 |
| About Bioline | All Journals | Testimonials | Membership | News |
|
||||||
|
||||||
Australasian Biotechnology, Conference PaperInnovations and Broad Horizons for Environmental Biotechnology Gary S. Sayler The University of Tennessee, The Center for Environmental Biotechnology, Knoxville, Tennessee 37996 Code Number:AU98031 Over the next thirty years, it has been predicted that worldwide markets for environmental technology will exceed $400 billion annually. These markets are extremely broad and are encompassed by a vision of environmental sustainability. Within this vision, environmental biotechnology can play a distinct role. Yet, this role is not clearly defined and the market share that environmental biotechnology can capture remains unpredictable. What is clear, is that environmental biotechnology has opportunities to contribute new solutions and directions in remediation of contaminated environments, minimizing future waste release and creating pollution prevention alternatives. To take advantage of these opportunities, innovative new strategies, which advance the use of molecular biological methods and genetic engineering technology, must be developed. These methods would improve the understanding of existing biological processes in order to increase their efficiency, productivity, and flexibility. Examples of the development and implementation of such strategies include: 1) the use of recombinant organisms in soil bioremediation, 2) genetic and molecular process monitoring and control for optimization of waste treatment systems, bioprocessing and bioremediation, and 3) novel gene discovery technology and wireless integrated circuit biosensor biotechnology for direct environmental analysis, and system improvement. While the research and development landscape is expected to shift from bioremediation technology to more complex topics in environmental sustainability, the resulting transition creates enormous opportunities for innovative biotechnical approaches to capture a significant share of the future environmental technology market. Environmental biotechnology is operationally defined as the use of living organisms, their parts and processes, for socio-economic benefit in environmental protection, restoration, and sustainability. The breadth of this definition is wide, covering not only issues of chemical pollution of the environment, but also issues such as analysis and exploitation of biological and genetic diversity, environmental sensors and diagnostics, chemical and biological agent detection, and bioprocessing in complex systems, among others. In general, the biological focus of environmental biotechnology has been microorganisms, primarily bacteria and to a lesser extent fungi; however, there has been major growth in the higher eukaryotes, in particular, plants relative to bioremediation, waste treatment, and soil fertility and natural products. As early as 1986, a fundamental research agenda for advancing environmental biotechnology was defined (1). While this agenda was somewhat focused on issues relative to waste management, it was, and is, broadly applicable to all areas of the technology. This four part agenda (later expanded to five parts) is summarized as follows:
It should be readily apparent from this agenda that environmental biotechnology is not the purview of any one scientific or engineering discipline. As in the case for traditional biotechnology, a non-hierarchical approach to this agenda is best accomplished by strong, integrated multidisciplinary interactions among molecular biologists, life and environmental scientists and engineers, and policy (both public and regulatory) specialists. This research agenda has proven to be sufficiently flexible to accommodate both the explosive growth of information and tools available from developments in molecular biology, as well as the changing economic and regulatory driving forces for R & D and commercialization. To illustrate the factors influencing dynamics of both R & D and applications of environmental biotechnology, we can use the example of waste management and bioremediation of contaminated environments. In the mid 1980's, the development of technology was viewed as the new cost effective process for the destruction of chemical pollutants in soil and other contaminated environmental matrices. To some extent, this bioremediation alternative to expensive soil incineration or landfill technology was oversold, given the fact that scaling up laboratory results to field processes was largely only successful for relatively easy to degrade contaminants such as fuel hydrocarbons. In addition, genetic engineering research needed for difficult to degrade pollutants was hampered by the distinct uncertainties that regulatory risk evaluation may preclude use of recombinant microbial processes in the environment. The regulatory driving force for remediation was cleanup to prescribed endpoints of chemical contamination: endpoints often difficult to achieve by bioremediation or kinetically limited by physical variables making bioremediation less advantageous on a cost basis. An example of such unsolved problems are the issues associated with in situ and ex situ TCE bioremediation, which, consequently, has limited the technology to non-source contaminants and thus, as a technical solution to groundwater contaminants, has only been embraced by only a limited number of bioremediation practitioners (2). A dramatic shift is occurring in the driving force for environmental biotechnology, both in hazardous waste contamination and in other areas of application of the technology (3). This shift is to economic and cost benefit factors rather than prescribed regulatory cleanup chemical standards. These factors or economic driving forces can be described in the following way. Causing pollution and discharging wastes is an economic drain on industry in a globally