Continuous Biodegradation of High Strength Phenol by Immobilized Bacteria in Packed-Bed Column Reactor

V. Arutchelvan, Professor of Civil Engineering, Annamalai University, Annamalainagar-608002, India, Tel: +91 4144 238884, Email: arul.au@gmail.com
V. Kanakasabai, Professor of Civil Engineering, Annamalai University, Annamalainagar-608002, India, Tel: +91 4144 239731, Email: vkhdce@yahoo.com
S. Nagarajan, Professor of Chemistry, Annamalai University, Annamalainagar-608002, India, Tel: +91 4144 238734, Email: drsn@sify.com

Phenols are toxic to all types of organisms. Two bacterial strains capable of utilizing phenol as a sole carbon source were isolated from the phenol bearing industrial wastewater. Based on the biochemical test results the organisms were identified as Pseudomonas cepacia and Bacillus brevis. The batch studies revealed that both organisms were very efficient in phenol degradation. The well-acclimatized cultures of P. cepacia and B. brevis degraded 2500 and 1750 mgl^-1 of phenol in 144 h, respectively in batch process. In order to enhance the rate of degradation, the isolated organisms were immobilized in over-burnt clay cylindroids. These cylindroids are rigid, non-toxic, non-biodegradable, locally available, economical, easy to dispose and eco-friendly. Phenol was continuously degraded in packed-bed column reactor with the immobilized cells. The effect of varying substrate concentration, Hydraulic Retention Time (HRT) and COD removal was studied. The organisms P.cepacia and B.brevis degraded about 2000 and 1250 mgl^-1 of phenol concentration within 12 h of HRT, respectively. The COD removal efficiency was about 92% for P.cepacia and 90% for B.brevis corresponding to the maximum phenol degradation. The SEM study shows the presence of the organisms inside the porous matrix.

Enhanced Bioremediation of Chlorinated VOCs Using Sodium Lactate and Dibasic Ammonium Phosphate

Mark T. Becker, URS Corporation, 12 Commerce Drive, Cranford, NJ 07016, Tel: 908-709-3971, Fax: 098-272-3940, Email: mark_becker@urscorp.com.
Fayaz Lakhwala, URS Corporation, 12 Commerce Drive, Cranford, NJ 07016, Tel: 908-709-3951, Fax: 098-272-3940, Email: fayaz_lakhwala@urscorp.com.
Vincent L. Pace, Atlantic Richfield Company, 28100 Torch Parkway, MC 2N, Warrenville, IL 60555, Tel: 630-836-5630, Fax: 630-836-6336, Email: Vincent.Pace@bp.com.
Stephanie Fiorenza, Atlantic Richfield Company, 501 Westlake Park Blvd., Floor 20, Room 20.101C, Houston, TX  77079, Phone: 281-366-7484, Fax, 281-366-7094, Email: Stephanie.Fiorenza@BP.com.
Anthony Kaufman, URS Corporation, 12 Commerce Drive, Cranford, NJ 07016, Tel: 908-709-3905, Fax: 098-272-3940, Email: Anthony_Kaufman@urscorp.com

Historic discharges at a former industrial facility in New Jersey created a plume of chlorinated ethenes in groundwater, including trichloroethene (TCE), cis-1,2-dichloroethene (c-DCE) and vinyl chloride (VC).  The presence of degradation products c-DCE and VC suggest that reductive dechlorination is occurring, but after several years of monitoring, concentrations of these compounds have not decreased significantly.  Groundwater quality data indicated that a lack of total organic carbon (TOC) and nutrients such as phosphate and nitrogen may be limiting degradation processes.  A field pilot test was conducted to enhance the on-going reductive dechlorination process.  Amendments were manually injected into the plume weekly for a period of three months.  The amendments consisted of sodium lactate (WilClear^TM) solution and dibasic ammonium phosphate (DAP).  The sodium lactate was diluted to ease injection and the crystalline DAP was mixed into the sodium lactate solution.  Amendments were injected via gravity feed through tubing into injection wells.  After each injection, a submersible pump was used to circulate groundwater from contaminated monitoring wells into injection wells to distribute the amendments throughout the plume.  Monitoring data collected during this pilot test indicates that the injections were effective at enhancing the reductive dechlorination of target contaminants.  Concentrations of TCE decreased 86% to 96% while c-DCE concentrations remained constant and VC production was limited.  Dissolved oxygen concentrations in all monitoring wells within the plume decreased to 0 mg/l and redox potential decreased from 64 mV and 209 mV to –155mV and –157 mV, respectively.  Production of acetic and propionic acids and methane increased and peaked 3 months after the injections ceased.  Within six months of the end of the injection program, however, contaminant and geochemical parameters had rebounded to pre-injection levels.  Based on the results of the sodium lactate/DAP injections, a workplan is being prepared to inject a longer lasting lactate-based substrate.

