual disappearance of 2-hydroxy-4-methylquinoline, followed by the appearance and persistence of a second metabolite tentatively identified as a hydroxy-4-methylcoumarin. Currently, there is no indication that 4-methylquinoline degradation proceeds differently in the consortium culture compared with Lep1 alone. This is the first report of 4-methylquinoline biodegradation under aerobic conditions.     

Aerobic metabolic potential of microbial populations indigenous to deep subsurface environments

Subsurface sediment samples, collected from three boreholes ranging in depths from 0.1 to 260 m, were used in substrate mineralization studies to examine the aerobic metabolic potential of microbial populations indigenous to the deep subsurface. Mineralization was measured by quantifying the amount of ¹⁴CO₂ released from radiolabeled acetate, phenol, or 4‐methoxybenzoate added to subsurface sediments at 10 μg g^‐1. Mineralization of the three compounds was observed in all but a few of the subsurface samples and did not decrease with depth. In addition, mineralization data collected from similar geologic formations from the different boreholes indicated that there was significant lateral continuity of microbial activity. Regression analyses were performed to determine which environmental factors were related to microbial metabolic potential. Mineralization was positively correlated with heterotrophic abundance as measured by plate counts. Other parameters that appeared to influence metabolic potential included pH and clay content.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer.

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

一株金黄杆菌对2-吡啶甲酸的好氧生物降解

【背景】2-吡啶甲酸具有高毒性、致癌性,能长期稳定存在于水体中,从而对环境造成危害。【目的】开发一种能够高效经济处理含2-吡啶甲酸废水的技术。【方法】筛选一株在好氧条件下以2-吡啶甲酸为唯一碳、氮、能源的菌株,考察该菌株的降解性能,建立降解动力学模型。【结果】经过16S r RNA基因序列分析,该菌株被鉴定为金黄杆菌(Chryseobacterium sp.),命名为ZD2。当2-吡啶甲酸初始浓度为100、200、400、600和800 mg/L时,ZD2完全降解2-吡啶甲酸的时间分别为10、18、22、78和114 h。零级动力学模型较好地描述了2-吡啶甲酸的降解行为,当初始浓度为100-400 mg/L时,降解速率常数随着浓度的增加而增加,并于400 mg/L时达到最大;600-800 mg/L时,降解速率常数开始下降,呈现抑制作用。【结论】菌株ZD2对2-吡啶甲酸的降解效果较好,能够为处理实际的2-吡啶甲酸工业废水提供理论依据。     

Aerobic biodegradation of selected monoterpenes

 Batch experiments were conducted to assess the biotransformation potential of four hydrocarbon monoterpenes (d-limonene, α-pinene, γ-terpinene, and terpinolene) and four alcohols (arbanol, linalool, plinol, and α-terpineol) under aerobic conditions at 23°C. Both forest-soil extract and enriched cultures were used as inocula for the biodegradation experiments conducted first without, then with prior microbial acclimation to the monoterpenes tested. All four hydrocarbons and two alcohols were readily degraded. The increase in biomass and headspace CO₂ concentrations paralleled the depletion of monoterpenes, thus confirming that terpene disappearance was the result of biodegradation accompanied by microbial growth and mineralization. Plinol resisted degradation in assays using inocula from diverse sources, while arbanol degraded very slowly. A significant fraction of d-limonene-derived carbon was accounted for as non-extractable, dissolved organic carbon, whereas terpineol exhibited a much higher degree of utilization. The rate and extent of monoterpene biodegradation were not significantly affected by the presence of dissolved natural organic matter. Received: 27 November 1995/Received last revision: 15 March 1996/Accepted: 17 March 1996     

Aerobic biodegradation of alkylphenol ethoxylates

Primary aerobic biodegradation of alkylphenol ethoxylates (APEOs) was studied using a new simple and fast porphyrin method, which did not require the extraction step. Extent of primary biodegradation of a nonylphenol ethoxylates (NP-10) was excess of 92% after 1.5 days, and reached 99% after 2 days, which was similar to the results obtained using modified CTAS (thiocyanate active substances) method. Degradation of benzene ring of NP-10 was studied using UV-absorbance at 277 nm in chloroform. Results showed that only little of benzene ring was degraded.     

Aerobic biodegradation of vinyl chloride in groundwater samples.

Studies were conducted to examine the biodegradation of 14C-labeled vinyl chloride in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labeled material being degraded after 108 days and approximately 65% being mineralized to 14CO2.     

Identification of microorganisms isolated from jet fuel systems

Seventy-two samples from jet aircraft fuel systems were examined for microbial contamination. Ten contaminated samples yielded 43 microorganisms which were classified into nine genera of bacteria and three genera of fungi. The predominant types, comprising about 37% of the isolated cultures, were identified as Bacillus spp. The remaining cultures were distributed among 11 genera, each of which represented 2 to 9% of the total isolates. Four cultures could not be assigned to a genus on the basis of the diagnostic criteria used. Only five isolates, in the genera Pseudomonas and Hormodendrum (Cladosporium), grew abundantly in a mineral salts solution with JP-4 fuel as the sole source of carbon. The presence of fuel utilizers in a fuel system may be a better index to potential problems that have been correlated with microbial contamination than the presence of aerobic sporeforming bacilli.     

