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Phytoremediation - Terry Lab UCB |
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TERRY LAB CASE-STUDIES Use of constructed wetlands to decontaminate wastewater
______________________________________________________________ Use of constructed wetlands to decontaminate wastewater Constructed wetlands are effective and reliable wastewater treatment systems. They have long been used to clean up wastewaters contaminated with conventional pollutants such as high nitrogen, phosphorus, or total suspended solids. Constructed wetlands are virtually the only means of cleaning up large volumes of water contaminated with very low levels of pollutants, e.g. toxic trace elements. They offer an efficient alternative to conventional water treatment systems because they are relatively inexpensive to construct and operate, easy to maintain, and quite tolerant of fluctuating hydrologic and contaminant loading rates. They have other benefits in that they provide green space, wildlife habitat, and recreational and educational areas. Furthermore, they are one of the most productive ecosystems in terms of biomass, especially when dominated by aquatic macrophytes. This is largely because of ample light, water, nutrients, and the presence of plants that have developed morphological and biochemical adaptations enabling them to take advantage of these optimum conditions. Our research has extended the use of constructed wetlands to include the remediation of waters contaminated with heavy metals and other toxic trace elements. (i) The Chevron Wetland: Perhaps our most striking success is the demonstration that constructed wetlands can cleanup the toxic element, selenium (Se), from large volumes of industrial wastewater1. Research conducted at the Chevron oil refinery at Richmond, CA, demonstrated that 89 percent of the selenium from oil refinery wastewater was removed by a 90-acre constructed wetland that processed approximately 2 to 3 million gallons (~10 million liters) of refinery effluent per day. This study revealed that 20 to 30% of the toxic selenium entering the wetland was released to the atmosphere in non-toxic volatile forms, a process known as “biological volatilization”. This is extremely important as the selenium removed by volatilization cannot enter the local food chain. This research stimulated substantial media interest including articles in the San Francisco Chronicle, the San Jose Mercury, the Sacramento Bee, Science News, as well as television reports: KRON, CNN and the BBC (UK) all sent teams to Berkeley to film the wetland story. (ii) The Corcoran Wetland: The success of the research at the Chevron wetland led to our next wetland project at Corcoran, 60 miles north of Bakersfield. In this experiment, we built 10 quarter-acre wetland cells to test the idea that constructed wetlands can be used to solve one of the most serious problems confronting agriculture in California, i.e., the problem of what to do with selenium and heavy metal contaminated irrigation drainage water. Huge amounts of selenium-polluted water are being accumulated in thousands of evaporation ponds throughout California and other western states. Agricultural drainage water differs from industrial wastewater in that it is highly saline and contains several toxic metals in addition to selenium. Each of the 10 wetland cells was planted with different plant species in mono- and mixed-cultures to determine the best plant species composition for maximum selenium removal. The best of these wetlands removed 85% of the selenium from the inflow as well as removing strontium and vanadium2, 3. (iii) Tennessee Valley Authority and Allegheny Power Services wetlands: Electric utilities are also mandated by the Clean Water Act to clean up their aqueous discharges. We carried out two separate studies, one at the Tennessee Valley Authority wetland in Alabama, and the other at the Allegheny Power Services wetland in Pennsylvania. The goal of this research was to develop optimal design criteria for building constructed wetlands. Both wetlands were shown to be capable of removing substantial amounts of toxic heavy metals including Mn and Fe, with the removal efficiency often exceeding 90%. We have also shown that there are substantial differences among various wetland plant species in their ability to remove specific trace elements4. For example, we found that species of the algae, Chara and Spirogyra, were particularly effective phytoextractors of several toxic trace elements. We have also identified duckweed, brass button, water hyacinth, smartweed, brass button, umbrella plant, water zinnia, water lettuce and mare’s tail to be among the top plant species accumulating high concentrations of certain toxic trace elements5-7. (iv) Indiana site restoration project: In some situations, effluents may be so toxic that they cannot be tested in a constructed wetland directly. For example, we were approached by managers of an electric utility in Indiana who were interested to know whether their wastewater, which was contaminated with highly toxic levels of selenocyanate, arsenic, and boron, could be treated by using constructed wetlands. To answer this question, we used miniature wetland cells (mesocosms) set up in a greenhouse. Our results showed that the contaminants were substantially depleted and that it would be safe to treat this toxic effluent using a constructed wetland system8. Interestingly, the principal contaminant, selenocyanate, was detoxified as