Water Resources Research Center

PROJECT TITLE:

Sequential Anaerobic-Aerobic Biodegradation of PCBs and PAHs in Phytoremediation Cuttings Using Slurry Reactors and Composters

PRINCIPAL INVESTIGATOR:

Roger Babcock, Ph. D., Water Resources Research Center, U.H. Civil Engineering

1. STATEMENT OF RESEARCH INTEREST

The use of phytoextraction for clean-up of soils contaminated with heavy metals is an attractive alternative to excavation and treatment/removal operations that is quite well documented (Raskin, 2000). The use of plants to extract toxic organic compounds such as polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs) from contaminated soils is not well documented and constitutes a current research topic. However, there is preliminary evidence that phytoextraction of PCBs has occurred at a Haiku Valley contamination site. Currently, there is no evidence that PAHs have been phytoextracted by the plant species at the site. In addition, there is evidence that a rhizosphere concentration phenomenon is occurring at the Haiku site whereby PCBs are present at higher concentrations in the vicinity of plant roots than in the bulk soil (analyses for PAHs in the rhizosphere has not been done).

The transfer of contaminants such as heavy metals, PCBs, and PAHs into plant tissue via phytoextraction followed by harvesting the plant biomass must be considered as the first step in an overall treatment scheme. Additional treatment and/or disposal of the harvested biomass must be carefully considered. Harvested biomass can be incinerated or buried at approved facilities or treated biologically. Treatment options that include biological mineralization are often the most cost effective and are usually favorable compared to incineration or burial without treatment. In addition to phytoextraction, there are other possible methods for clean-up of soils contaminated with PCBs and PAHs. These include direct biological treatment of the soil either in situ or following excavation under aerobic and/or anaerobic conditions. Different methods such as soil slurry reactors, bioventing, composting and many other processes have been investigated.

The complete biodegradation of PCBs is not simple because it requires the actions of an acclimated consortium of microorganisms. PCBs with five or fewer chlorine atoms are subject to some degree of biotransformation (less than complete biodegradation) under aerobic conditions (Quensen, 1990). This aerobic degradation has been shown to occur by a consortium under co-metabolic conditions (Adriaens, 1989, 1990). Under anaerobic conditions, reductive dechlorination of highly chlorinated PCBs (including Aroclor 1254 and 1269) is possible (Morris, 1992). This suggests a two-stage treatment process in which anaerobic reductive dechlorination is used to initiate degradation of the heavily chlorinated PCB congeners, followed by aerobic degradation of mono- and di-chlorinated congeners (terminal products of the higher chlorinated congeners).

Sequential anaerobic dechlorination followed by aerobic mineralization has been documented (e.g. Bowlds, 1992; Anid, 199 1; Guiot, 1998) but not extensively. Similarly, multiple ring PAHs such as benzo(a)pyrene are generally very slow to degrade aerobically but may be susceptible to sequential anaerobic-aerobic degradation. To date, documentation of research in which phytoextracted contaminants have been biodegraded in an anaerobic or aerobic process has not been found in the literature.

Reductive dechlorination of PCBs is achieved through a consortium of select microorganisms that may not be present in natural soils that are not contaminated with PCBs. Thus, the best place to find the requisite organisms is at contaminated sites. There is evidence that analog substrate enrichment (with biphenyl) may speed-up degradation of some PCBs such as Aroclor 1242 (Brunner, 1985) but biphenyl did not seem to help with Aroclor 1254 (Rhee, 1993). There may be other so-called "Inducer compounds" (Babcock, 1993) which may provide enhanced degradation rates for Aroclor 1254. Some researchers have found that it may be essential to have sediment present for significant PCB dechlorination (Monis, 1992). The sediment may provide some carbon or growth factor or immobilized cell support. Little data are available on composting of PCB contaminated soils.

However, it is logical that a sequential anaerobic-aerobic composting operation could biodegrade PCB-contaminated soils and/or biomass. PAH-contaminated soils have been composted with some degree of success (McFarland, 1992).

This study will consist of laboratory-scale research to investigate two different technologies for remediation of PCB and PAH contaminated soil. Technology 1 is based upon the premise that phytoextraction will be a viable method for removal of soil-bound PCBs (and PAHs) and will consist of slurry reactors operated in sequential anaerobic-aerobic mode to treat actual plant cuttings from the Haiku site with and without PCB and PAH spikes. Technology 2 is based upon the premise that it may be most feasible to treat a combination of plant biomass and rhizosphere soil directly and will consist of an enclosed composter that will be operated in sequential anaerobic-aerobic mode. Laboratory reactor systems will be constructed and operated with adequate controls (spiked water, killed biomass). The model compounds to be utilized in any spiking experiments will be Aroclor 1254 and benzo(a)pyrene. The reactor systems will be operated for at least 6 months.

DESCRIPTION AND SCOPE OF WORK

The primary objective of this research project will be to demonstrate PCB mineralization (Aroclor 1254) in the presence of phyto-plant cuttings during sequential anaerobic-aerobic treatment under controlled laboratory conditions in both slurry-type reactors and composters. A secondary objective will be to determine the fate of benzo(a)pyrene under the reaction conditions found to be acceptable for PCB mineralization.

PCBs are a family of compounds produced by chlorination of biphenyl which produces a mixture of "congeners." PCBs were manufactured in complex mixtures by several companies including Monsanto which used the trade name "Aroclor." The Aroclor product number reflected composition, i.e. Aroclor 1254 indicates 12 carbon atoms and 54% chlorine. Aroclor 1254 is considered one of the more toxic PCB mixtures due to its high chlorine content. In this study, commercial grade Aroclor 1254 will be utilized as the model compound mixture for spiking experiments. This will be in addition to the actual aged/weathered PCB mixture present in the Haiku site soils and plant cuttings. The second model compound will be commercial grade benzo(a)pyrene.