Bioremediation of VPH and EPH Contaminated Groundwater Using in-situ Submerged Oxygen Curtain (iSOC) Technology

Jacqueline Lees, ENSTRAT, Inc., 420 Maple Street, Marlboro, MA 01752, Tel: 508-460-6100 x19, Fax: 508-460-8115, Email: jlees@enstrat.net
James F. Begley, MT Environmental Restoration, 24 Bay View Avenue, Plymouth, MA 02360, Tel: 508-732-0121, Fax: 508-732-0122, Email: jbegley@cape.com

An iSOC bioremediation system for residual aliphatic and aromatic volatile and extractable petroleum hydrocarbons (VPH and EPH) contaminated groundwater was installed and operated at a Massachusetts site. While more soluble petroleum compounds such as BTEX are know to be readily degraded under aerobic conditions, the degradation of other less soluble and less bioavailable VPH and EPH compounds is not as well understood. This case study will examine the performance of an in-situ bioremediation system for residual VPH and EPH contaminated groundwater in an area downgradient of a source area excavation.

Extracellular Electron Shuttling (EES) Compounds in Bioremediation

Student Presenter

Caitlin Bell , Rm4162, 205 North Mathew Street, Civil Engineering Building, Department of Civil and Environmental Engineering, University of Illinois, Urbana, IL, 61801. Tel: 217-333-8121  Email: bell5@uiuc.edu
Kevin T. Finneran, Rm3221, 205 North Mathew Street, Civil Engineering Building, Department of Civil and Environmental Engineering, University of Illinois, Urbana, IL, 61801. Tel: 217-244-7956  Email: finneran@uiuc.edu

Electron shuttling compounds are a class of molecules that accept electrons from microbial respiratory pathways and transfer electrons abiotically to more electronegative electron acceptors.  Recent reports suggest that extracellular electron shuttling (EES) molecules may serve as "redox active" agents in bioremediation applications for organic and inorganic contaminants; humic substances, extracellular quinones, and reactive Fe(II) have been investigated in most detail.  Our research has focused on humic substance mediated biodegradation of electronegative contaminants, catalyzed by prokaryotes and eukaryotes.  Recent data suggest that white and brown rot fungi will reduce the synthetic humic analogue, anthraquinone-2,6-disulfonate (AQDS), in the presence of oxygen.  Gloeophyllum trabeum reduced approximately 4mM AQDS in 12 days in aerobic, aqueous cultures, which is comparable to anaerobic cultures with AQDS.  Fe(III) was also reduced in the presence of oxygen; Fe(II) re-oxidation kinetics were limited by fungal biomass.  The reduced EES were stable in air, suggesting that electron transfer to contaminants such as the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) may be possible in aerobic environments.  To date reductive strategies are applied in anaerobic systems; these data indicate that fungal reductive processes may be possible despite aerobic geochemical conditions.

Chlorinated contaminants are also environmentally relevant electron acceptors.  To date research has focused on direct dechlorination by specific microorganisms, which are not present in all environments.  Preliminary data suggest that highly chlorinated compounds (tetrachloroethylene (PCE) and trichloroethane (TCA)) may accept electrons from the reduced EES AQDS.  Bioremediation strategies for chlorinated compounds predicated on EES will be broadly applicable to all environments, as the microorganisms involved have been identified in all environmental media.  Future studies will investigate EES mediated transformation of polychlorinated biphenyls, which are hydrophobic and inaccessible in aqueous media.  EES may overcome the mass transfer limitations by cycling electrons from microbial respiration to adsorbed contaminant mass, without the need for direct cell-PCB contact.

The Effect of Aggregate and Initial Contaminant Concentration on the Biodegradation Rate of Total Petroleum Hydorcarbons

Ramona Boodoosingh, Department of Civil and Environmental Engineering, Tufts University , Medford, MA 02155, Email: rboodoosingh@yahoo.com
Denise Beckles, Department of Chemistry, University of the West Indies, St. Augustine, Trinidad, W.I.
Christopher Swan, Department of Civil and Environmental Engineering, Tufts University, Medford, MA 02155
Anne-Marie Desmarais, Department of Civil and Environmental Engineering, Tufts University, Medford, MA 02155

Bioremediation is one of the most cost-effective, and environmentally friendly methods to remediate many different types of waste materials widely used by many private and public entities. Though the parameters that affect the rate of bioremediation differ for each project, some parameters can be more easily controlled in the field than others.  For example, effective microbial action is crucial to the success of bioremediation.  However, microbes can be detrimentally affected by an initial contaminant concentration that is too high.  In addition, the type of bulking agent or aggregate used affects the ability of the mixture to retain moisture, the amount of drainage, aeration and ultimately the length of time for biodegradation.

