Bioremediation

Can J Microbiol, 1999 Jul, 45(7), 623 - 6
Sphingomonas sp . strain Lep1: an aerobic degrader of 4-methylquinoline; Pfaller SL et al.; Strain Lep1, isolated from a bacterial consortium capable of aerobic degradation of 4-methylquinoline (4-MQ), was chosen for further characterization as it was the only member of the consortium able to grow on 4-MQ in pure culture . Lep1 was identified as a Sphingomonas sp . based on phylogenetic analysis of 16S rDNA . Furthermore, the presence of sphingolipids and 2-hydroxy fatty acids in the membrane, and a 63% G + C ratio supports the placement of Lep1 in this genus . Additional genetic, physiological, and ecological characterization of bacteria such as Lep1 will allow for the potential exploitation of degradative strains for purposes of bioremediation of contaminated soils.

Biodegradation, 1999 Jun, 10(3), 201 - 17
Intrinsic bioremediation of a petroleum hydrocarbon-contaminated aquifer and assessment of mineralization based on stable carbon isotopes; Bolliger C et al.; This study presents a stepwise concept to assess the in situ microbial mineralization of petroleum hydrocarbons (PHC) in aquifers . A new graphical method based on stable carbon isotope ratios (delta 13C) was developed to verify the origin of dissolved inorganic carbon (DIC) . The concept and the isotope method were applied to an aquifer in Student, Switzerland, in which more than 34,000 liters of heating oil were accidentally released . Chemical analyses of ground water revealed that in this aquifer locally, anaerobic conditions prevailed, and that PHC mineralization was linked to the consumption of oxidants such as O2, NO3-, and SO4(2-) and the production of reduced species such as Fe2+, Mn2+, H2S and CH4 . However, alkalinity and DIC balances showed a quantitative disagreement in the link between oxidant consumption and DIC production, indicating that chemical data alone may not be a reliable assessment tool . delta 13C ratios in DIC have been used before for bioremediation assessment, but results were reported to be negatively influenced by methanogenesis . Using the new graphical method to display delta 13C data, it was possible to identify anomalies found in methanogenic monitoring wells . It could be shown that 88% of the DIC produced in the contaminated aquifer originated from microbial PHC mineralization . Thus, the new graphical method to display delta 13C ratios appears to be a useful tool for the assessment of microbial hydrocarbon mineralization in a complex environment.

Mol Microbiol, 1999 Jun, 32(5), 907 - 12
Microbial genomes; Pallen MJ; Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry . It is hard for the traditional bacteriologist to grasp the scale and pace of the venture . Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced . Indeed, we will often have more than one sequence from a species or genus--for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus . However, genome sequencing risks becoming expensive molecular stamp-collecting without the tools to mine the data and fuel hypothesis-driven laboratory-based research . Bioinformatics, twinned with the new experimental approaches forming functional genomics', provides some of the needed tools . Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings . Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.

Plasmid, 1999 Sep, 42(2), 73 - 91
Genetic exchange between bacteria in the environment; Davison J; Nucleotide sequence analysis, and more recently whole genome analysis, shows that bacterial evolution has often proceeded by horizontal gene flow between different species and genera . In bacteria, gene transfer takes place by transformation, transduction, or conjugation and this review examines the roles of these gene transfer processes, between different bacteria, in a wide variety of ecological niches in the natural environment . This knowledge is necessary for our understanding of plasmid evolution and ecology, as well as for risk assessment . The rise and spread of multiple antibiotic resistance plasmids in medically important bacteria are consequences of intergeneric gene transfer coupled to the selective pressures posed by the increasing use and misuse of antibiotics in medicine and animal feedstuffs . Similarly, the evolution of degradative plasmids is a response to the increasing presence of xenobiotic pollutants in soil and water . Finally, our understanding of the role of horizontal gene transfer in the environment is essential for the evaluation of the possible consequences of the deliberate environmental release of natural or recombinant bacteria for agricultural and bioremediation purposes .

Biotechnol Bioeng, 1999 Oct 5, 65(1), 100 - 7
Cometabolism of chlorinated solvents and binary chlorinated solvent mixtures using M . trichosporium OB3b PP358; Aziz CE et al.; The mutant methanotroph, Methylosinus trichosporium OB3b PP358, which constitutively expresses soluble methane monooxygenase (sMMO), was used to study the degradation kinetics of individual chlorinated solvents and binary solvent mixtures . Although sMMO's broad specificity permits a wide range of chlorinated solvents to be degraded, it creates the potential for competitive inhibition of degradation rates in mixtures because multiple chemicals are simultaneously available to the enzyme . To effectively design both ex-situ and in-situ groundwater bioremediation systems using strain PP358, kinetic parameters for chlorinated solvent degradation and accurate kinetic expressions to account for inhibition in mixtures are required . Toward this end, the degradation parameters for six prevalent chlorinated solvents and the verification of enzyme competition model for binary mixtures were the focus of this investigation . M . trichosporium OB3b PP358 degraded trichloroethylene (TCE), chloroform, cis-1,2-dichloroethylene (c-DCE), trans-1,2-dichloroethylene (t-DCE), and 1, 1-dichloroethylene (1,1-DCE) rapidly, with maximum substrate transformation rates of >20.8, 3.1, 9.5 24.8, and >7.5 mg/mg-day, respectively . 1,1,1-trichloroethane (TCA) was not significantly degraded . Half-saturation coefficients ranged from 1 to greater than 10 mg/L . Competition experiments were carried out to observe the effect of a second solvent on degradation rates and to verify the applicability of the Monod model adjusted for competitive inhibition . Binary mixtures of 0.3->0.5 mg/L TCE with up to 5 mg/L c-DCE and up to 7 mg/L 1,1,1-TCA were studied with 20 mM of formate and no growth substrate . No competition was observed at any of these concentrations . Additional competition experiments, using binary mixtures of t-DCE with TCE and t-DCE with c-DCE, were conducted at higher concentrations (i.e., 7-18 mg/L) and enzyme competition was observed . Predictions from a competitive inhibition model compared well with experimental data for these mixtures .

Appl Environ Microbiol, 1999 Aug, 65(8), 3354 - 9
Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(pPOB) and two modified Pseudomonas strains; Halden RU et al.; Pseudomonas pseudoalcaligenes POB310(pPOB) and Pseudomonas sp . strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium . Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB . Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB . Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%) . Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10(6) to 10(8) CFU/g (dry weight) of soil . Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant . Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil . This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria . In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to Pseudomonas sp . strain B13 occurred at frequencies of 5 x 10(-7) and 10(-1) transconjugant per donor, respectively . Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil . Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.

