Grantee Research Project Results
Final Report: Biostimulation of BTX Degradation with Environmentally Benign Aromatic Substrates
EPA Grant Number: R823420Title: Biostimulation of BTX Degradation with Environmentally Benign Aromatic Substrates
Investigators: Alvarez, Pedro J.
Institution: University of Iowa
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $246,342
RFA: Exploratory Research - Engineering (1995) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Land and Waste Management
Objective:
Bioremediation shows great promise as an approach to hazardous waste management. The presence and expression of appropriate catabolic capacities, however, can limit the success of bioremediation. We seek to overcome such limitations by exploiting favorable substrate interactions. The general hypothesis driving this project is that controlled addition of non-toxic aromatic substrates (e.g., benzoate) can result in faster and more complete degradation of priority pollutants (e.g., benzene, toluene, and xylenes, collectively known as BTX). A stimulatory substrate could induce catabolic enzymes, act as primary substrates for cometabolism, or serve as growth substrates for the proliferation of desirable phenotypes. This would be particularly beneficial when the target compounds are present at insufficient concentrations to support steady microbial growth, or to establish and sustain in situ reactive zones to attenuate BTX migration and protect downgradient groundwater resources. The specific objectives of this research project were:
(1) To advance our knowledge of the substrate interactions and population shifts that occur under multiple substrate conditions (e.g., waste mixtures), and to obtain basic criteria to enhance beneficial interactions during bioremediation;
(2) To determine how geochemical factors such as dissolved oxygen concentrations affect oxygenase enzyme induction, longevity, and activity, using well defined systems with axenic cultures from our collection of indigenous microorganisms; and
(3) To characterize the kinetics of secondary substrate utilization and to model the degradation of trace contaminants in flow-through systems that are amended with (primary) stimulatory substrates.
Summary/Accomplishments (Outputs/Outcomes):
These results supports the notion that biostimulation by structural analogue addition might enhance several bioremediation approaches, including enhanced polishing capabilities of engineered remediation projects and a more efficient establishment and sustenance of in situ reactive zones. We also investigated substrate interactions and enzyme activity and longevity under different substrate and electron acceptor conditions, as discussed below.
Flow-through aquifer columns were used to evaluate the efficacy of using benzoate as a biostimulatory substrate to enhance the aerobic biodegradation of benzene, toluene, and o-xylene (BTX), fed continuously at low concentrations (about 0.2 mg/L each). When used as a co- substrate, benzoate addition (1 mg/L) enhanced BTX degradation kinetics and attenuated BTX breakthrough relative to acetate-amended (2 mg/L) or unamended control columns. The benzoate-amended column also experienced an increase in predominance of pseudomonad species capable of degrading BTX. The feasibility of injecting benzoate to enhance the growth of BTX degraders and establish a buffer zone downgradient of a BTX plume was also investigated. Using pristine aquifer material without previous exposure to BTX, aquifer columns were fed benzoate (2 mg/L), acetate (4 mg/L), or mineral medium without supplemental substrates during a two-day acclimation stage. All columns were subsequently fed BTX alone, and their breakthrough was monitored. Previous exposure to benzoate, but not to acetate, shortened the acclimation period to BTX degradation and enhanced the short-term bioattenuation potential of the indigenous consortium. This suggests that benzoate could be used to establish and sustain in situ reactive zones to attenuate BTX migration and protect downgradient groundwater resources.
This work also investigated substrate interactions between ethanol and BTX. Ethanol is frequently found along with BTX compounds in groundwater contaminated with gasoline. Yet, little is known about its effect on bioremediation of the toxic BTX contaminants. Aquifer microcosms were used to investigate the effect of ethanol on microbial degradation of representative BTX compounds under electron acceptor conditions commonly found in intrinsic bioremediation projects. Under aerobic conditions, ethanol retarded BTX biodegradation and exacerbated the biochemical oxygen demand (BOD). Anoxic conditions developed quickly when the BOD exerted by ethanol exceeded the available oxygen. This led to the persistence of benzene, which was also recalcitrant in denitrifying, sulfidogenic, and methanogenic microcosms during 99 days of incubation. Toluene was degraded under all anaerobic electron acceptor conditions tested, although the onset of relatively fast degradation always commenced after ethanol had been removed. Toluene degradation was not significantly affected by ethanol in denitrifying microcosms containing excess nitrate. Yet, no toluene degradation occurred when nitrate was limiting because nitrate was depleted while ethanol was being degraded. Ethanol also adversely affected toluene degradation in iron-reducing and methanogenic microcosms. Curiously, ethanol enhanced toluene degradation in sulfate reducing microcosms. This was hypothesized to be due to a low initial concentration of toluene degraders and their incidental growth during ethanol degradation. Albeit, the preferential utilization of ethanol and the accompanying depletion of electron acceptors suggest that ethanol would have a negative effect on passive BTX bioremediation. This is particularly important for the fate of benzene, which is the most toxic of the BTX and the most recalcitrant under anaerobic conditions.