A two-month field study was conducted in Trinidad and Tobago on crude oil contaminated waste.  This paper will look at the effect of two parameters, initial concentration and type of bulking agent, on the rate of biodegradation of Total Petroleum Hydrocarbons (TPH) in the oily waste being treated in this tropical environment.  A nutrient-supplying bioremediation agent was used to enhance the bioremediation process.  The results of this study can assist in the design of effective bioremediation projects in tropical environments.

Isolation and 16S rDNA Characterization of two Soil Bacteria Capable of Degrading Quinalphos an Organophosphorus Insecticide

Student Presenter

Sharungbam Geeta Devi, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre. Mumbai. - 400085, Tel: 91-22-25593831, Fax: 91-22-25515050, Email: geetasharungbam@gmail.com, sharung@sify.com
B.P. Kapadnis, Department of Microbiology, University of Pune, Pune - 411007, Tel: 91-20-25690643, Fax: 91-20-25690087 Email: bpkap@hotmail.com
R.L. Deopurkar, Department of Microbiology, University of Pune, Pune - 411007, Tel: 91-20-25690643, Fax: 91-20-25690087, Email: writetodeopurkar@unipune.ernet.in
S.P. Kale, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai. - 400085, Tel: 91-22-25593830, Fax: 91-22-25515050, Email: skale@apsara.barc.ernet.in, sharadkale@gmail.com

Quinalphos is an organophosphorus insecticide and acaricide with contact and stomach actions. No enough studies has been done on biodegradation of quinalphos. Hence attempts were made in the present study to isolate and identify bacteria from insecticide contaminated soils, which are capable of degrading quinalphos by utilizing it as a sole source of carbon and energy. An enrichment culture technique was used to isolate bacteria responsible for the enhanced biodegradation of quinalphos from soil previously exposed with various pesticides. Microscopic examination and physiological tests were followed by 16S rDNA sequence analysis to characterize the two bacterial isolates. One of the isolated bacteria was identified as a Pseudomonas sp. and another as Bacillus sp. Both Pseudomonas sp and Bacillus sp were found to be very active in degrading quinalphos, upto 92% degradation at original concentration of 12mg L^-1 of quinalphos within 15days was observed.Mix cultures study with Pseudomonas sp. and Bacillus sp. showed 83.71% degradation in10 days.  The degradation of quinalphos was maximum in the range of pH 7.0 and pH 8.0.  With glucose and in Nutrient broth, in 10days Pseudomonas sp. and Bacillus sp. showed  complete degradation. This shows that slight addition of rich carbon source stimulate the degradation of quinalphos by both Pseudomonas sp. and Bacillus sp.. Our findings showed that indigenous microorganism are a better means for degradation studies since Pseudomonas sp. and Bacillus sp. which we had isolated form the natural habitats are a potential degrader of quinalphos. These isolates could be used to remediate quinalphos contaminated sites.

Degradation of Oil-Derived Hydrocarbons by Bacteria Isolated from Oil-Contaminated Soils

F.G. Dias, R. Filho Lima and L.R. Durrant, Departamento de Ciências de Alimentos – Faculdade de Engenharia de Alimentos (FEA), Universidade Estadual de Campinas (UNICAMP) Cx. Postal: 6121, CEP:13083-862 Phone: +55 19 37882172  Email: durrant@fea.unicamp.br

Oil and its derivatives continue to be our main energy source.  Constantly we receive information through the media of the ecological disasters and social problems caused by oil spills or by its derivatives.  It is estimated that of the 3.2 billion tons of oil produced per year, 3.2 million tons enter the marine environment.  This work had the objective to evaluate the capacity of Bacillus circulans, Chromobacterium sp and Enterobacter aerogenes, isolated from oil-contaminated soils, to grow and degrade oil-derived hydrocarbons. They were cultivated for 12 days at 30º C and 150 rpm, in a medium containing minerals and 0.05% (final concentration) of one of the following hydrocarbons: tridecane, tetradecane, pristine, hexadecane, phenyldecane, phenanthrene, naphthalene, pyrene and benzo[a]pyrene. The remaining hydrocarbons were extracted with hexane and analyzed by gas chromatography.  Bacillus circulans degraded tetradecane (24%), tridecane (23%), hexadecane (24%), pristine (24%), phenyldecane (25%), naphthalene (100%), phenanthrene (25%), pyrene (100%) and benzo[a]pyrene (100%). A Chromobacterium sp degraded the same hydrocarbons in the ratios of 20%; 21%; 21%; 21%;  22%;  100%;  100%;  100%;  100% respectively.  Enterobacter aerogenes degraded 37%; 33%; 35%; 37%; 34%; 100%; 23%; 100%; 100% of the hydrocarbons present in the culture media, respectively. Our results indicate that these three bacterial strains have potential to be used in future studies of oil-derivatives contaminated environments.

We acknowledge financial support from FAPESP-SP, BRAZIL.