Appl Microbiol Biotechnol, 1999 Jun, 51(6), 877 - 82
Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria; Newcombe DA et al.; Bioaugmentation has previously been unreliable for the in situ clean-up of contaminated soils because of problems with poor survival and the rapid decline in activity of the bacterial inoculum . In an attempt to solve these problems, a 500-l batch fermenter was investigated for its ability to deliver inoculum repeatedly to contaminated soils via irrigation lines . In a field experiment, mesocosms were filled with 350 kg soil containing 100 mg kg-1 atrazine, and inoculated one, four or eight times with an atrazine-degrading bacterial consortium that was produced in the fermenter . After 12 weeks, no significant degradation of atrazine had occurred in soil that was inoculated only once; whereas, mesocosms inoculated four and eight times mineralized 38% and 72% of the atrazine respectively . Similar results were obtained in a laboratory experiment using soil contaminated with 100 mg kg-1 {14C}atrazine . After 35 days, soil that was inoculated once with 10(8) cfu ml-1 of the consortium or with the atrazine-degrading bacterium, Pseudomonas sp . strain ADP, mineralized 17% and 35% of the atrazine respectively . In comparison, microcosms inoculated every 3 days with the consortium or with Pseudomonas sp . (ADP) mineralized 64% or 90% of the atrazine over this same period . Results of these experiments suggest that repeated inoculation from an automated fermenter may provide a strategy for bioaugmentation of contaminated soil with xenobiotic-degrading bacteria.

Crit Rev Microbiol, 1999, 25(2), 85 - 119
Degradation of pesticides by actinomycetes; De Schrijver A et al.; Actinomycetes have considerable potential for the biotransformation and biodegradation of pesticides . Members of this group of Gram-positive bacteria have been found to degrade pesticides with widely different chemical structures, including organochlorines, s-triazines, triazinones, carbamates, organophosphates, organophosphonates, acetanilides, and sulfonylureas . A limited number of these xenobiotic pesticides can be mineralized by single isolates, but often consortia of bacteria are required for complete degradation . Cometabolism of pesticides is frequently observed within this group of bacteria . When compared with pesticide degradation by Gram-negative bacteria, much less information about molecular mechanisms involved in biotransformations of pesticides by actinomycetes is available . Progress in this area has been seriously hampered by a lack of suitable molecular genetic tools for most representatives of this major group of soil bacteria . Overcoming this constraint would enable a better exploitation of the biodegradation and biotransformation abilities of actinomycetes for applications such as bioremediation and construction of transgenic herbicide-resistant crops.

Poult Sci, 1999 Jul, 78(7), 956 - 64
The use of poultry litter as co-substrate and source of inorganic nutrients and microorganisms for the ex situ biodegradation of petroleum compounds; Williams CM et al.; The purpose of this investigation was to determine the feasibility of utilizing poultry litter as a source of microorganisms, C co-substrate, N, and P to enhance the biodegradation of petroleum compounds in contaminated soil . An initial laboratory-scale study utilized soil contaminated with approximately 3,000 mg/kg (ppm) total petroleum hydrocarbons (TPHC) as diesel fuel . Biotreatment units, each containing 10 L of contaminated soil, were supplemented (0, 1, 10, and 20%, total weight basis) with broiler litter containing 3.65% N and 1.89% P . Petroleum-degrading microorganisms previously enriched from broiler and turkey litter were also inoculated into the litter-treated units . A significant first order rate of TPHC biodegradation was measured for all treatment units containing broiler litter (P < 0.05) . Based on these results, a subsequent study was conducted at the site of a commercial facility permitted to treat soil (ex situ) contaminated with hazardous compounds . Soil treatment plots, each containing approximately 1 ton of soil contaminated with approximately 1,200 to 1,600 mg/kg diesel fuel were established . Each plot was replicated four times and the experiment was conducted for 35 d . Treatments were as follows: control, soil only; soil + commercial blend of bioremediation nutrients; soil + commercial fertilizer; soil + poultry litter (1% by volume); soil + poultry litter (10% by volume); soil + pelleted poultry litter (10% by volume) . The results showed that the remediation of soil contaminated with petroleum compounds is significantly (P < 0.05) enhanced when supplemented with poultry litter (pelleted or nonpelleted) in concentrations of 10% soil volume . These results demonstrate the potential for a specialized market for the use of poultry litter.

Appl Microbiol Biotechnol, 1999 May, 51(5), 682 - 5
Fermented whey--an inexpensive feed source for a laboratory-scale selenium-bioremediation reactor system inoculated with Thauera selenatis; Bledsoe TL et al.; It is critical that an inexpensive electrondonor/carbon-source be found for selenium bioremediation using the selenate-respiring bacterium, Thauera selenatis . Since acetate is a preferred substrate for growth of this organism, a method was developed for fermenting the lactose in whey to large amounts of acetate . Indigenous whey microorganisms fermented the whey lactose in this manner when grown in continuous culture at a very slow dilution rate (D = 0.05 h-1) . The successful use of the fermented whey lactose as the carbon-source/electron-donor feed for a laboratory-scale selenium-bioremediation reactor system, inoculated with T . selenatis, treating selenium-contaminated drainage water was also demonstrated . Selenium oxyanions and nitrate were reduced by 98%.

Microb Ecol, 1999 Jul, 38(1), 69 - 78
Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia; Piehler MF et al.; > Abstract Successful stimulation of N2 fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution . This study explored the biodegradation of petroleum pollution by indigenous N2 fixing marine microbial consortia . Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N . It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N2 fixing consortia and enhance biodegradation of petroleum . Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia . Structure and function of the microbial consortia were determined by measurement of N2 fixation (acetylene reduction), hydrocarbon biodegradation (14C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (3H-thymidine incorporation) . Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC . Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N2 fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity . Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition . These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N2 fixing microbial consortia to degrade petroleum hydrocarbons.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p69.html

Curr Opin Biotechnol, 1999 Jun, 10(3), 234 - 9
Bioremediation of petroleum hydrocarbon contaminants in marine habitats; Head IM et al.; Bioremediation is being increasingly seen as an effective, environmentally benign treatment for shorelines contaminated as a result of marine oil spills . Despite a relatively long history of research on oil-spill bioremediation, it remains an essentially empirical technology and many of the factors that control bioremediation have yet to be adequately understood . Nutrient amendment is a widely accepted practice in oil-spill bioremediation but there is scant understanding of the systematic effects of nutrient amendment on biodegradative microbial populations or the progress of bioremediation . Recent laboratory and field research suggests that resource-ratio theory may provide a theoretical framework that explains the effects of nutrient amendment on indigenous microbial populations . In particular, the theory has been invoked to explain recent observations that nutrient levels, and their relative concentration, influence the composition of hydrocarbon-degrading microbial populations . This in turn influences the biodegradation rate of aliphatic and aromatic hydrocarbons . If such results are confirmed in the field, then it may be possible to use this theoretical framework to select bioremediation treatments that specifically encourage the rapid destruction of the most toxic components of complex pollutant mixtures.

Trends Biotechnol, 1999 Jun, 17(6), 219 - 25
Sustainable development: how can biotechnology contribute?
Zechendorf B.
Sustainable development has become a priority for the world's policy makers . Among the broad range of technologies with the potential to reach the goal of sustainability, biotechnology could take an important place, especially in the fields of food production, renewable raw materials and energy, pollution prevention, and bioremediation . However, technical and economic problems still need to be solved . In some cases, the environmental impact of biotechnological applications has been misjudged; in other cases, expectations cannot yet be matched.