Each PCB mixture (i.e. Aroclor 1254) consists of >60 different congeners making analytical quantification challenging. Many PCB fate studies have not adequately quantified PCB degradation products or demonstrated biodegradation. It is not adequate to simply monitor the disappearance of one or two specific congeners because without adequate controls, abiotic losses (adsorption, volatilization, and photolysis) could account for some or all of the removal. In this study, we will monitor the concentration of a whole series of PCB congeners. If biodegradation occurs as predicted, during the first treatment step (anaerobic dechlorination), the quantity of highly chlorinated congeners (tetra-, penta-, hexa-) will systematically decrease and a concomitant increase in lower chlorinated (mono-, di-, and tri-) congeners will remain (this will provide a chromatogram that looks distinctly different from one describing abiotic losses).

Then during the second treatment step (aerobic mineralization), the quantity of tri-, di-, and monochlorinated congeners will sequentially decrease. There may be some compounds that accumulate, however, most should be mineralized. The best way to conclusively demonstrate mineralization would be to use C14 labeled PCBs, to collect off-gases, and determine the mass of C14 present in the gas phase, the liquid phase, and incorporated into cell mass using scintillation counting. However, since we do not want to use radioactive chemicals, we will use a combination of GC/ECD and GC/MS/'MS to accurately quantify liquid-phase and adsorbed phase PCB and daughter product concentrations throughout the treatment phases. in addition, adequate controls will be used to determine abiotic losses (spiked water, killed biomass). This study will not quantify toxicity of plant cuttings or soil before or after biotreatment.

Slurry reactor experiments will be conducted in 160-mL serum bottles with Teflon-lined septa and aluminum crimp tops. The bottles will be placed in a temperature-controlled orbital shaker. Serum bottles will contain mineral media, a carbon source, a PCB spike, a microbial inocula, and phyto-cuttings. The mineral media composition is given in Table 1. Methanol or acetone will be added to anaerobic bottles as a carbon source to induce anaerobic activity which will be measured by gas production. Biphenyl will be added to some aerobic bottles to determine if analog enrichment will enhance biodegradation rates and/or reduce initial lag periods. The PCB spike will consist of commercial grade Aroclor 1254 at either 500, 1 00, 1 0, or 0 mg/L. Anaerobic microbial inocula will consist of either 1-2 grams of contaminated soil, a few ml of sludge from an anaerobic digester or a combination of both.

An anaerobic control will not receive an inoculum. Aerobic microbial inocula will consist of either 1-2 grams of contaminated soil, a few ml of activated sludge, or possibly a culture of Pseudomonas strain LB400 if available. An aerobic control will not receive an inoculum. Some aerobic bottles will be started concurrently with the anaerobic bottles in an attempt to develop enrichment cultures capable of degrading tri-, di-, and mono-chlorinated PCB congeners. These bottles will be spiked with mixtures of commercially available 4-chlorobiphenyl, 4,4'-dichlorobiphenyl, and 3,4-dichlorobiphenyl. Gas production volume will be measured by periodic insertion of a needle connected to a gas-tight syringe. The anaerobic and aerobic slurry reactors will be operated for at least 6 months.

Composting experiments will be conducted in stoppered 125-mL Erlenmeyer flasks. The flasks will be placed in a temperature-controlled orbital shaker. Composter flasks will contain contaminated soil (with root material), a carbon source, a bulking agent (compost, straw, etc.), a PCB spike, a microbial inocula, and phyto-cuttings. The composters will be operated similarly to the slurry reactors.

Table 1. Mineral media composition

Mineral					mg/L
(NH₄)2HPO₄					80
NH₄CI					1000
K₂HPO₄					200
NaCl₂					10
CaCl₂					10
MgCl					250
CoCl₂ * 6H₂O					1.5
CuCl₂* 2H₂O					0.2
Na₂MoO₄* H₂O					0.23
ZnCl₂					0.19
NiSO₄* 6H₂O					0.2
FeSO₄ * 7H₂O					1.0
AlC1₃* 6H₂O					0.4
H₃BO₃					0.38

The workplan is as follows:

2. Create adsorption "Isotherms" using Aroclor 1254 and several different shredded plant cuttings from the Haiku site. Repeat with benzo(a)pyrene. This will provide an estimate of the absorbability of the PCB and PAH on the plant tissue.

3. Operate anaerobic dechlorination slurry reactor bottles with plant cuttings and added Aroclor 1254 and necessary nutrients, inducer compounds (i.e. biphenyl) and/or microorganisms as needed; monitor degradation kinetics and degradation product composition using gas chromatography with ECD and/or MS detection. After acceptable performance with PCBs is achieved, add benzo(a)pyrene to reactor(s) and determine its biodegradation under the same conditions.

4. Operate aerobic slurry reactor bottles with effluent from anaerobic reactors (and spiked with mono- and di-chlorinated biphenyls); add nutrients, inducers and microorganisms as necessary; monitor degradation kinetics and degradation product composition gas chromatography with ECD and/or MS detection.

5. Operate laboratory composter flasks with PCB contaminated soil mixed with bulking agent, added nutrients, inducer compounds and microorganisms as necessary in anaerobic-aerobic sequential mode; monitor degradation kinetics and degradation product composition.

6. Estimate scalability and full scale costs.