Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus

Lavendamycin methyl ester (LME) is a derivative of a highly functionalized aminoquinone alkaloid lavendamycin and could be used as a scaffold for novel anticancer agent development. This work demonstrated LME production by cultivation of an engineered strain of Streptomyces flocculus CGMCC4.1223 ΔstnB1, while the wild-type strain did not produce. To enhance its production, the effect of shear stress and oxygen supply on ΔstnB1 strain cultivation was investigated in detail. In flask culture, when the shaking speed increased from 150 to 220 rpm, the mycelium was altered from a large pellet to a filamentous hypha, and the LME production was almost doubled, while no significant differences were observed among varied filling volumes, which implied a crucial role of shear stress in the morphology and LME production. To confirm this suggestion, experiments with agitation speed ranging from 400 to 1,000 rpm at a fixed aeration rate of 1.0 vvm were conducted in a stirred tank bioreactor. It was found that the morphology became more hairy with reduced pellet size, and the LME production was enhanced threefolds when the agitation speed increased from 400 to 800 rpm. Further experiments by varying initial k _L a value at the same agitation speed indicated that oxygen supply only slightly affected the physiological status of ΔstnB1 strain. Altogether, shear stress was identified as a major factor affecting the cell morphology and LME production. The work would be helpful to the production of LME and other secondary metabolites by filamentous microorganism cultivation.     

Effect of agitation speed on the morphology of Aspergillus niger HFD5A-1 hyphae and its pectinase production in submerged fermentation

AIM: To investigate the impact of agitation speed on pectinase production and morphological changing of Aspergillus niger(A. niger) HFD5A-1 in submerged fermentation. METHODS: A. niger HFM5A-1 was isolated from a rotted pomelo. The inoculum preparation was performed by adding 5.0 m L of sterile distilled water containing 0.1% Tween 80 to a sporulated culture. Cultivation was carried out with inoculated 1 × 107 spores/m L suspension and incubated at 30 ℃ with different agitation speed for 6 d. The samples were withdrawn after 6 d cultivation time and were assayed for pectinase activity and fungal growth determination. The culture broth was filtered through filter paper(Whatman No. 1, London) to separate the fungal mycelium. The cell-free culture filtrate containing the crude enzyme was then assayed for pectinase activity. The biomass was dried at 80 ℃ until constant weight. The fungal cell dry weight was then expressed as g/L. The 6 d old fungal mycelia were harvested from various agitation speed, 0, 50, 100, 150, 200 and 250 rpm. The morphological changing of samples was then viewed under the light microscope and scanning electron microscope.RESULTS: In the present study, agitation speed was found to influence pectinase production in a batch cultivation system. However, higher agitation speeds than the optimal speed(150 rpm) reduced pectinase production which due to shear forces and also collision among the suspended fungal cells in the cultivation medium. Enzyme activity increased with the increasing of agitation speed up to 150 rpm, where it achieved its maximal pectinase activity of 1.559 U/m L. There were significant different(Duncan, P 0.05) of the pectinase production with the agitation speed at static, 50, 100, 200 and 250 rpm. At the static condition, a well growth mycelial mat was observed on the surface of the cultivation medium and sporulation occurred all over the fungal mycelial mat. However with the increased in agitation speed, the mycelial mat turned slowly to become a single circular pellet. Thus, it was found that agitation speed affected the morphological characteristics of the fungal hyphae/mycelia of A. niger HFD5A-1 by altering their external as well as internal cell structures.CONCLUSION: Exposure to higher shear stress with an increasing agitation speed could result in lower biomass yields as well as pectinase production by A. niger HFD5A-1.     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10⁹ BHK-21 cells from 4 × 10⁶ cells in 13 day with 1,051 mL culture medium.     

An investigation of agitation speed as a factor affecting the quantity and monomer distribution of alginate from <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> ATCC<Superscript>®</Superscript> 9046

Alginate is a copolymer of β-d-mannuronic and α-l-guluronic acids. Distribution of these monomers in the alginate structure is one of the important characteristics that affect the commercial value of the polymer. In the present work, the effect of agitation speed in the range of 200–700 rpm on alginate production by Azotobacter vinelandii ATCC^® 9046 was investigated at a dissolved oxygen tension of 5% of air saturation. Experiments were conducted in a fermentor operated in batch mode for 72 h while the production of biomass and alginate, the consumption of substrate and the change in culture broth viscosity and monomer distribution of the polymer were monitored. Results showed that the growth rate of the bacteria increased from 0.165 to 0.239 h^?1 by the increase of mixing speed from 200 to 400 rpm. On the other hand, alginate production was found to be the most efficient at 400 rpm with the highest value of 4.51 g/l achieved at the end of fermentation. The viscosity of culture broth showed similar trends to alginate production. Viscosity was recorded as 24.61 cP at 400 rpm while it was only 4.26 cP at 700 rpm. The MM- and GG-block contents were almost equal in most of the culture times at 400 rpm. On the other hand, GG-blocks dominated at both low and high mixing speeds. Knowing that GG-blocks make rigid and protective gels with divalent cations, due to the higher GG-block content, the gel formation potential is higher at 200 rpm as well at 700 rpm, which might originate from the unfavorable environmental conditions that the bacteria were exposed to.     

Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage

Mechanical stress caused by agitation is one of the factors that can affect hematopoietic stem cell expansion in suspension bioreactors. Therefore, we have investigated the effects of agitation on umbilical cord blood hematopoietic stem cell (UCB-HSC) growth and differentiation. A comparison was made between various agitation rates (20, 40 and 60 rpm) in spinner-flask and cells cultured in glass petri dish as a static culture. Moreover, the fluid dynamic at various agitation rates of spinner-flask was analyzed to determine shear stress. The spinner-flask contained a rotational moving mixer with glass ball and was kept in tissue culture incubator. To reduce consumption of cytokines, UCB-serum was used which widely decreased the costs. Our results determined that, agitation rate at 40 rpm promoted UCB-HSCs expansion and their colony forming potential. Myeloid progenitors were the main type of cells at 40 rpm agitation rate. The results of glucose consumption and lactic acid production were in complete agreement with colony assay and expansion data and indicated the superiority of culture in spinner-flask when agitated at 40 rpm over to other agitation speeds and also static culture. Cell viability and colony count was affected by changing the agitation speed. We assume that changes in cell growth resulted from the effect of shear stress directly on cell viability, and indirectly on signaling pathways that influence the cells to differentiate.     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

Bioconversion of tea polyphenols to bioactive theabrownins by Aspergillus fumigatus

Theabrownins (TB) are water-soluble phenolic compounds associated with the various health benefits of Pu-erh tea, a post-fermented Chinese dark tea. This work reports on the production of theabrownins from infusions of sun-dried green tea leaves using a pure culture of Aspergillus fumigatus isolated from a solid-state Pu-erh tea fermentation. A theabrownins yield of 158 g kg^?1 sun-dried green tea leaves was obtained in 6 days at 45 °C in an aerobic fermentation. In a 2 l fermenter, the yield of theabrownins was 151 g kg^?1 sun-dried green tea leaves in 48 h of aerobic culture (45 °C, 1 vvm aeration rate, 250 rpm agitation speed). Extracellular polyphenol oxidase and peroxidase of A. fumigatus contributed to this bioconversion. Repeated batch fermentation process was used for producing theabrownins but was less productive than the batch process.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture

The influence of agitation and aeration on growth and on production of glucose oxidase of Asp. niger has been studied. It was found that both rate of growth and glucose oxidase production was higher at an agitation speed of 700 rpm than at 460 rpm. Further increase in speed of agitation resulted in neither a higher rate of growth nor a higher glucose oxidase activity. Total glucose oxidase activity was highest in a medium containing 5% sugar (at an agitation speed of 700 rpm) and did not get higher when the sugar concentration of the medium was increased to 7%. When pure oxygen was bubbled through the culture the rate of growth of the culture (in the linear phase) was 95 mg. mycelial dry wt./100 ml./hr., and only 61 mg. when air was applied. The glucose oxidase activity of oxygenated culture was double the activity of aerated culture. Viscosity of the homogenized culture became higher with higher concentration of mycelia. The viscosity of oxygenated culture was found to be lower than that of aerated culture.     

Biodegradation of 4-chlorophenol by <Emphasis Type="Italic">Arthrobacter chlorophenolicus A6</Emphasis>: effect of culture conditions and degradation kinetics

Among known microbial species, Arthrobacter chlorophenolicus A6 has shown very good potential to treat phenolic wastewaters. In this study, the levels of various culture conditions, namely initial pH, agitation (rpm), temperature (°C), and inoculum age (h) were optimized to enhance 4-chlorophenol (4-CP) biodegradation and the culture specific growth rate. For optimization, central composite design of experiments followed by response surface methodology (RSM) was applied. Results showed that among the four independent variables, i.e., pH, agitation (rpm), temperature (°C), and inoculum age (h) investigated in this study, interaction effect between agitation and inoculum age as well as that between agitation and temperature were significant on both 4-CP biodegradation efficiency and culture specific growth rate. Also, at the RSM optimized settings of 7.5 pH, 207 rpm, 29.6°C and 39.5 h inoculum age, 100% biodegradation of 4-CP at a high initial concentration of 300 mg l⁻¹ was achieved within a short span of 18.5 h of culture. The enhancement in the 4-CP biodegradation efficiency was found to be 23% higher than that obtained at the unoptimized settings of the culture conditions. Results of batch growth kinetics of A. chlorophenolicus A6 for various 4-CP initial concentrations revealed that the culture followed substrate inhibition kinetics. Biokinetic constants involved in the process were estimated by fitting the experimental data to several models available from the literature.     