Appl Biochem Biotechnol, 1999 Feb, 76(2), 79 - 97
Metabolite formation and toxicity measurements in evaluating bioremediation of a jet-fuel-contaminated aquifer; Long SC et al.; The metabolic capabilities of subsurface, jet-fuel-contaminated, aquifer microbial communities were characterized using an ecological approach to biotreatment assessment . A multifaceted experimental design was used that incorporated quantification of metabolite formation and toxicity screening along with the typical microbial activity measurements and pollutant degradation measurements used for assessing bioremediation potential . For select experiments, dissolved oxygen levels and pH in microcosm systems were also monitored . Results suggest that a sizable, metabolically active microbial community exists in both contaminated and uncontaminated areas of the study site . Time course metabolism analyses indicated that the microbial communities were capable of degrading all three test compounds (amino acids, decane, and toluene) without any apparent adaptation period . Measurements of mineralization, cellular uptake, and metabolite formation indicated that metabolite formation was the predominant fate of the target pollutants in the microcosms . The results of toxicity screening time courses indicated that under oxygen-limiting conditions, the potential for the accumulation of toxic, acidic metabolites that could adversely affect the rates and extent of bioremediation existed . The experimental results indicate that the microbial communities at the site possess the metabolic potential for in situ biodegradation of the jet fuel . Care must be taken in the design of an in situ biodegradation treatment system (for this site and perhaps other petroleum-contaminated aquifers) to avoid the development of microaerophilic or oxygen-depleted zones, which could result in possible self-poisoning owing to acidic metabolite accumulation.

Lett Appl Microbiol, 1999 May, 28(5), 350 - 4
Bacterial surface display of an anti-pollutant antibody fragment; Dhillon JK et al.; A peptidoglycan-associated lipoprotein (PAL) fused to an antibody fragment (scFv) specific to the herbicide and environmental pollutant atrazine, has been successfully targeted to the cell surface of Escherichia coli . Anti-atrazine binding could be observed via an atrazine-alkaline phosphatase conjugate . Cells containing the PAL fusion grew with little cellular toxicity when compared with the control . In contrast, expression of anti-atrazine antibody fragments alone caused the cells to lyse after 4 h . The surface display of anti-pollutant antibodies may have a future role in the bioremediation of contaminated water or the development of pollutant-specific, whole-cell biosensors.

J Hazard Mater, 1999 Mar 19, 65(3), 305 - 15
Bioremediation of phenols and polycyclic aromatic hydrocarbons in creosote contaminated soil using ex-situ landtreatment; Guerin TF; Soil from a former creosoting plant containing phenols and polycyclic aromatic hydrocarbons, was remediated using an ex-situ landtreatment process . Total 16 USEPA priority PAH and total phenol were reduced from 290 mg/kg and 40 mg/kg to < 200 mg/kg and 2 mg/kg, respectively . The bioremediation process involved soil mixing, aeration, and slow release fertilizer addition . The indigenous populations of PAH and phenol utilizing populations of microorganisms were shown to increase during the treatment process, indicating that biostimulation was effective . The most extensive degradation was apparent with the 2- and 3-ring PAH, with decreases of 97% and 82%, respectively . The higher molecular weight 3- and 4-ring PAH were degraded at slower rates, with reductions of 45% and 51%, respectively . Six-ring PAH were degraded the least with average reductions of < 35% . The residual concentrations of PAH and total phenol obtained in the study allowed the treated soil to be disposed of as low level contaminated landfill.

Nat Biotechnol, 1999 May, 17(5), 491 - 4
Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase; French CE et al.; Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains . Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems . To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives . Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds . Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings . This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil.

Trends Biotechnol, 1999 May, 17(5), 200 - 4
Bacteria designed for bioremediation; Timmis KN et al.; Although many environmental pollutants are efficiently degraded by microorganisms, others persist and constitute a severe health hazard . In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms . Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.

Appl Environ Microbiol, 1999 May, 65(5), 2103 - 11
Lignin-modifying enzymes of flavodon flavus, a basidiomycete isolated from a coastal marine environment
Raghukumar C, D'Souza TM, Thorn RG, Reddy CA.
A basidiomycetous fungus Flavodon flavus (Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of 14C-labeled synthetic lignin to 14CO2 in 24 days . When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase . Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen . In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal . LME production was also seen in media prepared with artificial seawater . Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3) . The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase . F . flavus showed effective degradation of various dye pollutants in media prepared with or without artificial seawater . This is the first report on the production of all three major classes of LMEs by F . flavus and points to the bioremediation potential of this organism in terrestrial as well as marine environments.

Appl Microbiol Biotechnol, 1999 Mar, 51(3), 375 - 81
Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3; Gebendinger N et al.; A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities . The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts . Cyanazine competitively inhibited atrazine degradation . The maximum atrazine degradation rate (Vmax) was 41 times higher and the half-saturation constant for the inhibitor (Ki) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines . Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity . Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate . These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers . Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants.

Chemosphere, 1999 May, 38(12), 2897 - 912
Biodegradation of low aqueous concentration pentachlorophenol (PCP) contaminated groundwater; Schmidt LM et al.; Bioremedial treatment to remove low level organic contamination to regulatory standards has met with limited success . In this study source water from a contaminated surficial aquifer at a former wood treatment facility was used to evaluate the potential for indigenous microorganisms to degrade low level (< 1.0 mg) pentachlorophenol (PCP) to a regulatory drinking water standard of 0.001 mg/L . PCP degradation was evaluated in series of batch reactors in a two phase study to (a) determine the rate and extent of PCP removal and (b) evaluate the impact of nutrient amendment (N and P) on removal rate . All reactors with the exception of the abiotic control demonstrated PCP removal to a level < 0.002 mg/L within a maximum period of 32 d with and without nutrient amendment . A regression analysis of reactive phosphate (ortho-P) concentration versus removal rate produced an R2 of 0.94 (p = 0.006) indicating a significant correlation between the level of available phosphate and PCP degradation rate . Selective bacterial enumeration (for PCP degrading bacteria) revealed PCP-degrading bacteria increased in abundance prior to and in conjunction with the degradation phase to a density of between 10(3) to 10(4) CFU/ml . Isolates were also analyzed for total fatty acids using Fatty Acid Methyl Ester (FAME) methodology and the results indicated that PCP degrading bacteria were present in the aquifer and consisted of predominately fluorescent, oxidase positive Pseudomonas species . Overall, data indicate that autochthonous microbes are capable of removing low level PCP (< 1.0 mg/L) to approach if not reach the regulatory standard of 0.001 mg/L with the addition of oxygen, with or without nutrient amendment . Results of this research can be applied to full-scale implementation of in-situ or ex-situ bioremediation of groundwater at former wood treatment facilities.

Curr Opin Biotechnol, 1999 Apr, 10(2), 137 - 41
Engineering of improved microbes and enzymes for bioremediation; Chen W et al.; Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants . Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation . A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes . The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.

