gp‐41 protein production by E. coli in unconventional bioreactor coupled with magnetic field generator

BACKGROUND: In this work recombinant gp‐41 protein production by E. coli in an unconventional bioreactor coupled with a magnetic field generator was studied. The submerged fermentation process was carried out by fed‐batch operation, recycling the culture medium externally through a stainless steel spiral U‐shape tube inserted on the inside of a magnetic field generator. The exposure time and magnetic field induction were varied in a range from 1 to 12 h and 10 to 100 mT, respectively, according to a central composite design with centered face. RESULTS: It was found that the inhibitory or stimulatory effect of magnetic field on cell growth and protein production depended on exposure time and magnetic field strength. The production of recombinant gp41 protein was 20% higher than the control. CONCLUSION The procedure here presented could be an easy‐to‐use approach to improve the efficiency of recombinant protein production by E. coli. Copyright © 2007 Society of Chemical Industry     

Using viscosity‐time plots of Escherichia coli cells undergoing chemical lysis to measure the impact of physiological changes occurring during batch cell growth

BACKGROUND: Viscosity–time plots for plasmid‐bearing E. coli cells undergoing alkaline lysis are reported in this study. The plots demonstrate generic features that reflect the progress of fermentation and allow an assessment of the genomic DNA denaturation following cellular release into the alkaline solution. This rheological analysis could offer useful insights to the state of fermentation or the selection of operational specifications and predictions of the performance of subsequent downstream operations. RESULTS: Studies showed a distinct change in the rheological profile throughout the batch fermentation, with different viscosity versus time profiles for lag, exponential and stationary microbial growth phases. The DNA denaturation time was found to increase with fermentation time from about 120 s after 3 h of fermentation to about 180 s after 7 h of fermentation. CONCLUSION: The increase of denaturation time was mainly caused by a rise in the genomic content of cells during the exponential growth phase. The viscosity–time profiles were found to provide a good indication of the cellular contents, reflecting the physiological changes occurring during a batch fermentation process. Copyright © 2009 Society of Chemical Industry     

Simultaneous saccharification and fermentation of sludge‐containing cassava mash for batch and repeated batch production of bioethanol by Saccharomyces cerevisiae CHFY0321

BACKGROUND: The aim of this study was to examine the repeated batch production of bioethanol from sludge‐containing cassava mash as starchy substrate by flocculating yeast to improve volumetric bioethanol productivity and to simplify the process of a pre‐culture system. RESULTS: For the repeated batch production of bioethanol using cassava mash, the optimal recycling volume ratio was found to be 5%. The repeated batch fermentation was completed within 36 h, while the batch fermentation was completed after 42 h. Volumetric productivity, final ethanol concentration, and ethanol yield were attained to 2.15 g L^?1 h^?1, 83.64 g L^?1, and 85.15%, respectively. Although cell accumulation in the repeated batch process is difficult due to the cassava mash, the repeated batch process using Saccharomyces cerevisiae CHFY0321 could exhibited 10‐fold higher initial viable cell number (1.7 × 10⁷ CFU mL^?1) than that of the batch process. CONCLUSION: The liquefied cassava powder was directly used for the repeated batch process without removal of sludge. Repeated batch bioethanol production by simultaneous saccharification and fermentation using self‐flocculating yeast could reduce process costs and accelerate commercial applications. This result was probably due in part to the effect of the initial viable cell density. Copyright © 2008 Society of Chemical Industry     

Performance evaluation of batch and unsteady state fed‐batch reactor operations for the production of a marine microbial surfactant

