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| USDA SUCCESSFUL PROPOSAL | ||||||||||
Topic
Number: DARPA 91-111
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| Keywords: | |
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| biomimetic | sulfate-reducing |
| hazardous | methanogenic |
| waste | solvents |
| remediation | hydrocarbons |
The activities of DoD and its contractors result in the generation of large amounts of hazardous wastes. Many of the constituents of concern are waterborne or have become waterborne as a result of leaks or spills. Among the most troublesome of these was tes are organic solvents, heavy metals and salts. Even at low concentrations, these constituents are often toxic, tend to be resistant to conventional treatment methods and are persistent in the environment.
This research will focus on biodegradation opportunities for the following common waste constituents:
Aromatic hydrocarbons
benzene
toluene
ethylbenzene
xylenes
phenols
cresols
Halogenated hydrocarbons
tetrachloroethylene (PCE)
trichloroethylene (TCE)
1,1,1-trichloroethane (TCA)
Heavy metals
Inorganic salts
Benzene, toluene and xylenes are components of fuels and often the focus of groundwater cleanup efforts. Phenols and cresols are used in paint stripping and carbon (smut) removal operations. Halogenated hydrocarbons are used as solvents and in vapor deg reasing operations. Heavy metals and inorganic salts are present in metal stripping and electroplating effluents.
In wastewater streams and in contaminated groundwater, the above constituents are often found together. It is appropriate, then, to investigate remediation processes that handle a variety of constituents.
It appears that a two- or three-stage process could be used to biodegrade many of the hazardous wastes generated by DoD activities. In the initial stage, sulfate-reducing bacteria (SRB) would be used to accomplish the following transformations:
o-Cresol -> CO(2)
m-Cresol -> CO(2)
p-Cresol -> CO(2)
Phenol -> CO(2)
PCE -> TCE
TCE -> dichloroethylene (DCE) -> vinyl chloride (VC)
TCA -> CO2
Heavy metals would be precipitated as metal sulfides and removal of dissolved hydrogen sulfide would increase the pH of the waste stream. Either kinetic control (control of mean cell residence time) or control of sulfate concentration could be used to pr event methanogenesis in the first stage. Hydrogen sulfide produced by sulfate reduction could either be oxidized to sulfur for sale or to sulfate for recycling as an electron acceptor if the waste stream were deficient in sulfate.
In the second stage, methogens would be used to accomplish the following transformations:
Phenol ->CO2 + Methane (CH4)
TCE -> VC
DCE -> VC
Benzene -> CO2 + CH4
Toluene -> CO2 + CH4
Ethylbenzene -> CO2 + CH4
Xylenes -> CO2 + CH4
Methane produced by the process could be used either as an energy source for reactor
temperature control or as a substrate for aerobic methanotrophic bacteria.
A third aerobic stage might be required to convert vinyl chloride (VC) and other residual organics to carbon dioxide. Either a conventional heterotrophic or a methanotrophic culture could be used. Key to the feasibility of such a process is understandin g the kinetics and stoichiometry of each process step.
The overall goal of the Phase I research is to identify novel hazardous waste bioremediation pathways and to refine them sufficiently to focus subsequent research efforts in central research issues.
Quantify the stoichiometries of the transformations of interest.
What are the chemical stoichiometries of each of the chemical transformations?
What biomass yield data are available?
Quantify the energetics of the transformations of interest.
What are the free energy changes involved?
What implications do identified free energy changes have for the kinetics of the reactions?
Quantify the kinetics of each of the transformations of interest.
What doubling time or maximum specific growth rate data are available?
Under what conditions (temperature, pH, etc.) were the data collected?
Screen specific microorganisms and mixed cultures for their ability to metabolize/biodegrade the compounds of interest.
Can selected well-known microorganisms that are available from culture collections metabolize/biodegrade the compounds?
Can mixed cultures obtained from local hazardous wastesites metabolize/biodegrade the compounds?
Can mixed cultures obtained from local extreme environments metabolize/biodegrade the compounds?
Document the findings of Phase I research in a final report.
Each research task will address one of the research objectives presented above.