Another component of this study was the effect of dissolved oxygen concentration during growth on the expression, activity, and longevity of toluene dioxygenase in Pseudomonas putida F1. Many priority pollutants is often limited by the availability of oxygen. Yet, the effect of dissolved oxygen concentration on the expression, activity, and longevity of catabolic enzymes is poorly understood. Furthermore, data on enzyme decay rates are extremely scarce. These critical knowledge gaps motivated us to study the longevity of BTX degradation activity in resting cells grown and induced at different dissolved oxygen concentrations.
Toluene dioxygenase (TDO) in Pseudomonas putida F1 (PpF1) is an ideal enzyme to use as a model for bioremediation studies. It is common in nature and has a broad substrate specificity, enabling it to catalyze the insertion of two molecules of molecular oxygen into the aromatic nucleus of many aromatic contaminants such as benzene, toluene, and xylenes (BTX). PpF1 was used as a model bacterium to investigate how low dissolved oxygen (DO) levels affect catabolic enzyme expression and biodegradation activity, and to determine how long the enzyme stays active after the inducing substrate (i.e., toluene) has been removed. Studies of TDO expression were conducted using resting PpF1 cells grown under different dissolved oxygen concentrations. Cells were grown in a continuous culture fermenter equipped with a dissolved oxygen monitor and air flow regulators which permitted the control of dissolved oxygen concentration in the growth medium. Toluene vapors were supplied as the sole carbon and energy source using a separate air flow line. Induced cells were harvested and subsequently washed and resuspended in air-saturated sterile mineral medium prior to conducting biodegradation assays. Activity of TDO was assessed over time by measuring the rate of toluene disappearance (corrected for abiotic controls) in the presence of chloramphenicol (350 mg/L). Chloramphenicol is a bacteriostatic antibiotic that inhibits de novo enzyme synthesis. This ensures a constant level of enzyme activity during the biodegradation assay. Toluene degradation activity assays were thus conducted on previously induced cells grown under different DO concentrations and allowed to rest for different durations. Dissolved oxygen concentration was only a factor during growth, as all in vivo enzyme activity assays were conducted under fully aerobic conditions.
Results show that the activity of the TDO enzyme present in resting cells decreases exponentially with cell age. This was confirmed by in vivo activity assays conducted with PpF1 cells harvested from two different fermentation runs. In both runs, the decay coefficient for enzyme activity was larger than typical coefficients for cell decay, indicating that TDO activity decays at a rate faster than cells die. An analysis of variance (ANOVA) of the data showed that the DO concentration during growth affected neither the specific toluene degradation activity nor its longevity for a given run. Therefore, DO levels during growth do not affect TDO enzyme activity. This implies that, similar to the activated sludge process, aerobic BTX bioremediation can proceed effectively if the dissolved oxygen concentration is maintained between 1 and 2 mg/L. Thus, attempts to fully saturate the DO concentration in aquifers may not be justified due to a low incremental benefit to cost ratio. Enzyme expression occurred even at DO levels below which denitrification commonly occurs (i.e. < 0.3 mg/L). This suggests the possibility of aerobic transformations under denitrifying conditions.
Engineered BTX bioremediation efforts rely on a non-specific stimulation of indigenous microorganisms. Thus, adding non-toxic inducers could enhance current bioremediation practices by enhancing our ability to biostimulate specific microbial populations and target specific degradation pathways. To explore this possibility, we studied which non-toxic structural analogues could induce which of the five known (aerobic) BTX degradation pathways (i.e., the tol, tod, tom, tbu, and tmo pathways). We hoped that some npn-toxic aromatic substrates (i.e., benzoate, salicylate, phenylalanine, tryptophan, and tyrosine) would exhibit a high specificity in stimulating the induction of particular BTX degradation pathways. Specific inducers of particular genetic operons would offer two direct benefits to bioremediation: (a) increased specificity of the target population to be stimulated, and (b) (because known degradative pathways are stimulated) predictability of substrate range, intermediates and end products. The reference strains used in this study were Pseudomonas putida mt-2, which has the tol plasmid, Pseudomonas putida F1, which has the tod gene, Burkholderia cepacia G4, with the tom gene, Burkholderia pickettii PKO1, with the tbu gene, and Acromobacter xylosoxidans KR1, with the tmo gene expressing toluene para-monooxygenase. Pseudomonas fluorescens NG3, which we isolated on naphthalene, was also tested. To test enzyme induction specificity and efficacy, pure cultures were fed separate structural analogues. When growth occurred, cells were harvested, washed, and resuspended at an OD600 of 0.5 for subsequent enzyme activity analysis. In vivo enzyme activity was quantified by measuring the rate of toluene disappearance in batch reactors amended with 350 mg/L chloramphenicol. Chloramphenicol (CA) is a bacteriostatic antibiotic that inhibits de novo protein synthesis by binding to the 50s ribosome, thereby blocking translation. CA inhibits the synthesis of new enzymes when toluene is added, but it does not adversely affect toluene degradation activity by previously induced cells. Thus, CA ensures constant enzyme and cell concentrations during the assay. Positive controls for this assay consisted of toluene-grown cells. Negative controls consisted of cells grown on pyruvate, which were not induced.