Degradation of Dyes by Bacteria Isolated from the Activated Sludge of a Textile Industry

E. Dias-Franciscon and L.R. Durrant, Departamento de Ciências de Alimentos – Faculdade de Engenharia de Alimentos (FEA), Universidade Estadual de Campinas (UNICAMP) Cx. Postal: 6121, CEP:13083-862 Phone: +55 19 37882172  Email: durrant@fea.unicamp.br

Reactive azo dyes are widely used in the textile industries. They are characterized by the presence of one or more azo bond, -N=N-, which function as a chromophoric group. Under usual dyeing conditions these dyes do not bind completely to the fibers resulting in colored effluents.  Dye removal is desired, not only by esthetic reasons, but also because many azo dyes and their derived products are toxics to aquatic life and mutagenic to humans. Although activated sludge process has been extensively used by the textile industries this treatment is not effective and these dyes are adsorbed by the biomass, being later disposed in landfills, causing contamination of these wide areas. 

Trying to solve these problems, three bacterial strains were isolated from activated sludge of textile industries near Campinas (SP, Brazil). Isolates were cultivated with different dyes: Reactive azo-dye Remazol Red RR (C.I. RBN 198) and Remazol Reactive Blue (C.I. RB 220) and degradation of the dyes was determined by UV-visible spectrophotometry and confirmed by liquid chromatography and also COD reduction (Chemical Oxygen Demand). Complete degradation of RBN 198 and RB 220 was found after 24 hs and 168 hs, respectively, by Trichococcus sp. and COD reduction was 41 and 68%, respectively. In the case of Enterobacter sp, RBN 198 dye was 95 % and RB 220 completely degraded, while COD was found to be 76 and 63 % respectively. A Bacillus sp degraded 75% of RB 220 and 84% of the RBN used, with COD reductions of 85 and 88%, respectively.  

The ability to decolorize these dyes and reduce of COD by these strains, suggest that they may have potential to be applied in the treatment of effluents and sites contaminated by dyes.

We acknowledge financial support from CNPQ - BRAZIL.

Physiological Approaches to Increase Molar Hydrogen Yield in Anaerobic, Fermentative Bacterial Cultures

Student Presenter

Jennifer L. Hatch, Environmental Engineering and Science Program, Civil and Environmental Engineering, University of Illinois, 205 North Mathews Avenue, Urbana, IL 61801, Tel: 217-333-8121, Email: jhatch2@uiuc.edu
Kevin T. Finneran, Assistant Professor, Environmental Engineering and Science Program, Civil and Environmental Engineering, University of Illinois, 3221 Newmark Laboratory, 205 North Mathews Avenue, Urbana, IL 61801, Tel: 217-244-7956, Email: finneran@uiuc.edu

Most approaches to increase hydrogen production focus on reactors without regard to increasing the hydrogen yield per mol of substrate consumed.  Optimized reactor designs are necessary; however, increasing the molar hydrogen yield is necessary before hydrogen-based fuel strategies can be commercially applied in a cost-effective manner.  This research aims to circumvent organic acid synthesis and increase hydrogen production by altering the electron transfer pathway in fermentative bacteria.  Carbon and hydrogen metabolisms of a fermentative pure culture (Clostridium beijerinckii) will be altered along with culture conditions to develop strains of microorganisms and co-cultures which will over-produce hydrogen. 

This approach in the over-production of hydrogen is alteration of the redox potential of the proton/H₂ couple so that electron transfer to protons becomes favorable relative to organic acid synthesis.  By utilizing electron transfer molecules such as quinones to increase the relative redox potential of the H⁺/H₂ redox couple, it is possible to cycle electrons from glucose oxidation through the electron carriers to hydrogenase and protons; this circumvents the organic acid synthesis pathway and increases hydrogen yield.

C. beijerinckii was grown anaerobically in glucose-rich medium in order to perform resting cell suspensions under non-growth conditions in anaerobic bicarbonate buffer.  250 mM biologically reduced AQDS (AH₂QDS) increased hydrogen production by 50% above the expected stoichiometry for C. beijerinckii.  Adding 5-100nM NAD⁺ increased the rate of hydrogen production as well as the yield by 75% for C. beijerinckii.  With increasing concentrations of AH₂QDS, hydrogen production was seen to increase, but with addition of NAD⁺, decreased concentrations proved to increase hydrogen yield.

The extent of AQDS and acetate cycling between Geobacter metallieducens (GS-15) grown on AQDS media and C. beijerinckii will be evaluated on batch and reactor scales while monitoring hydrogen production.  The effects of several different environmental samples such as agricultural waste and digester fluid for substrate will also be investigated in a reactor using pure and mixed cultures.  Ultimately, hydrogen from this process will be recovered for use in fuel cells.  