Biodegradation, 1998, 9(5), 319 - 26
Intrinsic capacities of soil microflorae for gasoline degradation; Solano-Serena F et al.; A methodology to determine the intrinsic capacities of a microflora to degrade gasoline was developed, in particular for assessing the potential of autochtonous populations of polluted and non polluted soils for natural attenuation and engineered bioremediation . A model mixture (GM23) constituted of the 23 most representative hydrocarbons of a commercial gasoline was used . The capacities of the microflorae (kinetics and extent of biodegradation) were assessed by chromatographic analysis of hydrocarbon consumption and of CO2 production . The degradation of the components of GM23 was assayed in separate incubations of each component and in the complete mixture . For the microflora of an unpolluted spruce forest soil, all hydrocarbons of GM23 except cyclohexane, 2,2,4- and 2,3,4-trimethylpentane isomers were degraded to below detection limit in 28 days . This microflora was reinforced with two mixed microbial communities selected from gasoline-polluted sites and shown to degrade cyclohexane and 2,2,4-trimethylpentane . With the reinforced microflora, complete degradation of GM23 was observed . The degradation patterns of individual components of GM23 were similar when the compounds were present individually or in the GM23 mixture, as long as the concentrations of 2-ethyltoluene and trimethylbenzene isomers were kept sufficiently low (< or = 35 mg.l-1) to remain below their inhibitory level.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 231 - 9
Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants; Keasling JD et al.; Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municipal wastewater, and may play a key role in heavy metal tolerance and bioremediation . However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity . We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters . Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals . Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals . Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle .

Biotechnol Bioeng, 1999 Apr 5, 63(1), 56 - 68
Trichloroethene degradation in a two-step system by methylosinus trichosporium OB3b . Optimization of system performance: use of formate and methane; Sipkema EM et al.; The breakdown of dissolved TCE in a two-step bioremediation system is described . In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE . A special design allowed both for the addition of air (uG,s = 0.01-0 . 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor . In two-step systems presented thus far, only formate was used (excess, 20 mM) . We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results . Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate . A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition . Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system . The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application . The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor .

Biotechnol Bioeng, 1998 May 20, 58(4), 345 - 55
Cyclodextrin effects on the ex-situ bioremediation of a chronically polychlorobiphenyl-contaminated soil
Fava F, Di Gioia D, Marchetti L.
The possibility of enhancing the intrinsic ex-situ bioremediation of a chronically polychlorinated biphenyl-contaminated soil by using cyclodextrins was studied in this work . The soil, contaminated with a large array of polychlorinated biphenyls and deriving from a dump site where it has been stored for about 10 years, was found to contain indigenous cultivable aerobic bacteria capable of utilising biphenyl and chlorobenzoic acids . The soil was amended with inorganic nutrients and biphenyl, saturated with water, and treated in aerobic batch slurry- and fixed-phase reactors . Hydroxypropyl-beta-cyclodextrin and gamma-cyclodextrin, added to both reactor systems at the concentration of 10 g/L at the 39th and 100th days of treatment, were found to generally enhance the depletion rate and extent of the soil polychlorobiphenyls . Despite some abiotic losses could have affected the depletion data, experimental evidence, such as the production of metabolites tentatively characterized as chlorobenzoic acids and chloride ion accumulation in the reactors, indicated that cyclodextrins significantly enhanced the biological degradation of the soil polychlorobiphenyls . This result has been ascribed to the capability of cyclodextrins of enhancing the availability of polychlorobiphenyls in the hydrophilic soil environment populated by immobilised and suspended indigenous soil microorganisms . Both cyclodextrins were metabolised by the indigenous soil microorganisms at the concentration at which they were used . Therefore, cyclodextrins, both for their capability of enhancing the biodegradation of soil polychlorobiphenyls and for their biodegradability, can have the potential of being successfully used in the bioremediation of chronically polychlorinated biphenyl-contaminated soils .

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 125 - 30
Induction and enhancement of stress proteins in a trichloroethylene-degrading methanotrophic bacterium, Methylocystis sp . M; Uchiyama H et al.; The responses of the trichloroethylene-degrading bacterium Methylocystis sp . M to six different water-pollutants, carbon starvation, and temperature-shock (heat and cold) were examined using 2-dimensional gel electrophoresis . Twenty-eight polypeptides were induced, and these stress-induced proteins were classified into three groups . Some of the chemically induced proteins were the same as those induced by carbon starvation and temperature-shock . Two of the polypeptides were induced by trichloroethylene . Trichloroethylene-stress protein synthesis required 1-2 h at a concentration of trichloroethylene that had no effect on growth . Furthermore, 25 stress-enhanced polypeptides were observed, and one of these was enhanced by trichloroethylene . Based on these results, we discuss applications of chemical-stress induction of proteins to establish effective bioremediation and bioassay by methanotrophs.

Appl Microbiol Biotechnol, 1999 Jan, 51(1), 1 - 12
Bacterial catabolic transposons; Tan HM; The introduction of foreign organic hydrocarbons into the environment in recent years, as in the widespread use of antibiotics, has resulted in the evolution of novel adaptive mechanisms by bacteria for the biodegradation of the organic pollutants . Plasmids have been implicated in the catabolism of many of these complex xenobiotics . The catabolic genes are prone to undergo genetic rearrangement and this is due to their presence on transposons or their association with transposable elements . Most of the catabolic transposons have structural features of the class I (composite) elements . These include transposons for chlorobenzoate (Tn5271), chlorobenzene (Tn5280), the newly discovered benzene catabolic transposon (Tn5542), and transposons encoding halogenated alkanoates and nylon-oligomer-degradative genes . Transposons for the catabolism of toluene (Tn4651, Tn4653, Tn4656) and naphthalene (Tn4655) belong to class II (Tn3 family) elements . Many catabolic genes have been associated with insertion sequences, which suggests that these gene clusters could be rapidly disseminated among the bacterial populations . This greatly expands the substrate range of the microorganisms in the environment and aids the evolution of new and novel degradative pathways . This enhanced metabolic versatility can be exploited for and is believed to play a major part in the bioremediation of polluted environments.

J Recept Signal Transduct Res, 1999 Jan-Jul, 19(1-4), 703 - 15
Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein; Kotrba P et al.; The lamB gene was inserted at with DNA fragments encoding N-terminal beta- and C-terminal alpha-domains of human metallothionein 1A (HMT1A) . The hybrid LamB proteins were expressed as full-length products . Virtually whole pool of hybrid LamB proteins was found localized in the outer membrane of E . coli to and cells expressing LamB variants retained sensitivity to lambda phage, indicating their correct folding . Expression of hybrid LamB proteins increased natural ability of E . coli accumulate bivalent heavy metals ions with the highest efficiency observed for cadmium . The order of amount of cadmium accumulated is alpha-domain of HMT1A > HMT1A >> beta-domain of HMT1A . This correlates with affinity for cadmium and stability of metallothionein and its individual domains . This confirms suitability of LamB vehicle for surface display of various bioactive molecules and suggests possibility of engineering of cell surface for bioremediation of heavy metals.

