Physical and metabolic interactions of Pseudomonas sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during growth on crude oil and effect of a chemical surfactant on them.

Methods to enhance crude oil biodegradation by mixed bacterial cultures, for example, (bio)surfactant addition, are complicated by the diversity of microbial populations within a given culture. The physical and metabolic interactions between Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 were examined during growth on Bow River crude oil. The effects of a nonionic chemical surfactant, Igepal CO-630 (nonylphenol ethoxylate), also were evaluated. Strain F9-D79 grew attached to the oil-water interface and produced a mycolic acid-containing capsule. Crude oil emulsification and surface activity were associated with the cellular fraction. Strain JA5-B45 grew in the aqueous phase and was unable to emulsify oil, but cell-free supernatants mediated kerosene-water emulsion formation. In coculture, stable emulsions were formed and strain JA5-B45 had an affinity for the capsule produced by strain F9-D79. Igepal CO-630 inhibited F9-D79 cells from adhering to the interface, and cells grew dispersed in the aqueous phase as 0.5-microm cocci rather than 2.5-microm rods. The surfactant increased total petroleum hydrocarbon removal by strain JA5-B45 from 4 to 22% and included both saturated compounds and aromatics. In coculture, TPH removal increased from 13 to 40% following surfactant addition. The culture pH normally increased from 7.0 to between 7.5 and 8.5, although addition of Igepal CO-630 to F9-D79 cultures resulted in a drop to pH 5.5. We suggest a dual role for the nonylphenol ethoxylate surfactant in the coculture: (i) to improve hydrocarbon uptake by strain JA5-B45 through emulsification and (ii) to prevent strain F9-D79 from adhering to the oil-water interface, indirectly increasing hydrocarbon availability. These varied effects on hydrocarbon biodegradation could explain some of the known diversity of surfactant effects.

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on crude oil.

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on Bow River crude oil was investigated using solid phase microextraction (SPME). Inoculum treatments were examined in relation to C(5)-C(11) hydrocarbon degradation. Up to 1600 mg/l biomass (dry weight) was tested without achieving significant volatile hydrocarbon partitioning and affecting analysis. Inoculum age rather than concentration had the most profound impact on biodegradation. When late log phase crude oil-grown inocula were used, C(5)-C(11) biodegradation reached 55-60%; methylcyclohexane and other branched compounds eluting before n-C(8) were recalcitrant. Increasing the late log inoculum concentration from 0.63 to 63 mg/l resulted in a twofold increase in degradation rate without improving the substrate range. Methylcyclohexane recalcitrance was correlated with reduced levels of hydrocarbon-degrading bacteria and volatile hydrocarbon evaporation from the inoculum flasks. A decreased lag phase prior to degradation was observed when using early stationary phase cultures as inocula and most compounds up to C(11), including methylcyclohexane, were biodegraded.

Enzyme treatment to reduce solids and improve settling of sewage sludge.

The effect of microbial enzymes in reducing the disposable solid content of sludge was investigated. A mixture of industrial cellulase, protease, and lipase, in equal proportion by weight, reduced total suspended solids (TSS) by 30-50% and improved settling of solids. An increase in solid reduction was observed with increasing enzyme concentration. The effect of combinations of enzyme treatments indicated that two-enzyme combinations of protease and cellulase produced better solid reduction than individual enzymes and that lipase further augmented this effect. Among the individual enzymes, protease produced a more settleable sludge as compared to cellulase and lipase. Adjustment of the pH of the enzymatically treated sludge to the acidic range (pH 2-4) further improved solid reduction, and adjustment to the alkaline range (pH 10-12) improved settleability.

Microbial treatment of a styrene-contaminated air stream in a biofilter with high elimination capacities.

A styrene-utilizing mixed microbial culture was isolated and utilized in a biofilter for the biological treatment of a contaminated air stream. Biofilter media consisted of composted wood bark and yard waste. The biofilters were acclimated at 120 s residence time and further evaluated at 60 and 30 s gas residence times. The biofilters received organic loading rates of up to 350 g/m(3) h. The styrene volumetric removal rate was a function of the organic loading rate and increased with increasing loading rates. Average volumetric removal rates of 69-118 g/m(3) h observed in our studies were higher than reported values for styrene biofilters. Average styrene removal efficiencies ranged from 65% to 75% (maximum 100%). Axial analysis of styrene concentration along the column indicated that the bulk of the styrene removal occurred in the first section of the biofilter. Analyses of the media indicated that the moisture content of the first section (50-55% w/w) was significantly lower than in the second and third sections (65-70% w/w). The pressure drops across the biofilter were low due to the high concentration of large media particles. The total pressure drops were 1-3, 4-6, and 10-16 mm for the 120-, 60-, and 30-s residence time periods, respectively.