The stoichiometry of a reaction indicates what changes will occur and to what extent. An understanding of the stoichiometry of a microbially-mediated reaction allows the yield of the reaction, i.e., the ratio of the mass of products formed to the mass of reactants consumed, to be estimated.
This task will bring together the findings of a number of investigations that have studied the stoichiometries of microbially-mediated transformations of the compounds of interest. For example, Bak and Widdel (1986) have reported that the stoichiometry f or complete oxidation (mineralization) of phenol by the sulfate-reducing bacterium (SRB) Desulfobacterium phenolicum is as follows:
2C6 H6O + 7SO4-2+ (2H +) + 6H20 -> (12HCO3-) + 7H2S
Similarly, Ramand and Suflita (1991) have proposed the following stoichiometry for complete mineralization of m-cresol by SRB:
C7H8O + 4.25 (SO4-2) + 3H2O -> (7HCO3-) + 4.25 (HS-) + (2.7H+)
The following reaction stiochiometry of phenol transformation to C02 and CH4 has been proposed by Healy & Young (1978):
C6H6O + 4H2O -> 2.5CO2 + 3.5CH4
The yield of biomass (growth yield) from a microbially mediated reaction is another aspect of stoichiometry. Biomass yield is generally expressed in terms of dry weight of biomass produced per unit weight (or mole) of substrate consumed. Published bioma ss yield data will be reviewed and summarized.
Another aspect of stoichiometry is
the energetics or thermodynamics of the oxidation-reduction (redox) reactions mediated by
microorganisms. Analysis of the redox reactions involved can, in turn, shed light on the
free energy available for microbial grow th. Because in most biological systems, each step
in the oxidation of a substrate involves the transfer of two electrons, free energy
changes in such reactions are often expressed as free energy change per two electrons (e-)
transferred at unit concentra tions of reactants and products and at pH 7.0. Energetics
data will be obtained from the literature and derived from reaction stoichiometries using
free energy data from Thauer et al. (1977) and other sources.
While free energy changes have often been correlated with biomass yields (Bailey and
Ollis, 1986), both Middleton and Lawrence (1977) and Snoeyink and Jenkins (1980) have
pointed out that the maximum rate at which various microorganisms can grow (Umax ) is
related to the energy available from the redox reaction that is catalyzed. For example,
the maximum specific growth rate associated with the following microbially catalyzed
reaction are clearly correlated with a progressively lower amount of energy available to
the microorganisms from substrate oxidation: aerobic heterotrophic oxidation,
heterotrophic denitrification, nitrate oxidation, ammonia oxidation, heterotrophic sulfate
reduction, and heterotrophic methane fermentation. Thus, energetics data are helpful in
developing a relative, qualitative understanding of the kinetics of microbially mediated
transformations and shed light on the potential for the use of kinetic control in process
design. The calculated free energy change associated with a biologically-catalyzed
chemical reaction can also be used to estimate the true growth yield of the organism
involved. The following example calculation illustrates how we will develop the expected
free energy changes under standard conditions for the tra nsformations of concern to this
research. Complete oxidation of acetate to carbon dioxide occurs via the following
reaction:
CH3COO + (S04_2) -> (2HCO3_) = HS-
This reaction is the sum of the following two half reactions:
(CH3COO-) + 4H20 -> (2HCO3-) + (4H2 + H+) and (SO4-) + (H+) + 4H2 -> (HS_) + 4H2O AG0' = +104.6 kJ/reaction
Kinetics describe the rate at which chemical and biological processes occur. In bioprocess engineering, process rates characterize the rates at which microorganisms grow under specified environmental conditions. Two parameters, the maximum specific grow th rate (Umax) and the half saturation coefficient (Ks), are used to characterize bioprocess rates. Typically, the maximum specific growth rate is the maximum rate of increase of microorganism mass divided by the mass of microorganisms in a system under a specified set of growth conditions unconstrained by the availability of a limiting nutrient. It is related to the minimum microorganism doubling time (Td,min) as follows:
Td,min = (ln/2)/Umax
The half saturation coefficient is the limiting substrate concentration at which the specific growth rate is equal to half of Umax. In microbial transformations of toxic substrates, a third parameter, Ki, the inhibition coefficient, is also useful.
