Biocatalytic transformation of [(2-Hydroxyethyl)thio]acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxydans ssp. xylosoxydans (SH91).

A Gram-negative bacterium, Alcaligenes xylosoxydans ssp. xylosoxydans (SH91), consumed thiodiglycol (TDG), the nontoxic hydrolysis product of sulfur mustard, as a primary carbon source and transformed TDG to commercially relevant chemical precursors, [(2-hydroxyethyl)thio]acetic acid (HETA) and thiodiglycolic acid (TDGA). Aerobic fed batch and repeated batch experiments were run to compare the molar yields of HETA and TDGA that result under different operating policies. In repeated batch experiments, 35% of the TDG was converted to HETA. Under the conventional batch process and a repeated fed batch process, the HETA yields were reduced (21% and 18%, respectively), while the yield of TDGA was increased (47% and 31%,respectively). This work demonstrated that cell growth associated biocatalytic transformations were manipulated to achieve a desired byproducts profile through an understanding of the specific reaction and cell growth kinetics and by altering the reaction operating policy accordingly.

Framework for online optimization of recombinant protein expression in high-cell-density Escherichia coli cultures using GFP-fusion monitoring.

A framework for the online optimization of protein induction using green fluorescent protein (GFP)-monitoring technology was developed for high-cell-density cultivation of Escherichia coli. A simple and unstructured mathematical model was developed that described well the dynamics of cloned chloramphenicol acetyltransferase (CAT) production in E. coli JM105 was developed. A sequential quadratic programming (SQP) optimization algorithm was used to estimate model parameter values and to solve optimal open-loop control problems for piecewise control of inducer feed rates that maximize productivity. The optimal inducer feeding profile for an arabinose induction system was different from that of an isopropyl-beta-D-thiogalactopyranoside (IPTG) induction system. Also, model-based online parameter estimation and online optimization algorithms were developed to determine optimal inducer feeding rates for eventual use of a feedback signal from a GFP fluorescence probe (direct product monitoring with 95-minute time delay). Because the numerical algorithms required minimal processing time, the potential for product-based and model-based online optimal control methodology can be realized.

Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor.

Synthesis of an operon fusion protein was investigated in batch and fed-batch cultures at high cell densities of recombinant Escherichia coli JM105 [pBAD-GFP::CAT]. Glucose-limited growth was achieved without accumulation of inhibitory byproducts allowing high cell densities (110 g L(-1) DCW) to be attained. This was believed to be the highest reported value for dry cell mass of E. coli strain JM105 expressing two recombinant proteins. Transcription of the two reporter genes, green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT), was under the control of the p(BAD) promoter of the araBAD (arabinose) operon. Each protein was independently translated via separate ribosome binding sites. CAT served as a model recombinant protein product to illustrate the noninvasive quantitative reporting ability of GFP during high cell density fermentations. Expression of GFP was monitored on-line using an intensity-based optical sensor. A linear correlation between the on-line GFP intensity and the enzymatic activity of CAT allowed for in vivo real-time quantitative monitoring of a fermentation product under conditions of high biomass concentration and high productivity.

Observations of metabolite formation and variable yield in thiodiglycol biodegradation process : impact on reactor design.

The complete microbial degradation of thiodiglycol (TDG), the primary hydrolysis product of sulfur mustard, by Alcaligenes xylosoxydans ssp. xylosoxydans (SH91) was accomplished in laboratory-scale stirredtank reactors. An Andrews substrate inhibition model was used to describe the cell growth. The yield factor was not constant, but a relationship with initial substrate concentration has been developed. Using a substrate-inhibition and variable-yield kinetic model, we can describe the cell growth and substrate consumption in batch and repeated batch fermentations. Several reactor-operating modes successfully degrade TDG concentration to below 0.5 g/L. According to the experimental results, the two-stage repeated batch operation has the best degradation efficiency, and it also can degrade 500 mM TDG (= 60 g/L) to 5 mM (= 0.7 g/L) in <5 d. A hypothesis for explaining variable-yield and byproduct formation based on the capacity and utilization of metabolic loads is presented.

Bioreactor strategies for the treatment of growth-inhibitory waste: an analysis of thiodiglycol degradation, the main hydrolysis product of sulfur mustard.