Aerobic biodegradation of 2,2'-dithiodibenzoic acid produced from dibenzothiophene metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2'-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2'-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2'-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B12, and it degraded 2,2'-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2'-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2'-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2'-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Adaptation to and biodegradation of xenobiotic compounds by microbial communities from a pristine aquifer

The ability of subsurface microbial communities to adapt to the biodegradation of xenobiotic compounds was examined in aquifer solids samples from a pristine aquifer. An increase in the rates of mineralization of radiolabeled substrates with exposure was used as an indication of adaptation. For some compounds, such as chlorobenzene and 1,2,4-trichlorobenzene, slight mineralization was observed but no adaptation was apparent during incubations of over 8 months. Other compounds demonstrated three patterns of response. For m-cresol, m-aminophenol, and aniline intermediate rates of biodegradation and a linear increase in the percent mineralized with time were observed. Phenol, p-chlorophenol, and ethylene dibromide were rapidly metabolized initially, with a nonlinear increase in the percent mineralized with time, indicating that the community was already adapted to the biodegradation of these compounds. Only p-nitrophenol demonstrated a typical adaptation response. In different samples of soil from the same layer in the aquifer, the adaptation period to p-nitrophenol varied from a few days to as long as 6 weeks. In most cases the concentration of xenobiotic added, over the range from a few nanograms to micrograms per gram, made no difference in the response. Most-probable-number counts demonstrated that adaptation is accompanied by an increase in specific degrader numbers. This study has shown that diverse patterns of response occur in the subsurface microbial community.     

Aerobic thermophilic biodegradation of microbial cells

Summary Results are presented comparing the extent of solubilization/biodegradation of whole yeast cells by mixed thermophilic bacterial cultures under conditions of oxygen, excess and oxygen limitation. The process was most effective at a low dissolved oxygen concentration as suggested by solids removal data and by the production of often considerable quantities of carboxylic acids. The temperature optimum was also investigated and, under oxygen limited conditions, the most consistant results were obtained for operation at 65°C reflecting the true thermophilic nature of the process microbes. An operating temperature of 70°C probably exceeded the optimum for effective functioning of the thermophilic microbes and resulted in a less efficient process, whilst an operating temperature of 60°C was intermediate with respect to its effectiveness.     

Functional adaptation of microbial communities from jet fuel-contaminated soil under bioremediation treatment: simulation of pollutant rebound

To investigate the link between the functionality and the diversity of microbial communities under strong selective pressure from pollutants, two types of mesocosms that simulate natural attenuation and phytoremediation were generated using soil from a site highly contaminated with jet fuel and under air-sparging treatment. An increase in the petroleum hydrocarbon concentration from 4900 to 18,500 mg kg(-1) dw soil simulated a pollutant rebound (postremediation pollutant reversal due to residual contamination). Analysis of soil bacterial communities by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments showed stronger changes and selection for a phylogenetically diverse microbial population in the mesocosms with pollutant-tolerant willow trees. Enumeration of the main subfamilies of catabolic genes characteristic to the site detected a rapid increase in the degradation potential of both systems. A marked increase in the abundance of genes encoding extradiol dioxygenases with a high affinity towards various catecholic substrates was found in the planted mesocosms. The observed adaptive response to the simulated pollutant rebound, characterized by increased catabolic gene abundance, but with different changes in the microbial structure, can be explained by functional redundancy in biodegrading microbial communities.     

Anaerobic biodegradation tests and gas emissions from subsurface flow constructed wetlands

Anaerobic tests with gravel from horizontal subsurface flow constructed wetlands (SSF) used for the treatment of urban wastewater were developed in order to evaluate the anaerobic biodegradability of their effluents. Two types of assays were conducted. The reactors used for the first type were glass vials of 45 mL, that were used for only one measurement, requiring starting experiments with a number of reactors equal to the measurements to be made. For the second type of experiments multiple measurements were done in the same reactors, by using flasks of 2.2L. The COD of the SSF effluents used for the tests ranged from 60 to 130 mg/L. The evolution of CO(2) in the headspace of the reactors was used as indicator of anaerobic biodegradation rates. CO(2) mass emission rates ranged from 0.005 to 0.015 micromol/mL day. CH(4) generation was not detected in the tests in relation with the refractory properties of the effluent organic matter of the studied SSF. In situ measurement of CO(2) and CH(4) emissions from the gravel of the SSF ranged from 0.106 to 0.464 and from 0.039 to 0.107 mmol/m(2)h, respectively. Several CO(2) fluxes measured in the field were quite consistent with the emissions observed in the laboratory. The developed tests can help to understand the performance of SSF and improve their operation.     

Aerobic biodegradation and detoxification of wastewaters from the olive oil industry

Growth and polyphenol biodegradation by three microorganisms namely Geotrichum sp., Aspergillus sp. and Candida tropicalis were studied on olive mill wastewater (OMW). These three microorganisms were selected for their tolerance to the polyphenols. The biodegradation process of OMW was investigated in batch regime by conducting experiments where the initial concentration of chemical oxygen demand (COD) was varied. Furthermore, some tests were performed to determine the most important nutrients necessary for aerobic degradation of OMW. Average COD removals were 55.0%, 52.5% and 62.8% in wastewaters fermented with Geotrichum sp., Aspergillus sp. and C. tropicalis, respectively. The maximum removal of polyphenols was 46.6% (Geotrichum sp.), 44.3% (Aspergillus sp.) and 51.7% (C. tropicalis). In addition, significant decolorization was evident.