The quality of life on Earth is linked inextricably to the overall quality of the environment. In response to growing pressures on air, water, and land resources, global attention has focused in recent years on finding new ways to sustain and manage the environment. Biotechnology is an essential tool in this endeavor because it can provide new approaches for understanding, managing, preserving, and restoring the environment.
Biotechnology can be used to assess the well-being of ecosystems, transform pollutants into benign substances, generate biodegradable materials from renewable sources, and develop environmentally safe manufacturing and disposal processes. Researchers are just beginning to explore biotechnological approaches to problem solving in many areas of environmental management and quality assurance, such as
Environmental biotechnology is not a new field; composting and wastewater treatment technologies are familiar examples of "old" environmental biotechnologies. However, recent developments in molecular biology, ecology, and environmental engineering now offer opportunities to modify organisms so that their basic biological processes are more efficient and can degrade more complex chemicals and higher volumes of waste materials. Notable accomplishments of the "new" environmental biotechnology include the cleanup of water and land areas polluted with petroleum products.
While some success has been achieved, the potential benefits of the new environmental biotechnology are far from fully realized. Advances in this arena are delayed not only by legal and social barriers (which may be formidable) but also by a dearth of basic scientific knowledge about organisms that may be used in biotechnologies and the ecological systems in which these technologies are to be employed. Only new knowledge acquired through Federally supported basic research can provide the foundation for new environmental applications of biotechnology, facilitate the development of these technologies by the commercial sector, and ensure adequate evaluation and safe application of products without blocking innovation with regulatory requirements.
Research in environmental biotechnology has unique international aspects. International cooperation will be needed to help generate new scientific knowledge in this arena, assure U.S. access to the requisite technologies and genetic resources, and establish markets for the resulting U.S. products and processes worldwide. In addition, environmental biotechnology has tremendous potential for use in developing nations seeking low- cost solutions to environmental problems, such as municipal waste disposal, conversion of agricultural wastes to energy sources, and cleanup of polluted areas.
Here, a research program is proposed in one area of environmental biotechnology -- bioremediation, an emerging approach to rehabilitating areas fouled by pollutants or otherwise damaged through ecosystem mismanagement. A novel research framework is described and specific opportunities are identified that offer potential for significant advances. Five research priorities are identified:
Bioremediation is addressed as one example of an environmental biotechnology. Because the knowledge required for bioremediation is similar to that needed for the development of many other environmental biotechnologies, the research approach described here is likely to have wide application.
The United States has a large number of identified polluted areas, including land, fresh water, and marine sites that, by law, must be cleaned up. Estimates for the cleanup of Federal lands alone may be $450 billion. The extent of contaminated non- Federal agricultural acreage, mining areas, industrial sites, and aquifers and other water bodies is unknown, but the magnitude of the problem is undoubtedly large and clean-up expenses could be astronomical. It has been estimated that cleanup of both Federal and non-Federal lands could cost $1.7 trillion using conventional approaches, which would produce noxious waste by-products and thereby impose additional clean-up or environmental costs.(1)
Due to its comparatively low cost and generally benign environmental impact, bioremediation offers an attractive alternative and/or supplement to more conventional clean-up technologies. Bioremediation has been successful at many sites contaminated with petroleum products. However, it is not always the technology of choice because efficacy and the rate of degradation at any particular site cannot be predicted reliably. Improved predictive and process validation capabilities would help stimulate wider use of this technology. Research also could lead to development of biotechnologies to remediate areas contaminated by metals, pesticides, radioactive elements, other toxic materials, and mixed wastes.
These types of studies could be especially productive at this time. Recent developments in biology have provided new tools and approaches for monitoring the environment and engineering organisms with the capacity to degrade environmental pollutants. These developments have created unprecedented opportunities for significant advances.(2) Indeed, bioremediation is expected to become an industry with annual sales of more than $500 million by the year 2000.(3)
The United States is among several nations developing bioremediation technologies. Maintaining and enhancing the U.S. position in this arena will require continued investment in the generation of new knowledge needed for the development of new technologies. Investment in bioremediation research has the dual benefits of solving important environmental problems while stimulating the growth of the U.S. bioremediation industry.
