Residual Substrate Concentration Research Articles

Effect of Endogenous Methane Production: A Step Forward in the Validation of Biochemical Methane Potential (BMP) Tests

This work evaluates the influence of the inoculum type, the pre-consumption of the residual substrate and the ratio of blanks’ headspace volume to working volume (Hv Wv−1, 0.6 to 10) on Biochemical Methane Potential (BMP) measurements when methane is monitored by gas chromatography. Different inocula were tested: digested sewage sludge—DSS, granular sludge—GS and fresh dairy manure—DM. Microcrystalline cellulose was used as the substrate. BMP surpassed the maximum theoretical value (BMPmax = 414 L kg−1) when methane produced in the blanks was not discounted, showing that degassing cannot stand alone as an alternative to the procedure of discounting the inoculum’s background production. Still, when the residual substrate concentration is high (e.g., in DM), degassing is mandatory because methane produced from its digestion will conceal the methane produced from the substrate in the BMP determination. For inocula with a low residual substrate (e.g., GS), short degassing periods are recommended in order to avoid detrimental effects on methanogenic activity. For moderate residual substrate concentrations (e.g., DSS), BMP values closer to BMPmax (90–97%) were achieved after degassing and discounting the blanks with lower Hv Wv−1. For higher Hv ∙ Wv−1, less accurate quantification occurred, likely due to error propagation. Proper inoculum pre-incubation time and discounting the methane production from blanks with low Hv Wv−1 (adjusted according to the estimated background methane) are essential for accurate BMP determinations.

Toward Improved Bioremediation Strategies: Response of BAM-Degradation Activity to Concentration and Flow Changes in an Inoculated Bench-Scale Sediment Tank.

Compound-specific isotope analysis (CSIA) can reveal mass-transfer limitations during biodegradation of organic pollutants by enabling the detection of masked isotope fractionation. Here, we applied CSIA to monitor the adaptive response of bacterial degradation in inoculated sediment to low contaminant concentrations over time. We characterized Aminobacter sp. MSH1 activity in a flow-through sediment tank in response to a transient supply of elevated 2,6-dichlorobenzamide (BAM) concentrations as a priming strategy and took advantage of an inadvertent intermittence to investigate the effect of short-term flow fluctuations. Priming and flow fluctuations yielded improved biodegradation performance and increased biodegradation capacity, as evaluated from bacterial activity and residual concentration time series. However, changes in isotope ratios in space and over time evidenced that mass transfer became increasingly limiting for degradation of BAM at low concentrations under such stimulated conditions, and that activity decreased further due to bacterial adaptation at low BAM (μg/L) levels. Isotope ratios, in conjunction with residual substrate concentrations, therefore helped identifying underlying limitations of biodegradation in such a stimulated system, offering important insight for future optimization of remediation schemes.

Effect of the Progressive Increase of Organic Loading Rate in an Anaerobic Sequencing Batch Reactor for Biodiesel Wastewater Treatment

The wastewater from the biodiesel industry is an environmental problem, and from a sanitation resources perspective, the anaerobic sequencing batch reactor (ASBR) is an interesting alternative for wastewater treatment. A better understanding of ASBR operation behavior under the progressive increase of the organic loading rate (OLR) is crucial for upscaling. The objective of this study was to monitor an ASBR operating with an OLR ranging from 1.3 to 9.3 kgCOD m−3 d−1. The average chemical oxygen demand (COD) removal efficiencies of the ASBR were 52, 41, 47, and 11% for phases 1, 2, 3, and 4, respectively. The apparent kinetic coefficient, i.e., the rate of degradation of organic matter, was between 0.10 and 1.80 h−1, considering the kinetic model that considers the residual substrate concentration, which was the one that best fit the obtained data. The progressive increase in applied OLR modified the microbial biomass diversity, which in turn influenced the degradation kinetics of the organic matter. In addition, the values of the applied OLR of 5.1 kgCOD m−3 d−1 and a food to microorganism ratio (F/M) of 0.6 kgCOD kgVSS−1 d−1 were shown to be limiting values that promoted the overload of ASBR.

Homogeneous solid dispersion (HSD) system for rapid and stable production of succinic acid from lignocellulosic hydrolysate.

