301 chapter eight Future needs for research and development Jeffrey W. Talley Contents 8.1 Introduction 301 8.2 Polycyclic aromatic hydrocarbons 301 8.3 Chlorinated solvents 303 8.4 Polychlorinated biphenyls 304 8.1 Introduction During the course of project CU-720, researchers and engineers in Federal Integrated Biotreatment Research Consortium (FIBRC) conducted basic research and developed treatment processes, many of which were scaled up to pilot- or field-scale implementation. In the course of this work, a number of research and developmental issues were identified as worthy of following up but were not pursued due to a desire to move the technology with the most potential to the pilot or field scale. The following issues were identified in each of the thrust areas as frontiers of science and technology in bioremediation. 8.2 Polycyclic aromatic hydrocarbons In theory, soils contaminated with polycyclic aromatic hydrocarbons (PAHs) may be treated utilizing various cleanup strategies. However, many pro- posed strategies have significant economic and feasibility problems. What is needed is an effective technology that supports the economics of disposal, eliminates adverse contaminant impacts, and supports the reuse of treated contaminated soils. Regardless of whether the biotreatment system is passive L1656_C008.fm Page 301 Monday, June 20, 2005 1:47 PM © 2006 by Taylor & Francis Group, LLC 302 Bioremediation of Recalcitrant Compounds (augmented natural attenuation) or engineered (in-place treatment), a prag- matic solution is to focus active biotreatment on the available contaminant fraction. Research is needed to identify the factors affecting the bioavailabil- ity of PAHs on soil and how these factors affect treatment rates and accept- able toxicological endpoints. Research is then needed to use the bioavailabil- ity information to develop a technical base for enhancing natural recovery processes involved in in situ biotreatment of PAH-contaminated soils. Such research could result in providing guidelines for the assessment and predic- tion of the bioavailability of PAHs for in situ biotreatment. Beyond availability, the issue of residual contamination is still unre- solved in the case of biological treatment of PAHs, where an understanding of the complex interactions between hydrophobic organic contaminants and soil is key to establishing realistic risk assessment criteria. Cleanup criteria based only on the chemical properties of the contaminant lead to an over- prediction of the risks associated with the contamination. However, leaving an immobile, bound residue in the soil after cleanup needs to be justified scientifically. On the other hand, if residuals are released, the rate should be sufficiently slow to allow consumption by the microbial community. Thus, an alternative environmental endpoint (cleanup level) may be appropriate rather than basing decisions solely on total concentration of contaminant in the soil or sediment. Thermal programmed desorption (TPD) is one new technology available to study the first of these issues, the mechanism of PAH binding to the soil, the process that affects their bioavailability in the environment. Phytoremediation (defined as the use of green plants to remove, contain, or decrease toxicity) is a cost-effective technology with many advantages over highly engineered solutions, including public acceptance. Only in the last decade has research been conducted on phytoremediation of PAHs. Recent studies have shown that plants are capable of removing not just the lower-molecular-weight PAHs, such as anthracene, but also HMW com- pounds, such as chrysene and benzo(g,h,i)pyrene. In contrast to phytoreme- diation of metals, which is an extraction process, large organic compounds such as the PAHs are degraded in the rhizosphere (root zone) of the plant. The rhizosphere comprises the top 3 to 6 ft of soil. Various elements of the rhizosphere appear to play a role in the degradation. First, the soil around the roots is very different in chemistry and physical structure from the bulk soil. The root system of the plant is a moist, aerobic environment, which promotes microbial activity. Second, an exudate is released from the roots into the surrounding soil. The exudate is composed of simple sugars, amino acids, enzymes, aliphatic and aromatic compounds, and vitamins and has been shown to increase the dissipation of soil-bound PAHs when applied to soil as a single treatment. Third, there are active microbial and fungal com- munities associated with the root system. These are enhanced by the root exudate in ways that are not completely understood. Growth, and the devel- opment of biomass, is certainly one response, but in some plant species the L1656_C008.fm Page 302 Monday, June 20, 2005 1:47 PM © 2006 by Taylor & Francis Group, LLC Chapter eight: Future needs for research and development 303 exudate appears to favor selection of microorganisms that degrade the con- taminant. Plants can be incorporated into current treatment strategies in several ways. The most recent development is an alternative agricultural technique, plant-based biotechnology. The goal of this “molecular farming” is to pro- duce large quantities of root exudate to be used as a soil amendment. As these exudates vary between species and soil conditions, a systematic study needs to be undertaken before this technology is feasible. The most promising method of incorporating