Appl Environ Microbiol, 1999 Mar, 65(3), 1335 - 9
Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria; Allen CC et al.; The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp . strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation . Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used . The effects of the nonpolar solvent 2,2,4, 4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined . HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates . The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene . Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate . However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions . The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results . The surfactant inhibited growth of the wild-type strain S . yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.

Microb Ecol, 1999 Feb, 37(2), 116 - 128
Moving Waves of Bacterial Populations and Total Organic Carbon along Roots of Wheat; Semenov AM et al.; > Abstract To determine if spatial variation in soluble carbon sources along the root coincides with different trophic groups of bacteria, copiotrophic and oligotrophic bacteria were enumerated from bulk soil and rhizosphere samples at 2 cm intervals along wheat roots 2, 3, and 4 weeks after planting . There was a moderate rhizosphere effect in one experiment with soil rich in fresh plant debris, and a very pronounced rhizosphere effect in the second experiment with soil low in organic matter . We obtained wavelike patterns of both trophic groups of bacteria as well as water-soluble total organic carbon (TOC) along the whole root length (60 or 90 cm) . TOC concentrations were maximal at the root tip and base and minimal in the middle part of the roots . Oscillations in populations of copiotrophic and oligotrophic bacteria had two maxima close to the root tip and at the root base, or three maxima close to the tip, in the middle section, and at the root base . The location and pattern of the waves in bacterial populations changed progressively from week to week and was not consistently correlated with TOC concentrations or the location of lateral root formation . Thus, the traditional view that patterns in bacterial numbers along the root directly reflect patterns in exudation and rhizodeposition from several fixed sources along the root may not be true . We attributed the observed wavelike patterns in bacterial populations to bacterial growth and death cycles (due to autolysis or grazing by predators) . Considering the root tip as a moving nutrient source, temporal oscillations in bacterial populations at any location where the root tip passed would result in moving waves along the root . This change in concept about bacterial populations in the rhizosphere could have significant implications for plant growth promotion and bioremediation.

Appl Environ Microbiol, 1999 Feb, 65(2), 648 - 51
Detection of methanotrophs in groundwater by PCR; Cheng YS et al.; Methanotrophic bacteria have significant potential for bioremediation, which would require methods for monitoring the presence and activity of these organisms in environmental samples . In this study, PCR was used to detect methanotrophic bacteria . Primers were designed on the basis of a partial sequence of pmoA, which encodes one of the proteins of the particulate methane monooxygenase . Specific amplification of a portion of pmoA was obtained with template DNA isolated from lab strains of methanotrophs . A pmoA product was also obtained by using DNA from groundwater . The identity of the PCR product was confirmed by sequencing or by amplification with a nested primer . Reverse transcriptase PCR detected pmoA mRNA.

Annu Rev Microbiol, 1998, 52, 423 - 52
New perspectives on microbial dehalogenation of chlorinated solvents: insights from the field; Lee MD et al.; A variety of microbial dechlorination mechanisms have been demonstrated in laboratory microcosms, pure cultures, and in situ sedimentary environments . New perspectives on in situ processes from these efforts allow the design of more realistic bioremediation strategies that complement natural processes regardless of whether the strategy used is one of engineered accelerated bioremediation or natural attenuation . Since 1994 the scientific community has acquired considerable knowledge regarding natural attenuation of organochlorine compounds . Natural attenuation of chlorinated solvents has been documented at a number of field sites . Reductive dechlorination driven by co-contaminants or naturally occurring organics as substrates in combination with aerobic or co-metabolic degradation contains certain chlorinated solvent plumes . Although natural attenuation is not a panacea, at sites where it is applicable, it offers a scientifically sound, cost-effective method to remediate groundwater contaminated with chlorinated solvents.

Arch Environ Contam Toxicol, 1999 Feb, 36(2), 152 - 7
Soil-based phytotoxicity of 2,4,6-trinitrotoluene (TNT) to terrestrial higher plants
Gong P, Wilke B, Fleischmann S.
Seed germination and early stage seedling growth tests were conducted to determine the ecotoxicological threshold of 2,4, 6-trinitrotoluene (TNT) in two soils of different properties . Soils were amended up to 1,600 mg TNT kg-1 soil and four representative species of higher plants, two dicotyledons (Lepidium sativum L., common name: cress; and Brassica rapa Metzg., turnip) and two monocotyledons (Acena sativa L., oat; and Triticum aestivum L., wheat), were assessed . Cumulative seed germination and fresh shoot biomass were measured as evaluation endpoints . Phytotoxicity of TNT was observed to be affected by soil properties and varied between plant species . Cress and turnip showed higher sensitivity to TNT than did oat and wheat . The lowest observable adverse effect concentration (LOAEC) of TNT derived from this study was 50 mg kg-1 soil . In contrast to high TNT concentrations, low levels of TNT, i.e . , 5-25 mg kg-1 soil for cress and turnip and 25-50 mg kg-1 for oat and wheat, stimulated seedling growth . Oat was capable of tolerating as much as 1,600 mg TNT kg-1 and demonstrated a potential ability of TNT detoxification in one of the soils tested, suggesting that this plant might be useful in the bioremediation of TNT contaminated soils.

Crit Rev Microbiol, 1998, 24(4), 335 - 73
Methanotrophs, Methylosinus trichosporium OB3b, sMMO, and their application to bioremediation; Sullivan JP et al.; One of the most problematic groups of the USEPA and EU priority pollutants are the halogenated organic compounds . These substances have a wide range of industrial applications, such as solvents and cleaners . Inadequate disposal techniques and accidental spillages have led to their detection in soil, groundwater, and river sediments . Persistence of these compounds in the environment has resulted from low levels of biodegradation due to chemical structural features that preclude or retard biological attack . Research has indicated the idea that treatment systems based on methanotrophic co-metabolic transformation may be a cost-effective and efficient alternative to physical methods because of the potential for high transformation rates, the possibility of complete compound degradation without the formation of toxic metabolites, applicability to a broad spectrum of compounds, and the use of a widely available and inexpensive growth substrate . A substantial amount of work concerning methanotrophic cometabolic transformations has been carried out using the soluble form of methane monooxygenase (sMMO) from the obligate methanotroph Methylosinus trichosporium OB3b . This NADH-dependent monooxygenase is derepressed when cells are grown under copper stress . sMMO has a wider specificity than the particulate form . sMMO has been shown to degrade trichloroethylene (TCE) at a rate of at least one order of magnitude faster than obtained with other mixed and pure cultures, suggesting it has a wider application to bioremediation . Furthermore, sMMO catalyzes an unusually wide range of oxidation reactions, including the hydroxylation of alkanes, epoxidation of alkenes, ethers, halogenated methanes, cyclic and aromatic compounds including compounds, that are resistant to degradation in the environment . However, the practical application of methantrophs and Methylosinus trichosporium OB3b to the treatment of chlorinated organics has met with mixed success . Although oxidation rates are rapid, compound oxidation with M . trichosporium OB3b is difficult . This fastidious organism grows relatively slowly, which limits the speed with which sMMO expressing biomass can be generated . Furthermore, product toxicity toward the cell, affecting the stability of the enzyme when transforming certain compounds has been observed, for example, by the products of 1,2,3 trichlorobenzene hydroxylation (2,3,4- and 3,4,5-trichlorophenol) and of TCE degradation (chloral hydrate) . Because of this toxicity and the inability of sMMO to further oxidize its own hydroxylation products, the ability of methane monoxygenase to carry out the monooxygenation of a wide variety of substituted aromatics and polyaromatics cannot be fully exploited in M . trichosporium OB3b . Many of these problems could be overcome by the use of either a mixed downstream heterotrophic population of organisms that could accommodate the products of hydroxylation or to express sMMO in an organism that could metabolize the products of hydroxylation . The latter of these two approaches would have several advantages . The main benefit would be the removal of the need for methane, which is required to induce sMMO in M . trichosporium OB3b, and supply carbon and energy to the cells that continuously oxidise the target compound, but also acts as a competitive inhibitor of sMMO . Instead, the recombinant could utilize the products of sMMO-mediated hydroxylation as a carbon source.

