Quantification of toluene dioxygenase induction and kinetic modeling of TCE cometabolism by Pseudomonas putida TVA8.

As measured by the toluene-induced bioluminescent response of Pseudomonas putida TVA8 in batch experiments, toluene dioxygenase (Tod) enzyme activities are dependent on toluene concentration between 0 and 30 mg/L. To provide a measure of the Tod activity for use in Michaelis-Menten competitive-inhibition kinetics, a correlation between toluene concentration and induced Tod activity as measured by an induced bioluminescent response of P. putida TVA8 is presented as a nondimensional Tod activity parameter. A packed-bed, radial-flow bioreactor (RFB) using the bioreporter P. putida TVA8A serves as the model system for studying the effect of the enzyme activity parameter on model predictions of vapor-phase toluene oxidation and trichloroethylene (TCE) cometabolism. Mass balances were performed on a differential section of the RFB to describe the radial transport of vapor-phase toluene and TCE through a bulk gas phase and the concomitant biological reaction in a stationary biofilm phase. The finite-element Galerkin weak-statement formulation with first-order basis functions was used to find the optimum solution to the highly nonlinear, coupled equations. For this RFB system with toluene concentrations less than 1 mg/L in the bulk gas phase, the Tod activity parameter enables accurate predictions of steady-state TCE degradation rate (0.27 microg TCE/min).

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism.

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.

Induction of the tod operon by trichloroethylene in Pseudomonas putida TVA8.

Bioluminescence, mRNA levels, and toluene degradation rates in Pseudomonas putida TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE). TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations. Compared to uninduced TVA8 cultures, todC1 mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures. Compared to uninduced P. putida F1 cultures, todC1 mRNA levels increased 4.4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures. Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures.

Production of ethanol from starch by co-immobilized Zymomonas mobilis-glucoamylase in a fluidized-bed reactor.

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilized Zymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. The Z. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of kappa-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35 degrees C and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.

Kinetics and response of a Pseudomonas fuorescens HK44 biosensor.

The reporter bacterium, Pseudomonas fluorescens HK44 (HK44), was characterized in an immobilized state to investigate utility for deployment as a remote sensor in the subsurface. A packed-bed reactor with alginate-immobilized HK44 simulated hydrodynamic conditions such as might be found in a subsurface environment. The reporter bacterium, HK44, harbors a reporter plasmid, pUTK21, which contains a transcriptional fusion between the nahG gene in the lower pathway of the catabolic plasmic NAH7 and a luxCDABE gene cassette. The upper nah pathway and the lux pathway in pUTK21 are induced by salicylate. The lux enzymes catalyze the light reaction. HK44 demonstrated a quantitative relationship between salicylate concentration and degradation. Light intensity mimicked salicylate concentration, whereas degradation was first order in biomass and first order in salicylate concentration, with a degradation constant of 2.23 x 10(-2) dm(3) g(-1) min(-1).

Performance of coimmobilized yeast and amyloglucosidase in a fluidized bed reactor for fuel ethanol production.

The performance of coimmobilized Saccharomyces cerevisiae and amyloglucosidase (AG) was evaluated in a fluidized-bed reactor. Soluble starch and yeast extracts were used as feed stocks. Conversion of soluble starch streams to ethanol has potential practical applications in corn dry and wet milling and in developmental lignocellulosic processes. The biocatalyst performed well, and demonstrated no significant loss of activity or physical integrity during 10 wk of continuous operation. The reactor was easily operated and required no pH control. No operational problems were encountered from bacterial contaminants even though the reactor was operated under nonsterile conditions over the entire course of experiments. Productivities ranged between 25 and 44 g ethanol/L/h/. The experiments demonstrated that ethanol inhibition and bed loading had significant effects on reactor performance.

Development of a membrane-based vapor-phase bioreactor.

A vapor-phase bioreactor has been developed utilizing porous metal membranes in a cylindrical design employing radial flow as opposed to traditional axial flow for the vapor stream. The system was evaluated for the biodegradation of p-xylene (p-xylene) from a water-saturated air stream by Pseudomonas putida ATCC 23973 immobilized onto sand. The biocatalyst was placed in the annular space between two cylindrical, porous stainless-steel membranes. Details of the reactor system are presented along with biological data verifying system performance. The feed flow rate and p-xylene concentration were varied between 60 and 130 cm(3)/min and 15-150 ppm, respectively. Continuous reactor operation was maintained for 80-200 h with removal efficiencies (based onp-xylene disappearance) between 80 and 95%. The effluent concentration histories were compared to determine the operating range of the bioreactor.

Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures.

Biodegradation of chlorinated aliphatics and aromatic compounds in total-recycle expanded-bed biofilm reactors.

