Activity and stability of a recombinant plasmid-borne TCE degradative pathway in suspended cultures

The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min. mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h.     

Modeling trichloroethylene degradation by a recombinant pseudomonad expressing toluene ortho-monooxygenase in a fixed-film bioreactor

Burkholderia cepacia PR123(TOM23C), expressing constitutively the TCE-degrading enzyme toluene ortho-monooxygenase (Tom), was immobilized on SIRANtrade mark glass beads in a biofilter for the degradation and mineralization of gas-phase trichloroethylene (TCE). To interpret the experimental results, a mathematical model has been developed which includes axial dispersion, convection, film mass-transfer, and biodegradation coupled with deactivation of the TCE-degrading enzyme. Parameters used for numerical simulation were determined from either independent experiments or values reported in the literature. The model was compared with the experimental data, and there was good agreement between the predicted and measured TCE breakthrough curves. The simulations indicated that TCE degradation in the biofilter was not limited by mass transfer of TCE or oxygen from the gas phase to the liquid/biofilm phase (biodegradation limits), and predicts that improving the specific TCE degradation rates of bacteria will not significantly enhance long-term biofilter performance. The most important factors for prolonging the performance of biofilter are increasing the amount of active biomass and the transformation capacity (enhancing resistance to TCE metabolism). Copyright 1998 John Wiley & Sons, Inc.     

TCE degradation in a methanotrophic attached-film bioreactor

Trichloroethene was degraded in expanded-bed bioreactors operated with mixed-culture methanotrophic attached films. Biomass concentrations of 8 to 75 g volatile solids (VS) per liter static bed (L(sb)) were observed. Batch TCE degradation rates at 35 degrees C followed the Michaelis-Menten model, and a maximum TCE degradation rate (q(max)) of 10.6 mg TCE/gVS . day and a half velocity coefficient (K(S)) of 2.8 mg TCE/L were predicted. Continuous-flow kinetics also followed the Michaelis-Menten model, but other parameters may be limiting, such as dissolved copper and dissolved methane-q(max) and K(S) were 2.9 mg TCE/gVS . day and 1.5 mg TCE/L, respectively, at low copper concentrations (0.003 to 0.006 mg Cu/L). The maximum rates decreased substantially with small increases in dissolved copper. Methane consumption during continuous-flow operation varied from 23 to 1200 g CH(4)/g TCE degraded. Increasing the influent dissolved methane concentration from 0.01 mg/L to 5.4 mg/L reduced the TCE degradation rate by nearly an order of magnitude at 21 degrees C. Exposure of biofilms to 1.4 mg/L tetrachloroethene (PCE) at 35 degrees C resulted in the loss of methane utilization ability. Tests with methanotrophs grown on granular activated carbon indicated that lower effluent TCE concentrations could be obtained. The low efficiencies of TCE removal and low degradation rates obtained at 35 degrees C suggest that additional improvements will be necessary to make methanotrophic TCE treatment attractive. (c) 1993 John Wiley & Sons, Inc.     

A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase

A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358. The two-stage bioreactor system was successfully operated for 20 days. PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell. Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop. Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air. Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min. The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d. These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams.     

Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates.

Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.     

Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates

Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.     

Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene

Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation.     

Continuous vapor-phase trichloroethylene biofiltration using hydrocarbon-enriched compost as filtration matrix

Two sources of finished compost material were examined for the capacity to support trichloroethylene(TCE)-degrading microbial populations in a gas-phase bioreactor. Gaseous hydrocarbon was passed through the bioreactor to stimulate cometabolic oxidation of TCE. Significant differences in TCE removal efficiencies were observed between the two compost types, and between hydrocarbon-stimulated and non-stimulated compost. At an average column retention time of 5.6 min, deciduous leaf debris compost removed more than 95% of a 5–50 ppm (by vol.) TCE gas stream, whereas less than 15% removal was observed under similar conditions with a woodchip and bark compost. Trichloroethylene removal efficiency varied with the hydrocarbon-stimulation regime employed, although propane and methane stimulated TCE degradation equally well. Amendment of compost with granular activated carbon substantially increased biological TCE removal. Differences in TCE removal efficiencies observed between the two compost types and between hydrocarbon-stimulated and non-stimulated composts were investigated in terms of changes in the overall heterotrophic microbial populations by using community-level physiological profile analysis. Received: 14 April 1997 / Received revision: 21 July 1997 / Accepted: 25 August 1997     

Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4

Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates. Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C. Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE. The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates. The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein. Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation. In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased. TCE degradation was observed up to 300 microM TCE with no significant decreases in rates. On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein. These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors.     

Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4.

Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates. Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C. Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE. The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates. The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein. Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation. In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased. TCE degradation was observed up to 300 microM TCE with no significant decreases in rates. On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein. These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors.     

Biodegradation of Trichloroethylene in Continuous-Recycle Expanded-Bed Bioreactors

Experimental bioreactors operated as recirculated closed systems were inoculated with bacterial cultures that utilized methane, propane, and tryptone-yeast extract as aerobic carbon and energy sources and degraded trichloroethylene (TCE). Up to 95% removal of TCE was observed after 5 days of incubation. Uninoculated bioreactors inhibited with 0.5% Formalin and 0.2% sodium azide retained greater than 95% of their TCE after 20 days. Each bioreactor consisted of an expanded-bed column through which the liquid phase was recirculated and a gas recharge column which allowed direct headspace sampling. Pulses of TCE (20 mg/liter) were added to bioreactors, and gas chromatography was used to monitor TCE, propane, methane, and carbon dioxide. Pulsed feeding of methane and propane with air resulted in 1 mol of TCE degraded per 55 mol of substrate utilized. Perturbation studies revealed that pH shifts from 7.2 to 7.5 decreased TCE degradation by 85%. The bioreactors recovered to baseline activities within 1 day after the pH returned to neutrality.     