Enhancing Bioremediation of DNAPL TCE in Carbon-Poor, Slightly Oxidizing Groundwater within Glacial Soils

Lucas A. Hellerich, Ph.D., P.E., Metcalf & Eddy, Inc., 860 North Main Street Extension, Wallingford, CT 06492, Tel: 203-269-7310, Fax: 203-269-8788, Email: lucas.hellerich@m-e.aecom.com 
John L. Albrecht, L.E.P., Metcalf & Eddy, Inc., 860 North Main Street Extension, Wallingford, CT 06492, Tel: 203-269-7310, Fax: 203-269-8788, Email: john.albrecht@m-e.aecom.com
Dave Hart, C/O Noranda Aluminum P.O. Box 70, New Madrid, MO 63869, Tel: 573-643-6763, Fax: 573-643-6715, Email: Dave.Hart@falconbridge.com

This paper describes a field pilot study of enhanced bioremediation (reductive dechlorination) of trichloroethene (TCE) at a site located in western Connecticut.  Geology at the test location consisted of glacial till overlying weathered gneissic bedrock.  The estimated static groundwater velocity was 0.17 ft/day.  The concentration of TCE in the study area varied spatially, but was up to 768 mg/L (~ 70% solubility of TCE), indicating the presence of DNAPL.  Low levels of the degradation products cis-1,2-dichloroethene (cis-1,2-DCE) (330 µg/L), vinyl chloride (85 µg/L), and ethene (7.7 µg/L) were detected, suggesting the occurrence of microbially-mediated reductive dechlorination.  Groundwater chemistry was characterized as follows: circum-neutral pH; slightly oxidizing (~ 2 mg/L dissolved oxygen); low total organic carbon (TOC) content (2.7 mg/L TOC); moderate sulfate (43 mg/L); and measureable methane (45 µg/L) and dissolved hydrogen (19 nM), indicating the potential for methanogenic activity.  Methanogenic bacteria were detected at moderate levels.  Dehalococcoides spp. bacteria, the only known bacteria capable of completely reducing TCE to ethene, were also detected, but at levels several orders of magnitude lower than the methanogens.

A dilute solution (10% – 15%) of EOS^TM emulsified soybean oil, amended with a bromide tracer, was injected into the subsurface, providing a source of carbon and reducing power for the naturally-occurring microbial populations.  The injectate was chased with potable water to facilitate distribution in the saturated subsurface.  Reducing conditions, characterized by negative oxidation-reduction potential and decreased DO levels, were achieved within several weeks of the injection event.  The distribution of EOS^TM was evaluated using TOC and bromide concentrations, and indicated the presence of preferential flow paths in the subsurface.  Aqueous TOC concentrations ranged from 22 mg/L to 17,000 mg/L within the test volume.  Chlorinated ethenes, geochemical parameters, electron acceptors, and microbial populations were measured as a function of time.  An evaluation of the efficacy of enhancing reductive dechlorination at the site was performed.

The Biodegradation of Petroleum Hydrocarbons in Three Ecuadorian Crude Oils

Bo Liu, NewFields Environmental Forensics Practice, LLC, 100 Ledgewood Place, Suite 302, Rockland, MA 02370, Tel: (781) 681-5040, Fax: (781) 681-5048, Email: bliu@newfields.com
Gregory S. Douglas, NewFields Environmental Forensics Practice, LLC, 100 Ledgewood Place, Suite 302, Rockland, MA 02370, Tel: (781) 681-5040, Fax: (781) 681-5048, Email: gdouglas@newfields.com
Jeffery H. Hardenstine, NewFields Environmental Forensics Practice, LLC, 100 Ledgewood Place, Suite 302, Rockland, MA 02370, Tel: (781) 681-5040, Fax: (781) 681-5048, Email: jhardenstine@newfields.com

When crude oil is released to the environment its hydrocarbon composition begins to change due to physical/chemical/biological process collectively known as weathering.  Physical weathering such as evaporation and water washing may occur relatively rapidly, however the most important process related to the long term degradation and removal of the oil from the environment is biodegradation.  A laboratory study has been performed to evaluate the compositional changes and biodegradation potential of three crude oils.  Nutrients and naturally occurring bacteria were added to crude oil samples from Ecuador and incubated in the laboratory at 30°C for up to twenty weeks.  The bacterial inocula, used in this study were developed with indigenous bacteria from soils in petroleum producing regions in Ecuador.  These tests showed that volatile hydrocarbons, including gasoline range organics (GRO, C₅-C₁₂ range TPH) such as benzene, toluene, ethylbenzene and xylenes (BTEX), in the crude oil were degraded by naturally occurring bacteria within 1-7 days.  A second group of compounds, semi-volatile hydrocarbons, were also measured in the crude oil.  This group of compounds includes petroleum hydrocarbons such as diesel range organics (DRO, C₁₀-C₂₈ range TPH) and the USEPA 16 priority pollutant polycyclic aromatic hydrocarbons (PAH) such as naphthalene, fluorene, phenanthrene and chrysene.  Laboratory tests showed that semi-volatile hydrocarbons were significantly degraded within 2 weeks.  Approximately 84%-89% of the DRO and 97% of the total USEPA Priority Pollutant PAHs within the semivolatile range of crude oil were biodegraded after twenty weeks.  Crude oil residues found in site soils from Ecuador appear to be degraded to a degree similar to that documented in this laboratory study.  These similarities indicate that significant biodegradation has occurred—either naturally or as the result of remediation activities—at these sites.  