BACKGROUND: Biosurfactants are microbially derived surface‐active and amphipathic molecules produced by various microorganisms. These versatile biomolecules can find potential applications in food, cosmetics, petroleum recovery and biopharmaceutical industries. However, their commercial use is impeded by low yields and productivities in fermentation processes. Thus, an attempt was made to enhance product yield and process productivity by designing a fed‐batch mode reactor strategy. RESULTS: Biosurfactant (BS) production by a marine bacterium was performed in batch and fed‐batch modes of reactor operation in a 3.7 L fermenter. BS concentration of 4.61 ± 0.07 g L^?1 was achieved in batch mode after 22 h with minimum power input of 33.87 × 10³ W, resulting in maximum mixing efficiency. The volumetric oxygen flow rate (K_La) of the marine culture was about 0.08 s^?1. BS production was growth‐associated, as evident from fitting growth kinetics data into the Luedeking‐Piret model. An unsteady state fed batch (USFB) strategy was employed to enhance BS production. Glucose feeding was done at different flow rates ranging from 3.7 mL min^?1 (USFB‐I) to 10 mL min^?1 (USFB‐II). USFB‐I strategy resulted in a maximum biosurfactant yield of 6.2 g l^?1 with an increment of 35% of batch data. The kinetic parameters of USFB‐I were better than those from batch and USFB‐II. CONCLUSION: Comparative performance evaluation of batch and semi‐continuous reactor operations was accomplished. USFB‐I operation improved biosurfactant production by about 35% over batch mode. USFB‐I strategy was more kinetically favorable than batch and USFB‐II. © 2012 Society of Chemical Industry     

Fed‐batch fermentation of olive mill wastewaters for lipase production

In the Mediterranean basin countries, huge amounts of olive mill wastewaters (OMW) are produced by the olive oil industry. It constitutes a serious environmental problem, nevertheless its composition turns OMW into a potential growth medium to lipolytic microorganisms. The aim of this work was to study lipase production as well as OMW degradation in fed‐batch cultures of Candida cylindracea CBS 7869, Candida rugosa CBS 2275 and Yarrowia lipolytica W29 (ATCC 20460). Besides the improvement of lipase production, the fed‐batch approach enhanced the effluent degradation, since it led to good COD and lipids reduction, both higher than 50%. C. rugosa achieved the highest value of lipase productivity (130 U L^?1 h^?1), in parallel with highest lipids reduction (77%). This study demonstrates that OMW are becoming a competitive and valuable growth medium in fermentation processes with lipolytic microorganisms. The fed‐batch strategy used proved to be an efficient approach to enhance lipase production from OMW and to reduce significantly the final organic load of the medium. Copyright © 2012 Society of Chemical Industry     

Effects of Oxygen Supply Modes on the Production of Human Growth Hormone in Different Scale Bioreactors

Recombinant Escherichia coli has been studied as a main host for recombinant protein productions, but it is still difficult to cultivate E. coli in a large industrial‐scale process due to the oxygen supply limitation. In this study, E. coli BL(21) harboring a new constructed plasmid (pEHUb‐hGH) was used for producing recombinant human growth hormone (r‐hGH) in 5‐L and 30‐L scale fermentors by supplying air and high purity oxygen, respectively, where the high purity oxygen was produced from a vacuum pressure swing adsorption (PSA). The impact of oxygen supply modes, i.e., air and high purity oxygen, on cell growth and r‐hGH production was investigated in different scale fermentors. In the case of high purity oxygen supply, the final cell density and r‐hGH concentrations were 63.0 and 4.8 g/L in the 5‐L fermentor, 51.6 and 4.0 g/L in the 30‐L fermentor, respectively. In addition, the productivity of r‐hGH was doubled in the 5‐L fermentor, and increased 4‐fold in the 30‐L fermentor, compared to the results obtained in the case of the air supply. The supply of high purity oxygen eliminated the oxygen limitation and acetate formation effectively, and apparently, did not affect the degradation of r‐hGH. This shows that the recombinant E. coli cultivation with high purity oxygen produced from PSA may provide an effective method for large‐scale industrial production of recombinant proteins.     

Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase

The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm^?3 to 10 g dm^?3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7‐fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9‐fold. After implementation of a fed‐batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8‐, 2.9‐, 5.3‐ and 6.4‐fold, respectively, in relation to the previously‐mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins. © 2002 Society of Chemical Industry     

On-line estimation of biomass and intracellular protein for recombinant Escherichia coli cultivated in batch and fed-batch modes

One of the difficulties encountered in the control of bioprocesses is the lack of reliable on-line sensors for key state variables. On-line measurements of the recombinant biomass and intracellular protein are the key requirements to achieving an effective process operation. This paper investigates the suitability of using on-line estimation to predict concentrations of biomass and protein for recombinant Escherichia coli cultivated in batch and fed-batch modes. Various pairs of on-line accessible measurements of glucose, lactate, and acetate are fed back into the on-line estimator to train the tuning parameters in order to predict concentrations of biomass and intracellular protein. Such predictions are consistent with experimental data using off-line measurements.     