it passed through the mesocosm. Subsequent research showed that the wetland plants had physiological and biochemical characteristics that enabled them to mediate the uptake and conversion of selenocyanate into nontoxic forms9. (v) Conclusion: As demonstrated above, our research has clearly established that constructed wetlands provide an attractive technology for the treatment of polluted wastewater from industry and from agriculture. We strongly believe that the use of constructed wetlands will revolutionize wastewater treatment, and lead to the creation of new wetland habitat, while continuing to maintain sustainable water supplies and natural ecosystems. Use of phytoremediation to decontaminate soil Many agricultural and industrial sites have soils that are contaminated with toxic heavy metals, metalloids (e.g., selenium, arsenic), or organic pollutants (e.g., trichloroethylene, perchlorate). These sites may be remediated or restored using different phytoremediation approaches. Phytoextraction utilizes the ability of certain plants to remove contaminants from soil and water and accumulate them in plant tissues that may then be harvested and removed from the site. Phytostabilization on the other hand, uses plants to immobilize contaminants chemically and physically at the site, thereby preventing their movement to ground waters or to the atmosphere (i.e., through soil erosion and wind). Phytovolatilization makes use of plants and their associated microbes to convert contaminants to a volatile form and remove them from the local ecosystem. Phytovolatilization has the major advantage in that there is no hazardous waste to dispose as in phytoextraction. Phytodetoxification involves the ability of plants to change the chemical species of the contaminant to a less toxic form, e.g., plants can take up toxic hexavalent chromium and convert it to non-toxic trivalent chromium. (i) Use of Indian mustard to decontaminate agricultural soils: Indian mustard is an example of a plant that is being used to remediate many different types of contaminated sites. It is particularly useful for phytoremediation because it is fast-growing, produces a large biomass, tolerates fairly toxic soils, and can be grown several times in one growing season. We have increased the capacity of this plant for phytoremediation by genetically engineering it to remove selenium, cadmium and other toxic trace elements from contaminated soil. We have developed over one hundred lines of transgenic plants that overexpress key enzymes responsible for metal and/or metalloid sequestration and volatilization by plants. Specifically, we developed transgenic plants with a superior capacity for the phytoremediation of selenium and/or heavy metals by overexpressing the enzymes ATP sulfurylase (APS), γ-glutamyl cysteine synthetase (ECS), or glutathione synthetase (GS). In fact, our laboratory was the first laboratory worldwide to develop and successfully field test genetically engineered plants with an enhanced capacity for phytoremediation - specifically, we enhanced the ability of Indian mustard plants for the removal of Se from contaminated soil10. The field research showed that the APS transgenics accumulated almost five-fold more Se in their aboveground tissues than wildtype while ECS and GS transgenic plants accumulated 3- and 2.5-fold more Se than WT, respectively11. This research has since been followed up with a second field study that showed that transgenic plants overexpressing selenocysteine methyltransferase and selenocysteine lyase were also successful in improving phytoremediation of Se under field conditions12. The APS, ECS and GS plants were also successful in promoting the increased accumulation of toxic heavy metals10, 12-19. (ii) Selenium phytoremediation using genes from hyperaccumulator plants: A quite different approach to genetically engineering plants for improved phytoremediation is to introduce genetic traits from slow-growing, hyperaccumulator species into a fast-growing, high biomass species. Several researchers have attempted to combine genes in this way in order to develop the ideal plant for phytoremediation, i.e., one with a high growth rate and biomass which can also hyperaccumulate the pollutant. Our laboratory was the first to achieve this using a gene from the selenium hyperaccumulator, Astragalus bisulcatus, a plant able to accumulate Se to concentrations in excess of 4000 parts per million. A. bisulcatus contains a gene that encodes the enzyme selenocysteine methyltransferase (SMT)20. This enzyme confers tolerance to Se by methylating the toxic amino acid, selenocysteine, to non-toxic methylselenocysteine. This prevents selenocysteine from being incorporated into proteins and thereby altering their structure and function. Our research showed that the fast-growing and high biomass Indian mustard transgenics overexpressing SMT were able to accumulate and volatilize Se at substantially higher rates than wildtype (unaltered) plants. (iii) Cadmium phytoremediation: Metal-tolerant plants are known to accumulate and store high concentrations of heavy metals by binding them to peptides called phytochelatins. We have over-expressed the genes for two important enzymes involved in phytochelatin synthesis, i.e. g-glutamyl cysteine synthase (ECS) and glutathione synthetase (GS) in Indian mustard plants18, 19. The resulting transgenic plants had an increased ability to take up cadmium (Cd) compared to wildtype plants. The transgenic ECS and GS plants were also more tolerant to