phytoremediation into soil PAH remediation is to develop a new paradigm of treatment that utilizes rhizospheric degradation as part of a treatment train. Once contaminated soil has been treated by land farming, for example, a plant cover would be established. The advantages are: • Plant cover reduces both wind and water erosion from the treated soil. • The improved soil structure allows continued bioremediation. •Naturally occurring nitrogen-fixing microorganisms reduce the need for chemical fertilizers, reducing costs. • The roots establish an optimal environment and furnish energy for growth of microorganisms. • The exudate contains oxidative enzymes that contribute to PAH deg- radation. Current research indicates that the establishment of plants on contami- nated sites may be an economic, effective, low-maintenance approach to complete soil PAH remediation. 8.3 Chlorinated solvents The Solvent Extraction Residual Biotreatment (SERB) technology developed here has been specifically targeted for contamination at sites containing separate phase dense nonaqueous phase liquids (DNAPLs). Molecular probes for two microorganisms capable of reductive dechlorination of chlo- roethenes have been developed. The experience over the last two decades with recalcitrant compounds suggests that there are perhaps many more microorganisms in nature capable of reductive dechlorination that have so far not been discovered. The techniques developed and described here can be used to continue to search for such potential microorganisms and for their isolation, cultivation, and monitoring. Since chloroethenes are still commonly used, the potential for fresh spills and environmental contaminations exists. These being mostly man-made chemicals, the fresh sites are not likely to possess extensive dechlorination capability. In such cases, it may be desirable to deliver known dechlorinators in the environment. In order to ensure successful introduction of the microbes in the surface and confidently utilize the SERB technology, it is L1656_C008.fm Page 303 Monday, June 20, 2005 1:47 PM © 2006 by Taylor & Francis Group, LLC 304 Bioremediation of Recalcitrant Compounds necessary to develop a better understanding of methods of delivery of the microorganisms in the desired locations. Our understanding of the move- ment and survival of augmented microorganisms in the subsurface needs improvement as well. Pilot-scale and full-scale demonstrations at different sites are needed to refine the techniques developed and presented in the design guidance manual. The design guidance document that describes the SERB technology should be promoted among technology vendors and the user community. It is suggested that this be done using the Air Force model for the promotion and improvement of in situ bioventing technology. Additional considerations needing attention are: • Further characterization of shifts in microbial ecology in response to SERB • Evaluation of mixtures of optimal cosolvent/electron donor solutions •Modeling of impact of source removal on long-term economics for site remediation costs 8.4 Polychlorinated biphenyls Metabolic engineering of pathways involved in aerobic biodegradation of mono- and dichlorinated polychlorinated biphenyls (PCBs) has shown that genetically stable genetically engineered microorganisms (GEMs) capable of growing on PCBs can be generated. Such organisms have been found to be able to survive in contaminated soils for a considerable time, especially when delivered in a suitable carrier. Biomarkers for these GEMs have also been developed for tracking them in engineered systems. The metabolic engineer- ing approach that produces microbial strains that thrive on complex recal- citrant contaminants, rather than strains that transform contaminants using cometabolism, is very interesting. The ability of GEMs to extract energy from recalcitrant compounds simplifies management of treatment strategies for such contamination. This approach should be pursued for other, more com- plex PCBs. The process of anaerobic dechlorination of highly chlorinated PCBs is very slow, requiring several years, and it is a major bottleneck in any demonstration project. The microbial strains capable of doing anaerobic dechlorination have not been isolated and characterized. Their isolation from the contaminated sites and maintenance in laboratories/culture collections are highly desirable in order to promote this technology. Similarly, the GEMs generated in the present work should also be registered with suitable culture collections in order to make them more widely available to potential users. Due to the slow nature of the degradation process, pilot demonstration of the technology has been lagging. This should be more extensively demonstrated. L1656_C008.fm Page 304 Monday, June 20, 2005 1:47 PM © 2006 by Taylor & Francis Group, LLC . Bioremediation of Recalcitrant Compounds necessary to develop a better understanding of methods of delivery of the microorganisms in the desired locations. Our understanding of the move- ment and. processes, many of which were scaled up to pilot- or field-scale implementation. In the course of this work, a number of research and developmental issues were identified as worthy of following up. of optimal cosolvent/electron donor solutions •Modeling of impact of source removal on long-term economics for site remediation costs 8. 4 Polychlorinated biphenyls Metabolic engineering of