Abstract
Biotechnology has a growing place in the remediation of hazardous waste sites throughout the world, and especially in Asia where population density is high and land and fresh water are scarce. In-situ bioremediation has been demonstrated already to be highly effective for petroleum hydrocarbons (alkanes, aromatics, polychlorophenols) and organophosphate pesticides in soils and for gasoline by-products (benzene, toluene, xylene) and chlorinated solvents (trichloroethylene) in groundwater. Heavy metals and PCBs are not suitable for bioremediation. Environmental biotechnology includes solid-phase and slurry-phase bioremediation for contaminated soils and sitespecific bioreactors for contaminated groundwater. Specific examples are presented. From a policy point of view, accumulated wastes must be detoxified,preferably at sites where they already exist. We cannot continue to rely on their removal and disposal “elsewhere”. For current waste streams, we must minimize the volumes and toxicity. Environmental biotechnology will play a key role.
Keywords: Bioremediation, biotechnology, environmental biotechnology, groundwater contamination, hazardous chemicals, micro-organisms, soil contamination.
Address for reprints: Gilbert S Omenn, MD, PhD, Profesmr and man, School of Public Health & Community Medicine, SC-30, Universityofwashington, Seattle, Washington 98195, USA.
Environmental Biotechnology: Biotechnology Solutions for a Global Environmental Problem, Hazardous Chemical Wastes* Gilbert S Omenn, MD, PhD School of Public Health &Community Medicine, University of Washington, Seattle, USA *Presented, in part, 17 January 1990 at the International Symposium on Biotechnology for Solving Global Environmental Problems, Tokyo, Japan Bioindustry Development Center, and 3 July, 1990 at the International Symposium on Health, Environment, and Social Change, Taipei, Taiwan.
naut William Anders. In this photograph, we see a tiny fragile globe floating in the vastness of space - a closed, vulnerable system upon which all our lives depend. The imuact on
countries of Asia, land and water are especially precious resources. Since the 1940s, rapid growth of the chemical industry has fueled economic growth and improvements in standards of living and has produced millions of tons of hazardous and toxic wastes - petroleum products, lubricants, protective coatings, pesticides, dielectric fluids, flame retar-
and Development, may surpass even the 16th-century discovery that the Earth is not the center o f the Universe’. The intrusion of human civilizations has presented increasing challenges to Planet Earth. These challenges include retention of infrared radiation or global warming due to greenhouse gases, depletion of stratospheric ozone, loss ofbiological diversity, deforestation, desertification, acid precipitation, threat of major epidemics among humans (including AIDS) or crops, and threat of nuclear war. Among the risks to life on this Planet, the chemical contamination Of ’Oils and water systems deserves our attention, since water is the most distinctive and enabling feature for life on Earth. In the densely populated
dant and forgiving, have become threatened by “out of sight, out of mind” attitudes toward disposal of these chemicals, their residues, and off-grade mixtures. These chemical wastes accumulate in landfills, holding ponds, lakes, rivers, groundwater and soil environments, and the atmosphere. The generation of chemical wastes is highly correlated with the population and standard of living. Industrialization in Third World countries has brought hazardous chemical problems to sometimesunprepared societies2. As the Bhopal accident demonstrated, hazardous chemical processes may be managed inadequately. Prevention of worker exposures and prevention of emissions to air and water may be considered luxuries, or not considered at all.
Introduction
Our generation is the first to have seen its own planet from a distance, photographed most memorably as the “Earthshot” by Apollo 8 astro-
40 Downloaded from aph.sagepub.com at FUDAN UNIV LIB on May 13, 2015
Asia-Pacific Journal of Public Health 1992/1993 Vol. 6 No. 2 Furthermore, many chemicals, especially pesticides, that have been banned or restricted in industrialized countries are being exported to Third World countries. Ill-advised subsidies for purchase stimulate gross overuse of pesticides, with a worldwide epidemic of acute poisonings and with adverse agricultural effects3. In 1982, the United Nations General Assembly approved a resolution (with one dissent) that “Products which have been banned for domestic consumptionand/or sale because they have been judged to endanger health and environment should be sold abroad by companies, corporations, or individuals onlywhen a request for such products is received from an importing country or when the consumptionofsuch productsisofficially permitted in the importing country”. In theory, these nations may wish to accept certain (low-level) risks, which other countries choose to avoid, but they seldom make such explicit decisions and they lack the expertise to make detailed risk assessments. Less developed countries may also be recipients of wastes from more developed countries needing disposal sites; the Eastern European countries have abused their land and water resources in this way. The ever-growing human population forces us to ask “How can we help Nature’s cycles keep up with the waste streams we introduce on Planet Earth?” Throughout the world efforts are increasing to recycle wastes and reduce the amounts of municipal and industrial wastes produced4. The guiding principle in managing accumulated hazardous wastes must be to detoxify them, preferably at the sites where they exist. We cannot continue to rely on removal of wastes to disposal sites, usually landfills, at those places we collectively call “Elsewhere”.
Technologies for clean-up of soils and groundwater contaminated with hazardous chemicals include several types of incineration, chemical and physical treatment to solidify or vitrify and thereby immobilize the chemicals, vapor extraction and air stripping with absorption, and a variety of b i o r e m e d i a t i o n approaches. Biotechnology has a growing place in the remediation of hazardous waste sites in the United States and, surely, in other countries throughout the Asia-Pacific region. The main bioremediation technologies in the field today involve engineering applications of basic principles of aerobic metabolism: optimizing the concentrations of oxygen and nutrients and assuring adequate moisture within a given environment to enhance the indigenous microbial population. Generally, there is little or no knowledge of the organisms present at the site. Better understanding of microbial ecology and genetic enhancement of biodegradation should accelerate progress in this field.