Cometabolic degradation of trichloroethylene in a bubble column bioscrubber

A bubble column bioreactor was used as bioscrubber to carry out a feasibility study for the cometabolic degradation of trichloroethylene (TCE). Phenol was used as cosubstrate and inducer. The bioreactor was operated like a conventional chemostat with regard to the cosubstrate and low dilution rates were used to minimize the liquid outflow. TCE degradation measurements were carried out using superficial gas velocities between 0.47and 4.07 cm s(-1) and TCE gas phase loads between 0.07 and 0.40 mg L(-1) Depending on the superficial gas velocity used, degrees of conversion between 30% and 80% were obtained. A simplified reactor model using plug flow for the gas phase, mixed flow for the liquid phase, and pseudo first order reaction kinetics for the conversionof TCE was established. The model is able to give a reasonable approximation of the experimental data. TCE degradation at the used experimental conditions is mainly limited by reaction rate rather than by mass transfer rate. The model can be used to calculate the reactor volume and the biomass concentration for a required conversion. (c) 1995 John Wiley & Sons Inc.     

Effect of substrate concentration on dual-species biofilm population densities of Klebsiella oxytoca and Burkholderia cepacia in porous media

The long-term operation of bioremediation technologies relies on the success of the contaminant-degrading microorganism(s) to compete for available resources with microorganisms already present in an aquifer or those that may contaminate a bioreactor. Though research has been performed studying the interaction of multiple species in batch and chemostat reactors, little work has been done looking at multi-species interactions in environments that more closely resemble field-scale applications. The research presented herein examined the interaction of Burkholderia cepacia PR1-pTOM(31c), an aerobic trichloroethylene (TCE)-degrading bacterium, with Klebsiella oxytoca, a facultative bacterium, in a flow-through porous media (PM) reactor. Growth characteristics and population distributions in PM were compared to previously reported values from batch and chemostat reactors. The faster growing organism in batch experiments (K. oxytoca) did not always have the greater population density in dual-species PM experiments. The biofilm population distribution was influenced by substrate concentration, with B. cepacia having a greater dual-species population density than K. oxytoca at a low (30 mg/L dissolved organic carbon [DOC]) substrate concentration and K. oxytoca having a greater population density at a high (700 mg/L DOC) substrate concentration. This change in species population distribution with change in substrate concentration, which was not observed in batch reactors, was also observed in chemostat reactors. Therefore, manipulation of substrate concentration enabled the control of species dominance to the advantage of the TCE degrading population in this dual-species PM system and may provide a mechanism to enhance bioremediation scenarios involving TCE or other contaminants of concern.     

Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for Enhanced 1-naphthol synthesis and chloroform degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase alpha-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V(max) of 9.3 versus 4.5 nmol.min(-1).mg of protein(-1) and unchanged K(m)), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 +/- 0.07 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1) [mean +/- standard deviation]) at 91 microM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V(max) of 2.61 versus 0.95 nmol.min(-1).mg of protein(-1) and unchanged K(m)) and chloride release (0.034 +/- 0.002 versus 0.012 +/- 0.001 nmol.min(-1).mg of protein(-1)). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 +/- 0.3 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1)). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Cytotoxicity associated with trichloroethylene oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)) lost 95% of their acetate-dependent O(2) uptake activity (a measure of general respiratory activity), yet toluene-dependent O(2) uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 micromol (mg of cells(-1)) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)), up to 90% were Tol(-) variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.     

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in biofilm cultures

The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term. Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. Copyright 1998 John Wiley & Sons, Inc.     

Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 +/- 0.3 versus 1.39 +/- 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 +/- 0.03 versus 0.029 +/- 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 +/- 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the alpha-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.     

Propane-Induced biodegradation of vapor phase trichoroethylene

Microbial degradation of trichloroethylene (TCE) has been demonstrated under aerobic conditions with propane. The primary objective of this research was to evaluate the feasibility of introducing a vapor phase form of TCE in the presence of propane to batch bioreactors containing a liquid phase suspension of Mycobacterium vaccae JOB5 to accomplish degradation. The reactor system consisted of three phases: a vapor phase introducing air, propane, and TCE; a liquid phase of the microbial suspension; and a solid phase in the form of the microorganisms. Long-term and initial rate experiments were conducted on three culture sets to evaluate microbial response. In two long-term test fed propane and approximately 0.1 mg/L and 1 mg/L of TCE, respectively, propane utilization was more efficient at the high TCE concentration (600 mmol propane/mmol TCE versus 11,900 mmol propane/mmol TCE), because the propane degradation rate was approximately the same for both tests (6.73 mg/L . h and 7.85 mg/L . h for the high and low tests). In addition, TCE utilization decreased after complete propane consumption. Initial rate tests on culture sets fed propane only revealed that cells with a history of exposure to a high concentration of TCE had the highest specific growth rate, but the lowest half-saturation constant (7.60e(-3) h(-1) and 0.10 mg/L, respectively). Tests fed variable TCE concentrations (0.031 to 5.378 mg/L in the liquid phase) with no propane showed TCE depletion but no biomass growth. The tests revealed that the TCE removal increased as the TCE concentration increased, indicating a greater removal efficiency at the higher concentrations. Tests with a constant initial propane concentration and variable liquid phase TCE concentration revealed that specific propane utilization was essentially the same. (c) 1995 John Wiley & Sons, Inc.     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.