Appl Environ Microbiol, 1999 Jan, 65(1), 264 - 9
Pleiotropic effects of adaptation to a single carbon source for growth on alternative substrates
Velicer GJ.
It is frequently assumed that populations of genetically modified microorganisms will perform their intended function and then disappear from the environment due to inherent fitness disadvantages resulting from their genetic alteration . However, modified organisms used in bioremediation can be expected to adapt evolutionarily to growth on the anthropogenic substrate that they are intended to degrade . If such adaptation results in improved competitiveness for alternative, naturally occurring substrates, then this will increase the likelihood that the modified organisms will persist in the environment . In this study, bacteria capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were used to test the effects of evolutionary adaptation to one substrate on fitness during growth on an alternative substrate . Twenty lineages of bacteria were allowed to evolve under abundant resource conditions on either 2,4-D or succinate as their sole carbon source . The competitiveness of each evolved line was then measured relative to that of its ancestor for growth on both substrates . Only three derived lines showed a clear drop in fitness on the alternative substrate after demonstrable adaptation to their selective substrate, while five derived lines showed significant simultaneous increases in fitness on both their selective and alternative substrates . These data demonstrate that adaptation to an anthropogenic substrate can pleiotropically increase competitiveness for an alternative natural substrate and therefore increase the likelihood that a genetically modified organism will persist in the environment.

Appl Environ Microbiol, 1999 Jan, 65(1), 95 - 101
Effect of toxic metals on indigenous soil beta-subgroup proteobacterium ammonia oxidizer community structure and protection against toxicity by inoculated metal-resistant bacteria; Stephen JR et al.; Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide . Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation . In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the Proteobacteria (beta-subgroup ammonia oxidizers) . In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria . Effects on nitrogen cycling were measured by monitoring the NH3 and NH4+ levels in soil samples . The gene encoding the alpha-subunit of ammonia monooxygenase (amoA) was selected as a functional molecular marker for the beta-subgroup ammonia oxidizing bacteria . Community structure comparisons were performed with clone libraries of PCR-amplified fragments of amoA recovered from contaminated and control microcosms for 8 weeks . Analysis was performed by restriction digestion and sequence comparison . The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR . All amoA gene fragments recovered grouped with sequences derived from cultured Nitrosospira . These comprised four novel sequence clusters and a single unique clone . Specific changes in the community structure of beta-subgroup ammonia oxidizers were associated with the addition of metals . These changes were not seen in the presence of the inoculated metal-resistant bacteria . Neither treatment significantly altered the total number of beta-subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls . Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.

Biosens Bioelectron, 1998 Nov 15, 13(11), 1197 - 203
In situ mapping of community-level cellular response with catalytic microbiosensors; Peteu SF et al.; Chemotaxis, the migration of cells in the direction of a spatial chemical gradient, is important in disease progression, microbial ecology, and bioremediation . The ability to map chemoattractant gradients and the corresponding cellular growth and motility patterns is essential to the study of chemotaxis . Microelectrodes and microbiosensors have the potential to measure chemoattractant gradients with high spatial resolution . In this study, Clark-type amperometric microelectrodes and microbiosensors were used to measure solute concentrations gradients generated by a chemotactic band of Escherichia coli in a semi-solid gel . A computerized image analysis system was used to simultaneously measure the cellular concentration profile across the chemotactic band . The experimental results compared favorably with a mathematical model of solute and cell transport in the gel . Scanning electron micrographs (SEM) of micro(bio)sensor tips taken after 6 months of use showed evidence of degradation, including adhesion of foreign particles to the glass body, the adhesion of a small gel capsule to the sensor tip, and separation of the bio-interface from the tip . A needle-type microbiosensor was constructed to better protect the tip and hence increase the ruggedness of the microbiosensors.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 397 - 402
Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli; Wang RF et al.; The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories . Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C . elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C . elegans 6-phosphogluconate dehydrogenase gene . The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing . The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence . The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences . Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences . The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid-E . coli system (pQE30) . The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity . Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer . Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity . The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis . The C . elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree . Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.

J Biotechnol, 1998 Dec 11, 66(2-3), 91 - 9
Perspectives in agrobiotechnology; Tengerdy RP et al.; This review surveys the most important and promising contributions of agricultural biotechnology to the development of sustainable, environment-friendly agriculture . It deals with the recent achievements of genetic technology for the development of new transgenic microbial, plant and animal products . It also deals with the newest developments and perspectives of microbial intervention in agricultural practices, such as biofertilizers, biocontrol agents, and various microbiological products used in modern agriculture . The review surveys the outlook for a waste-free, environment-friendly sustainable agricultural practice, including waste management, recycling and bioremediation technologies . The review lists the most important marketable agrobiotechnological products, and their present and projected sales volume.

Riv Biol, 1998, 91(2), 221 - 32
Biological filtering and ecological machinery for self-purification and bioremediation in aquatic ecosystems: towards a holistic view; Ostroumov SA; According to one of the approaches to the definition of criteria for the phenomenon of life, the key attribute is the ability of the system for some self-regulating and self-supporting . Part of such holistic functions of aquatic ecosystems as self-regulating and self-supporting is their cleaning the water via a multitude of various mechanisms . The goal of this paper is to present some fundamental elements of the theory of ecosystem self-purification which emphasizes the importance of the four functional biological filters that are instrumental in purification and upgrading the quality of water in aquatic ecosystems . These functional filters are: (1) direct water filtering by aquatic organisms that are filter-feeders; (2) the filter (represented mainly by communities of aquatic plants/periphyton) which prevents input of pollutants and biogenic elements (N, P) from land into water bodies; (3) the filter (represented by benthic organisms) which prevents re-entry of pollutants and biogenic elements from the bottom sediments into the water; (4) the filter (represented by microorganisms attached to particles which are suspended in the water) that provides microbiological treatment of water column . New experimental data by the author reveal the role of man-made effects on the ecological machinery which purifies water . The analysis and discussion lead to the holistic theory of the natural process of bioremediation of aquatic ecosystems.