Principles of Bioremediation Microorganisms canuse manychemicals as food and energy sources. With full aerobic metabolism, usingoxygen as the final electron acceptor, the endproducts are carbon dioxide and water. With some chemicals, especially
Table 1 outlines some of the most prevalent a n d important chemicals contaminating soils or ground-water and the present capability of biotechnology to detoxify these chemicals iii sitir. In general, aerobic biodegradation is best suited
Table 1. High-priority chemicals by type of contamination Soils
Groundwater
Petroleum hydrocarbons aliphatic (alkanes) aromatic (benzene, ctc) chloro-aromatics (PCP) Polychlorobiphenyls(PCBs)
++++
Chlorinated Pesticides Organophosphate pesticides
On-Site Remediation On-site remediation requires extensive knowledge of the hydrogeology of the site, the ecology of the soil and water compartments, and the effectiveness of various alternative and complementary technologies.
man-made chemicals, oxidation may be incomplete or may be better carried out by anaerobic organisms, whichusenitrateorsulfateasthe final electron acceptor. Some organisms can biotransform chemicals but obtain no energy yield; these organisms need additional substrate to grow, a co-metabolic scheme. To assess the feasibility of biotreatment, threeaspects must becombined: * microbial physiology, biochemistry, and genetics, to understand the metabolic processes leading to detoxification and the genetics controlling the enzyme functions involved; * microbial ecology, toappreciate the microenvironments in which the treatment may be performed and the structure and function of indigenous or inoculated microbial communities; and * field site engineering, to implement the desired biodegradation scheme, maintaining optimal growth conditions and combining with physical or chemical methods, as n e c e ~ s a r y ~ - ~ .
Heavy Metals
Chlorinated Solvents (TCE) Gasoline (BTX) Triazine herbicides Heavy Metals
Biotechnology
+/-
+++ -
++
++++
+
-I?+
Rating of Biotechnology Effectiveness: - ,ineffective; +, limited effectiveness, to + + + + ,demonstrated highlyeffective. Sentinel compounds indicated: PCP, polychlorophenols; TCE, trichloroethylene; BTX, benzene/tolucnc/xylene
Downloaded from aph.sagepub.com at FUDAN UNIV LIB on May 13, 2015
Asia-Pacific Journal of Public Health I992/1993 Vol. 6 No. 2 for remediation of sites contaminated with petroleum hydrocarbons and polar solvents, such as alcohols a n d ketones. A n a e r o b i c biodegradation is best applied to reductive dechlorination of chlorinated aliphatic and aromatic hydrocarbons, such as tetrachloroethane. Because anaerobic degradation may result in the accumulation of partially oxidized organic compounds, this treatment technique is often coupled with aerobic biological treatment. Metals are not usuallygood targets for bioremediation - or for physical methods either. However, the redox state ofcertain metals, including cadmium, chromium, arsenic, and mercury, can be transformed by microorganisms to a state better suited to separation or isolation, or one that is less toxic. Solid-Phase Bioremediation for Contaminated Soil The traditional form of bioremedia‘tion of contaminated soil has been known for years as “land farming”, often little more than dumping contaminatedwasteonto land and letting Nature work its way, as in the biocycling of natural compounds. The main advances in modem “solid-phase bioremediation” come from optimization of conditions, by addition of nutrients, active aeration, and enhanced release of chemicals absorbed to soil particles. This technique has been used to clean up fuel oils, diesel fuels, pesticides, and other types of easily degraded substrates. Solidphase biotreatment of petroleumcontaminated and creosotecontaminated soils is the most widely used and most cost effective biotreatment technology at the present time. Forexample, at a site with extensive hydrocarbon contamination of soil (up to 16,000 ppm), indigenous organisms were activated by spraying the nitrogen and phosphate, and oxygenating, reducing all components below the target of 100 ppm total concentration. This action was sufficient to permit disposal at a Class 111 landfill, rather than Class I (most hazardous) landfill, as previously scheduled to be done. Complete biodegradation
greenhouse. An overhead spray irriwas demonstrated in the laboratory to gation system within the greenhouse be feasible, but was not called for in provides moisture and distributes this case. A more complicated case was a nutrients and microbial inocula, as site with substantial amounts of 4-6 needed, as large batches of soil are ring polycyclic aromatic hydrocarbrought into the treatment bed for bons (PAHs). It was necessary to add detoxification. The soil treatment an organism capable ofbiodegrad-ing facility is attached to an air managethese more recalcitrant, higher molecment system for the volatiles, with ular weight PAH compounds. Constia vapor-phase bioreactor and then tutive strain B600 of Beijeriitckin B 1, activated carbon, in series. Contamiisolated by Gibson and Mahaffey, was nated leachate is collected in a useful in degradingphenanthreneand sump, then pumped to an on-site bioother high MW ring compounds, esreactor. pecially since it did not require inducThe nature and concentration of tion of activity by low MW PAH the contaminants and the regulatory substrates’. Similarly, P S C ~ I ~ U I I ~ requirements OI~~S in a given country or strain DBMIOl with constitutive state will determine whether elabobiodegradative activity against rate closed systems are required, or polycyclic aromatic hydrocarbons, some emissions of vol8tiles into the including dibenzofuran and air would be permissible. benzo(a)pyrene, was isolated from a creosote-contaminated site, evaluSlurry-Phase Bioremediation of ated in the laboratory, and inocuContaminated Soil lated in the field at a test site in Another approach with soils is solidCalifornia. phase treatment in an aqueous slurry, The solid phase system can be essentially a large bioreactor. This apmodified to control volatiles and leaproach is especially appropriate for chate. For example, a treatment bed control of manufacturing effluents has been lined with an 80-mm highand for groundwater clean-up. An exdensity liner having heat-welded ample is treatment ofa field contamiseams (Figure 1)’. nated with polychlorophenols up to The liner is covered with sand, 8000 ppm. In this case, aeration and which protects it and provides proper nutrients had littleeffect over 13 days, drainage for contaminated water but inoculation of the soil slurries leaching from soils placed on the with a consortium of five organisms treatment bed. Lateral perforated selected by chemostat yielded prompt drainage pipes on top ofthe synthetic reduction of PCP levels. There was liner in the sand bed collect the soil stoichiometric release of chloride, leachate. The whole treatment bed is demonstrating full detoxification. covered by a modified plastic film Figure 1. Schemefor solid-phasebiorcmediationof contaminatedsoils, brought into
a controlled environment on-site. From Bourquin (1989).