Continuous bio-production of succinic acid was reported in homogeneous solid dispersion (HSD) system utilizing porous coconut shell activated carbon (CSAC) as immobilization carrier. The aim of the present work was to implement the HSD system to increase the area of cell immobilization and the rate of succinic-acid production from the lignocellulosic medium. The ratio of the two enzymes (cellulase-to-hemicellulase) was initially optimized to break down the lignocellulose into its free monomers, wherein the best ratio was determined as 4:1. Succinic-acid production was evaluated in the HSD system by varying the substrate loading and dilution rate. The results showed that high productivities of succinic acid were obtained when 60g/L glucose was fed over a dilution rates ranging from 0.03 to 0.4/h. The titer of succinic acid decreased gradually with higher dilution rate, whereas the residual substrate concentration increased with it. Critical dilution rate was determined to be 0.4/h at which the best productivity of succinic acid of 6.58g/Lh and its yield of 0.66g/g were achieved using oil palm fronds (OPF) hydrolysate. This work lends evidence to the use of CSAC and lignocellulosic hydrolysate to further exploit the potential economies of scale.

The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae

Backgroundl-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains.ResultsChemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10−3 and 1.8 g L−1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L−1l-arabinose and 20 g L−1d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2.ConclusionIts high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

A Kinetic Study of Biogas Produced from Cow and Elephant Dungs Using the Residual Substrate Concentration Approach

Kinetic investigations of biogas produced from Cow dung and Elephant dung were carried out. Three samples tagged: 1 (100% Cow dung), 2 (50% Cow dung and 50% Elephant dung) and 3 (100% Elephant dung) were investigated in prototype batch bio-digesters using anaerobic digestion process. In addition, the kinetic studies of the three samples investigated were carried out. Results obtained revealed that Sample ‘2’, the co-digestion of Cow and Elephant dung gave the best cumulative biogas production of 3.92*10-4 g/cm3 and average yield of 0.084 5 over a period of 33 days. The kinetic of the process followed a shifting order (0-1); the kinetic parameters obtained can be used to size bio-digester and also monitor the rate of biogas produced. Therefore, the co-digestion of Elephant and Cow dung in equal proportion can be used to increase yield of biogas.

A dynamic method for the investigation of induced state metabolic capacities as a function of temperature

BackgroundScience-based recombinant bioprocess designs as well as the design of statistical experimental plans for process optimization (Design of Experiments, DoE) demand information on physiological bioprocess boundaries, such as the onset of acetate production, adaptation times, mixed feed metabolic capabilities or induced state maximum metabolic rates as at the desired cultivation temperature. Dynamic methods provide experimental alternatives to determine this information in a fast and efficient way. Information on maximum metabolic capabilities as a function of temperature is needed in case a reduced cultivation temperature is desirable (e.g. to avoid inclusion body formation) and an appropriate feeding profile is to be designed.ResultsHere, we present a novel dynamic method for the determination of the specific growth rate as a function of temperature for induced recombinant bacterial bioprocesses. The method is based on the control of the residual substrate concentration at non-limiting conditions with dynamic changes in cultivation temperature. The presented method was automated in respect to information extraction and closed loop control by means of in-line Fourier Transformation Infrared Spectroscopy (FTIR) residual substrate measurements and on-line first principle rate-based soft-sensors. Maximum induced state metabolic capabilities as a function of temperature were successfully extracted for a recombinant E. coli C41 fed-batch bioprocess without the need for sampling in a time frame of 20 hours.ConclusionsThe presented method was concluded to allow the fast and automated extraction of maximum metabolic capabilities (specific growth rate) as a function of temperature. This complements the dynamic toolset necessary for science-based recombinant bacterial bioprocess design and DoE design.

Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms

Concentrations of biosynthate (microbial biomass plus extracellular proteins) and residual substrate were inferred using elemental analysis for batch cultures of Clostridium thermocellum. Inferring residual substrate based on elemental analysis for a cellulose (Avicel)-grown culture shows similar results to residual substrate determined by quantitative saccharification using acid hydrolysis. Inference based on elemental analysis is also compared to different on-line measurements: base addition, CO2 production, and Near Infra Red optical density (OD850 ). Of these three on-line techniques, NIR OD850 has the best correlation with residual substrate concentration and is the most practical to use. Both biosynthate and residual substrate concentration demonstrate typical sigmoidal trends that can easily be fitted with a five-parameter Richards curve. The sigmoidal character of the inferred concentrations and on-line data, especially the CO2 production rate, suggest that there is a maximum in cell-specific rates of growth and substrate utilization during batch fermentations of crystalline cellulose, which is not observed during grown on cellobiose. Using a sigmoidal fit curve, the instantaneous specific growth rate was determined. While soluble substrate grown cultures show a constant growth rate, cultures grown on solid substrate do not. Features of various approaches are compared, with some more appropriate for rapid general indication of metabolic activity and some more appropriate for quantitative physiological studies.