Some kinetic data on the transformations of interest are presented directly in the literature, often in terms of Td, min. These data and data derived from reported results of subs rate utilization experiments will be gathered and analyzed. The analysis w ill reveal gaps in the data base required to formulate mass balance models of selected microbially mediated transformations.
Bioremediation is feasible only in those instances wherein microorganisms have evolved (or can be induced to develop) metabolic pathways for transforming the compounds of interest. Previous investigators have identified a variety of microorganisms and co nsortia that have developed such capabilities. For example, the following microorganisms are known to capable of degrading the indicated compounds:
Denitrifying bacteria
Other anaerobic bacteria
Methanotrophic bacteria
Ammonia-oxidizing bacteria Nitrosomonas europea: TCE Other aerobic bacteria
In addition, the common and familiar bacterium, Escherichia coli K-12 strain 25290, has been found to dehalogenate carbon tetrachloride (Criddle et al. 1990).
Based on the above listing, it is clear that the ability to transform the compounds of interest is probably characteristic of a wide variety of microorganisms. In this task, we will screen a variety of anaerobic microorganisms for their ability to meta bolize selected toxic compounds because the least is known about that class of microorganisms (Grady, 1990). Specific microorganisms will be obtained from culture collections, such as the American Type Culture Collection (ATCC), and mixed cultures will b e obtained from local hazardous waste sites and other extreme environments.
Likely candidates from culture collections are as follows:
Sulfate-reducing bacteria
Methanogenic bacteria
In addition, anaerobic mixed cultures will be collected from two local sites known to be contaminated by chlorinated hydrocarbons and from Silver Bow Creek in Butte, MT, part of the largest Superfund site in the United States.
Screening will be accomplished by incubating small, inoculated batches of defined media at 37'C for an extended period. The cultures will be initiated early in the study because extended acclimation periods (e.g., weeks or months) have been reported (Cor apcioglu, 1991).
For sulfate-reducing cultures, the defined media recommended by Widdel and Pfennig (1984) will be used with the normal electron donor replaced by low concentrations of selected toxic compounds. Particular attention will be given to ensuring that the full spectrum of trace elements reported to be required for metabolism of hydrocarbons. The medium will be designed to indicate the production of sulfide by ferrous sulfide precipitation.
For methanogens, the prereduced defined medium recommended by Healey and Young (1979) and their serum bottle variation of the Hungate technique for growing anaerobic bacteria will be used. Growth will be indicated by the production of methane.
For both sulfate-reducing and methanogenic culture, biodegradation potential of all of the aromatic and chlorinated hydrocarbons listed above will be tested as single substrates at three low concentrations. The supernatant of sterilized controls and thos e cultures where bacterial growth is indicated will be analyzed by gas chromatography to provide an estimate of the extent of toxic electron donor removals.
Six copies of a final report on the Phase I project will be submitted to DoD in accordance with the negotiated delivery schedule. The final report will include a completed SF 298 as the first page identifying the purpose of the work, a brief description of the work carried out, the findings or results, and potential applications of the effort. The balance of the report should indicate in detail the project objectives, work carried out, results obtained, and estimates of technical feasibility.
Recent research by the principal investigator and others has identified a number of promising hazardous waste biodegradation pathways after which treatment processes could be modeled. Related work on both anaerobic an aerobic pathways is summarized below . This review emphasizes pure culture work as it is most likely to elucidate pathways.
Our PI is the inventor of an innovative process for bioremediation of hazardous wastes that contain heavy metals and sulfates. The BIOCAT TM process is the subject of his doctoral thesis (Hunter, 1989). That process was developed over a 3-year period us ing the same approach to process development that we propose herein. The process is called biocatalyzed demineralization of acidic metal sulfate solutions and comprises the steps of (1) acid phase anaerobic digestion of biomass to produce volatile acids and a partially stabilized sludge, (2) use of the volatile acids as carbon sources and electron donors for biological sulfate reduction for removal of acidity, metals and sulfate from acid mine drainage, and to produce acetate, (3) use of the acetate solu tion as feed for methane phase anaerobic digestion to produce methane and to reduce the organic content of the effluent of the process, and for use of the methane to satisfy the energy requirements of the process. Single substrate and multiple substrate chemostat studies were conducted by our PI to determine if kinetic control (i.e., a relatively short mean cell residence time) could be used to ensure partial oxidation of higher molecular weight volatile acids (propionic and butyric) and production of ac etate during the sulfate reduction step. The studies confirmed that propionate produced during acid phase anaerobic digestion can be used as an electron donor for sulfate reduction (during which it is converted to acetate) and that the acetate is availab le as a substrate for a subsequent methane production step.