Key to the success of the investigations proposed in this chapter is the overall structure of the research program. The complex environmental milieu in which bioremediation and other environmental technologies will be employed demands an holistic research strategy; the traditional, piecemeal approach will not be adequate. A research program designed using an holistic approach would
Bioremediation research generally is conducted at one of three scales: laboratory, pilot scale, or field trial. To help ensure that results achieved at the first two scales can be translated to the field, the research program should be conceived as a continuum, with investigators working at each scale involved throughout the research conceptualization and planning process. The aim is to translate research findings from the laboratory into viable technologies for remediation in the field.
The biological and physical complexity of the field environment, where research findings ultimately will be tested and applied, demands a multidisciplinary research team composed of microbiologists, ecologists, engineers, hydrologists, and other specialists.
A major impediment to achieving the full potential of bioremediation is the lack of access to field sites for iterative research on basic mechanisms, technology development, and process verification. Field research sites should be identified that accommodate long-term experimentation on a large spatial scale.
Many polluted sites contain mixtures of wastes of such chemical complexity that a suite of biochemical processes is required for degradation. Waste mixtures may contain sanitary (household) waste plus hazardous organic or inorganic materials. Hazardous waste is defined legally as toxic, corrosive, reactive, or ignitable. "Mixed waste" generated by Federal weapons and research complexes is a combination of radioactive and hazardous waste. Therefore, research focusing on the degradation of mixtures of wastes by groups of organisms is likely to be most realistic and efficacious.
Different types of organisms can be bioremediation agents. For example, the use of plants to concentrate pollutants (phytoremediation) is an emerging research area. This chapter, however, examines bioremediation by microorganisms, the most biochemically diverse and least understood group of organisms on Earth.
Microorganisms (primarily bacteria and fungi) are nature's original recyclers. Their capability to transform natural and synthetic chemicals into sources of energy and raw materials for their own growth suggests that expensive chemical or physical remediation processes might be replaced or supplemented with biological processes that are lower in cost and more environmentally benign.
Microorganisms therefore represent a promising, albeit largely untapped resource for new environmental biotechnologies. Research continues to verify the bioremediation potential of microorganisms. For example, a recent addition to the growing list of bacteria that can sequester or reduce metals is Geobacter metallireducens, which removes uranium, a radioactive waste, from drainage waters in mining operations and from contaminated groundwaters. Even dead microbial cells can be useful in bioremediation technologies. These discoveries suggest that further exploration of microbial diversity is likely to lead to the discovery of many more organisms with unique properties useful in bioremediation.(4)
A tiny fraction of the microbial diversity of the Earth has been identified, and an even smaller fraction has been examined for its biodegradation potential.(5) Research aimed at characterizing this diversity likely would lead to the discovery of novel mechanisms for biodegradation of pollutants. Once the biochemical and ecological nature of microbial biotransformations of pollutants is understood, new bioremediation technologies based on these mechanisms will be possible.
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Expanded research is needed on the basic ecology of microorganisms and interactions among microbial community members. In nature, microorganisms seldom exist or act as single species; instead, they act collectively as consortia. Research examining bioremediation of polychlorinated biphenyls (PCBs) in the Hudson River, for example, revealed that both anaerobic and aerobic bacteria, acting together, were responsible for degrading the pollutant. (Anaerobes grow in the absence of oxygen, while aerobes require it.) Additional research of this type will provide a framework for understanding how microbial communities respond to various environmental stresses; how to accelerate in situ bioremediation by native microbial communities; and whether the introduction of engineered microbes with enhanced bioremediation potential can survive and function within established communities and help remediate the site. Such studies are likely to provide corollary insights into aspects of microbial biochemistry important for bioremediation as well as the roles of microorganisms in biogeochemical cycling.
Physiology/Biochemistry Research
PRIORITY: Determine the biochemical mechanisms, including enzymatic pathways, involved in aerobic and particularly anaerobic degradation of pollutants.