Testing alternative kinetic models for utilization of crystalline cellulose (Avicel) by batch cultures of Clostridium thermocellum

Descriptive kinetics of batch cellulose (Avicel) and cellobiose fermentation by Clostridium thermocellum were examined with residual substrate and biosynthate concentrations inferred based on elemental analysis. Biosynthate was formed in constant proportion to substrate consumption until substrate was exhausted for cellobiose fermentation, and until near the point of substrate exhaustion for cellulose fermentation. Cell yields (g pellet biosynthate carbon/g substrate carbon) of 0.214 and 0.200 were obtained for cellulose and cellobiose, respectively. For cellulose fermentation a sigmoidal curve fit was applied to substrate and biosynthate concentrations over time, which was then differentiated to calculate instantaneous rates of growth and substrate consumption. Three models were tested to describe the kinetics of Avicel utilization by C. thermocellum: (A) first order in cells, (B) first order in substrate, and (C) first order in cells and substrate, and second order overall. Models (A) and (B) have been proposed in the literature to describe cultures of cellulolytic microorganisms, whereas model (C) has not. Of the three models tested, model (c) provided by far the best fit to batch culture data. A second order rate constant equal to 0.735 L g C(-1) h(-1) was found for utilization of Avicel by C. thermocellum. Adding an endogenous metabolism term improved the descriptive quality of the model as substrate exhaustion was approached. Such rate constants may in the future find utility for describing and comparing cellulose fermentation involving other microbes and other substrates.

Multiple Objective Optimisation of Batch Cultivation of Saccharomyces Cerevisiae in Mixing Systems

ABSTRACTMultiple objective optimisation is a natural extension of the traditional optimisation of a single objective function. On the one hand, if the multiple objective functions are commensurate, minimizing a single objective function, it is possible to minimize all the criteria and the problem can be solved using traditional optimisation techniques. On the other hand, if the objective functions are incommensurate or competing, then the minimization of one objective function requires a compromise in another objective function. Here we discuss the problems of multiple objective optimisation of batch cultivation of Saccharomyces cerevisiae in different mixing systems (impulse and vibromixing). The multiple objective optimisation problems are transformed to a single objective function with weight coefficients. A combined algorithm is applied for solving the single optimisation. The applied multiple objective optimisation of the process showed a vast increase in the productivity and, respectively, decrease in the residual substrate concentration.

Mixed culture polyhydroxyalkanoates production from sugar molasses: The use of a 2-stage CSTR system for culture selection

Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers. The use of mixed microbial cultures (MMC) and low cost feedstocks have a positive impact on the cost-effectiveness of the process. It has typically been carried out in Sequencing Batch Reactors (SBR). In this study, a 2-stage CSTR system (under Feast and Famine conditions) was used to effectively select for PHA-storing organisms using fermented molasses as feedstock. The effect of influent substrate concentration (60–120 Cmmol VFA/L) and HRT ratio between the reactors (0.2–0.5 h/h) on the system’s selection efficiency was assessed. It was shown that Feast reactor residual substrate concentration impacted on the selective pressure for PHA storage (due to substrate-dependent kinetic limitation). Moreover, a residual substrate concentration coming from the Feast to the Famine reactor did not jeopardize the physiological adaptation required for enhanced PHA storage. The culture reached a maximum PHA content of 61%. This success opens new perspectives to the use of wastewater treatment infrastructure for PHA production, thus valorizing either excess sludge or wastewaters.

Prediction of multi-components (chlorine, biomass and substrate concentrations) in water distribution systems using artificial neural network (ANN) models

Artificial Neural Networks (ANN) models are used to predict residual chlorine, substrate and biomass concentrations in a Water Distribution System (WDS). ANN models with different architectures are developed: a one output ANN model (predicting chlorine, substrate and biomass individually), a two output ANN model (predicting chlorine + substrate, chlorine + biomass or substrate + biomass) and a three output ANN model (chlorine + substrate + biomass). This study is carried out for the Bangalore City and North Marin WDSs. Data for these WDSs is obtained from the multi-component reaction transport model. The models are compared using the correlation coefficient (R) and the Mean Absolute Error (MAE). The models developed are able to predict, reasonably well, the temporal variations in the chlorine, substrate and biomass concentrations. Error analysis is carried out to determine the robustness of the models.