competitive world and is generally a waste of resources or finished product regardless of the regulatory penalty of pollution discharge. Secondly, depending on ultimate end use or natural resource use objectives; less stringent cleanup approaches and endpoints may be cost effective without significant added risk to health or the environment. As a result of these new driving forces, environmental biotechnology as a research and development endeavor or as an industry can expect new and changing opportunities. These changing opportunities are reflected in Figure 1, which attempts a gross prediction of the commercial and R&D spending for general environmental technology early into the next century (4). Figure 1. Transition of environmental biotechnology (4) As indicated by figure 1, the long-term trend for applications and research in environmental biotechnology should focus on environmental sustainability. Environmental sustainability is defined as the utilization and management of natural resources in a manner that is consistent with their steady state availability for future generations. Bioremediation, waste treatment, and pollution prevention and recycling are all components of this mission for environmental sustainability. However, it is clear, especially in the US, that as clean up and remediation of past environmental contamination is accomplished, bioremediation must shift to a niche specific focus with fewer available R&D dollars and there must be a clear cost benefit advantage over other remediation practices. While waste and wastewater treatment technology are often viewed esoterically with limited prospect for biotechnology, in reality there is a great deal of R&D and commercial potential in improving the performance, operating characteristics, stability and flexibility of biological waste treatment. This is true for not only traditional industrial and domestic wastewater treatment, but also systems for gas phase bioconversion of pollutants, wetland treatment of contaminants, as well as in composting and other solid waste, disposal systems that can be re-engineered for treatment applications. An example is re-engineering of solid waste landfills for in situ treatment with biogas recovery and eventual reclamation of the site for alternative land use applications. Closely coupled with waste treatment is pollution prevention which seeks to 1) limit waste going to treatment facilities, 2) make more effective linkage of waste treatment to chemical specific waste streams and effluents, and 3) developing processes and products with lessened risk of environmental discharge, damage and/or persistence in the environment. This later area, which encompasses product life cycle analysis, has broad implications in environmental biotechnology, not only in accurately predicting the fate of chemicals in the environment, but also in developing alternative feedstocks and processes en route to consumer and industrial products. This area will dramatically expand opportunities in environmental biotechnology for new agents in bioprocessing of specialty and commodity chemicals, as well as biological replacements for synthetic chemicals, e.g. enzyme replacement of synthetic detergents or bioleaching as a replacement for chemical bleaching of paper pulp (5). Across the spectrum of environmental sustainability, environmental biotechnology has sought to expand the use of molecular biological methods to exploit and recover biological resources from the environment and to analyze the diversity, health, and resilience of natural and recovering ecosystems. These molecular methods cover the complete range of tools employed by traditional biotechnology, including the genetic engineering of recombinant organisms for release to the environment for purposes in bioremediation. Some currently in use and developing molecular methods in environmental biotechnology are summarized as follows:
Many of these molecular technologies have been broadly applied by environmental biotechnology in the area of bioremediation research and these uses are examples for broader applications in the field. A pertinent example for the use of nucleic acids recovered from soil as target DNA for probe hybridization is derived from a large field investigation at a U.S. Air Force Base, Columbus, Mississippi (CAFB) (6). This is the site for a major natural attenuation study of fuel hydrocarbons in a groundwater aquifer. Natural attenuation is the unaided physical, chemical, and biological reduction of contaminant concentration in the environment. Biotechnically, natural attenuation has been embraced as a cost effective alternative remediation strategy if it can be demonstrated that the process of attenuation ensures that contaminants do not transport beyond the boundaries of the site and that potable water or ecologically sensitive zones are not impacted. Biotechnically, there is the need to predict the relative rate of contaminant removal due to biodegradation and to provide monitoring capability of the biological component of attenuation to ensure that the contaminated site can sustain biodegradation over the long term course of natural attenuation. At the CAFB, site regulatory permission was granted to introduce a decane carrier hydrocarbon, containing benzene, toluene, xylene, and naphthalene on aquifer sediments in the groundwater flow path. Suites of bacterial biodegradative gene probes were used to track the dynamic fluctuation of microbial process associated with biodegradation of the test contaminants. This was accomplished by extracting DNA from aquifer solids recovered from bore hole drillings at multiple depths above and along the contaminant gradient as hydrocarbons plumes were transported over time. Table 1 is an example of the types of genetic probes that can be employed in such field analysis. Table 1. Relevant characteristics of DNA probes used for the CAFB study.