Solid-phasetreatment process Atyp~calsection A-A’
I rr
r Treatment zone
r Spray nozzle
Leacha:e col!ecllonsyslem Synthetr.llner t- Cornpaned secondary 5011 liner
r
SyS!em area overhead enclosure
,-Eanh It11 term
1 To Ireatmen1and
recycle 01dsspo-1
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Addition of organisms was essential and was effective in reducing levels to below 0.5 ppb. Similar good results wereobtained inasoil-slurrybiotreatment for trichloroethylene (TCE). The bioreactor scheme decreases acclimation time, increases biodegradation rates, allows greater process control, increases contact between microorganisms and contaminants, and facilitates inoculation with specific cultures. The percent solids in the aqueous slurry can be adjusted based on the concentration of contaminants, the rate of biodegradation in feasibility studies, and the physical natureofthesoils. The per-unitcost of treatment using slurry-phase bioremediation is higher than that ofother biotreatments, but much less expensive than incineration. It is important to monitor the biodegradation process to prove that detoxification has actually been accomplished. Also, it is necessary to assess adsorption ofcompounds, such as benzo(cY)pyrene, in high-clay soils, since microorganisms may be unable to attack compounds unless desorption is facilitated. Biotreatment of Contaminated Groundwater Natural microorganisms may be employed either by direct injection or incorporated into bioreactors. Bioreactors provide a practical means of
assuringa controlled environment for biodegradation, thereby avoiding questions about release of organisms into the general environment. Such questions are still an obstacle for use of genetically-engineered organisms in the United States’.’’. For groundwater sites contaminated with chlorinated or mixed solvents, a typical approach involves empirical development in the laboratory ofa site-specific bioreactorwith a consortium of indigenous organisms plus laboratory stock cultures. Then the bioreactor can be taken into the field in combination with an air stripper, so that non-biodegradable solvents are air-stripped while the biodegradable solvents are treated in a continuous stir bioreactor (Figure 2). For example, a Superfund site in California was contaminated with short-chain chlorinated hydrocarbons and soluble organic compounds such as acetone, 2-butanone, alcohols, and glycols. The contaminated groundwater was first pumped into an air stripper at 50 gallons/minute and then into a 10,000 gallon bioreactor, with a site-specific microbial consortium. The emuent from the bioreactor is directly discharged under national permits. Discharge levels less than 5 ppb for chlorinated hydrocarbons, 500 ppb for acetone and ketones, and 1 ppm for alcohols and glycols have been
Figure 2. Scheme for biotreatment of contaminated groundwater. From Bourquin (1989).
I
.r
Carbon
To surface
Air stripper
achieved continuously during more than three years of Operation of the system. This case illustrates the use of combined technologies. Neither air stripping nor biotreatment alone was effective in reducing the contaminants to acceptable levels. The combination was cost-effective and prev e n t e d any m o v e m e n t o f t h e groundwater plume. A particularly common groundwater contaminant is trichloroethylene (TCE). Nelson et al, while at the US Environmental Protection Agency, isolated a Psetrdoitioiias strain G4 which grows on phenol and in which phenol induces a monooxygenase which biodegrades TCE, as well as phenol”. This organism has been employed in a bioreactor through which TCE-contaminated groundwater is pumped. It would not be permissible to add phenol, a toxic compound, as a “nutrient” for this strain in the environmental compartment. However, Nelson and Bourquin, now at Ecova Corporation, a leading environmental biotechnology company, have discovered a nontoxic, fully metabolized substitute substratelinducer to replace phenol. With this proprietary system, they have been able to utilize the G4 strain by direct injection in a suitable well-demarcated groundwater site (Bourquin, personal communication). These kinds of metabolic manipulations presage genetic manipulations. An alternative geneticallyengineeredsolution, which eliminates the need for phenol or another inducer altogether, is the construction of a constitutive toluene monooxygenase-producing E. coli strain; Winter et a1 at Amgen, Inc, used recombinant DNA techniques to introduce the toluene-oxidizing pathway genes from Pseticloitioiias itieitclociita KR-1 into the E. coli hostI2. This organism has not yet been utilized in field trials. Prospects for GeneticallyEngineered Organisms The modern manipulations feasible with recombinant DNA biotechnology permit modifications of 43