Much of the information relevant to bioremediation has come from studies of the genetics and physiology of aerobic bacteria. As a result, the best-known biochemical processes related to bioremediation are oxygen dependent. This characteristic limits their effectiveness in many polluted underground and underwater sites with minimal or no oxygen. Both biological and physical strategies for improving the supply of oxygen in such sites have been proposed, and research on aerobic organisms must be continued. However, long-term success in dealing with a wide array of polluted sites with little or no oxygen will require information that can be obtained only through increased research on the genetics, physiology, and biochemistry of anaerobic organisms.
In nature, biodegradation in sites with little or no oxygen is mediated by anaerobic and microaerophilic microorganisms. Because of the difficulty of isolating and culturing such organisms in the laboratory, their metabolic diversity and their potential use in environmental biotechnology only recently have been appreciated. These technical obstacles are being overcome with improved cultivation methods, new technologies for identification of microorganisms, and new methods for studying their metabolism in situ.
New knowledge about anaerobic microorganisms has expanded opportunities for exploiting their metabolic diversity in bioremediation. Since the discovery in 1982 that some anaerobes can dehalogenate carbon compounds, microbes with this capability have been detected in a wide variety of anaerobic environments, such as the Hudson River sediments. However, to exploit anaerobes for bioremediation, more knowledge is needed about the biology of diverse anaerobic microbes, including how they respond to fluctuating oxygen levels.
Because the concentration of oxygen in soil and water environments is often highly variable (in time and space), biodegradation in nature probably is mediated by both aerobic and anaerobic microorganisms. This phenomenon can be exploited through the development of anaerobic-aerobic technologies for controlled bioremediation. In other words, the environment can be modified either spatially or temporally to allow the development of various types of biodegradative microbial communities, thereby fostering the degradation of pollutants vulnerable to different agents. Anaerobic-aerobic processes can be developed to exploit the full range of microbial metabolic activity for cleanup of environments contaminated with multiple pollutants.
PRIORITY: Expand understanding of microbial genetics as a basis for enhancing the capabilities of microorganisms to degrade pollutants.
The successful use of microorganisms in bioremediation depends on the development of a basic understanding of the genetics of a broad spectrum of microorganisms, many of them not yet isolated or studied in any detail. Microorganisms adapted to degrade specific pollutants have been found among populations growing naturally at polluted sites. However, the genetic mechanisms underlying specific adaptations are poorly understood.
In the past, researchers have been unable to conduct genetic studies on these hard-to-culture organisms, but recent developments in molecular biology now make it possible to isolate and study genes of almost any organism. Scientists now can analyze genes that govern a wide variety of metabolic processes, including the degradation of environmental pollutants. Such genetic analyses provide information about mechanisms underlying the operation and evolution of degradation pathways.
While the isolation and characterization of novel microorganisms and genes are important in developing bioremediation strategies, knowledge about the general features of the microbial genome also are needed. Bacteria exhibit a high degree of genetic plasticity, or changeability. Analysis of complete microbial genomes will reveal the nature of this plasticity and suggest how genetic engineering can be used to modify organisms to impart the characteristics needed for bioremediation.
New knowledge about the diversity of microorganisms and the organization of their genes also will help explain how environmental factors influence the expression of genes and the regulation of microbial metabolism. Research has revealed that genes expressed when one compound is present can play a role in the metabolism of a second compound. For example, some bacteria were found to degrade highly toxic trichloroethylene (TCE) when the less toxic compound toluene was present. Mutant organisms also have been isolated that can degrade TCE in the absence of toluene due to genetic changes that cause the TCE-degrading gene to be "turned on" continuously. However, bacteria capable of TCE degradation may lack other traits -- such as tolerance for heavy metals, salts, and acid soils -- needed for their use in bioremediation. Genes for these traits could be added through genetic engineering, thereby increasing the degradation efficiency of native microorganisms.
