A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion

Abstract. A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 μM parathion (146 mg l−1) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis–Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 μM p-nitrophenol (83 mg l−1) and P. putida KT2440 pSB337 inhibited by 1,000 μM p-nitrophenol (139 mg l−1). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.

[1]  J. Oakeshott,et al.  Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture , 2000, Applied and Environmental Microbiology.

[2]  R. Mandelbaum,et al.  Rapid atrazine mineralization under denitrifying conditions by Pseudomonas sp. strain ADP in aquifer sediments , 1998 .

[3]  S. Harayama,et al.  Construction of Bacterial Consortia That Degrade Arabian Light Crude Oil , 1996 .

[4]  H. Hasman,et al.  Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens , 2000, Journal of bacteriology.

[5]  Ashok Mulchandani,et al.  Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase , 1997, Nature Biotechnology.

[6]  D. Focht,et al.  Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating , 1992, Applied and environmental microbiology.

[7]  H. Schweizer.  A method for construction of bacterial hosts for lac-based cloning and expression vectors: alpha-complementation and regulated expression. , 1994, BioTechniques.

[8]  Y. Oh,et al.  Construction of a bacterial consortium for the biofiltration of benzene, toluene and xylene emissions , 1997 .

[9]  A. Solanas,et al.  Isolation and characterization of a 9-fluorenone-degrading bacterial strain and its role in synergistic degradation of fluorene by a consortium , 1998 .

[10]  D. Springael,et al.  Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers , 2000, Applied and Environmental Microbiology.

[11]  J. Keasling,et al.  Construction and characterization of F plasmid-based expression vectors. , 1998, Biotechnology and bioengineering.

[12]  D. Janssen,et al.  Formation and detoxification of reactive intermediates in the metabolism of chlorinated ethenes. , 2001, Journal of biotechnology.

[13]  J. Keasling,et al.  Dual Labeling with Green Fluorescent Proteins for Confocal Microscopy , 2000, Applied and Environmental Microbiology.

[14]  John F. Andrews,et al.  A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates , 1968 .

[15]  J. Keasling,et al.  Metabolic Engineering of an Aerobic Sulfate Reduction Pathway and Its Application to Precipitation of Cadmium on the Cell Surface , 2000, Applied and Environmental Microbiology.

[16]  J. So,et al.  Improved transformation of Pseudomonas putida KT2440 by electroporation , 1995 .

[17]  P. Adriaens,et al.  Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl , 1989, Applied and environmental microbiology.

[18]  K. Timmis,et al.  Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans , 1999, Journal of Industrial Microbiology and Biotechnology.

[19]  K. Timmis,et al.  Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS , 1997, Applied and environmental microbiology.

[20]  E. Katsivela,et al.  An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water , 1999, Applied Microbiology and Biotechnology.

[21]  Dorian Liepmann,et al.  Development of engineered biofilms on poly- L-lysine patterned surfaces , 2001, Biotechnology Letters.

[22]  V H Edwards,et al.  The influence of high substrate concentrations on microbial kinetics , 1970, Biotechnology and bioengineering.

[23]  F. Richards,et al.  THE PURIFICATION AND PROPERTIES OF NEUROSPORA MALATE DEHYDROGENASE. , 1965, Archives of biochemistry and biophysics.

[24]  Hak-Sung Kim,et al.  Degradation of Chloronitrobenzenes by a Coculture of Pseudomonas putida and a Rhodococcussp , 1999, Applied and Environmental Microbiology.

[25]  J H Luong,et al.  Generalization of monod kinetics for analysis of growth data with substrate inhibition , 1987, Biotechnology and bioengineering.

[26]  J. Ramos,et al.  Metabolism of Nitrate Esters by a Consortium of Two Bacteria , 1996, Nature Biotechnology.

[27]  W. Mulbry,et al.  Purification and characterization of three parathion hydrolases from gram-negative bacterial strains , 1989, Applied and environmental microbiology.

[28]  L. Newman,et al.  Metabolism of polyhalogenated compounds by a genetically engineered bacterium , 1994, Nature.

[29]  W. Mulbry,et al.  Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta , 1986, Applied and environmental microbiology.

[30]  V. de Lorenzo,et al.  Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene. , 2001, Journal of biotechnology.

[31]  J. Keasling,et al.  Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds , 2000, Applied and Environmental Microbiology.

[32]  D. Crowley,et al.  Survival and activity of an atrazine-mineralizing bacterial consortium in rhizosphere soil , 1996 .