Curtis A. Lajoie

Bioluminescent reporter bacterium for toxicity monitoring in biological wastewater treatment systems

Toxic shock due to certain chemical loads in biological wastewater treatment systems can result in death of microorganisms and loss of floc structure. To overcome the limitations of existing approaches to toxicity monitoring, genes encoding enzymes for light production were inserted to a bacterium (Shk 1) isolated from activated sludge. The Shk I bioreporter indicated a toxic response to concentrations of cadmium (0.01 to 10,000 mg/L), 2,4-dinitrophenol (0.01 to 1,000 mg/L), and hydroquinone (0.01 to 10,000 mg/L) by reductions in initial levels of bioluminescence on exposure to the toxicant. The decrease in bioluminescence was more severe with increasing toxicant concentration. Bioluminescence did not decrease in response to ethanol concentrations up to 10,000 mg/L or to pH conditions between 6.1 and 7.9. A continuous toxicity monitoring system using this bioreporter was developed for influent wastewater and tested with hydroquinone. The reporter exhibited a rapid and proportional decrease in bioluminescence in response to increasing hydroquinone concentrations.

Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors

Degradation of polychlorinated biphenyls (PCBs) in the environment is limited by their aqueous solubility and the degradative competence of indigenous populations. Field application vectors (FAVs) have been developed in which surfactants are used to both increase the solubility of the PCBs and support the growth of surfactant-degrading strains engineered for PCB degradation. Surfactant and PCB degradation by two recombinant strains were investigated. Pseudomonas putida IPL5 utilizes both alkylethoxylate [polyoxyethylene 10 lauryl ether (POL)] and alkylphenolethoxylate [Igepal CO-720 (IGP)] surfactants as growth substrates, but only degrades the ethoxylate moiety. The resulting degradation products from the alkyl- and alkylphenolethoxylate surfactants were 2-(dodecyloxy)ethanol and nonylphenoldiethoxylates, respectively. Ralstonia eutropha B30P4 grows on alkylethoxylate surfactants without the appearance of solvent-extractable degradation products. It also degrades the 2-(dodecyloxy)ethanol produced by strain IPL5 from the alkylethoxylate surfactants. The extent of degradation of the alkylethoxylate surfactant (POL) was greater for strain IPL5 (90%) than for B30P4 (60%) as determined by the cobaltothiocyanate active substances method (CTAS). The recombinant strain B30P4::TnPCB grew on biphenyl. In contrast, the recombinant strain IPL5::TnPCB could not grow on biphenyl, and PCB degradation was inhibited in the presence of biphenyl. The most extensive surfactant and PCB degradation was achieved by the use of both recombinant strains together in the absence of biphenyl. PCB (Aroclor 1242) and surfactant (POL) concentrations were reduced from 25 ppm and 2000 ppm, respectively, to 6.5 ppm and 225 ppm, without the accumulation of surfactant degradation products. Given the inherent complexity of commercial surfactant preparations, the use of recombinant consortia to achieve extensive surfactant and PCB degradation appears to be an environmentally acceptable and effective PCB remediation option.

Molecular site assessment and process monitoring in bioremediation and natural attenuation

A variety of modern biotechnical approaches are available to assist in optimizing and controlling bioremediation processes. These approaches are broad-ranging, and may include genetic engineering to improve biodegradative performance, maintenance of the environment, and process monitoring and control. In addition to direct genetic engineering strategies, molecular diagnostic and monitoring technology using DNA gene probing methods and new quantitative mRNA analytical procedures allows direct analysis of degradative capacity, activity, and response underin situ conditions. Applications of these molecular approaches in process developments for trichloroethylene (TCE), polychlorinated biphenyls (PCB), and polynuclear aromatic hydrocarbons (PAH) bio-oxidation in soils, aquifer sediments, and ground-water treatment reactors have been demonstrated. Molecular genetic technologies permit not only the development of new processes for bioremediation, but also new process monitoring, control strategies, and molecular optimization paradigms that take full advantage of vast and diverse abilities of microorganisms to destroy problem chemicals.