Recombinant microorganisms with expanded degradation capabilities have been developed recently. Researchers in several countries studied a number of microorganisms, each of which degraded a restricted range of pollutants, and characterized the genes involved. Investigators then combined genes from different species into one strain of bacteria that can degrade multiple types of pollutants.(7) Similar applications of modern genetic techniques should make it possible to tailor bacteria for bioremediation of sites contaminated with specific combinations of toxic compounds.
FROM THE LABORATORY TO THE FIELD:
THE RESEARCH CONTINUUM
PRIORITY: As a standard practice, conduct microcosm/mesocosm studies of new bioremediation techniques to determine in a cost-effective manner whether they are likely to work in the field, and establish dedicated sites where long-term field research on bioremediation technologies can be conducted.
PRIORITY: Develop, test, and evaluate innovative biotechnologies, such as biosensors, for monitoring bioremediation in situ; models for the biological processes at work in bioremediation; and reliable, uniform methods for assessing the efficacy of bioremediation technologies.
Once organisms or groups of organisms with bioremediation potential have been identified through laboratory screening or genetically engineered microcosm/mesocosm studies -- scale-up studies conducted in large bioreactors or on small, protected areas of land or water -- can indicate whether the organisms are likely to perform as desired at field scales. This intermediate step permits control of the pollutant concentration, the numbers of degradative agents, and the physiochemical environment.
More specifically, the dissipation of the pollutant can be quantified and the breakdown products measured and identified using labels and tracers that would be too costly to employ in the field. Changes in the degradative capacity and ecological adaptation of the degrading microorganism can be monitored, and environmental conditions that favor bioremediation can be assessed. If non-native organisms are being evaluated, then their ability to survive and compete with the native microbes can be determined. In addition, potential adverse effects of the bioremediation strategies studied can be detected and mitigated.
Many bioremediation applications benefit from testing and scale- up from the laboratory to the mesocosm level. An example is the use of bioreactors for the immobilization of metals from water or the treatment of industrial and municipal waste, before the techniques are used in the field. When degradation is examined under intentionally varied environmental conditions, the results can suggest whether biodegradation is likely to occur in the field at an acceptable rate with or without oxygen and nutrient augmentation. It is useful to employ systems of various sizes, ranging from small laboratory microcosms, such as soil columns and biofilms, to soil lysimeters (large, instrumented in situ blocks of soil isolated by physical barriers).
While the results obtained from laboratory bioreactors cannot be extrapolated directly to predict field performance, research at the microcosm/mesocosm scale can reveal genetic and biochemical changes in organisms exposed to the target pollutants. This approach may save considerable time and resources by suggesting whether a remediation system has any chance to succeed in the field.
The successful development and application of bioremediation technologies depends on field-based research to verify the efficacy of planned approaches under natural conditions. Field research is a complex undertaking, in part because any number of problems can arise. For example:
A further challenge in field evaluations of bioremediation technologies is the need for data collection over long periods of time and at various spatial scales. It can be difficult, if not impossible, to find suitable field sites. A recent report from a workshop sponsored by the American Academy of Microbiology (AAM) concluded that
A major problem in the development of bioremediation technology is the lack of field sites that are well- characterized with respect to contaminants, geohydrology, and geochemistry; such sites are urgently needed for understanding the natural events that are taking place and also for the transfer of technology developed in the laboratory to field conditions. An integrated interdisciplinary approach is essential for the application and verification of bioremediation, and this can only be achieved under environmental conditions. Although some sites may already exist, their openness and flexibility of use is unlikely to support more than a few efforts in the bioremediation community.(8)
Although some Federal programs are beginning to address the critical need for field experimentation identified by the two AAM reports, there are no long-term, interdisciplinary bioremediation research programs conducted at dedicated field sites. To accelerate the discovery and development of effective bioremediation technologies, several long-term research sites should be established. Coordinated, interdisciplinary research should be conducted to explore the bioremediation-related characteristics of these sites. Then, multidisciplinary groups of researchers should define cooperatively the basic questions to be answered, the scope of the work to be accomplished, and the methods of validation of field results.
