Current Research

Beyond Parent Compound Disappearance in the Bioremediation of Polycyclic Aromatic Hydrocarbon-Contaminated Soil; National Institute of Environmental Health Sciences (grant P42ES005948); 4/1/11-3/31/16.

This is Project 5 in the UNC Superfund Research Program. Polycyclic aromatic hydrocarbons (PAHs) are among the chemicals of most concern at Superfund sites and at many industrial sites that are not subject to regulation under the Superfund program. Remediation goals for these sites are usually based on the removal of the PAHs to site-specific levels, with an assumption that PAH removal corresponds to a reduction in risk to human health and the environment. Although bioremediation is an established technology for removing PAHs from contaminated soil, previous studies have shown that it does not always lead to a reduction in toxicity. The causes of toxicity and the mechanisms by which toxicity might be avoided or diminished are not well-understood. We hypothesize that metabolites produced by PAH-degrading bacteria are responsible at least in part for the toxicity that can result from bioremediation. The overall objective of this project is to fill key gaps in knowledge that will inform and improve field applications of bioremediation that lead to true reductions in risk.

Bioremediation conditions influence the community of PAH-degrading microorganisms in contaminated soil, which in turn influences both PAH removal and the extent to which metabolites might accumulate. We are exploring the effects of bioremediation conditions on PAH removal and soil toxicity. We have used two laboratory-scale platforms to perform bioremediation of soil from a former manufactured-gas plant site in Salisbury, North Carolina: (1) a small, semi-continuous slurry-phase bioreactor (simulating above-ground treatment), and (2) a conntinuous-flow column system (simulating in situ bioremediation). To date we have observed that bioremediation in either the bioreactor or the column system can lead to increases in genotoxicity of the soil as a result of bioremediation. Among the conditions to be evaluated for its potential effect on genotoxicity is the addition of a hydrophobic surfactant at a low dose, which we recently demonstrated can improve the bioavailability and biodegradation of PAHs that remained in a field-contaminated soil after conventional bioremediation (see below).

In addition to studying the influence of bioremediation conditions on the genotoxicity of PAH-contaminated soil, we are using high-throughput pyrosequencing and other molecular methods to identify the PAH-degrading bacteria most likely to influence PAH removal, metabolite accumulation, and toxicity in the treated soil. In addition, pyrosequencing will be used to evaluate the influence of surfactant addition on the microbial community in the laboratory bioreactor. Our ultimate goal is to study the genomes of selected organisms to identify genes associated with PAH metabolism. Differences in the ability to metabolize various PAHs will be correlated to sequence differences in these genes so that genetic determinants of metabolite accumulation can be identified. Another goal is to identify the compounds in bioremediated soil that may be causing increases in genotoxicity.

Selected Previous Projects

An Integrated System for Energy Recovery and Nitrogen Removal from Swine Waste; Carolina Public Health Solutions; 7/1/09-6/30/11.

The regional capacity to manage and dispose of the waste generated at swine production farms in North Carolina has been overwhelmed, leading to serious impairment of water quality and impacts on human health. Ammonia emitted to the atmosphere from swine waste contributes to severe odors near hog farms as well as respiratory illnesses, both from ammonia itself and from the fine particulate matter formed from ammonia. Deposition of ammonia from the atmosphere and application of treated liquid waste to agricultural fields also impairs ground- and surface-water quality. The traditional approach to managing hog waste has been to store it in open anaerobic “lagoons,” from which the waste is either sprayed on agricultural fields as fertilizer or used to flush waste from the barns. In 2007, the State of North Carolina banned the construction of new lagoons and spray fields on swine farms and now requires an 80% reduction in ammonia emissions for new waste management systems.

In addition to ammonia, hog waste also contains high concentrations of organic matter, which can undergo microbial conversion to methane under anaerobic conditions. Methane is a potent greenhouse gas (GHG) that is also emitted into the atmosphere from open lagoons, but if captured can be a source of energy. We are evaluating the feasibility of integrating the removal of ammonia and total nitrogen from hog waste with methane recovery from anaerobic lagoons. We have installed a trailer containing a pilot nitrogen removal system at a farm that already has a covered lagoon for methane recovery. The pilot system consists of a biological process to convert ammonia to nitrate (nitrification) and subsequent conversion of nitrate to inert N2 gas (denitrification). Compared to the few existing systems for nitrogen removal on hog farms, our proposed scheme would maximize the amount of organic matter available for conversion to methane, substantially remove ammonia, and reduce the concentration of nitrate remaining in the waste when applied to an agricultural field.