During that project, our PI conducted a thorough review of the growth requirements, reaction stoichiometries, biomass yields, energetics and growth kinetics of sulfate-reducing bacteria that will form the basis of work on this project. He also proved tha t kinetic control could be used to ensure that microbial sulfate-reduction occurred in the first of a series of reactors with methanogenesis occurring in a downstream reactor. By means of kinetic control, an essentially pure culture was maintained in the sulfate-reducing reactor even though the growth medium contained multiple substrates capable of degradation by a wide variety of microorganisms. Moreover, kinetic control allowed the products of microbial sulfate reduction step to be amenable to microbial conversion to methane in a subsequent process step.
Grbic-Galic & Vogel (1986) reported that a methanogenic consortia was able to use relatively high concentrations of benzene or toluene as a sole carbon and energy source. They were rapidly transformed into hydroxylated intermediates under strictly anaero bic methanogenic conditions. Aromatic intermediate products in toluene-fed cultures included p-cresol, o-cresol and benzene. Metabolism of benzene produced phenol as the major metabolite.
Both anaerobic (Evans & Fuchs, 1988) aerobic pathways have been reported for biodegradation of phenolic compounds. Tschech & Fuchs (1987) have isolated strains of a nitrate-reducing Pseudomonas sp. capable of growth on phenol and p-cresol. Pcresol was m etabolized via anaerobic oxidation of the methyl group through the alcohol and the aldehyde to the carboxyl yielding 4-hydroxybenzoate.
Bak & Widdel (1986) have isolated a sulfate-reducing bacteria, Desulfobacterium phenolicum, that is capable of complete oxidation of phenol to carbon dioxide with sulfate as the terminal electron acceptor. The organism was also capable of using p-cresol (but not o-cresol) as an electron donor.
Haggblom et al. (1990) found that metabolism of p-cresol proceeds through the initial oxidation of the aryl methyl group under methanogenic, sulfate-reducing and nitrate-reducing conditions. Similarly, Suflita et al. (1989) found that o-cresol is anaerob ically oxidized to o-hydroxybenzoate. M-cresol metabolism proceeds through an initial carboxylation reactions under methanogenic conditions (Roberts et al., 1990) and under sulfate-reducing conditions (Ramanand & Suflita, 1991). In general, biodegradati on of cresol isomers is favored under sulfate-reducing conditions (Smolenski & Suflita, 1987).
Saleh et al. (1964) quantified the effect of a variety of chlorinated hydrocarbons on sulfate-reducing bacteria (SRB) growth. The inhibitory concentrations of a number of compounds was surprisingly high ( > 1,000 mg/1). Fathepure et al. (1987) reported that a SRB identified then as strain DCB-1 and later named Desulfomonile tiedii was able to dechlorinate PCE and TCE at a significant rate. A sequential reductive dechlorination pathway (PCE --> TCE --> DCE --> VC) was proposed. Even high PCE dechlorina tion rates were achieved when DCB-1 was grown in a methanogenic culture. Dolfing (1990) reported that reductive dechlorination by DCB-1 is coupled to ATP production and growth. Growth requirements of the bacterium were described by De Weerd et al. (1990 ). Bagley & Gossett (1990) described PCE transformation to TCE and DCE by sulfate-reducing enrichment cultures.
Vinyl chloride, a toxic product of biodegradation of higher chlorinated aliphatic hydrocarbons, is incompletely biodegraded under methanogenic conditions (Barrio-Lage et al., 1990) and is essentially completely (99%) degraded under aerobic conditions (Dav is & Carpenter, 1990)
The proposed approach will identify ways of applying recent basic research into the pathways of biodegradation of toxic compounds to the hazardous waste remediation needs of the DoD. It will combine information from the fields of microbiology and reactor engineering to identify and develop promising remediation concepts.