Integral to the research conducted at these field sites would be assessment of the efficacy of the biotechnologies employed. As new bioremediation data are collected and verified, new field experiments could be planned and discoveries transferred to the private sector for development and commercialization.
A coordinated program of long-term research at field laboratories could facilitate the development of risk assessment baseline information, including bioavailability data; promote the transfer of all findings to industry and other potential user groups; generate quantitative information on mechanisms of passive remediation as a bioremediation alternative; foster the development of biosensors for process characterization and validation; and, perhaps most importantly, establish a database that could be used to predict conditions under which bioremediation can be achieved.
Efficacy Testing Methods and Data Evaluation
One barrier to the acceptance and use of bioremediation technologies is the lack of established methods for determining efficacy in the field. Evaluation of bioremediation in the field is difficult due to the complexities of the natural environment, the competing abiotic processes, and the vast temporal and spatial scales involved. The AAM has identified the lack of adequate performance standards as a key factor undermining user confidence in bioremediation. The recent growth of the bioremediation industry has intensified the problem of determining the effectiveness of a large number of competing processes.
The development of standardized evaluation protocols would be facilitated by research comparing various methods for obtaining evidence. Possible measures include numbers and activities of bacteria, changes in inorganic carbon concentration, or the extent of transformation of pollutants into forms that can be biodegraded more easily. Although innovation might be inhibited by premature setting of standards or overregulation of the fledgling bioremediation industry, methods for standardized efficacy testing and data evaluation should be developed as an integral part of the industry's growth. Ideally, to ensure objectivity, a government agency not directly involved in site cleanup or regulation would help develop standardized testing methods and materials.
As efficacy studies are carried out, a large volume of historical data will be generated. The quality and comparability of such data must be evaluated and controlled carefully.
Biosensors: One Promising Assessment Technology
Currently, in situ degradation processes cannot be measured or validated directly; researchers must rely on tracers and gas generation to assess bioremediation processes. Increased investment in biosensor research could lead, over the long term, to improved tools for efficacy assessment. The development of biosensors promises to revolutionize the way pollutants are detected and monitored in the environment.
Unlike standard methods, which rely on analytical chemistry in measuring the total concentration of a pollutant, biosensors can detect the fraction available to microorganisms. Biosensors also have the advantage of being nondestructive and located on-line, meaning that samples do not have to be removed and transported to a laboratory for analysis. Biosensors may utilize either whole bacterial cells or specific molecules (e.g., enzymes or biomimetics) as a detection system. Combinations of biosensors in arrays can be exploited to deal with a diversity of toxicants and pollutants.
One type of biosensor involves linking a gene such as the mercury resistance gene (mer) or the toluene degradation (tol) gene to genes that code for bioluminescence within living bacterial cells. The biosensor cells can signal that extremely low levels of inorganic mercury or toluene are present in contaminated waters and soils by emitting visible light, which can be measured with fiber-optic fluorometers.
A second type of biosensor employs molecular detectors, which consist of enzymes, nucleic acids, antibodies, or other "reporter" molecules attached to synthetic membranes. Antibodies specific for an environmental contaminant can be coupled to changes in fluorescence to increase sensitivity of detection. Fluorescent or enzyme-linked immunoassays have been derived for a variety of contaminants, including pesticides and PCBs. With sustained long-term research, molecular arrays could be constructed on synthetic membranes and other matrices that would allow the simultaneous detection of a range of contaminants in a variety of environmental substrates.
Enzyme redesign is an emerging area of research that holds great promise for improving the bioremediation potential of microorganisms. Enzyme models can be created and manipulated by supercomputers to change primary enzyme structure and then predict resulting changes in enzymatic specificity and activity. Changes that would enhance bioremediation activity then can be engineered into bacteria, and those bacteria tested for a desired result in microcosms and mesocosms.
In the absence of reasonable parameters for the biological processes involved in bioremediation, models also can be developed to indicate whether physical factors also are at work. Models could be used to distinguish the important variables from those with only minor effects and to extrapolate results for one geographic region to another, based on predicted patterns of interactions between physical and biological factors.