Bioavailability and Biodegradation of Polycyclic Aromatic Hydrocarbons; National Institute of Environmental Health Sciences; 4/1/06-3/31-11 (F. Pfaender, Co-PI).

In this multi-component project we studied the biodegradation of polycyclic aromatic hydrocarbons (PAHs), a large class of pollutants found in tars, oil, and other materials derived from fossil fuels at many contaminated sites across the United States. PAHs are hydrophobic chemicals and therefore are not very soluble in water. Accordingly, the rate at which PAHs dissolve into water is one factor that can control the rate at which they are biodegraded by microorganisms. The bioavailability of PAHs is also a factor to consider in the potential risk of human exposure to PAHs, either during contact with contaminated soil or sediment or through ingestion of groundwater that has been contaminated with PAHs released from contaminated soil. Interventions intended to enhance the bioavailability of PAHs with the objective of maximizing biodegradation by soil microorganisms can also lead to undesirable outcomes, such as the formation of toxic metabolic byproducts.

This was Project 5 in the UNC Superfund Research Program. Our overall objective was to evaluate the relationships among PAH bioavailability and biodegradation. An additional objective was to increase our knowledge of the microbial ecology of PAH biodegradation by using a cultivation-independent molecular tool (stable-isotope probing; see below) to identify microorganisms responsible for degrading a range of PAHs in relevant, complex systems. This knowledge is important in eventually being able to assess the potential for an indigenous microbial community to degrade specific PAHs in contaminated environments.

Most of the research in this project was conducted through the use of laboratory-scale soil columns that were filled with contaminated soil from a site in Salisbury, North Carolina, which had been used in the past to produce manufactured gas from coal. Artificial groundwater was pumped through the columns to simulate conditions that might exist in the field. Two columns were operated in parallel: one served as a control, in which nothing but water flowed through the column. The second column was used to evaluate biostimulation through the addition of pure oxygen and inorganic nutrients to stimulate the aerobic bacteria that are usually most responsible for rapid PAH biodegradation.

In this project, we also demonstrated that further removal of PAHs remaining in contaminated soil after conventional bioremediation in a bioreactor (see below) could be achieved by adding a relatively hydrophobic nonionic surfactant, Brij 30. Surfactant addition has been proposed for a number of years to increase the bioavailability of hydrophobic contaminants such as PAHs, primarily through solubilization of the contaminants in surfactant micelles. We observed, however, that the removal of PAHs remaining after biological treatment in the bioreactor was enhanced at surfactant doses that would not have led to PAH solubilization. The surfactant at such a low dose did improve the desorption of the PAHs, which is likely what led to the increase in PAH biodegradation. Minimizing the dose of surfactant to improve the bioedgradation of hydrophobic contaminants is important, because there is a direct relationship between the required dose and cost.

Who’s Doing What in a Complex Bioreactor? Stable Isotope Probing of Specific Degraders in Engineered Biological Treatment Processes; National Science Foundation

Molecular biological tools are now being used widely to study complex microbial communities, including those found in bioreactors used for waste treatment or bioremediation. Most of these tools can identify which organisms are present but cannot connect their presence with a specific function. A technique developed by Radajewski et al. (Nature, 403:646-649, 2000) permits the direct identification of microbes that can grow on a specific carbon source. The technique involves incubation of a sample from the system of interest with a 13C-labeled carbon source. Organisms that can grow on the carbon source will assimilate the "heavy" (isotopically labeled) carbon into cellular macromolecules, including the nucleic acids. Heavy DNA or RNA can then be separated from the unlabeled nucleic acids by density-gradient ultracentrifugation. Once the labeled nucleic acids are isolated, they can be amplified by polymerase chain reaction and subjected to conventional molecular techniques for community analysis. The difference in this case is that only the organisms capable of growing on the carbon source of interest will be identified. Knowing which organisms are involved in degrading specific chemicals in complex environmental systems is the first step towards being able to quantify them. In turn, being able to quantify relevant organisms in these systems will make it possible to assess the potential for in situ bioremediation, to determine the need to add relevant microorganisms to some systems, and to evaluate the efficacy of various bioremediation strategies.

In this project we used the stable-isotope probing (SIP) method to identify organisms in a slurry-phase bioreactor that can grow on the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene. The bioreactor is being used to treat PAH-contaminated soil from a former manufactured-gas plant site (see below). We found that different bacteria are associated with growth on each of the three different PAHs. This was somewhat surprising, as PAH-degrading bacteria isolated from contaminated environments typically can grow on a range of different PAHs; therefore, we expected that there would be some overlap among the organisms capable of degrading each PAH we evaluated. Research Associate Dr. David Singleton developed the application of SIP to PAHs and oversaw the research carried out under this project.