The microbial degradation of thiodiglycol, the primary hydrolysis product of sulfur mustard, by a pure culture of Alcaligenes xylosoxydans ssp. xylosoxydans (SH91) was accomplished in laboratory scale stirred tank reactors. This is a major component of the overall biodegradation process proposed for the complete mineralization of sulfur mustard. Several configurations were evaluated for degradation efficiency including batch, repeated batch, continuous stirred tank reactor (CSTR), and two-stage series CSTR. The repeated batch reactor provided the highest degradation rate of thiodiglycol. Further, this method degraded thiodiglycol in the liquid broth to below the detection limits (0.03 mM). Both batch and repeated batch experiments were simulated by an unstructured mathematical model. Simulation results were in agreement with the experimental data, particularly at low TDG concentration (around 30 mM). This study demonstrates the degradation of thiodiglycol using bioreactors and, more generally, is an experimental study of bioreactor designs for the degradation of growth-inhibitory substances.

Reactor comparisons for the biodegradation of thiodiglycol, a product of mustard gas hydrolysis.

An environmentally benign method for the mineralization of sulfur mustard has been proposed involving chemical hydrolysis of sulfur mustard to thiodiglycol, and then the biological degradation of thiodiglycol to generate biomass and gaseous carbon dioxide. Alcaligenes xylosoxidans (SH91) was isolated based on its ability to utilize thiodiglycol as a sole carbon source. This article compares different biological reactor designs and experimentally assesses their relative effectiveness in degrading thiodiglycol using pure cultures of SH91. The reactor configurations studied are batch, continuous stirred-tank reactor (CSTR), and CSTR with cell recycle. From the results, it is clear that the CSTR with cell recycle offers superior performance for a given residence time or volume. These pure culture data are necessary for accurate design of a pilot-scale system where mixed cultures will be employed because of a possible incomplete chemical hydrolysis step.

The effect of cellular energetics on foreign protein production.

Escherichia coli strain F-122 was used to determine if there are additional physiological effects, other than decreasing energetic efficiency accompanied by the excretion of the acetate, on foreign protein production. This organism was the host for expressing HIV582-beta-galactosidase fusion protein under the control of the trp promoter, with ampicillin resistance. By comparing parallel batch cultures with and without acetate addition, it was found that the presence of acetate in the media did not influence beta-galactosidase activity. In these experiments, it appears that the low protein productivity often observed during acetate formation is the result of inefficient cell metabolism, rather than acetate acting as a specific inhibitor of protein production.

Optimization for a recombinant E. coli fed-batch fermentation.

The operating strategy that produces the maximum foreign protein expression for a fed-batch process is desired. This is achieved by using a feasible quadratic programming (FSQP) algorithm with a structured model that describes cell growth and product formation for recombinant E. coli. Optimization calculations for a fed-batch culture have not been performed with a model of this complexity up to this point. A constraint on the maximum cell concentration was included. For a fixed value of batch time, the results show that the optimal time profile of feed flow rate can increase the yield of foreign protein by 12-29% over a constant feed rate policy. Also, it was found that the computation time for the FSQP algorithm can be reduced significantly by considering suboptimal profiles of the feed rate, with a minor effect on calculated protein yield.

A metabolic model of cellular energetics and carbon flux during aerobic Escherichia coli fermentation.

An integrated metabolic model for the production of acetate by Escherichia coli growing on glucose under aerobic conditions was presented previously (Ko et al., 1993). The resulting model equations can be used to explain phenomena often observed with industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low dissolved oxygen concentration, a high specific growth rate, or a combination of these conditions. However, several questions still need to be addressed. First, cell composition is growth rate and media dependent. Second, the macromolecular composition varied between E. coli strains. And finally, a model that represents the carbon fluxes between the Embden-Meyerhof-Parnas (EMP) and the hexose monophosphate (HMP) pathways when cells are subject to internal and/or external stresses is still not well defined. In the present work, we have made an effort to account for these effects, and the resulting model equations show good agreement for wild-type and recombinant E. coli experimental data for the acetate concentration, the onset of acetate secretion, and cell yield based on glucose. These results are useful for optimizing aerobic E. coli fermentation processes. More specifically, we have determined the EMP pathway carbon flux profiles required by the integrated metabolic model for an accurate fit of the acetic acid profile data from a wild-type E. coli strain ML308. These EMP carbon flux profiles were correlated with a dimensionless measurement of biomass and then used to predict the acetic acid profiles for E. coli strain F-122 expressing human immunodeficiency virus-(HIV(528)) beta-galactosidase fusion protein. The effect of different macromolecular compositions and growth rates between these two E. coli strains required a constant scaling factor for improved quantitative predictions.