Process optimization for reverse micellar extraction of stem bromelain with a focus on back extraction

In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2‐ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter‐ion and iso‐propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04‐fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:845–855, 2014     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

An approach toward optimization of the influential growth determinants of opportunistic yeast isolate Pichia guilliermondii

The present study reports statistical optimization of growth conditions of an opportunistic fungal strain Pichia guilliermondii, isolated from the blood of patients suffering from bancroftian filariasis. Seven key determinants, namely, primary inoculums size (%), volume (mL) and pH of media, serum proportion, temperature (°C), incubation time (hr), and agitation speed (rpm) that influence in vitro growth of the pathogen were optimized statistically using response surface methodology (RSM). RSM with seven factors and two-level Box–Behnken design was employed for designing experimental run, prediction of case statistics, suitable exploration of quadratic response surfaces, and constructing a second-order polynomial equation. Analysis of variance (ANOVA) showed that primary inoculums size, volume of culture media, temperature, incubation time, and agitation speed exert most significant influence over fungal growth. The RSM study predicted that optimum fungal growth can be obtained using 10% primary inoculums size in 100 mL culture media with pH 6.0, 6.28% serum, 32.5°C temperature, and 24 hr of incubation, alongside agitation speed at 400 rpm. The desirability of the optimized growth model for P. guilliermondii is 99.123%, which indicated its accuracy and acceptability. Finally, the optimized growth module illustrated in the study could be useful in improving in vitro growth of clinically important P. guilliermondii.     

Influence of oxygen transfer on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> effects on growth rate and biodesulfurization capacity

The growth rate and desulfurization capacity accumulated by the cells during the growth of Pseudomonas putida KTH2 under different oxygen transfer conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different agitation conditions. During the culture, several magnitudes associated to growth, such as the specific growth rate, the dissolved oxygen concentration and the carbon source consumption have been measured. Experimental results indicate that cultures are influenced by the fluid dynamic conditions into the bioreactor. An increase in the stirrer speed from 400 to 700 rpm has a positive influence on the cell growth rate. Nevertheless, the increase of agitation from 700 to 2000 rpm hardly has any influence on the growth rate. The effect of fluid dynamics on the cells development of the biodesulfurization (BDS) capacity of the cells during growth is different. The activities of the intracellular enzymes involved in the 4S pathway change with dissolved oxygen concentration. The enzyme activities have been evaluated in cells at several growth time and different hydrodynamic conditions. An increase of the agitation from 100 to 300 rpm has a positive influence on the development of the overall BDS capacity of the cells during growth. This capacity shows a decrease for higher stirrer speeds and the activity of the enzymes monooxygenases DszC and DszA decreases dramatically. The highest value of the activity of DszB enzyme was obtained with cells cultured at 100 rpm, while this activity decreases when the stirrer speed was increased higher than this value.     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

Optimisation of the operational conditions of trichloroethylene degradation using Trametes versicolor under quinone redox cycling conditions using central composite design methodology

Extracellular radicals produced by Trametes versicolor under quinone redox cycling conditions can degrade a large variety of pollutant compounds, including trichloroethylene (TCE). This study investigated the effect of the agitation speed and the gas–liquid phase volume ratio on TCE degradation using central composite design (CCD) methodology for a future scale-up to a reactor system. The agitation speed ranged from 90 to 200 rpm, and the volume ratio ranged from 0.5 to 4.4. The results demonstrated the important and positive effect of the agitation speed and an interaction between the two factors on TCE degradation. Although the volume ratio did not have a significant effect if the agitation speed value was between 160 and 200 rpm, at lower speed values, the specific pollutant degradation was clearly more extensive at low volume ratios than at high volume ratios. The fitted response surface was validated by performing an experiment using the parameter combination in the model that maximised TCE degradation. The results of the experiments carried out using different biomass concentrations demonstrated that the biomass concentration had a positive effect on pollutant degradation if the amount of biomass present was lower than 1.6 g dry weight l⁻¹. The results show that the maximum TCE degradation was obtained at the highest speed (200 rpm), gas–liquid phase volume ratio (4.4), and a biomass concentration of 1.6 g dry weight l⁻¹.     

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.