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Asia-Pacific Journal of Public Health 1992/1993 Vol. 6 No. 2 existing strains of bacteria to assure higher biodegradative activity of key enzyme steps, to overcome substrate inhibition, to combine enzymes from unrelated metabolic pathways, to broaden substrate specificity, and to ensure complete mineralization of toxic intermediate^^,'^. Recombinant environmental biotechnology is an exciting area for basic and applied research. Molecular, microbiological, and ecological tools are available for empirical studies that should steadily advance our knowledge and narrow our uncertainties about the behavior and effects of both genetically engineered and indigenous organisms. Systematic risk assessment can be carried outI4. I confidently predict major advances in biotechnological solutions to these numerous kinds of hazardous waste clean-up challenges. And I urge that here in Taiwan, in the United States and Europe, and in Third World Countries we stimulate the use of biological systems in cleaning up polluted soil and water before these accumulations in the aggregate poison life on our Planet. International Cooperation As Rene Dubos stated, “Trend is not destiny”. Those of us with capabilities of biotechnology and engineering have the opportunity to modify severely negative trends in environmental contamination. Dubos also stated, “Think globally; act locally”. Wemust not be deterred by the huge scale of some of the environmental challenges; every desirable local act is a step in the right direction. Many international conferences have recognized that multinational actions are required to address global environmental risks. Significant cooperative efforts can be cited’’. Nine countries cooperate in the Nile River Basin; 18 in the Mediterranean Regional Sea Agreement, the model for what are ‘now 1 1 regional sea agreements with 120 participating countries; 34 in the Long-Range Transboundary Air Pollution Agreement; 61 nations in the London Dumping Convention; and hopefully all countries in the Montreal Accord on
restriction of chlorofluorocarbons. The LAW of the Sea, Outer Space, and Antarctica Treaties are other major cooperative efforts. The World Health Organization hasestablished in this Western Pacific Region a Centre for Promotion of Environmental Planning and Applied Studies (PEPAS), now I 1 years old, located in Kuala Lumpur16. Finally, there is an ongoing struggle to make environmental assessments a routine and effective part of World Bank and other economic development efforts under the important theme of “sustainable de~elopment”’*~*’~. Albert Schweitzer, in his introduction to Rachel Carson’s famous book Silent Spring, wrote in 1962 that “Man has lost the capacity to foresee and forestall”. His gloomy conclusion was that “Man will end by destroying theEarth”. We have the mission to respond to Schweitzer’s challenge: to show that we can at least try to foresee and forestall adverse consequences, while gaining benefits for the people, the economies, and the eco-systems of Planet Earth through Environmental Biotechnology. Acknowledgements
I am grateful to Dr A1 W Bourquin, Vice-president and Chief Scientist, Ecova Corporation, Redmond, Washington, and affiliate professor, D e p a r t m e n t o f Environmental Health, School ofpublic Health, University of Washington, for the two figures and field studies demonstrating applications of environmental biotechnology. References 1. World Commission on Environment and Development(Brundtland Commission): Final Report, Our
Common Future. Geneva, Switzerland, 1987. 2. Castleman BI, Navarro V. International mobility of hazardous products, industries, and wastes. Ann Rev PublicHealth 1987;7:1-19. 3. Repetto R. Paying the Price: Pesticide Subsidiesin Developing Countries. Washington D C World Resources Institute, 1985.
44 Downloaded from aph.sagepub.com at FUDAN UNIV LIB on May 13, 2015
4. World Resources Institute, Intema-
5.
6.
7.
8.
tional Institutefor Environmentand Development, and United Nations Environment Programme: World Resources 1988-9, An Asscssment of the Resource Base that Supports the Global Economy. New York Basic Books, 1989. Gibson DT. Microbial Degradation of Organic Compounds. Microbiology Series, Vol. 13. New York Marcell Dekker, Inc., 1984. Omenn GS, Hollaender A, editors. Genetic Control of Environmental Pollutants. New York: Plenum Press, 1984. Omenn GS, editor. Environmental Biotechnology, Reducing Risks from Environmental Chemicals through Biotechnology. New York: Plenum Press, 1988. Bourquin AW. Bioremediation of hazardous waste. Hazardous Materials, Sept/Oct 1989, pp. 16-23 and 48-59.
9. Tiedje JM, Colwell RR, Grossman YL, et al. The planned introduction ofgeneticallyengineered organisms: ecological considerations and recommendations.A report of the Ecological Society of America. Ecology
1989;70:298-315. 10. National Academy’ of Sciences
Council: Introduction of Recombinant DNA-Engineered Organisms into the Environment: Key Issues. Washington D C National Academy Press, 1987. 11. Nelson MJK, Pritchard PH, Bourquin AW. Preliminary development of a bench-scale treatment system for aerobic degradation of trichloroethylene. In: Omenn GS editor, Environmental Biotechnology: Reducing Risks of Environmental Chemicals through Biotechnology. New York: Plenum Press, 1988: 203-9. 12. Winter RB, Yen KM, Ensley BD.
Efficient degradation of triehloroethylene by a recombinant Escherichia coli. Biotech 1989;7:282-5. 13. Chakrabarty AM, Kamely D, Omenn GS, editors. Biotechnology and Biodegradation. Woodlands, Texas: Portfolio Publishing Co.,
1990. 14. Omenn GS, Bourquin AW. Risk assessment for biodegradation in pol-
lution control and pollution cleanup. In: ChakrabartyAM, Kamely D, Omenn GS, editors, Biotechnology and Biodegradation. Woodlands,
Asia-Pacific Journal of Public Health 199211993 Vol. 6 NO.2 Texas; Portfolio Publishing Co., 1990. 15. Mathews JT. International cooperation in environmentally soundeconomic development. In: R e p o r t o f Salzburg S e m i n a r
No. 259, Managing Global Environmental Risks, Salzburg, Austria, April 1987. 16. Guo PH. Promoting a health environment, the Centre for Promotion of Environmental Planning and Ap-
plied Research. World Health Nov. 1989;lO-11. 17. Clark WC, Munn RE. Sustainable Development of the Biosphere. Cambridge: Cambridge University Press, 1986.