Behavior of Autotrophic Perchlorate Reducing Bacteria and Analysis of Royal Demolition Explosive [RDX] in Water by High Performance Liquid Chromatography, HPLC/UV

José Roberto Rivera-Negrón, University of Puerto Rico - Mayagüez Campus, Chemical Engineering Department and Chemistry Department, PO Box 5692, Mayaguez, PR 00681, Tel: 787-432-9240, Email: negron.jr@gmail.com
Ashish K Sahu, University of Massachusetts, Civil and Environmental Engineering, 18 Marston Hall, Amherst, MA, 01003, Tel: 413-687-3347, Email: aksahu@acad.umass.edu
Sarina Ergas, University of Massachusetts, Civil and Environmental Engineering, 18 Marston Hall, Amherst, MA, 01003, Tel: 413-545-3424, Email: ergas@ecs.umass.edu

Perchlorate, (ClO₄^-) contamination in ground and surface waters can cause serious human health effects, severe ecological and environmental consequences. Perchlorate can disrupt thyroid function and may impact fetal and newborn development, resulting in changes in behavior and learning capability. Many times ClO₄^- is a co-contaminant with RDX. This research investigated biological reduction of perchlorate and RDX by autotrophic sulfur oxidizing bacteria. ClO₄^- was analyzed with Ion Chromatography (IC). RDX can enter the water from disposal of waste water from Army ammunitions plants, and it can enter water or soil from spills or leaks from improper disposal at hazardous waste sites. RDX can cause seizures in humans and animals when large amounts are inhaled or ingested. The Environmental Protection Agency (EPA) has determined that RDX is a possible human carcinogen (Class C). The RDX was measured with High Performance Liquid Chromatography (HPLC) with a Photo Diode Array Detector (PDA). The experiment was conducted following the Method 8330 from EPA. The bacteria population change was determined with a Bicinchoninic Acid (BCA) Protein Assay. Regarding the bacteria population, we saw an increase from 190 µg/ml to 270 µg/ml protein in the solution over 20 days.  Perchlorate was degraded from 9.2 ppm to 8.6 ppm. In the case of RDX alone with the bacteria, RDX degraded from an initially 475 ppb to below the MDL (about 5 ppb). Also the RDX, perchlorate and the bacteria, the concentration of RDX was decreased from 467 ppb to lower than the MDL. In conclusion the setup for the RDX analysis was accomplished and the bacteria can degrade both contaminants, ClO₄^- and RDX.

Natural Gene Transfer to Develop Resistance to Metal Toxicity in Microbial Communities at the Oak Ridge Reservation Field Research Center 

Safiyh Taghavi, Biology Department, Brookhaven National Laboratory, 40 Bell Avenue, Bldg 463, Upton, NY 11973, Tel: 631-344-5306, Fax: 631-344-3407, E-mail: taghavis@bnl.gov
David Moreels, Biology Department, Brookhaven National Laboratory, 40 Bell Avenue, Bldg 463, Upton, NY 11973, Tel: 631-344-4924, Fax: 631-344-3407, E-mail: dmoreels@bnl.gov
Craig Garafola, Biology Department, Brookhaven National Laboratory, 40 Bell Avenue, Bldg 463, Upton, NY 11973, Tel: 631-344-4924, Fax: 631-344-3407, E-mail: cgarafol@bnl.gov
Garry Crosson, Environmental Science Department, Brookhaven National Laboratory, 34 Railroad Street, Bldg 830, Upton, NY 11973Y, Tel: 631-344-4921, Fax: 631-344-4486, E-mail: gcrosson@bnl.gov
Jeffrey P. Fitts, Environmental Science Department, Brookhaven National Laboratory, 34 N Railroad Street, Bldg 830, Upton, NY 11973, Tel: 631-344-2777, Fax: 631-344-4486, E-mail: fitts@bnl.gov
Daniel van der Lelie (Principal Investigator), Biology Department, Brookhaven National Laboratory, 40 Bell Avenue, Bldg 463, Upton, NY 11973, Tel: 631-344-5349, Fax: 631-344-3407, E-mail: vdlelied@bnl.gov

The Field Research Center (FRC) at Oak Ridge National Lab is characterized by mixed pollution consisting of heavy metals, radionuclides and nitrate. The remediation of toxic metals depends on a method of in situ containment that decreases their mobility and bioavailability, which in the case of uranium is based on its reduction by dissimilatory sulfate and iron reducing organisms. However, the presence of high levels of heavy metals and nitrate can inhibit their activities: especially high levels of nitrate, used by anaerobic bacteria as an electron acceptor, will decrease the efficiency of U(VI) and metal reduction. Under these conditions a two-step approach is required. In a first step nitrate is removed via the activity of nitrate reducing bacteria, followed by a second step of U(VI) and metal reduction/precipitation. Unfortunately, the activity of nitrate reducing bacteria is inhibited under conditions of elevated heavy metals. It was therefore hypothesized that inoculation with nitrate reducing organisms, which were genetically engineered with heavy metal resistant genes, might improve nitrate reduction, either via establishment of the inoculum or via horizontal gene transfer of the heavy metal resistance determinant into the indigenous community of nitrate reducing bacteria.

In order to test our hypotheses, in situ simulating lab-scale column experiments were set-up with soil samples taken from the FRC site and percolated with a mineral medium contaminated with nickel. The columns were bio-augmented with Pseudomonas DM-Y2, which was engineered with nre isolated from heavy metal resistant Cupriavidus metallidurans 31A. The efficiency of nitrate reduction and U(VI) reduction by the established complex microbial community was evaluated by following the microbial community dynamics and by chemical analysis of the column effluents. In function of time the population was characterized by most probable counts of nitrate, sulfate and iron reducing organisms. The fraction of nickel resistant organisms was counted after spread plating on nickel containing selective media. Sequencing and cloning of the 16S rDNA genes of the eubacterial population was used to identify the predominant community members. Spectroscopic techniques were used to determine sulfate and nitrate concentrations, while iron, nickel and uranium concentrations and speciation were determined by ICP-MS and EXAFS. Based on our data we concluded that under conditions of Ni selection a shift in community composition occurred from a population dominated by β and γ-proteobacteria to a community dominated by Ni-resistant, nitrate reducing Arthrobacter, Pseudomonas and Sphingomonas species. In addition to nitrate reduction we also observed the establishment of a second redox-zone in which increased sulfate and U(VI) reduction occurred, even in the presence of nickel.

The Effect of PCB Partitioning between Aqueous and Organic Phases on Rate and Extent of Biodegradation

Student Presenter

Lars Rehmann, Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada, Tel: 613- 533-6000, Ext. 75170, Fax: 613-533-6637, Email: lrehmann@chee.queensu.ca
Andrew J. Daugulis, Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada, Tel: 613-533-2784, Fax: 613-533-6637, Email: andrew.daugulis@chee.queensu.ca

Octanol/water partitioning coefficients of environmentally significant compounds such as polychlorinated biphenyls (PCB) or polycyclic aromatic hydrocarbons (PAH) are in the order of 10^­­4 to 10⁵. This might result in extremely low aqueous phase concentrations of these compounds if an organic phase is present at contaminated sites.  It was the scope of this study to investigate whether or not the presence of an, otherwise non-toxic, organic phase can become the limiting factor in aerobic PCB degradation.

Burkholderia xenovorans LB400 is the most studied organism for aerobic PCB degradation, and its degradation kinetics for biphenyl were studied in a two-phase partitioning (TPPB). The use of a TPPB allowed it to operate a bioreactor at constantly low aqueous phase substrate concentrations near the half saturation concentration of the Monod model. The specific growth rate and the half saturation constant of the Monod model were estimated to be m_max = 0.25 h^-1 and K_S = 0.0001 g L^-1, with a yield coefficient of Y_X/S = 0.48 g biomass per g biphenyl. The estimated value of K_S is at the low end of the typical values for bacteria, which is expected for an organism utilizing a hydrophobic substrate. PCBs are degraded via the biphenyl pathway by the same enzymes as biphenyl, allowing the assumption that the half saturation constant for PCBs is in a similar range, most likely higher than the one for biphenyl. Hence, it was expected that low concentrations of PCBs could not be degraded in the presence of a large organic fraction. Experiments with a constant amount of PCBs and different amounts of two organic solvents showed that PCBs could only be degraded in the presence of a small organic fraction. The concentration in the aqueous phase became too low for successful biodegradation as soon as the organic fraction reached a certain level. It can therefore be concluded that reduced bioavailability of PCBs due to partitioning in an organic fraction can be the limiting factor during biodegradation of PCBs.