Lactic acid production by Lactobacillus sp. RKY2 in a cell-recycle continuous fermentation using lignocellulosic hydrolyzates as inexpensive raw materials

Continuous lactic acid fermentations were conducted using lignocellulosic hydrolyzates and corn steep liquor as inexpensive raw materials. Lactic acid concentrations decreased with increases in the dilution rate, whereas the residual substrate concentrations increased. However, lactic acid yields were maintained at more than 0.90 g g −1 over all cases experimented. The cell-recycle cultivation system exerted positive effects on fermentation efficiency, including volumetric productivity, which is attributable to the retention of cells in the bioreactor. The cell-recycle continuous fermentation of lignocellulosic hydrolyzates yielded a lactic acid productivity of 6.7 g l −1 h −1 for a dilution rate of 0.16 h −1 using 30 g l −1 of corn steep liquor and 1.5 g l −1 of yeast extract as nutrients. The productivity (6.7 g l −1 h −1) acquired by the cell-recycle continuous fermentation of lignocellulosic hydrolyzates was 1.6 times higher than the lactic acid productivity yielded in the continuous fermentation without cell-recycle system.

Development and application of kinetic models for the primary biodegradation of non-ionic surfactants

The growing concern for the environment is promoting the use of surfactant products from renewable sources such as fatty alcohol ethoxylates. The high production and use of these products implies the need to develop models that enable predictions of their behaviour in biodegradation processes. The biodegradation tests were carried out according to the OECD 301 E test for ready biodegradability. In this work, kinetic models of general application to surfactant biodegradation are developed, both for substrates that do not support growth and for those that do, considering a residual substrate concentration as not being biodegraded. The models were applied to three commercial non-ionic surfactants, fatty alcohol ethoxylates with different carbon-chain lengths and degrees of ethoxylation, also analysing the initial surfactant concentration.

A model forL-methionine production describing oxygen-productivity relationship

BACKGROUND: The state-time profile of cell mass, substrate and methionine concentrations of a methionine synthesis process shows strongly nonlinear features. A mathematical representation of this process was developed that conformed to systems analysis required for monitoring and controlling methionine production. The specific growth rate was defined by an exponential term to describe the lag phase in growth, extended before the onset of methionine production and substrate inhibition observed for this process. A switching function was used to describe the relation between methionine synthesis and dissolved oxygen concentration. In addition, the product formation kinetics of this model described the reutilization of methionine feedback regulation whenever the residual substrate concentration dropped below a critical value. RESULTS: The parameters for the model were determined from experimental data using a nonlinear regression technique. A complete nonlinear systems analysis of the model proved that using this model, the system was controllable and observable. The model prediction of methionine production in controlled and uncontrolled environments was satisfactory. Six statistical measures were employed to validate model prediction and its adequacy was shown through simulations. CONCLUSIONS: The proposed model for methionine production possesses the correct system architecture for application in process control. It predicts satisfactorily the relationship between methionine synthesis and dissolved oxygen, and time profiles of state variables. Copyright © 2008 Society of Chemical Industry

Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.

Mass production of bacterial communities adapted to the degradation of volatile organic compounds (TEX)

This study focuses on the mass cultivation of bacteria adapted to the degradation of a mixture composed of toluene, ethylbenzene, o-, m- and p-xylenes (TEX). For the cultivation process Substrate Pulse Batch (SPB) technique was adapted under well-automated conditions. The key parameters to be monitored were handled by LabVIEW software including, temperature, pH, dissolved oxygen and turbidity. Other parameters, such as biomass, ammonium or residual substrate concentrations needed offline measurements. SPB technique has been successfully tested experimentally on TEX. The overall behavior of the mixed bacterial population was observed and discussed along the cultivation process. Carbon and nitrogen limitations were shown to affect the integrity of the bacterial cells as well as their production of exopolymeric substances (EPS). Average productivity and yield values successfully reached the industrial specifications, which were 0.45 kg(DW)m(-3) d(-1) and 0.59 g(DW)g (C) (-1) , respectively. Accuracy and reproducibility of the obtained results present the controlled SPB process as a feasible technique.

Biological phosphate removal by model based continuous cultivation of Acinetobacter calcoaceticus