All of the tested non-toxic aromatic substrates supported the growth of the reference strains. None of them, however, induced BTX degrading enzymes. Nevertheless, even if a non- toxic aromatic substrates cannot induce BTX-degrading enzymes, its addition could still enhance BTX bioremediation. Specifically, adding structural analogues would increase the fraction of energy provided to the microbial community by aromatic substrates. This, in turn, could increase the fraction of the community capable of degrading aromatic compounds, including BTX. A fortuitous proliferation of BTX degraders would be conducive to faster BTX degradation rates and lower residual BTX concentrations. The specific proliferation of BTX degraders could be used to establish and sustain reactive (buffer) zones downgradient of BTX plumes, or to enhance anaerobic bioremediation processes in intrinsic bioremediation schemes. A mathematical model is currently being developed to characterize the kinetics of secondary substrate utilization that will be applicable for such analogue enrichment approaches.
Conclusions: In both intrinsic and active bioremediation projects, residual concentrations of carcinogenic compounds such as benzene could exceed applicable cleanup standards and remain a threat to public health. Possible reasons for residual contaminant concentrations include mass transfer and diffusion limitations, the requirement for a minimum substrate concentration to satisfy the maintenance energy demand and sustain a sufficient concentration of specific degraders, and the existence of a threshold substrate concentration below which induction of the necessary catabolic enzymes does not occur. Therefore, overcoming limitations associated with the presence and expression of appropriate catabolic capacities might be required in some bioremediation projects. This might be accomplished by adding supplemental substrates that increase the concentration of desirable phenotypes without repressing the required catabolic enzymes. These results show that the non-toxic aromatic substrates, benzoate, salicylate, phenylalanine, tryptophan and tyrosine do not induce BTX-degrading enzymes. Yet, these substrates can enhance BTX bioremediation by stimulating the growth of specific BTX degraders. On the other hand, the common oxygenate ethanol is likely to exacerbate the biochemical oxygen demand and exert a diauxic effect that inhibits BTX biodegradation. This work also showed that aerobic BTX biodegradation can proceed effectively hypoxic conditions. Thus, attempts to fully saturate the DO concentration in aquifers may not be justified due to a low incremental benefit to cost ratio.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 12 publications | 4 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Alvarez PJJ, Cronkhite LA, Hunt CS. Use of benzoate to establish reactive buffer zones for enhanced attenuation of BTX migration: Aquifer column experiments. Environmental Science & Technology 1998;32(4):509-515. |
R823420 (Final) |
not available |
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Corseuil HX, Alvarez PJJ. Natural bioremediation perspective for BTX-contaminated groundwater in Brazil: Effect of ethanol. Water Science and Technology 1996;34(7-8):311-318. |
R823420 (Final) |
not available |
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Corseuil HX, Hunt CS, dos Santos RCF, Alvarez PJJ. The influence of the gasoline oxygenate ethanol on aerobic and anaerobic BTX biodegradation. Water Research July 1998;32(7):2065-2072. |
R823420 (Final) |
not available |
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Vermace ME, Christensen RF, Parkin GF, Alvarez PJJ. Relationship between the concentration of denitrifiers and Pseudomonas spp. in soils: implications for BTX bioremediation. Water Research December 1996;30(12):3139-3145. |
R823420 (Final) |
not available |
Supplemental Keywords:
Adaptation, benzoate, ethanol, hypoxic, induction, toluene dioxygenase, RFA, Scientific Discipline, Toxics, Waste, Water, Remediation, Environmental Chemistry, Chemistry, HAPS, Bioremediation, Drinking Water, Engineering, EPCRA, Groundwater remediation, 33/50, bioremediation model, microbial degradation, Toluene, biodegradation, enzymes, Xylenes, Benzene, 1-(chloromethyl)-4-nitro-, environmental engineering, treatment, microbial risk management, contaminant release, biostimulation, Benzene (including benzene from gasoline), Xylenes (isomers and mixture), other - risk management, groundwaterProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.