a kb refers to kilobase pairsbbp refers to base pairs Figure 2. Relationship of degradation gene dynamics to chemical exposure to fuel hydrocarbon introduced into a groundwater aquifer. Over a period of 1-1/2 years, significant increases in degradative gene frequencies were observed among samples exposed to plumes of the various hydrocarbons (Figure 2). As plume movement in the groundwater appeared to slow, or even retreat, there were corresponding changes in gene abundance suggesting that, as a controlling factor, the biological performance of natural attenuation could indeed be measured at the genetic level and could be used to insure that the attenuation was indeed operative and effective. Related studies have been conducted on the bioremediation of polyaromatic hydrocarbons (PAH) in contaminated soils. Extended studies have shown that not only are genes for degradation elevated at these sites, but also the genes are expressed as messenger RNA (mRNA), which can be directly recovered from the site soil (7). This proves that the genes are active under prevailing conditions at the site. Recent developments in mRNA analysis of environmental samples now permits the use of technologies such as arbitrarily primed, reverse transcription- polymerase chain reaction used in a differential display format (AP/RT PCR-DD) to isolate cryptically expressed genes from environmental samples. Such powerful tools, originally developed for the human genome initiative, now provide information on novel genes expressed in the environment in response to natural or anthropogenic stress or biotechnical manipulations. An example of how this powerful new technology can be applied is given in Figure 3. Figure 3 demonstrates the use of differential display of mRNA to identify newly expressed genes in a bacterial strain exposed to salicylate. Recovery of the cDNA followed by sequence analysis revealed a new naphthalene dioxygenase homolog in a previous uncharacterized strain (8). Figure 3. Differential display of salicylate induced and uninduced RNA from P. Putida JS150 using primary primers. The issue of gene expression in the environment, and the ability to measure gene expression as a process and monitoring control strategy, has also been approached using transcriptional fusions with reporter genes. Several applications in the use of bioluminescent (lux) gene fusions have been demonstrated for both organic and inorganic chemicals in the environment. Whole cell bacterial biosensors that bioluminesce in response to chemicals such as naphthalene, toluene, and chlorobiphenyl are currently in use as tools to measure the presence of the specific chemicals in the environment. The bioavailability of the chemical contaminant and the ability of the environment to physiologically allow gene expression results in biodegradation of the chemical pollutants. Sensor technology has been developed using immobilized whole cells with fiber optic/photomultiplier based systems for remote on-line sensing of the chemicals. The most recent development in this area is the integration of the biosensing organism directly with a silicon oxide chip based light detection system to create a Bioluminescent Bioreporter Integrated Circuit (BBIC) for a new wireless biosensor technology. Similar advances are also occurring in the area of gene expression and detection, using oligonucleotide arrays immobilized on a matrix, to allow mass screening of target nucleic acid. A few years ago, the use of engineered microorganisms containing recombinant elements, such as lux reporter fusion, would seem impractical in terms of environmental use. However, in 1996, the University of Tennessee was given an EPA Consent Order, which enabled the field testing of recombinant bacteria for use in bioremediation processes monitoring and control (9). The organism, Pseudomonas fluorescens (HK44), contained a plasmid-introduced genotype for naphthalene catabolism and a lux transcription fusion for gene expression (10), and has been in field analysis at Oak Ridge National Laboratory since October 1996. Similarly, Alcaligenes and Pseudomonas strains containing recombinant genes for PCB bioremediation (11) are currently awaiting commercial field trials for