Batch cultivation of Acinetobacter calcoaceticus was carried out under statistically optimized culture conditions in order to study the kinetics of growth and phosphate uptake. It featured a biomass accumulation of 4.08 g L −1 with a consumption of 0.14 g L −1 phosphate (from initial phosphate concentration of 0.18 g L −1) in 11 h. Inhibition studies with respect to carbon, nitrogen and phosphate were also performed to quantify their limiting and/or inhibitory effect on specific growth rate. Using above kinetics/inhibition data, mathematical model equations were proposed and then model parameters were identified for batch cultivation of A. calcoaceticus. The batch model was extrapolated for continuous cultivation so as to predict, steady state concentrations of process variables and identify suitable nutrient feeding strategies for even better/quicker phosphate removal. In order to make a comprehensive study of the effect of dilution rate on culture growth and phosphate uptake, different dilution rates of 0.04, 0.09, 0.13, 0.19, 0.3, 0.4 h −1 were utilized in the model to predict transient and steady state data, which were experimentally verified to establish the validity of the model. It was observed that as the dilution rate increased, the steady state biomass concentration gradually decreased and residual substrate concentration increased, indicating the applicability of well-known Monod type growth pattern. Best biomass accumulation (3.5 g L −1) and phosphate uptake (∼76%) was obtained at D = 0.09 h −1 experimentally. Studies in a continuous stirred tank reactor (CSTR) demonstrated the feasibility to achieve faster phosphate removal without inherently slow anaerobic phase involvement.

Microbial in situ degradation of aromatic hydrocarbons in a contaminated aquifer monitored by carbon isotope fractionation

We present an approach for characterizing in situ microbial degradation using the 13C/ 12C isotope fractionation of contaminants as an indicator of biodegradation. The 13C/ 12C isotope fractionation of aromatic hydrocarbons was studied in anoxic laboratory soil percolation columns with toluene or o-xylene as the sole carbon and electron source, and sulfate as electron acceptor. After approximately 2 months' of incubation, the soil microbial community degraded 32 mg toluene l −1 and 44 mg o-xylene l −1 to less than 0.05 mg l −1, generating a stable concentration gradient in the column. The 13C/ 12C isotope ratio in the residual non-degraded fraction of toluene and o-xylene increased significantly, corresponding to isotope fractionation factors (αC) of 1.0015 and 1.0011, respectively. When the extent of biodegradation in the soil column was calculated based on the measured isotope ratios ( R t ) and an isotope fractionation factor (αC=1.0017) obtained from a sulfate-reducing batch culture the theoretical residual substrate concentrations ( C t ) matched the measured toluene concentrations in the column. This indicated that a calculation of biodegradation based on isotope fractionation could work in systems like soil columns. In a field study, a polluted, anoxic aquifer was analyzed for BTEX and PAH contaminants. These compounds were found to exhibit a significant concentration gradient along an 800-m groundwater flow path downstream of the source of contamination. A distinct increase in the carbon isotope ratio ( δ 13C) was observed for the residual non-degraded toluene (7.2‰), o-xylene (8.1‰) and naphthalene fractions (1.2‰). Based on the isotope values and the laboratory-derived isotope fractionation factors for toluene and o-xylene, the extent to which the residual substrate fraction in the monitoring wells had been degraded by microorganisms was calculated. The results revealed significant biodegradation along the groundwater flow path. In the wells at the end of the plume, the bioavailable toluene and o-xylene fractions had been almost completely reduced by in situ microbial degradation. Although indane and indene showed decreasing concentrations downstream of the groundwater flow path, suggesting microbial degradation, their carbon isotope ratios remained constant. As the physical properties of these compounds are similar to those of BTEX compounds, the constant isotope values of indane and indene indicated that microbial degradation did not lead to isotope fractionation of all aromatic hydrocarbons. In addition, physical interaction with the aquifer material during the groundwater passage did not significantly alter the carbon isotope composition of aromatic hydrocarbons.

Multicriteria optimization of gluconic acid production using net flow.

The biochemical process industry is often confronted with the challenge of making decisions in an atmosphere of multiple and conflicting objectives. Recent innovations in the field of operations research and systems science have yielded rigorous multicriteria optimization techniques that can be successfully applied to the field of biochemical engineering. These techniques incorporate the expert's experience into the optimization routine and provide valuable information about the zone of possible solutions. This paper presents a multicriteria optimization strategy that generates a Pareto domain, given a set of conflicting objective criteria, and determines the optimal operating region for the production of gluconic acid using the net flow method (NFM). The objective criteria include maximizing the productivity and concentration of gluconic acid, while minimizing the residual substrate. Three optimization strategies are considered. The first two strategies identify the optimal operating region for the process inputs. The results yielded an acceptable compromise between productivity, gluconic acid concentration and residual substrate concentration. Fixing the process inputs representing the batch time, initial substrate concentration and initial biomass equal to their optimal values, the remaining simulations were used to study the sensitivity of the optimum operating region to changes in the oxygen mass transfer coefficient, K(L) a, by utilizing a multi-level K(L) a strategy. The results show that controlling K(L) a during the reaction reduced the production of biomass, which in turn resulted in increased productivity and concentration of gluconic acid above that of a fixed K(L) a.