45 Downloaded from aph.sagepub.com at FUDAN UNIV LIB on May 13, 2015
Biotechnology has a growing place in the remediation of hazardous waste sites throughout the world, and especially in Asia where population density is high and land and fresh water are scarce. In-situ bioremediation has been demonstrated already to be highly effective for petroleum hydrocarbons (alkanes, aromatics, polychlorophenols) and organophosphate pesticides in soils and for gasoline by-products (benzene, toluene, xylene) and chlorinated solvents (trichloroethylene) in groundwater. Heavy metals and PCBs are not suitable for bioremediation. Environmental biotechnology includes solid-phase and slurry-phase bioremediation for contaminated soils and sitespecific bioreactors for contaminated groundwater. Specific examples are presented. From a policy point of view, accumulated wastes must be detoxified,preferably at sites where they already exist. We cannot continue to rely on their removal and disposal “elsewhere”. For current waste streams, we must minimize the volumes and toxicity. Environmental biotechnology will play a key role.
Keywords: Bioremediation, biotechnology, environmental biotechnology, groundwater contamination, hazardous chemicals, micro-organisms, soil contamination.
Address for reprints: Gilbert S Omenn, MD, PhD, Profesmr and man, School of Public Health & Community Medicine, SC-30, Universityofwashington, Seattle, Washington 98195, USA.
Environmental Biotechnology: Biotechnology Solutions for a Global Environmental Problem, Hazardous Chemical Wastes* Gilbert S Omenn, MD, PhD School of Public Health &Community Medicine, University of Washington, Seattle, USA *Presented, in part, 17 January 1990 at the International Symposium on Biotechnology for Solving Global Environmental Problems, Tokyo, Japan Bioindustry Development Center, and 3 July, 1990 at the International Symposium on Health, Environment, and Social Change, Taipei, Taiwan.
naut William Anders. In this photograph, we see a tiny fragile globe floating in the vastness of space - a closed, vulnerable system upon which all our lives depend. The imuact on
countries of Asia, land and water are especially precious resources. Since the 1940s, rapid growth of the chemical industry has fueled economic growth and improvements in standards of living and has produced millions of tons of hazardous and toxic wastes - petroleum products, lubricants, protective coatings, pesticides, dielectric fluids, flame retar-
and Development, may surpass even the 16th-century discovery that the Earth is not the center o f the Universe’. The intrusion of human civilizations has presented increasing challenges to Planet Earth. These challenges include retention of infrared radiation or global warming due to greenhouse gases, depletion of stratospheric ozone, loss ofbiological diversity, deforestation, desertification, acid precipitation, threat of major epidemics among humans (including AIDS) or crops, and threat of nuclear war. Among the risks to life on this Planet, the chemical contamination Of ’Oils and water systems deserves our attention, since water is the most distinctive and enabling feature for life on Earth. In the densely populated
dant and forgiving, have become threatened by “out of sight, out of mind” attitudes toward disposal of these chemicals, their residues, and off-grade mixtures. These chemical wastes accumulate in landfills, holding ponds, lakes, rivers, groundwater and soil environments, and the atmosphere. The generation of chemical wastes is highly correlated with the population and standard of living. Industrialization in Third World countries has brought hazardous chemical problems to sometimesunprepared societies2. As the Bhopal accident demonstrated, hazardous chemical processes may be managed inadequately. Prevention of worker exposures and prevention of emissions to air and water may be considered luxuries, or not considered at all.
Introduction
Our generation is the first to have seen its own planet from a distance, photographed most memorably as the “Earthshot” by Apollo 8 astro-
40 Downloaded from aph.sagepub.com at FUDAN UNIV LIB on May 13, 2015
Asia-Pacific Journal of Public Health 1992/1993 Vol. 6 No. 2 Furthermore, many chemicals, especially pesticides, that have been banned or restricted in industrialized countries are being exported to Third World countries. Ill-advised subsidies for purchase stimulate gross overuse of pesticides, with a worldwide epidemic of acute poisonings and with adverse agricultural effects3. In 1982, the United Nations General Assembly approved a resolution (with one dissent) that “Products which have been banned for domestic consumptionand/or sale because they have been judged to endanger health and environment should be sold abroad by companies, corporations, or individuals onlywhen a request for such products is received from an importing country or when the consumptionofsuch productsisofficially permitted in the importing country”. In theory, these nations may wish to accept certain (low-level) risks, which other countries choose to avoid, but they seldom make such explicit decisions and they lack the expertise to make detailed risk assessments. Less developed countries may also be recipients of wastes from more developed countries needing disposal sites; the Eastern European countries have abused their land and water resources in this way. The ever-growing human population forces us to ask “How can we help Nature’s cycles keep up with the waste streams we introduce on Planet Earth?” Throughout the world efforts are increasing to recycle wastes and reduce the amounts of municipal and industrial wastes produced4. The guiding principle in managing accumulated hazardous wastes must be to detoxify them, preferably at the sites where they exist. We cannot continue to rely on removal of wastes to disposal sites, usually landfills, at those places we collectively call “Elsewhere”.
Technologies for clean-up of soils and groundwater contaminated with hazardous chemicals include several types of incineration, chemical and physical treatment to solidify or vitrify and thereby immobilize the chemicals, vapor extraction and air stripping with absorption, and a variety of b i o r e m e d i a t i o n approaches. Biotechnology has a growing place in the remediation of hazardous waste sites in the United States and, surely, in other countries throughout the Asia-Pacific region. The main bioremediation technologies in the field today involve engineering applications of basic principles of aerobic metabolism: optimizing the concentrations of oxygen and nutrients and assuring adequate moisture within a given environment to enhance the indigenous microbial population. Generally, there is little or no knowledge of the organisms present at the site. Better understanding of microbial ecology and genetic enhancement of biodegradation should accelerate progress in this field.