Process for degradation of nitrobenzene: combining electron beam irradiation with biotransformation.

Electron beam irradiations of aqueous solutions containing 15-30 mg/L of nitrobenzene at 60 kGy dose removed 78% of the contaminant. Three mononitrophenols were detected as by-products of electron beam treatment of nitrobenzene. A mixed culture enriched on a mixture of 2-, 3-, and 4-nitrophenol degraded both the residual nitrobenzene and the nitrophenol products. Percentage removal of nitrobenzene increased with increasing electron beam dose. This observation led to the conceptual design of a two-stage electron beam microbial process for degradation of nitrobenzene. Three groups of pure isolates were characterized from the mixed culture based on their abilities to grow on cor- responding nitrophenol substrates: Group A, 2NP(-)3NP(-)4NP(+); Group B, 2NP(+)3NP(+)4NP(-); and Group C, 2NP(-)3NP(+)4NP(-). Bacteria that grew on 3-NP transformed nitrobenzene into ammonia in the electron beam-treated nitrobenzene samples.

Substrate selectivity of a 3-nitrophenol-induced metabolic system in Pseudomonas putida 2NP8 transforming nitroaromatic compounds into ammonia under aerobic conditions.

The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.

Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects.

Plants are valuable sources of a variety of chemicals including drugs, flavours, pigments and agrochemicals. Some of the biochemical reactions occurring in plant cells are complex and cannot be achieved by synthetic routes. In vitro plant cell and organ cultures and plant enzymes act as suitable biocatalysts to perform these complex reactions. A wide variety of chemical compounds including aromatics, steroids, alkaloids, coumarins and terpenoids can undergo biotransformations using plant cells, organ cultures and enzymes. The biocatalyst-mediated reactions are regiospecific and stereospecific. Reaction types include oxidations, reductions, hydroxylations, methylations, acetylations, isomerizations, glycosylations and esterfications. Genetic manipulation approaches to biotransformation offer great potential to express heterologous genes and to clone and overexpress genes for key enzymes. Biotransformation efficiencies can further be improved using molecular techniques involving site-directed mutagenesis and gene manipulation for substrate specificity.

Enzymatic asymmetric synthesis by decarboxylases.

Decarboxylation reactions using microbial cells or enzymes are increasingly being used for the synthesis of enantiomerically pure compounds because of their high degree of regio- and stereo-specificity. Pyruvate decarboxylase, benzoylformate decarboxylase and phenylpyruvate decarboxylase enzymes are capable of acyloin-type condensation reactions leading to formation of chiral alpha-hydroxy ketones, which are versatile building blocks in the pharmaceutical and chemical industries. Availability of three-dimensional structures of some decarboxylases in recent years has facilitated understanding of reaction mechanisms and the creation of mutants with enhanced activity and stability.

Cometabolic biotransformation of nitrobenzene by 3-nitrophenol degrading Pseudomonas putida 2NP8.

A strain of Pseudomonas putida (2NP8) capable of growing on both 2-nitrophenol and 3-nitrophenol, but not on nitrobenzene (NB), was isolated from municipal activated sludge. 2-Nitrophenol was degraded by this strain with production of nitrite. Degradation of 3-nitrophenol resulted in the formation of ammonia. Cells grown on 2-nitrophenol did not degrade nitrobenzene. A specific nitrobenzene degradation activity was induced by 3-nitrophenol. Ammonia, nitrosobenzene, and hydroxylaminobenzene have been detected as metabolites of nitrobenzene degradation by cells grown in the presence of 3-nitrophenol. These results indicated a NB cometabolism mediated by 3-nitrophenol nitroreductase.

Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol.

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated.

Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture.