soil PCB bioremediation. These strains, which have already been regulatory reviewed by the U.S. EPA for field trials, are undergoing scaleup testing in Canada for their potential biotechnical utility. Recent developments of such organisms have included creating lux sensor strains that function to address the issue of Aroclor (PCB) bioavailability as measurable endpoint of remediation success. Collectively, environmental biotechnology appears to be in healthy evolution. Broad applications of environmental biotechnology are being realistically evaluated and commercialized for use in bioremediation. Beyond this identified focus of bioremediation; environmental biotechnology is progressing in areas of biosensing, organism detection, and diagnostics waste and environmental processing and gene discovery. The broad application of molecular methods has resulted in a resurgent interest in environmental and ecological research that contributes fundamentally to environmental biotechnology. Based on 16S rRNA phylogenetic analysis of microbial communities, it is now clear that the biodiversity of microorganisms in natural environments is likely to be very high. Such phylogenetic biodiversity may reflect a comparable genomic biodiversity representing untapped natural resources that can be exploited for societal and environmental benefit. The tools of molecular biology permit, not only assessing this genomic diversity, but also recovering the information, genes, and organisms needed for the eventual transition to environmental sustainability. Acknowledgements This work was supported, in part, by US Department of Energy, grant number DE-FG05-94ER61870 and by the Waste Management Research and Education Institute, the University of Tennessee. References 1. Sayler, G.S. and R. Fox. 1991. Environmental Biotechnology: Perceptions, Reality and Applications. In: Environmental Biotechnology for Waste Treatment. Ed. G.S. Sayler, R. Fox, J. Blackburn. Plenum Press, NY. p.1-13. 2. Kato K. and K. L. Davis. 1996. Current Use of Bioremediation for TCE Cleanup: Results of a Survey. Remediation. p. 1-14. 3. Miller, D. 1997. Green Technology Trends: The Changing Context of the Environmental Technology Industry. In: Biotechnology in the Sustainable Environment. Eds. G. Sayler, J. Sanseverino, and K. Davis. Plenum Press. p. 5-12. 4. Sayler, G.S. 1997. Challenges and Opportunities in the Area of Environmental Biotechnology. In: Biotechnology in the Sustainable Environment. Eds. G. Sayler, J. Sanseverino, and K. Davis. Plenum Press. p. 1-4. 5. Nedwin, G.E. 1997. Green Chemistry: Using Enzymes as Benign Substitutes for Synthetic Chemicals and Harsh Conditions in Industrial Processes. In: Biotechnology in the Sustainable Environment. Eds. G. Sayler, J. Sanseverino, and K. Davis. Plenum Press. p.13-32. 6. Stapleton, R. and G. Sayler. 1998. Molecular Site Assessment of Natural Attenuation Potential for Petroleum Hydrocarbons. Microbial Ecology. In review. 7. Fleming, J. and G. S. Sayler. 1993. Quantitative Relationship between Catabolic Gene Frequency and Expression in Predicting PAH Degradation in Soils at Town Gas Manufactured Sites. Environmental Science and Technology 27:1068-1074. 8. Fleming, J., S. Yao, and G. Sayler. 1998. Optimization of Differential Display for Prokaryotes: Application to Pure Culture and Soil Microcosms. Appl. Environ. Microbiol. Submitted. 9. Sayler, G.S., C.D. Cox, R. Burlage, S. Ripp, D.E. Nivens, and U. Matrubutham. 1998. Releasing a Genetically Engineered Microorganism for Bioremediation: One-Year Field Experience. Environmental Science and Technology. In review. 10. King, J.M.H., P.M. DiGrazia, B. Applegate, R. Burlage, J. Sanseverino, P. Dunbar, F. Larimer, and G.S. Sayler. 1991. Rapid, Sensitive Bioluminescent Reporter Technology for Naphthalene Exposure and Biodegradation. Science. 249:778-781. 11. Lajoie, C.A., A.C. Layton, J.P. Easter, F. Menn, and G.S. Sayler. 1997. Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors. J. Industrial Microbiol. Biotechnol. Vol. 19, p. 252-262. Copyright 1998 Australian Biotechnology Association Ltd. The following images related to this document are available:Line drawing images[au98031c.gif] [au98031a.gif] [au98031b.gif] |
| |||||||||