high Cd levels compared to wildtype plants. (iv) Use of chloroplast engineered tobacco to cleanup mercury: This research (in collaboration with Prof. Henry Daniell at the University of Central Florida) showed that chloroplast engineered plants considerably enhanced the phytoremediation of mercury-contaminated soil. In contrast to engineering the plant nucleus, chloroplast engineering prevents the escape of potentially dangerous foreign genes via pollen to related weeds or crops. Our research showed that genetically engineered tobacco plants containing genes encoding two important bacterial enzymes (mercuric ion reductase, merA, and organomercurial lyase, merB) could efficiently remove and detoxify mercury (Hg) when the mercury was supplied as 400 μM phenylmercuric acetate (which mimics highly toxic methyl mercury). The transgenic tobacco plants showed an approximate 100-fold increase in the efficiency of Hg accumulation in shoots compared to wild-type plants. The transgenic plants were able to grow well with root concentrations of Hg up to three-fold higher, depending on the form supplied, and were also able to volatilize elemental mercury to permanently remove the contaminant from the system21, 22. Microbial remediation of toxic trace elements Microbial remediation can be carried out by free-living or rhizosphere (root-associated) microbes. Examples of various types of microbes (including microalgae) used in trace element remediation are presented below. (i) Use of bacteria to remediate selenium-contaminated wastewater: In order to identify free living bacteria with superior capacities for the remediation of contaminated water, we carried out a 16S rDNA phylogenetic analysis of a solar evaporation pond in the San Joaquin Valley, CA23. This pond contained extremely high levels of salt, Se and other potentially toxic elements. We hypothesized that microbes exposed to such harsh seleniferous conditions should be endowed with an unusual ability for Se remediation. This approach was very successful. For example, we were able to identify a previously unknown bacterial species that could not only tolerate enormously high concentrations of selenite (2M) and salt (32.5% sodium chloride), but also volatilized Se at extremely high rates. The bacteria we identified had several important applications. They could be used to: 1) bioaugment evaporation ponds to increase Se removal, 2) remediate agricultural drainage water in a bioreactor, and 3) provide a reservoir of genes (for potential incorporation into plants) encoding enzymes facilitating tolerance to extremely high concentrations of Se and salt. (ii) Use of microalgae to remediate Se-contaminated wastewater: In our pursuit of microbes capable of Se phytoremediation, we found a microalga (Chlorella sp.) that was able to volatilize Se at rates that were orders of magnitude higher than those previously obtained with higher plants. The Chlorella cultures reduced 87% of accumulated selenate to organic forms within 24 hours, dramatically better than higher plants which reduce selenate at much slower rates12. (iii) Use of root-associated bacteria to cleanup zinc/cadmium contaminated soil: Bacteria associated with plant roots can play important roles in the accumulation of toxic metals or metalloids by plants. Because of the considerable amount of interest in hyperaccumulator plants (discussed above), we investigated the possibility that root-associated microbes might be involved in the hyperaccumulation process of the well-known zinc and cadmium hyperaccumulator, Thlaspi caerulescens. This plant species accumulates zinc to concentrations greater than 10 mg Zn g-1 shoot dry weight without any symptoms of toxicity. The physiological and biochemical basis of this enhanced capacity to accumulate heavy metals was thought to reside strictly within the plant. However, researchers from our laboratory showed that microbes played a hitherto unknown and critical role in the hyperaccumulation of zinc by Thlaspi caerulescens, namely that bacteria were necessary to facilitate the solubilization of zinc from non-labile soils 17. In a different study, we also found that bacteria in the plant rhizosphere promoted the plant uptake of certain trace elements such as selenium and mercury. With respect to selenium, this stimulatory effect was partly due to a bacterially-mediated increase in root hair development, but was mostly due to rhizosphere bacteria, which stimulated the production of a heat-labile compound; this compound facilitated the transport of selenate across the plasma membrane into the root24. Phytodetoxification of contaminated water and soil Another major discovery we have made in the field of phytoremediation is that plants and microbes can detoxify certain toxic trace elements that contaminate industrial wastewater. These studies were performed using sophisticated high energy X-ray absorption spectroscopy carried out at the Stanford Synchrotron Radiation Laboratory at Stanford University. Several researchers have tried to devise tools for the fractionation and speciation of trace elements in biological tissues. Most attempts involved either indirect measurements or treatments that may alter the chemical state of the element under study. The synchrotron-based x-ray absorption spectroscopy (XAS) techniques used by our laboratory allow for direct measurement of the chemical species in vivo. This elegant technique is extremely specific and using XAS, we were able to accurately determine the forms in which trace elements are