Appl Microbiol Biotechnol, 1998 Oct, 50(4), 489 - 94
Repeated application of carvone-induced bacteria to enhance biodegradation of polychlorinated biphenyls in soil; Gilbert ES et al.; Carvone, the principal component of spearmint oil, induces biodegradation of polychlorinated biphenyls (PCB) by Arthrobacter sp . strain B1B . This study investigated the effectiveness of the repeated application of carvone-induced bacteria for bioremediation of Aroclor-1242-contaminated soil . Control treatments compared a single inoculation of carvone-induced cells, repeated applications of noninduced cells, and repeated applications of cell-free carvone/fructose medium . The results showed that repeated application of carvone-induced bacteria was the most effective treatment for mineralizing PCB, resulting in 27 +/- 6% degradation of Aroclor 1242 after 9 weeks; whereas a single application of cells resulted in no significant degradation . Addition of cell-free, carvone/fructose medium resulted in 10% degradation of PCB, which suggests that this treatment stimulated biodegradation of PCB by the indigenous microflora . The di- and trichlorobiphenyls were the most readily degraded congeners . More highly chlorinated congeners, which had been previously shown to be degraded in liquid culture, were not substantially degraded in soil, indicating that low bioavailability may have limited their degradation . With the development of new technology, which permits automated in situ fermentation and delivery of degrader microorganisms, the repeated application of carvone-induced bacteria may facilitate bioremediation of PCB-contaminated soils.

Plant Physiol, 1998 Nov, 118(3), 1049 - 56
The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics
Robineau T, Batard Y, Nedelkina S, Cabello-Hurtado F, LeRet M, Sorokine O, Didierjean L, Werck-Reichhart D.
Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants . On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin . In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas . CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds . Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation.

Nat Biotechnol, 1998 Oct, 16(10), 929 - 33
Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments; Lange CC et al.; Thousands of waste sites around the world contain mixtures of toxic chlorinated solvents, hydrocarbon solvents, and radionuclides . Because of the inherent danger and expense of cleaning up such wastes by physicochemical methods, other methods are being pursued for cleanup of those sites . One alternative is to engineer radiation-resistant microbes that degrade or transform such wastes to less hazardous mixtures . We describe the construction and characterization of recombinant Deinococcus radiodurans, the most radiation-resistant organism known, expressing toluene dioxygenase (TDO) . Cloning of the tod genes (which encode the multicomponent TDO) into the chromosome of this bacterium imparted to the strain the ability to oxidize toluene, chlorobenzene, 3,4-dichloro-1-butene, and indole . The recombinant strain was capable of growth and functional synthesis of TDO in the highly irradiating environment (60 Gy/h) of a 137Cs irradiator, where 5x10(8)cells/ml degraded 125 nmol/ml of chlorobenzene in 150 min . D . radiodurans strains were also tolerant to the solvent effects of toluene and trichloroethylene at levels exceeding those of many radioactive waste sites . These data support the prospective use of engineered D . radiodurans for bioremediation of mixed wastes containing both radionuclides and organic solvents.

Appl Environ Microbiol, 1998 Oct, 64(10), 3869 - 77
Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation; Dojka MA et al.; A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation . Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing . Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned . A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique . All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced . A total of 104 (94 bacterial and 10 archaeal) sequence types were determined . Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions . The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype . One of these sequence types is associated with the genus Syntrophus; Syntrophus spp . produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen . The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis . We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.

Appl Environ Microbiol, 1998 Sep, 64(9), 3451 - 7
Effect of nitrogen source on growth and trichloroethylene degradation by methane-oxidizing bacteria; Chu KH et al.; The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined . One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study . All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen . Both M . trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-beta-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions . The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources . Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M . trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells . TCE transformation capacities were found to correlate with cellular PHB content in all three cultures . The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.

J Appl Microbiol, 1998 Jul, 85(1), 1 - 8
Isolation of a novel pentachlorophenol-degrading bacterium, Pseudomonas sp . Bu34; Lee SG et al.; A pentachlorophenol (PCP)-degrading bacterium was isolated from possible PCP-contaminated soil from Pusan, Korea and identified as a member of the genus Pseudomonas . It used PCP as its sole source of carbon and energy . This micro-organism was capable of degrading PCP more effectively, certified by the increase in cell density and the decrease in PCP substrate . Pseudomonas sp . Bu34 was able to degrade a much higher concentration of PCP (4000 mg l-1) than any previously reported PCP-degrading bacteria and fungi and to grow in mineral salts solution containing one of a variety of chlorophenols . In non-acclimated strain Bu34, the cell number decreased from 87 to 99.9% in 75-4000 mg l-1 PCP at 24 h . In the acclimated strain the PCP toxic effect did not appear with 75 mg l-1 PCP treatment, but 1000-4000 mg l-1 PCP decreased the cell number of strain Bu34 by 25% to 24 h and then the cell number slightly increased at 48 h . Therefore, it suggested that the maximum resistance of acclimated strain Bu34 to PCP was 4000 mg l-1 PCP . We suggest that strain Bu34 could be used as a micro-organism for the bioremediation of highly PCP-contaminated soils, water or wood products.

FEBS Lett, 1998 Jul 24, 431(3), 343 - 6
Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis; Lamb SB et al.; The diverse substrate specificity of the cytochrome P450 (P450; CYP) enzyme superfamily offers the opportunity to develop enzymatic systems for environmental detoxification and biotransformations of drugs, pesticides and fine chemicals . Here we report on the immobilisation of a fusion protein between plant cytochrome P450-71B1 (CYP71B1) and its electron donor, plant NADPH cytochrome P450 reductase using an oil-in-water macro-emulsion, termed polyaphron, which contains a proportion of internal organic phase (phi) greater than 0.74 . Efficiency of P450 immobilisation was greater than 85%, and in this state enzymatic activity could be measured for more than 24 h at 15 degrees C . Chlortoluron, a recalcitrant herbicide pollutant in the environment, was shown to be metabolised, with the major metabolite (N-monodemethylated chlortoluron) being separated from the substrate due to partitioning into the aqueous phase . The turnovers exhibited superactivity compared with those obtained using free enzyme located in membranes prepared following heterologous expression in Saccharomyces cerevisiae and Escherichia coli . The potential to exploit the unprecedented catalytic diversity of the P450 superfamily in biocatalysis is discussed.

Nat Biotechnol, 1998 Aug, 16(8), 733 - 6
Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology; Kobayashi M et al.; Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, has been used in the industrial production of acrylamide and nicotinamide . Recent studies on NHases, which are roughly classified into iron and cobalt types according to the metal involved, have clarified the photoactivation mechanism, the novel ligand structure of the metal-binding sites, the unique mechanism of the enzyme hyper-induction, and the occurrence of an accessory gene involved in cobalt-containing NHase formation . These detailed analyses have led to the development of biotechnological applications of NHase, including biotransformation and bioremediation.

Curr Opin Biotechnol, 1998 Jun, 9(3), 300 - 4
Practical applications of marine bioremediation
Jones WR.
Bioremediation provides the potential for cost-effective, contaminant-specific treatments to reduce concentrations of individual or mixed environmental contaminants in the marine environment . Although there have been recent advances in our scientific understanding of bioremediation, in situ applications involving commercially available bacterial strains offer limited success . Bioaugmentation with indigenous microorganisms generally appears to be more effective, especially in combination with other site-specific amendments, although complete mineralization is rare.

Curr Opin Biotechnol, 1998 Jun, 9(3), 283 - 7
Deep ocean environmental biotechnology
Deming JW.
Major recent advances in deep-sea biotechnology have come in the form of continuing discoveries of novel microorganisms, unexpected genetic diversity, and new natural products of potential relevance to human health or environmental bioremediation . Continuing explorations of submarine hydrothermal vent environments have yielded new hyperthermophiles (maximal growth at 90 degreesC or greater) and more evidence that elevated hydrostatic pressure stabilizes cells and enzymes at high temperature . Vent samples have also yielded new mesophiles (optimal growth near 30 degreesC) that produce heparin-like exopolysaccharides or express extraordinary tolerance (removal by precipitation) of heavy metals . From the cold deep sea have come new findings of unexpected microbial diversity and the promise of industrially useful enzymes or secondary metabolites . New classes of predictive models are emerging to guide future exploration of microbial diversity in the deep ocean.

Crit Rev Microbiol, 1998, 24(2), 89 - 98
Systematics and environmental application of the genus Trichoderma; Esposito E et al.; Trichoderma, an anamorphic Hypocreaceae (class Ascomycetes), is common in the environment, especially in soils . Species of this genus have been used in the production of cellulolytic and hemicellulolytic enzymes, biological control of plant disease, biodegradation of chlorophenolic compounds, and soil bioremediation . They are also the cause of disease in commercially produced mushrooms . The species Trichoderma has not been clearly defined yet, despite being a very common fungus with an expanding number of applications . Therefore, we highlight the importance of the use of molecular techninques along with conventional methodologies based on morphological characters in order to achieve a "natural" taxonomic system for this group of fungi, as well as for any other complex group of fungi.

Adv Biochem Eng Biotechnol, 1998, 61, 1 - 21
Molecular adaptations in psychrophilic bacteria: potential for biotechnological applications; Russell NJ; Bacteria which live in cold conditions are known as psychrophiles . Since so much of our planet is generally cold, i.e . below 5 degrees C, it is not surprising that they are very common amongst a wide variety of habitats . To enable them to survive and grow in cold environments, psychrophilic bacteria have evolved a complex range of adaptations to all of their cellular components, including their membranes, energy-generating systems, protein synthesis machinery, biodegradative enzymes and the components responsible for nutrient uptake . Whilst such a systems approach to the topic has its advantages, all of the changes can be described in terms of adaptive alterations in the proteins and lipids of the bacterial cell . The present review adopts the latter approach and, following a brief consideration of the definition of psychrophiles and description of their habitats, focuses on those adaptive changes in proteins and lipids, especially those which are either currently being explored for their biotechnological potential or might be so in the future . Such applications for proteins range from the use of cold-active enzymes in the detergent and food industries, in specific biotransformations and environmental bioremediations, to specialised uses in contact lens cleaning fluids and reducing the lactose content of milk; ice-nucleating proteins have potential uses in the manufacture of ice cream or artificial snow; for lipids, the uses include dietary supplements in the form of polyunsaturated fatty acids from some Antarctic marine psychrophiles.

Anal Chem, 1998 Jul 1, 70(13), 2693 - 8
MALDI-TOF analysis of polymerase chain reaction products from methanotrophic bacteria; Hurst GB et al.; Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity . The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry . A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis . A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection . No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species . The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.

Curr Opin Biotechnol, 1998 Apr 1, 9(2), 135 - 40
Recombinant DNA techniques for bioremediation and environmentally-friendly synthesis
Keasling JD, Bang SW.
A number of new recombinant DNA techniques have been developed for genetically engineered microorganisms for biodegradation of environmental contaminants or for the synthesis of small molecules . These techniques include new expression vectors to carry the heterologous genes into the host organism, new mechanisms to control gene expression, containment mechanisms to control persistence of genetically-engineered microorganisms, application of site-directed and random mutagenesis to increase the substrate range or activity of biodegradative enzymes, and methods to track genetically-engineered microorganisms.

Appl Microbiol Biotechnol, 1998 May, 49(5), 489 - 99
Degradation of dioxin-like compounds by microorganisms; Wittich RM; Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF; PCDD/F, dioxins) have not been commercially produced in bulk amounts, as were polychlorinated biphenyls and other haloaromatic organics . Within the past two decades a lot of information has accumulated on the biodegradation of PCDD/F and other dioxin-like compounds because of their toxicity and because of significant environmental concern about many congeners of this class of chemicals . PCDD/F are subjected to reductive dehalogenations leading to less halogenated congeners, which can be attacked efficiently by fungal and bacterial oxidases and dioxygenases . In several cases these compounds can be utilized as carbon and energy sources . Pathways for their enzymatic degradation and the organisation of the corresponding degradative genes have been elucidated . Consequently, biotechnological applications will exploit the degradative potential of such microorganisms for bioremediation of contaminated sites.

Appl Environ Microbiol, 1998 Jul, 64(7), 2545 - 53
A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples; Widmer F et al.; Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation . Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms . The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples . Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes . A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed . The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100% . Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products . Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones . Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol . Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts . Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species . The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2350 - 6
Degradation and Fate of Carbon Tetrachloride in Unadapted Methanogenic Granular Sludge; Van Eekert MHA et al.; The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound . Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 microM) without any prior exposure to organohalogens . The maximum degradation rate was 1.9 micromol of CT g of volatile suspended solids-1 day-1 . The main end products of CT degradation were CO2 and Cl-, and the yields of these end products were 44 and 68%, respectively, of the initial amounts of {14C}CT and CT-Cl . Lower chlorinated methanes accumulated in minor amounts temporarily . Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed . Reduced components in the autoclaved sludge were vital for CT degradation . A major part (51%) of the CT was converted abiotically to CS2 . The amount of CO2 produced (23%) was lower and the amount of Cl- produced (86%) was slightly higher with autoclaved sludge than with living sludge . Both living and autoclaved sludges could degrade chloroform . However, only living sludge degraded dichloromethane and methylchloride . These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation . The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO2 . Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.

Biochemistry, 1998 Jun 23, 37(25), 8848 - 52
Design of a novel P450: a functional bacterial-human cytochrome P450 chimera; Shimoji M et al.; We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9 . The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular . The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase . This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas . Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.

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