Molecular diagnostics and chemical analysis for assessing biodegradation of polychlorinated biphenyls in contaminated soils

SummaryThe microbial populations in PCB-contaminated electric power substation capacitor bank soil (TVA soil) and from another PCB-contaminated site (New England soil) were compared to determine their potential to degrade PCB. Known biphenyl operon genes were used as gene probes in colony hybridizations and in dot blots of DNA extracted from the soil to monitor the presence of PCB-degrading organisms in the soils. The microbial populations in the two soils differed in that the population in New England soil was enriched by the addition of 1000 p.p.m. 2-chlorobiphenyl (2-CB) whereas the population in the TVA capacitor bank soil was not affected. PCB degradative activity in the New England soil was indicated by a 50% PCB disappearance (gas chromatography), accumulation of chlorobenzoates (HPLC), and14CO2 evolution from14C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to thebph gene probes, their ability to produce the yellowmeta-cleavage product from 2,3-dihydroxybiphenyl (2,3-DHB), and the degradation of specific PCB congeners by individual isolates in resting cell assays. Although the TVA capacitor bank soil lacked effective PCB-degrading populations, addition of a PCB-degrading organism and 10 000 p.p.m. biphenyl resulted in a >50% reduction of PCB levels. Molecular characterization of soil microbial populations in laboratory scale treatments is expected to be valuable in the design of process monitoring and performance verification approaches for full scale bioremediation.

An Integrated Surfactant Solubilization and PCB Bioremediation Process for Soils

Two decades after the manufacture and use of polychlorinated biphenyls (PCBs) were banned, PCB contamination remains widespread in the environment. Technologies available for PCB remediation are limited and often impractical for soils with dispersed PCB contamination. In this study, two remediation processes have been integrated for use on PCB-contaminated soils. This remediation strategy links in situ surfactant washing of PCBs from soil with aerobic biodegradation of the resulting surfactant-PCB solution by two field application vectors (F A Vs), Pseudomonas putida IFL5::TnPCB and Ralstonia eutropha B30F4::TnPCB, which utilize surfac-tants as growth substrates and cometabolize PCBs. A bench-scale demonstration of this process was performed using PCB-contaminated soils from an electric power substation site. In a 2-day recycling wash using a 1% (wt/vol) surfactant solution, greater than 70% of the PCBs were removed from the soil. In the biodegradation phase, greater than 90% of the surfactant and 35% of ...

Molecular diagnostics for polychlorinated biphenyl degradation in contaminated soils.

Molecular diagnostic methods using DNA hybridization with specific gene probes are being developed for the monitoring of microbial populations capable of polychlorinated biphenyl (PCB) degradation in contaminated soils. Evaluation of composite samples from contaminated electrical substation soil by gas chromatography (GC) indicated that the PCBs present in the soil (approximately 200 ppm) resulted from contamination with Aroclor 1248. The PCBs have been weathered or degraded so that the lower molecular weight PCB congeners are no longer present. Microbiological and molecular site characterizations are in progress to determine the abundance of PCB degradative organisms and catabolic genes present. Cloned DNA fragments for the bphC gene (2,3-dihydroxybiphenyl dioxygenase) from the biphenyl/chlorobiphenyl degradative pathways of different organisms were used as gene probes to identify indigenous microorganisms with bphC gene sequences. In colony hybridization experiments, positive signals with the pDA251 gene probe were detected in cultures from both contaminated and uncontaminated soils. The degradative abilities of indigenous microorganisms and an added PCB-degradative bacterial strain were also monitored with [14C]4-chlorobiphenyl mineralization assays and gas chromatography of PCB residues extracted from the soils. Enrichment of the contaminated soil with biphenyl and chlorobiphenyls did not stimulate the indigenous microorganisms to degrade the soil PCB. Nevertheless, enrichment of the contaminated soil with biphenyl and chlorobiphenyl and addition of the PCB-degrading strain Alcaligenes eutrophus GG4202 did result in additional degradation of the soil PCB. The results obtained from these experiments should assist in developing and monitoring a remediation plan for these PCB-contaminated soils.

Molecular Analysis and Control of Activated Sludge

Activated sludge is a common biological treatment method for both industrial and domestic wastewaters. It is often used for the treatment of waste streams that contain readily biodegradable substances that otherwise would exert a significant oxygen demand on receiving waters. Activated sludge is also used for the treatment of waste streams that contain potential environmental contaminants that are toxic to human or aquatic life.

An Integrated Treatment System for Polychlorinated Biphenyls Remediation

Bioremediation is an environmental biotechnology with promise for promoting a sustainable environment. Bioremediation makes use of natural processes and applies the metabolic properties of microorganisms for transforming contaminants to forms that are harmless in the environment. The added capability of biotechnology for tailoring microbial processes to specific problems expands the potential of bioremediation for encouraging a sustainable environment. The process for the biotransformation of polychlorinated biphenyls (PCB) described in this paper is a good example of the enhancement of bioremediation through the tools of biotechnology.