present in intact and functioning biological tissues. Three examples of important research findings using this technique are outlined below. (i) Chromium: We documented, for the first time, that many vegetable and wetland plants can remove toxic chromium (Cr) from the environment and convert it to a non-toxic form. Hexavalent chromium, Cr(VI), is a toxic element with many industrial uses. It also is a potent carcinogen and lethal at high doses. However, trivalent chromium, Cr(III), a variant of this metallic element, is not only non-toxic but essential for human and animal health. In 1996, Pacific Gas and Electric Co. settled a $400 million lawsuit brought by plaintiffs seeking damages for personal injuries allegedly suffered as a result of exposure to chromium in contaminated water arising from the company's gas compressor station in Hinckley, California. Our research demonstrated that plants absorb Cr(VI) and convert it to non-toxic Cr(III) in just a few hours. This phytodetoxification of Cr(VI) was shown for many species of crop plants25 and wetland plant species26. We also isolated many species of bacteria that can carry out the reduction of Cr(VI) to Cr(III). (ii) Manganese: Using XAS techniques, we demonstrated that cattails, a common wetland plant, possess the ability to oxidize toxic, bioavailable forms of manganese (Mn2+) to relatively insoluble, non-mobile Mn4+. The role of cattails at the APS wetland, which treats wastewater from an electric power plant, was critical for the removal of Mn by the wetland; 91% of the Mn entering the wetland in the wastewater was removed27. (iii) Selenium: We demonstrated that selenite (SeO32-), a mobile, bioavailable form of Se is precipitated to immobile Se0 by bacteria found in wetland sediments. We isolated many species of facultatively anaerobic bacteria from wetland sediments that can efficiently reduce selenite to elemental Se, thereby immobilizing it24. Our x-ray absorption speciation studies showed that some of these bacterial strains store elemental Se in their tissues. A bioreactor containing the most efficient selenite-reducing bacterial strain, and that accumulates Se0 in its tissues, would yield commercially useful Se0. (iv) Conclusion: These three discoveries would not be possible without the use of XAS speciation technology to determine the efficiency of phytoremediation of wastewater. They highlight the importance of using chemical speciation to determine the efficiency of phytoremediation or microbial bioremediation. On-farm treatment of contaminated drainage water (i) Agroforestry management: In collaboration with the California Department of Water Resources and the U.S. Bureau of Reclamation we field tested another technology for the treatment and disposal of agricultural drainage water, i.e., the so-called agroforestry drainage water disposal system. Agricultural drainage water is collected from one crop and then re-used by supplying to a succession of crops, including Eucalyptus trees and other salt-tolerant crops, before being deposited in a solar evaporator for the production of salt. By this means, drainage water is consumed totally, generating commercially useful crops, timber and salt. The Terry Lab established how Se volatilization from different components of the agroforestry system contributed to the mass Se removal by the whole system. Our research identified pickleweed (Salicornia) as a plant species that volatilizes Se at one of the highest rates attained in the field in any system28. Furthermore, we demonstrated that the amount of Se removed annually by volatilization is six times higher than the amount removed by plant uptake (up to 20% of the annual total Se input into the system). Phytostabilization and habitat restoration of contaminated soils In addition to our efforts to use plants to remove toxic elements from contaminated sites, we are also interested in using plants to stabilize the contaminants in situ (phytostabilization). This is to prevent their migration to the ground water or their indiscriminate dispersal to the atmosphere via soil erosion and wind. The goal is to determine suitable soil amendment treatments, as well as identifying appropriate plant species, necessary to revegetate a barren site. (i) Phytostabilization of an industrial site: Some industrial contaminated waste sites are so heavily polluted with heavy metals and other toxic materials that they are virtually impossible (or too expensive) to decontaminate. A chemical company near San Francisco Bay was faced with such a problem: their site was so contaminated with arsenic, mercury, lead, and selenium, as well as PCBs and PAHs, that it was unable to support any vegetation. There was a serious risk that the contaminants would be distributed well beyond the contaminated area through soil erosion and wind or by drainage to the groundwater. In order to phytostabilize the site, we took soil samples to the laboratory and determined various characteristics including pH, heavy metal content, and nutritional status. We then tested different types of soil amendments to improve soil quality so as to support vegetative growth. Using dolomite and organic fertilizer we were able to effectively reduce soil acidity and ameliorate soil conditions to obtain optimum plant growth. Among the various species tested, fawn tall fescue was the most tolerant species and was chosen for the phytorestoration of the contaminated site29.
Department of Plant and Microbial
Biology
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