An integrated metabolic modeling approach to describe the energy efficiency of Escherichia coli fermentations under oxygen-limited conditions: Cellular energetics, carbon flux, and acetate production.

An integrated metabolic model for the production of acetate by growing Escherichia coli on glucose under aerobic conditions is presented. The model is based on parameters which are easily determined by experiments. Forming the basis for this integrated metabolic model are the 12 principal precursor metabolites for biosynthetic pathways, the Embden-Meyerhof-Parnas pathway, the pentose phosphate cycle, the tricarboxylic acid cycle and the anapleurotic reactions, the Crabtree effect, the Pasteur effect, and the details of bacterial respiration. The result can be used to explain phenomena often observed in industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low oxygen concentration, a high specific growth rate, or a combination of these conditions.

Simple structured model for alpha-amylase synthesis by Bacillus amyloliquefaciens.

A predictive, simple, structured model describing the synthesis of alpha-amylase by Bacillus amyloliquefaciens was formulated. Three key intracellular processes were identified (i.e, translation, and excretion) along with two key intracellular components (i.e., mRNA and the intracellular form of the alpha-amylase enzyme). Nearly all the model parameters were estimated by means of performing independent experiments, primarily fed-batch experiments. The model was shown to predict transient system behavior in batch and in fed-batch operation with some limitation and minor model parameter revisions. Since a principal objective was to demonstrate that independent experimental parameter determination can be used to construct the predictive model, further fine-tuning of the parameters may be necessary before application for optimization and control purposes.

Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5].

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.

On the mechanism of growth of cells (Bacillus amyloliquefaciens) in the mixed aqueous two-phase system.

The growth of Bacillus amyloliquefaciens in the aqueous two-phase system, made up of polyethylene glycol, dextran, and water, was investigated. Generally, Bacillus partitions in the dextran phase, but the magnitude of the separation depends largely on the overall composition of polymers in the phase system. The kinetics of growth of Bacillus amyloliquefaciens was studied in the polyethylene glycol-rich continuous phase, dextran-rich dispersed phase, and in the mixed phase. From the kinetic data it appears that increasing the overall polymer composition causes the cells to adsorp at the interface. On the other hand, partition measurements indicate that increasing polymer concentrations make the cell partitioning more one-sided. This anomaly is explained by studying the interfacial adsorption of cells via dynamic surface tension measurements.

Cell growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens.

Growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.

Effects of recombinant plasmid size on cellular processes in Escherichia coli.

The effects of recombinant plasmid size on cell growth and viability, plasmid copy number, and synthesis of plasmid-encoded protein were investigated in Escherichia coli using plasmid pUC8 and four recombinant derivatives containing inserts of Drosophila melanogaster DNA of 1.7-6.0 kb. Growth in log phase was unaffected by plasmid size, but as plasmid size increased, maximum cell density decreased and, with the largest plasmid, cell death was accelerated after the stationary phase was reached. There was also a correlation between increasing plasmid size and decreased viability at high ampicillin concentrations, resistance to which is conferred by the plasmids. These effects were shown not to be due to transcription or translation of Drosophila sequences carried on the recombinant plasmids. Cells harboring the largest plasmid, pBS5 (8.7 kb), fared poorly in competition with plasmid-free cells in mixed cultures, compared with cells harboring pUC8 (2.7 kb). In addition, pBS5 was harbored at significantly fewer copies per cell than pUC8 at all phases of growth and supported much less production of the plasmid-encoded protein, beta-lactamase, than did pUC8. The results suggest that recombinant plasmid size may be an important parameter in the optimization of large-scale production of plasmid-encoded proteins.

Development of a direct digital-controlled fermentor using a microminicomputer hierarchical system.

A computerized fermentation system was developed for use in a research environment. Major goals in the design and construction of the system were flexibility and versatility. Direct digital control is employed for all low-level and high-level control loops. A microminicomputer hierarchical configuration is used to implement this control structure. The microcomputer is also utilized to simplify the interface with the fermentor, both at the hardware and minicomputer software levels. This computerized fermentation system provides accurate data acquisition, excellent control, and flexibility in the fermentation operation.