The effects of various hydrocarbon substrates, and a chemical surfactant capable of enhancing crude-oil biodegradation, on the community structure of a mixed-bacterial inoculum were examined in batch culture. Of 1000 TSA-culturable isolates, 68.6% were identified at the genus level or better by phospholipid fatty acid analysis over 7-day time course experiments. Cultures were exposed to 20 g/L Bow River crude oil with and without 0.625 g/L Igepal CO-630 (a nonylphenol ethoxylate surfactant), 5 g/L saturates, 5 g/L aromatics, or 125 g/L refinery sludge. A group of six genera dominated the cultures: Acinetobacter, Alcaligenes, Ochrobactrum, Pseudomonas/Flavimonas, Stenotrophomonas, and Yersinia. Species from four of the genera were shown to be capable of hydrocarbon degradation, and counts of hydrocarbon degrading and total heterotrophic bacteria over time were nearly identical. Pseudomonas/Flavimonas and Stenotrophomonas normally dominated during the early portions of cultures, although the lag phase of Stenotrophomonas appears to have been increased by surfactant addition. Acinetobacter calcoaceticus was the most frequently isolated microorganism during exposure to the saturate fraction of crude oil. Regardless of substrate, the culture medium supported a greater variety of organisms during the latter portions of cultures. Understanding the community structure and dynamics of mixed bacterial cultures involved in treatment of heterogeneous waste substrates may assist in process development and optimization studies.

Low surfactant concentration increases fungal mineralization of a polychlorinated biphenyl congener but has no effect on overall metabolism.

Three white rot fungi were compared for their ability to attack polychlorinated biphenyl (PCB) congeners in the presence and absence of the non-ionic Triton X-100 or the anionic Dowfax 8390 surfactants at half their critical micelle concentrations. Neither surfactant affected PCB biodegradation monitored by gas chromatography but the release of 14CO2 from 2,4',5-[U-14C]trichlorobiphenyl by Trametes versicolor was stimulated 12% by Triton X-100. Since mineralization is the complete metabolism of the congener and biodegradation was measured as substrate disappearance, Triton X-100 is proposed to aid intracellular solubilization of 2,4',5-trichlorobiphenyl for complete oxidation by T. versicolor.

Effect of surfactant solubilization on biodegradation of polychlorinated biphenyl congeners by Pseudomonas LB400.

A variety of commercial surfactants were tested to determine their effect on polychlorinated biphenyl (PCB) transformation by Pseudomonas LB400. Initial tests determined that most surfactants were fully or partially able to solubilize the PCB congeners 2,5,2'-chlorobiphenyl (CBP), 2,4,2',4'-CBP, 2,3,5,2',5'-CBP and 2,4,5,2',4',5'-CBP, at concentrations above the surfactants' critical micelle concentration (CMC). Surfactants were also found to have no negative effect on bacterial survival, as cell numbers were the same or higher after incubation in the presence of surfactants than after incubation without surfactants. A comparison of the extent of biotransformation of single PCB congeners by the bacterium revealed that, at surfactant concentrations above the CMC, the presence of an anionic surfactant promoted while nonionic surfactants inhibited PCB transformation, compared to a control with no surfactant. The rates of transformation of PCB congeners were also higher in the presence of the anionic surfactant compared to the control. The inhibitory effects of a nonionic surfactant, Igepal CO-630 at a concentration above its CMC, on transformation of 2,4,5,2',5'-CBP could be eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant CMC.

Microbial degradation of nitrobenzene and mono-nitrophenol by bacteria enriched from municipal activated sludge.

Using a mixture of three mono nitrophenols as sole carbon, nitrogen and energy sources, mixed cultures were enriched from municipal activated sludge to degrade both nitrophenols and nitrobenzene. Bacterial growth and degradation rate could be increased by supplementing the medium with 0.1% YE. Microorganisms were isolated from the nitrophenols enrichment, and they were identified as strains of Comamonas testosteroni and Acidovorax delafieldii. These strains showed broad degradation ability toward nitrophenols and nitrobenzene.

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.

Comparison of gas chromatography and mineralization experiments for measuring loss of selected polychlorinated biphenyl congeners in cultures of white rot fungi.

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4'-dichlorobiphenyl, 2,4',5-trichlorobiphenyl (2,4',5-TCB), 2,2',4,4'-tetrachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl. The congener tested for mineralization was 2,4',5-[U-14C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower,