[Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems].

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.

Cytotoxic potential of industrial strains of Bacillus sp.

The cytotoxic potential of selected strains of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis, used in the production of industrial enzyme products, has been assessed. Cytotoxicity was determined in Chinese hamster ovary (CHO-K1) cells by measuring total cellular metabolic activity using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Initially the MTT assay was validated against toxigenic strains of Bacillus cereus, to define the exact criteria for a toxigenic versus a nontoxigenic response. The assay proved sensitive to culture broths of both a diarrheagenic strain and an emetic strain of B. cereus. The enzyme-producing strains tested were nontoxic to CHO-K1 cells. Additionally it was demonstrated that our industrial strains did not react with antibodies against B. cereus enterotoxins by use of commercial antibody-based kits from Oxoid and Tecra. A short survey of the literature concerning the toxigenic potential of species within the subtilis group is included, as is a database search of known B. cereus enterotoxins against B. subtilis and B. licheniformis DNA sequences.

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates.

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).

Successive identification of biodegradation rates for multiple sequentially reactive contaminants in groundwater.

At the field scale, the biodegradation rate is usually estimated from analytical solutions to single species transport with first-order reactions, using measured data as input. Because many contaminants, e.g., chlorinated solvents, are degraded in a sequential pattern, with degradation products further reacting to produce new species, it is of great interest to quantify the transformation rate of every reaction. The conventional inverse solutions for identifying the transformation rates are limited to single species problems. In the present study, we propose a successive optimization approach to identify the biodegradation rate for each species by using a previously developed analytical solution to multi-species first-order reactive transport using data obtained at the field scale. By specifying a link between analytical solutions to sequentially reactive transport problems and optimization methods and assuming constant transport parameters (velocity, dispersivities, and retardation factors), the first-order transformation rates are optimized successively from parent species to its daughter species.

Nitrate reduction with Halomonas campisalis. Kinetics of denitrification at pH 9 and 12.5% NaCl.

Regeneration of ion exchange resins with NaCl produces brine containing high concentrations of nitrate that can be difficult to remove using standard biological, physical, or chemical technologies. In this study. Halomonas campisalis (ATCC #700597) (Mormile et al., 1999) was shown to completely reduce nitrate at 125 g/L NaCl and pH 9. This organism was also used in experiments to determine nitrate-reduction rates and biomass yields. Kinetic parameters were measured separately with glycerol, lactate. acetate, ethanol, and methanol. The specific nitrate-reduction rate coefficient was highest in cultures amended with acetate, while lactate and glycerol (a natural osmoticum in hypersaline environments) had lower reduction rates. No evidence of nitrate reduction was observed when ethanol or methanol was provided as an electron donor. Kinetic modeling provided values for nitrate and nitrite-reduction rate coefficients and for biomass yields. Measured rates and yields were similar to reported parameters obtained from non-halophilic nitrate-reducing cultures under low salt concentrations. Therefore, for highly saline solutions, the use of halophiles to selectively remove nitrate from these brines may represent a viable treatment option.

Bacterial reduction of chromium.

A mixed culture was enriched from surface soil obtained from an eastern United States site highly contaminated with chromate. Growth of the culture was inhibited by a chromium concentration of 12 mg/L. Another mixed culture was enriched from subsurface soil obtained from the Hanford reservation, at the fringe of a chromate plume. The enrichment medium was minimal salts solution augmented with acetate as the carbon source, nitrate as the terminal electron acceptor, and various levels of chromate. This mixed culture exhibited chromate tolerance, but not chromate reduction capability, when growing anaerobically on this medium. However, this culture did exhibit chromate reduction capability when growing anaerobically on TSB. Growth of this culture was not inhibited by a chromium concentration of 12 mg/L. Mixed cultures exhibited decreasing diversity with increasing levels of chromate in the enrichment medium. An in situ bioremediation strategy is suggested for chromate contaminated soil and groundwater.

Effects of nitrate and acetate availability on chloroform production during carbon tetrachloride destruction.

Fed batch experiments were performed to test the effects of electron donor and electron acceptor availability on the production of chloroform (CF) during carbon tetrachloride (CT) destruction by a denitrifying bacterial consortium. In one series of tests, acetate (electron donor) was present in excess while nitrate and nitrite (electron acceptor) were limiting. In the other series of tests, acetate was the limiting nutrient, and nitrate and nitrite were in excess. Under nitrate limiting conditions, 50% (+/-17%) of the CT transformed by the microorganisms was converted to CF. However, under acetate limiting conditions, only 4% (+/-4%) of the CT that was degraded appeared as CF. Previous research had suggested that denitrifying bacteria can degrade CT via two competing pathways. One of these pathways produces CF as the predominant end product. The second pathway produces CO(2) as the primary end product. The results shown here suggest that the first pathway is dominant when nitrate and nitrite are depleted while the second pathway, which produces little CF, dominates when nitrate or nitrite are available.

Development of a predictive description of an immobilized-cell, three-phase, fluidized-bed bioreactor.

A fully predictive mathematical description of a three-phase, tapered, fluidized-bed bioreactor is developed. This mathematical model includes the effects of the tapered bed, variable dispersion coefficient, and variable solid holdup upon the concentration profiles developed in the bed. In addition, the effect of the concentration profile which is developed inside the biocatalyst bead is included by means of an effectiveness factor calculation. Using accepted correlations for the dispersion coefficient and for the liquid, gas, and solid holdup in the bed, the model is fully predictive. The model was found to adequately predict experimental obtained concentration profiles. Then, the model was used to examine the various phase holdups through the bed and the degree to which the dispersion coefficient varied through the bed. The effect of changes in these calculated variables upon the reaction rate is discussed. (c) 1995 John Wiley & Sons, Inc.

Kinetics of nitrate inhibition of carbon tetrachloride transformation by a denitrifying consortia.

The kinetics of nitrate inhibition of carbon tetrachloride (CT) transformation were examined using a denitrifying consortium. Comparison of data from fed-batch experiments to the model reported by Hooker et al. indicate that the inhibition constant ranges between 3.2 and 21 mg/L, with an average of 8.8 mg/L. This range is much lower than the previously reported value of 169 mg/L. Simulations using the corrected parameter accurately reflect this new data and the data reported by Hooker et al. In contrast, the earlier reported coefficient value does not reflect the data reported in this work. (c) 1995 John Wiley & Sons, Inc.

Biological destruction of CCI(4): II. Kinetic modeling.

A denitrifying consortium capable of transforming carbon tetrachloride (CCI(4)) was cultured from an aquifer soil sample from the U.S. Department of Energy's Hanford Site in southeastern Washington State. A mathematical description of the kinetics of CCI(4) destruction by this microbial consortium is presented, and its prediction are compared to experimental data. The model successfully predicted the concentrations of acetate, nitrate, nitrite, biomass, and CCI(4) for all 12 experiments (a total of 60 concentration-vs.-time data sets). In addition, no statistically significant interactions exist between parameter values and individual test conditions. The ability of the model to predict the results of a treatability test for CCI(4) degradation in Hanford groundwater, without adjusting any model parameters, is discussed. (c) 1994 John Wiley & Sons, Inc.

Biological destruction of CCl(4): I. Experimental design and data.

A denitrifying consortium capable of transforming carbon tetrachloride (CCl(4)) was cultured from aquifer sediment from the U.S. Department of Energy's Hanford Site in southeastern Washington State. To understand the kinetics of the biological destruction of CCl(4) by these microbes, a set of experiments, the conditions of which were chosen according to a fractional factorial experimental design, were completed. This article reports on the experimental design along with the results for CCl(4), biomass, acetate, nitrate, and nitrite concentrations. These data indicate that growth is inhibited by high nitrite concentrations, whereas CCl(4) degradation is slowed by the presence of nitrate and/or nitrite. (c) 1994 John Wiley & Sons, Inc.

Size changes associated with metal adsorption onto modified bone gelatin beads.

Significant quantities of heavy metals will adsorb onto modified bone gelatin beads. As this adsorption occurs, the bead can undergo a substantial volume change. Research has shown that the equilibrium bead diameter was a function of the solution pH and the ion concentration in the solution. Here, we demonstrate that under certain conditions, the volume of the beads that absorbed the metal was only 35% of the bead volume when no metal was adsorbed. By taking advantages of these size changes, a fluidized-bed separator can be operated such that natural segregation of loaded beads occurs. This phenomenon may facilitate the design of continous separators for the recovery and concentration of heavy-metal-contaminated waters. These concepts are demonstrated using Cu(2+) adsorption onto such beads.