Principles of Bioremediation Microorganisms canuse manychemicals as food and energy sources. With full aerobic metabolism, usingoxygen as the final electron acceptor, the endproducts are carbon dioxide and water. With some chemicals, especially
Table 1 outlines some of the most prevalent a n d important chemicals contaminating soils or ground-water and the present capability of biotechnology to detoxify these chemicals iii sitir. In general, aerobic biodegradation is best suited
Table 1. High-priority chemicals by type of contamination Soils
Groundwater
Petroleum hydrocarbons aliphatic (alkanes) aromatic (benzene, ctc) chloro-aromatics (PCP) Polychlorobiphenyls(PCBs)
++++
Chlorinated Pesticides Organophosphate pesticides
On-Site Remediation On-site remediation requires extensive knowledge of the hydrogeology of the site, the ecology of the soil and water compartments, and the effectiveness of various alternative and complementary technologies.
man-made chemicals, oxidation may be incomplete or may be better carried out by anaerobic organisms, whichusenitrateorsulfateasthe final electron acceptor. Some organisms can biotransform chemicals but obtain no energy yield; these organisms need additional substrate to grow, a co-metabolic scheme. To assess the feasibility of biotreatment, threeaspects must becombined: * microbial physiology, biochemistry, and genetics, to understand the metabolic processes leading to detoxification and the genetics controlling the enzyme functions involved; * microbial ecology, toappreciate the microenvironments in which the treatment may be performed and the structure and function of indigenous or inoculated microbial communities; and * field site engineering, to implement the desired biodegradation scheme, maintaining optimal growth conditions and combining with physical or chemical methods, as n e c e ~ s a r y ~ - ~ .
Heavy Metals
Chlorinated Solvents (TCE) Gasoline (BTX) Triazine herbicides Heavy Metals
Biotechnology
+/-
+++ -
++
++++
+
-I?+
Rating of Biotechnology Effectiveness: - ,ineffective; +, limited effectiveness, to + + + + ,demonstrated highlyeffective. Sentinel compounds indicated: PCP, polychlorophenols; TCE, trichloroethylene; BTX, benzene/tolucnc/xylene
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Asia-Pacific Journal of Public Health I992/1993 Vol. 6 No. 2 for remediation of sites contaminated with petroleum hydrocarbons and polar solvents, such as alcohols a n d ketones. A n a e r o b i c biodegradation is best applied to reductive dechlorination of chlorinated aliphatic and aromatic hydrocarbons, such as tetrachloroethane. Because anaerobic degradation may result in the accumulation of partially oxidized organic compounds, this treatment technique is often coupled with aerobic biological treatment. Metals are not usuallygood targets for bioremediation - or for physical methods either. However, the redox state ofcertain metals, including cadmium, chromium, arsenic, and mercury, can be transformed by microorganisms to a state better suited to separation or isolation, or one that is less toxic. Solid-Phase Bioremediation for Contaminated Soil The traditional form of bioremedia‘tion of contaminated soil has been known for years as “land farming”, often little more than dumping contaminatedwasteonto land and letting Nature work its way, as in the biocycling of natural compounds. The main advances in modem “solid-phase bioremediation” come from optimization of conditions, by addition of nutrients, active aeration, and enhanced release of chemicals absorbed to soil particles. This technique has been used to clean up fuel oils, diesel fuels, pesticides, and other types of easily degraded substrates. Solidphase biotreatment of petroleumcontaminated and creosotecontaminated soils is the most widely used and most cost effective biotreatment technology at the present time. Forexample, at a site with extensive hydrocarbon contamination of soil (up to 16,000 ppm), indigenous organisms were activated by spraying the nitrogen and phosphate, and oxygenating, reducing all components below the target of 100 ppm total concentration. This action was sufficient to permit disposal at a Class 111 landfill, rather than Class I (most hazardous) landfill, as previously scheduled to be done. Complete biodegradation
greenhouse. An overhead spray irriwas demonstrated in the laboratory to gation system within the greenhouse be feasible, but was not called for in provides moisture and distributes this case. A more complicated case was a nutrients and microbial inocula, as site with substantial amounts of 4-6 needed, as large batches of soil are ring polycyclic aromatic hydrocarbrought into the treatment bed for bons (PAHs). It was necessary to add detoxification. The soil treatment an organism capable ofbiodegrad-ing facility is attached to an air managethese more recalcitrant, higher molecment system for the volatiles, with ular weight PAH compounds. Constia vapor-phase bioreactor and then tutive strain B600 of Beijeriitckin B 1, activated carbon, in series. Contamiisolated by Gibson and Mahaffey, was nated leachate is collected in a useful in degradingphenanthreneand sump, then pumped to an on-site bioother high MW ring compounds, esreactor. pecially since it did not require inducThe nature and concentration of tion of activity by low MW PAH the contaminants and the regulatory substrates’. Similarly, P S C ~ I ~ U I I ~ requirements OI~~S in a given country or strain DBMIOl with constitutive state will determine whether elabobiodegradative activity against rate closed systems are required, or polycyclic aromatic hydrocarbons, some emissions of vol8tiles into the including dibenzofuran and air would be permissible. benzo(a)pyrene, was isolated from a creosote-contaminated site, evaluSlurry-Phase Bioremediation of ated in the laboratory, and inocuContaminated Soil lated in the field at a test site in Another approach with soils is solidCalifornia. phase treatment in an aqueous slurry, The solid phase system can be essentially a large bioreactor. This apmodified to control volatiles and leaproach is especially appropriate for chate. For example, a treatment bed control of manufacturing effluents has been lined with an 80-mm highand for groundwater clean-up. An exdensity liner having heat-welded ample is treatment ofa field contamiseams (Figure 1)’. nated with polychlorophenols up to The liner is covered with sand, 8000 ppm. In this case, aeration and which protects it and provides proper nutrients had littleeffect over 13 days, drainage for contaminated water but inoculation of the soil slurries leaching from soils placed on the with a consortium of five organisms treatment bed. Lateral perforated selected by chemostat yielded prompt drainage pipes on top ofthe synthetic reduction of PCP levels. There was liner in the sand bed collect the soil stoichiometric release of chloride, leachate. The whole treatment bed is demonstrating full detoxification. covered by a modified plastic film Figure 1. Schemefor solid-phasebiorcmediationof contaminatedsoils, brought into
a controlled environment on-site. From Bourquin (1989).