Degradation of 2-aminobenzenesulfonate by a Two-Membered Bacterial Consortium

Student Presenter

Poonam Singh, Biotechnology laboratory, Department of Chemistry, I.I.T., Kanpur- 208016, India, Tel: + 91- 512-2597104, Fax: 91-512-2597436, Email: singhpoonam3012@rediffmail.com, spoonam@iitk.ac.in
Vivek Singh Chauhan, Biotechnology laboratory, Department of Chemistry, I.I.T., Kanpur- 208016, India, Tel: + 91- 512-2597104, Fax: 91-512-2597436, Email: viveksc@iitk.ac.in
Leela Iyengar, Biotechnology laboratory, Department of Chemistry, I.I.T., Kanpur- 208016, India, Tel: + 91- 512-2597104, Fax: 91-512-2597436, Email: leela@iitk.ac.in

Aminobenzenesulfonates (ABS) are used in the production of dyes, ion exchange resins, optical brightners and as additives to products like ink and engine oils. Consequently they are present in effluents from these production processes and find their way to rivers and surface waters. Due to their xenobiotic and highly polar nature, biodegradation of aminobenzenesulfonates require specialized bacterial strains. Reports on 2-ABS degradation, by either mixed or pure bacterial cultures, are very scarce. The present study is on the development of two-membered bacterial consortium for 2-ABS degradation.

Among several sources of inocula used in this study, enrichment could be developed only from the sludge derived from wastewater treatment plant for organic chemical manufacturing unit. This indicates that 2-ABS degrading organisms are still very rare in environment. Several transfers, over a period of six months, led to the development of two-membered consortium. During growth in batch culture, 2-ABS could be used as the sole carbon, energy, nitrogen and sulfate source. The relative number of one of these strains was much higher than the other one in 2-ABS grown culture. Although stiochiometric release of sulfate was observed, ammonia recovery was less than 40% for 800mg/L of 2-ABS. Mineralization was indicated by high TOC removals. Degradation was observed even upto 1500mg/L 2-ABS. The culture was highly specific for 2-ABS, as none of other susbstituted benzene sulfonates could be degraded. Thus it seems that mixtures of aromatic sulfonates can only be degraded by mixed bacterial consortia, which harbour diverse genes for congeneric substrates.

The role of these bacterial strains in 2-ABS degradation and their characterization by 16S rDNA sequence analysis are presently carried out.

Biodegradation of Weathered Oil in Soils with a Long History of TPH Contamination

Talaat Balba, Ph.D. Conestoga-Rovers & Associates, 2055 Niagara Falls Blvd, Niagara Falls, NY 14303, Tel: 716-297-6150, Fax: 716-297-2265, Email: tbalba@craworld.com
Sophia Dore, Ph.D. Conestoga-Rovers & Associates, 2055 Niagara Falls Blvd, Niagara Falls, NY 14303, Tel: 716-297-6150, Fax: 716-297-2265, Email: sdore@craworld.com
Donald Pope, B.S. Conestoga-Rovers & Associates, 2055 Niagara Falls Blvd, Niagara Falls, NY 14303 Tel: 716-297-6150, Fax: 716-297-2265, Email: dpope@craworld.com
Jennifer Smith, M.S. Conestoga-Rovers & Associates, 2055 Niagara Falls Blvd, Niagara Falls, NY 14303 Tel: 716-297-6150, Fax: 716-297-2265, Email: jjsmith@craworld.com
Alan Weston, Ph.D. Conestoga-Rovers & Associates, 2055 Niagara Falls Blvd, Niagara Falls, NY 14303 Tel: 716-297-6150, Fax: 716-297-2265, Email: aweston@craworld.com

Sites with a long history of exposure to petroleum hydrocarbons contain mainly long chain hydrocarbons or “weathered oil”.   This is because petroleum is a complex mixture of many organic compounds and these compounds biodegrade at different rates.  Under aerobic conditions the shorter hydrocarbon chains biodegrade first and the longer chains are more recalcitrant.

Long chain hydrocarbons are less soluble in water and more like tar in consistency than the shorter chain hydrocarbons, so a major problem with the degradation of these compounds is that bacteria that break down hydrocarbons do not come into contact with them.  These compounds are not bioavailable and therefore do not biodegrade.   

A petroleum refinery that has been active for many years has a large amount of weathered oil the surrounding soil and groundwater.   A treatability study was performed on samples from the Site to determine whether conditions could be manipulated to stimulate the biodegradation of the weathered oils.  Several sets of microcosms containing soil and groundwater were set up.   Along with nutrients and an oxygen source, a biodegradable surfactant was added to some of the sets in order to determine whether increasing the bioavailability of the hydrocarbons would enhance their degradation.

After the microcosms were set up, there was an initial increase in hydrocarbons in the aqueous phase and a decrease in hydrocarbons in the soil phase in the microcosms that had received surfactant.  This change was due the surfactant solubilizing hydrocarbons out of the soil and into the water.  As the experiment progressed, however, a decline in hydrocarbon levels in both soil and groundwater was observed.

It was determined that by manipulating the conditions and solubilizing the hydrocarbons, biodegradation of weathered oils could be stimulated.   Hydrocarbons could be removed from soils that had contained these weathered oils for many years without any appreciable degradation.     

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