Control and optimization of apheresis procedures in a COBE 2997 cell separator.

To obtain more efficient operation of a COBE Model 2997 clinical cell separator using either a Single Stage II (SS II) or a Dual Stage separation chamber, modifications were made to allow complete computer control. Product cell density was detected using an optical sensor and controlled by automatic feedback through a microcomputer interface. Control was accomplished by automatically adjusting the red blood cell (RBC) and plasma product flow rates using a proportional-integral (PI) algorithm. Results show that, using either chamber, the product cell density can be maintained at a preselected value for extended periods of time without operator intervention. This system allowed investigation of optimal operating regions for plateletpheresis and leukapheresis procedures. The effects of centrifuge rpm and controller set point on centrifuge operation were investigated using a second order factorial experimental design. Theoretical significance of model parameters was assessed with the aid of a hindered settling model and simple reasoning about the interface position relative to the collection port. The results suggest that, in either chamber, the optimum operating region for plateletpheresis procedures occurs at moderate controller set points and high centrifuge rpm. The resultant operating efficiency and product purity values are approximately 63 percent and 0.65 respectively in the SS II chamber and approximately 70 percent and 0.70 respectively in the Dual Chamber. In the SS II, the optimum operating region for leukapheresis procedures occurred at high controller set point values for any centrifuge rpm above 1200 with an operating efficiency near 100 percent. However, in the Dual Chamber, the optimum operating region for leukapheresis procedures occurred at high controller set points and high centrifuge rpm's, again providing an operating efficiency near 100 percent.

Minimizing the errors associated with the determination of effective diffusion coefficients when using spherical cell immobilization matrices.

Gel materials are often used to entrap biological catalysts. Several experimental methods have been proposed to estimate the diffusion coefficient of important chemical species within these materials. An error analysis for the bead method was performed and, contrary to previously reported results, when proper experimental conditions were employed, the error associated with the bead method was similar to that obtained using the other common methods.

Dynamic on-line optimization of a bioreactor.

An algorithm was developed which uses recursive least squares to identify a dynamic, discrete time model of a poorly defined system and uses both the dynamic and static portions of the identified model for on-line optimization. To test this new algorithm, a model of an continuous biochemical reactor was used as the "process." The objective, here, was to maximize ethanol production from the reactor by manipulating the feed rate to the reactor. The new algorithm, which uses dynamic information, was found to be superior to previously published algorithms which use only the steady-state portion of the identified model.

Interface dynamics in a centrifugal cell separator.

Experiments were performed with bovine blood to study the response of the plasma/cell interface position to pumping rate adjustments in two single-stage (SS I and SS II) cell separation chambers. Standard clinical instrumentation and equipment and on-line computer interfacing were used to monitor and control the interface position. The data provided a quantitative description of its dynamics in the SS I and the SS II chambers. In the SS I chamber, adjustments in the flow rate caused the interface to move very slowly and in a complex manner from one steady-state operation position to another. Such behavior made both manual and computer-controlled operation very difficult. By contrast, the SS II chamber was inherently unstable for most operating conditions. We demonstrated, however, that a feedback controller could be used easily to adjust or maintain the interface position, and this system moved from one steady-state operating condition to another 10 times as fast as the system using the SS I chamber. Also, the manner in which the controller allowed the system to respond to operator requests was much simplier than that for the SS I system.

The effects of RPM and recycle on separation efficiency in a clinical blood cell centrifuge.

A COBE blood cell centrifuge, model 2997 with a single stage channel, was modified to allow computer controlled sampling, and to allow recycle of red blood cells (RBCs) and plasma streams using bovine whole blood. The effects of recycle of the packed RBC and plasma product streams, and of the centrifuge RPM on platelet and white blood cell (WBC) separation efficiencies were quantified using a central composite factorial experimental design. These data were then fit using second order models. Both the model for the WBC separation efficiency and the model for the platelet separation efficiency predict that RPM has the greatest effect on separation efficiency and that RBC and plasma recycle have detrimental effects at moderate to low RPM, but have negligible impact on separation efficiency at high RPM.