Solid-phasetreatment process Atyp~calsection A-A’
I rr
r Treatment zone
r Spray nozzle
Leacha:e col!ecllonsyslem Synthetr.llner t- Cornpaned secondary 5011 liner
r
SyS!em area overhead enclosure
,-Eanh It11 term
1 To Ireatmen1and
recycle 01dsspo-1
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Addition of organisms was essential and was effective in reducing levels to below 0.5 ppb. Similar good results wereobtained inasoil-slurrybiotreatment for trichloroethylene (TCE). The bioreactor scheme decreases acclimation time, increases biodegradation rates, allows greater process control, increases contact between microorganisms and contaminants, and facilitates inoculation with specific cultures. The percent solids in the aqueous slurry can be adjusted based on the concentration of contaminants, the rate of biodegradation in feasibility studies, and the physical natureofthesoils. The per-unitcost of treatment using slurry-phase bioremediation is higher than that ofother biotreatments, but much less expensive than incineration. It is important to monitor the biodegradation process to prove that detoxification has actually been accomplished. Also, it is necessary to assess adsorption ofcompounds, such as benzo(cY)pyrene, in high-clay soils, since microorganisms may be unable to attack compounds unless desorption is facilitated. Biotreatment of Contaminated Groundwater Natural microorganisms may be employed either by direct injection or incorporated into bioreactors. Bioreactors provide a practical means of
assuringa controlled environment for biodegradation, thereby avoiding questions about release of organisms into the general environment. Such questions are still an obstacle for use of genetically-engineered organisms in the United States’.’’. For groundwater sites contaminated with chlorinated or mixed solvents, a typical approach involves empirical development in the laboratory ofa site-specific bioreactorwith a consortium of indigenous organisms plus laboratory stock cultures. Then the bioreactor can be taken into the field in combination with an air stripper, so that non-biodegradable solvents are air-stripped while the biodegradable solvents are treated in a continuous stir bioreactor (Figure 2). For example, a Superfund site in California was contaminated with short-chain chlorinated hydrocarbons and soluble organic compounds such as acetone, 2-butanone, alcohols, and glycols. The contaminated groundwater was first pumped into an air stripper at 50 gallons/minute and then into a 10,000 gallon bioreactor, with a site-specific microbial consortium. The emuent from the bioreactor is directly discharged under national permits. Discharge levels less than 5 ppb for chlorinated hydrocarbons, 500 ppb for acetone and ketones, and 1 ppm for alcohols and glycols have been
Figure 2. Scheme for biotreatment of contaminated groundwater. From Bourquin (1989).
I
.r
Carbon
To surface
Air stripper
achieved continuously during more than three years of Operation of the system. This case illustrates the use of combined technologies. Neither air stripping nor biotreatment alone was effective in reducing the contaminants to acceptable levels. The combination was cost-effective and prev e n t e d any m o v e m e n t o f t h e groundwater plume. A particularly common groundwater contaminant is trichloroethylene (TCE). Nelson et al, while at the US Environmental Protection Agency, isolated a Psetrdoitioiias strain G4 which grows on phenol and in which phenol induces a monooxygenase which biodegrades TCE, as well as phenol”. This organism has been employed in a bioreactor through which TCE-contaminated groundwater is pumped. It would not be permissible to add phenol, a toxic compound, as a “nutrient” for this strain in the environmental compartment. However, Nelson and Bourquin, now at Ecova Corporation, a leading environmental biotechnology company, have discovered a nontoxic, fully metabolized substitute substratelinducer to replace phenol. With this proprietary system, they have been able to utilize the G4 strain by direct injection in a suitable well-demarcated groundwater site (Bourquin, personal communication). These kinds of metabolic manipulations presage genetic manipulations. An alternative geneticallyengineeredsolution, which eliminates the need for phenol or another inducer altogether, is the construction of a constitutive toluene monooxygenase-producing E. coli strain; Winter et a1 at Amgen, Inc, used recombinant DNA techniques to introduce the toluene-oxidizing pathway genes from Pseticloitioiias itieitclociita KR-1 into the E. coli hostI2. This organism has not yet been utilized in field trials. Prospects for GeneticallyEngineered Organisms The modern manipulations feasible with recombinant DNA biotechnology permit modifications of 43