Ground-water contamination by chlorinated aliphatic compounds is a major cause for concern because of their toxicity. This study examined the biodegradation of trichloroethylene and aromatic compounds by microbial consortia enriched from contaminated subsurface sediments. The consortia were capable of utilizing methane and propane as sources of carbon and energy. Two continuously recycled expanded-bed bioreactors were inoculated with (1) the subsurface consortium, and (2) P. fluorescence, P. putida (strains pRB1401 and pWWO), and M. trichosporium OB3b. An uninoculated reactor containing 0.2% sodium azide and 0.5% formalin served as the control. Methane (5% v/v) and propane (3% v/v) were maintained by batch feeding through the course of the experiment. Greater than 97% degradation of trichloroethylene was observed over a period of 12 d. More than 99% of benzene, toluene, and xylene were degraded within the first 7 d. Dissolved oxygen levels were measured and found to be in the range 4.9-6.5 mg/L throughout the experiments.

Biodegradation of chlorinated aliphatic hydrocarbon mixtures in a single-pass packed-bed reactor.

Aliphatic chlorinated compounds, such as trichloroethylene (TCE) and tetrachloroethylene (PCE), are major contaminants of ground water. A single-pass packed-bed bioreactor was utilized to study the biodegradation of organic waste mixtures consisting of PCE, TCE, and other short-chain chlorinated organics. The bioreactor consisted of two 1960-mL glass columns joined in a series. One column was packed with sand containing a microbial consortia enriched from a contaminated site. The other column provided a reservoir for oxygen and a carbon source of methane/propane that was recirculated through the reactor. Sampling was accomplished by both direct headspace and liquid effluent concentration analyses. The reactor was operated in a single-pass mode. Greater than 99% degradation of trichloroethylene, approaching drinking water standards, was observed when the bioreactor residence time ranged from 1.9 to 3.2 d. Typically, when the reactor was pulse-fed with methane, propane, and air, 1 mol of TCE was degraded/110 mol of substrate utilized. Perturbation studies were performed to characterize reactor behavior. The system's degradation behavior was affected by providing different carbon sources, a pulse feeding regime, supplementing microbial biomass, and by altering flow rates.

Development of a differential volume reactor system for soil biodegradation studies.

A bench scale experimental system was developed for the analysis of polycyclic aromatic hydrocarbon (PAH) degradation by mixed microbial cultures in PAH contaminated Manufactured Gas Plant (MGP) soils and on sand. The reactor system was chosen in order to provide a fundamental protocol capable for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the important system variables and their interactions. The reactor design and peripherals are described. A plug flow differential volume reactor (DVR) was used in order to remove performance effects related to reactor type, as opposed to system structure. This reactor system could be well represented mathematically. Methods were developed for on-line quantitative determination of PAH liquid phase concentrations. The mathematical models and experimental data are presented for the biodegradation of naphthalene on artificial and MGP soils.

Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor.

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays. Naphthalene biodegradation rates were very high throughout the experimental run (95 to greater than 99% removed) resulting in very low or undetectable levels of naphthalene in the off-gas and reactor effluent. Attempts to reduce the rate of naphthalene biotransformation by either reducing the reactor temperature from 20 degrees C to 10 degrees C or the dissolved oxygen level (greater than 1 mg/L) were unsuccessful. Significant naphthalene biodegradation was observed at 4 degrees C. While variable, the microbial community as measured by population densities was not significantly affected by temperature changes. In terms of naphthalene biotransformation, the system was able to adapt readily to all perturbations in the reactor.

Breakthrough behavior of 17.5 mol% water in methanol, ethanol, isopropanol, and t-butanol vapors passed over corn grits.

Ground corn is now used in industry as an adsorbent to remove water from ethanol vapors. It is stable and inexpensive at 10 cents/lb (22 cents/kg). For regeneration it requires less than 2000 Btu/gal of 190 proof ethanol processed. If necessary, it could be readily saccharified and fermented into ethanol after use. This renewable resource has further exciting potential as an inexpensive adsorbent for water removal from other alcohols, including methanol, isopropanol, and t-butanol. Water sorption capacity in a fixed bed, nonisothermal adsorption column appears to be a function of the heat capacity of the non-adsorbed alcohol vapor, relative to the heat capacity of the corn adsorbent. Methanol, ethanol, isopropanol, and t-butanol containing 17.5 mol% water gave 105,151, 284, and 358 g anhydrous product/kg adsorbent, respectively, per adsorption cycle. This adsorbent, having operational temperature ranges between 80 and 100 degrees C, is indicated to be of potential utility in solvent recycle processes using these industrially important alcohols. Observed adsorption characteristics are discussed in terms of the alcohol properties of molecular size, heat capacity, and diffusivity. The adsorption mechanism is hypothesized to include transport of water molecules into the structure of adjacent starch molecules present in small spherical bodies (diameter of several microns) immobilized on the surface of the corn grit particles.