In her doctoral research, Dr. Sabrina Powell used SIP to study the enrichment of bacteria in the bioreactor microbial community after adding the compound salicylate. Because salicylate is known to stimulate PAH metabolism by some bacteria, we hypothesized that its addition to a soil undergoing bioremediation could select for PAH-degrading bacteria and potentially enhance the degradation of certain PAHs (see below). We also hypothesized that the method by which salicylate is added to a system (either as a single spike or continuously over a longer period) would influence which organisms are selected. Although different bacteria were selected under the different incubation conditions, there was relatively little effect on the selection of PAH-degrading bacteria. Salicylate addition as a spike selected for naphthalene-degrading bacteria, but the selected organisms under either incubation condition did not appear to be capable of degrading either the three-ring PAH phenanthrene or the five-ring, carcinogenic PAH benzo[a]pyrene.

Laboratory Investigation of a Thermophilic Anaerobic Digestion System to Treat Municipal Wastewater Sludge (Co-PI with Mark Sobsey); Brown and Caldwell, Inc. and Columbus, Georgia Water Works

Approximately 30 million pounds of dry solids from wastewater treatment are generated each day in the United States. Most of these solids are treated on-site and subsequently applied to agricultural lands in accordance with regulations developed by the U.S. Environmental Protection Agency. The land application regulations cover both chemical and biological contaminants in the biosolids. Depending on the concentration of pathogens, biosolids intended for land application are classified as either Class A or Class B. The Class A criteria require that the concentrations of three classes of pathogens - bacteria, enteric viruses and helminths (intestinal worms) - are below specified detection limits. All other biosolids are designated as Class B, with corresponding restrictions on the types of crops that can be grown on land to which Class B biosolids are applied, as well as restrictions on public access to the land. Because no such restrictions exist for the agricultural use of Class A biosolids, more farmers are likely to participate in land application programs for Class A biosolids. There is, therefore, a corresponding incentive for municipalities to produce Class A biosolids at the wastewater treatment plant.

The EPA regulations specify various methods by which Class A biosolids can be achieved. There are several sludge treatment processes pre-approved as achieving Class A product if certain operating conditions are met. In general, these processes rely on either chemical or thermal destruction of the pathogens in the sludge. Any process other than those pre-approved by EPA must be evaluated on a case-by-case basis to demonstrate that it can meet the Class A criteria. Processes proposed to achieve Class A status must be evaluated and approved by an EPA committee called the Pathogen Equivalency Committee (PEC). Class A equivalency can be sought and granted for either a specific treatment plant (site-specific equivalency) or for a generic process (national equivalency).

The purpose of this project was to evaluate whether thermophilic anaerobic digestion can achieve the Class A biosolids criteria in a new process called the Columbus Flow-Through Thermophilic Treatment (CBFT3) process. The CBFT3 process involves continuous or semi-continuous flow of sludge through a mixed thermophilic digester, followed by treatment in either a continuous plug-flow reactor or in a batch reactor. Its purpose is to achieve credit for the inactivation of pathogens that can occur in a mixed, continuous-flow digester. We constructed a laboratory system (above right) to provide semi-continuous operation of a 20-liter anaerobic digester held at constant temperature over the range of 51 - 55 °C. Temperature was controlled to within ± 0.1 °C and gas flow was measured continuously. Sludges from several municipal wastewater treatment plants were fed intermittently on a programmed cycle in which the feed and effluent draw-off pumps operated every 6 to 15 minutes to achieve a target hydraulic residence time. Because the most heat-resistant pathogens (helminths and enteric viruses) are not normally present in sludge at concentrations high enough to measure reliably, the pathogen surrogates Ascaris suum (a helminth) and vaccine-strain poliovirus were spiked into the feed sludge. In addition to these organisms, effluent biosolids were analyzed for fecal coliform, Salmonella, Clostridium perfringens (a heat-resistant spore-forming anaerobe), somatic coliphages and male-specific coliphages. Experiments were also conducted to quantify the kinetics of inactivation of Ascaris and poliovirus as a function of temperature.

Overall results from this project indicated that Ascaris is inactivated much more rapidly than was believed when the Class A biosolids criteria were developed, and that substantial inactivation of the pathogens occurred even in the mixed, continuous-flow digester. This project represented the first phase in the effort to obtain Class A equivalency for the CBFT3 process, and was the master's project for several students, including Phillip Crunk (far left in the photo at left). Since then, a full-scale prototype of the plug-flow reactor was installed at the South Columbus, Georgia, Water Resource Facility (see www.aaee.net/Website/E3ResGP.htm).

In a followup project that used the same laboratory digester system, we evaluated the inactivation of the pathogenic E. coli O157:H7 during thermophilic anaerobic digestion of manure from dairy cattle. This was the master's project for Nicole Van Abel (second from left in photo) and also was the basis of a project-based graduate class in 2003. As with the pathogenic species evaluated in the project described above, complete inactivation of E. coli O157:H7 was observed during digestion in a mixed, continuous-flow thermophilic digester. We observed, however, that a routine plating assay for serotype O157:H7 gave false positive results, suggesting that a more stringent immunoassay should be used to evaluate its removal in thermal processes used to treat manure or sludge.

Factors Influencing the Biodegradation of Polycyclic Aromatic Hydrocarbons in Contaminated Soil; National Institute of Environmental Health Sciences.

This was Project 4 in the UNC Superfund Research Program, which extended a recently-completed project titled Bacterial Biodegradation of High Molecular Weight Polycyclic Aromatic Hydrocarbons. In this project we focused mostly on the degradation of PAHs containing four or five rings, although some of our initial work was done on the three-ring compound phenanthrene. Seven of the four- and five-ring PAHs are designated as known human carcinogens, and these compounds also tend to be the most resistant to biodegradation when bioremediation is attempted at PAH-contaminated sites.

Limitations in the bioavailability of PAHs are typically invoked to explain why they often are not removed extensively during bioremediation of contaminated soil. Recent evidence on biodegradation of PAHs in field-contaminated soils has suggested, however, that factors other than bioavailability may govern the rate at which the high-molecular-weight PAHs are biodegraded. In this project, we evaluated the extent to which such factors can influence PAH degradation. We extended earlier work with pure cultures of PAH-degrading bacteria, then applied our knowledge of degradation mechanisms obtained with these organisms to the study of contaminated soils. To evaluate factors governing the biodegradation of PAHs in soil, we used a bench-scale slurry-phase bioreactor (right) to treat contaminated soil obtained from a former manufactured-gas plant site in Charlotte, NC.

Former students Dr. William Stringfellow and Rob Nagel isolated PAH-degrading bacteria from soil samples taken at eight different contaminated sites. We have shown that all 11 of the organisms tested were able to degrade at least partially the four-ring compounds benz[a]anthracene, chrysene, fluoranthene and pyrene, and the five-ring compound benzo[a]pyrene. Eight of these bacteria were able to mineralize (oxidize to carbon dioxide and water) benzo[a]pyrene; prior to this work, only two bacteria had been shown to be able to do so. Our results suggest that the inherent ability to degrade high molecular weight PAHs may be widespread among bacteria found at contaminated sites, so that other factors must be responsible for the apparent recalcitrance of these compounds. Dr. Chikoma Kazunga (left) followed up this work by studying the products formed from the incomplete metabolism of pyrene and fluoranthene. These products inhibit the degradation of phenanthrene and the mineralization of benzo[a]pyrene, chrysene, and benz[a]anthracene. Dr. Joanna Park found that these compounds are genotoxic, causing DNA damage both in vitro and in mammalian cells.

Effects of Bioavailability and Carbon Source Supplementation on Anaerobic Biodegradation of DDT and its Metabolites in Contaminated Soil; Ciba-Geigy Corporation.

DDT is a pesticide that was banned from use in the United States in the early 1970s. It remains in the environment, however, at many locations, including many Superfund sites. DDT is resistant to biodegradation, although research conducted in the 1960s and 1970s suggested that it could be broken down anaerobically in a process referred to as reductive dehalogenation. In this process, the chlorine atoms on a molecule are replaced with hydrogen atoms. For DDT, reductive dehalogenation leads to a product known as DDD and eventually other products.

We obtained soil from a Superfund site contaminated with DDT, DDD and another product of DDT degradation, DDE. We evaluated whether the addition of a nonionic surfactant could increase the rate of anaerobic degradation of the DDT and its metabolites. The surfactant markedly increased the rate of DDT degradation, but some DDD accumulated as a result. The amount of DDD that accumulated did not increase stoichiometrically with the amount of DDT degraded, suggesting that further degradation beyond DDD was occurring. In followup experiments conducted in the absence of soil, an inoculum obtained from the soil microcosms was able to remove DDT with virtually no accumulation of DDD. This project formed the basis for the doctoral research for Dr. Glenn Walters.