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Asia-Pacific Journal of Public Health 1992/1993 Vol. 6 No. 2 existing strains of bacteria to assure higher biodegradative activity of key enzyme steps, to overcome substrate inhibition, to combine enzymes from unrelated metabolic pathways, to broaden substrate specificity, and to ensure complete mineralization of toxic intermediate^^,'^. Recombinant environmental biotechnology is an exciting area for basic and applied research. Molecular, microbiological, and ecological tools are available for empirical studies that should steadily advance our knowledge and narrow our uncertainties about the behavior and effects of both genetically engineered and indigenous organisms. Systematic risk assessment can be carried outI4. I confidently predict major advances in biotechnological solutions to these numerous kinds of hazardous waste clean-up challenges. And I urge that here in Taiwan, in the United States and Europe, and in Third World Countries we stimulate the use of biological systems in cleaning up polluted soil and water before these accumulations in the aggregate poison life on our Planet. International Cooperation As Rene Dubos stated, “Trend is not destiny”. Those of us with capabilities of biotechnology and engineering have the opportunity to modify severely negative trends in environmental contamination. Dubos also stated, “Think globally; act locally”. Wemust not be deterred by the huge scale of some of the environmental challenges; every desirable local act is a step in the right direction. Many international conferences have recognized that multinational actions are required to address global environmental risks. Significant cooperative efforts can be cited’’. Nine countries cooperate in the Nile River Basin; 18 in the Mediterranean Regional Sea Agreement, the model for what are ‘now 1 1 regional sea agreements with 120 participating countries; 34 in the Long-Range Transboundary Air Pollution Agreement; 61 nations in the London Dumping Convention; and hopefully all countries in the Montreal Accord on
restriction of chlorofluorocarbons. The LAW of the Sea, Outer Space, and Antarctica Treaties are other major cooperative efforts. The World Health Organization hasestablished in this Western Pacific Region a Centre for Promotion of Environmental Planning and Applied Studies (PEPAS), now I 1 years old, located in Kuala Lumpur16. Finally, there is an ongoing struggle to make environmental assessments a routine and effective part of World Bank and other economic development efforts under the important theme of “sustainable de~elopment”’*~*’~. Albert Schweitzer, in his introduction to Rachel Carson’s famous book Silent Spring, wrote in 1962 that “Man has lost the capacity to foresee and forestall”. His gloomy conclusion was that “Man will end by destroying theEarth”. We have the mission to respond to Schweitzer’s challenge: to show that we can at least try to foresee and forestall adverse consequences, while gaining benefits for the people, the economies, and the eco-systems of Planet Earth through Environmental Biotechnology. Acknowledgements
I am grateful to Dr A1 W Bourquin, Vice-president and Chief Scientist, Ecova Corporation, Redmond, Washington, and affiliate professor, D e p a r t m e n t o f Environmental Health, School ofpublic Health, University of Washington, for the two figures and field studies demonstrating applications of environmental biotechnology. References 1. World Commission on Environment and Development(Brundtland Commission): Final Report, Our
Common Future. Geneva, Switzerland, 1987. 2. Castleman BI, Navarro V. International mobility of hazardous products, industries, and wastes. Ann Rev PublicHealth 1987;7:1-19. 3. Repetto R. Paying the Price: Pesticide Subsidiesin Developing Countries. Washington D C World Resources Institute, 1985.
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4. World Resources Institute, Intema-
5.
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tional Institutefor Environmentand Development, and United Nations Environment Programme: World Resources 1988-9, An Asscssment of the Resource Base that Supports the Global Economy. New York Basic Books, 1989. Gibson DT. Microbial Degradation of Organic Compounds. Microbiology Series, Vol. 13. New York Marcell Dekker, Inc., 1984. Omenn GS, Hollaender A, editors. Genetic Control of Environmental Pollutants. New York: Plenum Press, 1984. Omenn GS, editor. Environmental Biotechnology, Reducing Risks from Environmental Chemicals through Biotechnology. New York: Plenum Press, 1988. Bourquin AW. Bioremediation of hazardous waste. Hazardous Materials, Sept/Oct 1989, pp. 16-23 and 48-59.
9. Tiedje JM, Colwell RR, Grossman YL, et al. The planned introduction ofgeneticallyengineered organisms: ecological considerations and recommendations.A report of the Ecological Society of America. Ecology
1989;70:298-315. 10. National Academy’ of Sciences
Council: Introduction of Recombinant DNA-Engineered Organisms into the Environment: Key Issues. Washington D C National Academy Press, 1987. 11. Nelson MJK, Pritchard PH, Bourquin AW. Preliminary development of a bench-scale treatment system for aerobic degradation of trichloroethylene. In: Omenn GS editor, Environmental Biotechnology: Reducing Risks of Environmental Chemicals through Biotechnology. New York: Plenum Press, 1988: 203-9. 12. Winter RB, Yen KM, Ensley BD.
Efficient degradation of triehloroethylene by a recombinant Escherichia coli. Biotech 1989;7:282-5. 13. Chakrabarty AM, Kamely D, Omenn GS, editors. Biotechnology and Biodegradation. Woodlands, Texas: Portfolio Publishing Co.,
1990. 14. Omenn GS, Bourquin AW. Risk assessment for biodegradation in pol-
lution control and pollution cleanup. In: ChakrabartyAM, Kamely D, Omenn GS, editors, Biotechnology and Biodegradation. Woodlands,
Asia-Pacific Journal of Public Health 199211993 Vol. 6 NO.2 Texas; Portfolio Publishing Co., 1990. 15. Mathews JT. International cooperation in environmentally soundeconomic development. In: R e p o r t o f Salzburg S e m i n a r
No. 259, Managing Global Environmental Risks, Salzburg, Austria, April 1987. 16. Guo PH. Promoting a health environment, the Centre for Promotion of Environmental Planning and Ap-
plied Research. World Health Nov. 1989;lO-11. 17. Clark WC, Munn RE. Sustainable Development of the Biosphere. Cambridge: Cambridge University Press, 1986.
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