The Phase I effort will provide a foundation for process design/reactor engineering investigations to be conducted in Phase II by gathering and analyzing information on the stoichiometry, energetics and kinetics of specific biotransformations of interest. our approach is based on our firm belief that reactor engineering, the use of mathematical model to simulate process performance, is the key to rational process design. This approach was successfully used in development by our principal investigator of the BIOCAT process for acid mine drainage treatment and metals recovery described above.
In Phase II, batch or continuous culture experiments will be conducted to quantify stoichiometric and kinetic parameters that are not available in the literature. Both single-substrate and multiple-substrate studies will be conducted. The goal will be t o develop estimates of the parameters, Umax, Y, Ks and Ki in the following equation recommended by Grady (1990):
q = u/Y
q = (Umax/Y) (SI (Ks + Ss + Ss^2 /Ki)
where q = specific substrate-removal rate
The equation is appropriate for situations in which a substrate is inhibitory to it's own biodegradation at high concentration. It reduces to the Monod equation when Ki approaches infinity.
The proposed project appears to have potential use by the Federal Government in remediation of current DOD hazardous waste conditions. The waste constituents under study are either in current use or are present in existing groundwater contamination sites . For example, all of the chlorinated hydrocarbons and metals under consideration were in use at the Long Beach Naval Shipyard Plating Shop and Marine Machine Shop when the PI developed a wastewater management plan for the facility in 1975. TCE was a pr imary target of an in situ biodegradation study at Moffett Naval Air Station in Mountain View, California.
The proposed project appears to have potential commercial application in that the waste constituents under study are also produced by a variety of civilian activities. TCE, PCE and TCA, for example, are among the most common chlorinated hydrocarbons dete cted in groundwater (Wilson & Wilson, 1985).
Yellowstone Environmental Science, Inc. (YES Inc.) is a research and development firm founded in 1981 to create, develop and commercialize innovative products and processes for protection of the environment and public health. one our first inventions, a s ewer flow meter, has been licensed and will be in commercial production in October of this year. Our principal investigator on this project will be Dr. Robert M. Hunter.
Robert M. Hunter has over 18 years of experience managing multi-disciplinary research projects. For the last nine years, Dr. Hunter has focused on developing innovative solutions to specific public health and environmental problems. This talent was reco gnized by the U.S. Department of Energy when it selected him for funding in the 1986 Energy-Related Inventions Program.
Dr. Hunter has served as an expert consultant on specific environmental problems involving innovative technology, and he has served as a project manager on projects with large project teams and fees in excess of $560,000. He has exhibited that he has suc cessfully accomplished the following tasks that are pertinent to the proposed project:
Dr. Hunter is a professional engineer, a talented inventor, an experienced educator.
Mary Hunter, R.N., M.N., serves as president of YES Inc. Mary is a registered nurse practitioner with over 16 years of experience in public health and safety education. She has the final word on policy decisions and is actively involved in dayto-day man agement of YES Inc. She also accomplishes project tasks such as coordination, budget tracking, and report preparation.
Frank Stewart, M.S., P.E., has 17 years of experience in environmental engineering and engineering management. He has served as project engineer on a variety of hazardous waste investigations in Montana. His experience in hazardous waste remediation inc ludes computer modeling of contaminate migration at a Superfund Site, and numerous field investigations of petroleum and hazardous waste releases.
Frank M. Stewart, M.S., P.E.
Role: Senior research engineer Education:
Employment/experience:
YES Inc. maintains a fully-equipped 800 sf laboratory and a 320 sf office. The facilities where the proposed work will be performed meet environmental laws and regulations of federal, Montana and local governments for, but not limited to, the following g roupings: airborne emissions, waterborne effluents, external radiation levels, outdoor noise, solid and bulk waste disposal practices, and handling and storage of toxic and hazardous materials.
Equipment available for this project includes an incubator, a -precision temperature controlled water bath, optical .microscope, scales, pH meter, dispensers, misc. glassware, etc. Equipment available for Phase II includes chemostats, metering pumps, pH controllers, data logger, etc. Gas chromatography and GC/MS services are available locally.
No consultant involvement is proposed.
PRIOR, CURRENT OR PENDING SUPPORT
No prior, current or pending support for a similar proposal.