Xylitol production by Candida tropicalis from corn cob hemicellulose hydrolysate in a two‐stage fed‐batch fermentation process

BACKGROUND: Xylitol, a sugar alcohol widely used in food and pharmaceutical industries, can be produced through biological reduction of xylose present in hemicellulose hydrolysates by Candida tropicalis. However, the aeration rate and by‐products originating from hemicellulose hydrolysis strongly inhibit the production of xylitol in a fermentation process. A two‐stage fed‐batch fermentation system was developed to reduce these inhibitory effects and to improve xylitol production from corn cob hemicellulose hydrolysates by C. tropicalis. RESULTS: Results of batch fermentations indicated that high xylitol production could be obtained from C. tropicalis at an initial xylose concentration of 80 g L^?1 in corn cob hydrolysate medium at an aeration rate of 0.4 vvm at the micro‐aeration stage. In the two‐stage fed‐batch fermentation process, 96.5 g L^?1 xylitol was obtained after 120 h, giving a yield of 0.83 g g^?1 and a productivity of 1.01 g L^?1 h^?1, which were 12.16% and 65.57% higher than those in a batch fermentation. CONCLUSION: High xylitol production can be achieved in a two‐stage fed‐batch fermentation process, in which the negative effects of aeration rate and inhibitory compounds on xylitol formation can be considerably reduced. Copyright © 2011 Society of Chemical Industry     

Lipolytic enzyme production in batch and fed‐batch cultures of Ophiostoma piceae and Fusarium oxysporum

Lipase and esterase production by Ophiostoma piceae and Fusarium oxysporum were enhanced and extended by developing a fed‐batch process in stirred tank reactors. Fed‐batch strategy improved lipolytic enzyme production from Ophiostoma piceae in both 2 and 20 dm³ stirred tank reactors. However, fed‐batch fermentation of Fusarium oxysporum in the 2 dm³ reactor was more effective than both batch and fed‐batch fermentations in the 20 dm³ reactor. When a medium composed of only carbon and nitrogen source was intermittently fed to the cultures, the maximum specific lipase activity was improved by more than 80% and 35% in Ophiostoma piceae and Fusarium oxysporum cultures respectively. The maximum specific esterase activity was improved by 20% and 15% in Ophiostoma piceae and Fusarium oxysporum cultures respectively. The duration of production for both fungi extended from 144 to 216 h compared with a batch culture under the same condition. © 2000 Society of Chemical Industry     

Strain isolation and optimization of process parameters for bioconversion of glycerol to lactic acid

BACKGROUND: The crude glycerol from biodiesel production represents an abundant and inexpensive source which can be used as raw material for lactic acid production. The first aim of this investigation was to select a strain suitable for producing lactic acid from glycerol with a high concentration and productivity. The second aim was to obtain the optimum fermentation conditions, as a basis for large‐scale lactate production in the future. RESULTS: Eight bacterial strains, which could aerobically convert glycerol to lactic acid, were screened from soil samples. One of the strains, AC‐521, which synthesized lactic acid with a higher concentration, was identified based on its 16S rDNA sequences and physiological characteristics. These results indicated that this strain was a member of Escherichia coli. The optimal fermentation conditions for Escherichia coli AC‐521 were 42 °C, pH 6.5, 0.85 min^?1 (K_La). CONCLUSION: Escherichia coli AC‐521 suitable for producing lactic acid from glycerol with high concentration and productivity was identified. After 88 h of fed‐batch fermentation, both the lactic acid concentration and glycerol consumption reached maximum, giving 85.8 g L^?1 of lactic acid with a productivity of 0.97 g L^?1 h^?1 and a yield of 0.9 mol mol^?1 glycerol. Copyright © 2009 Society of Chemical Industry     

Optimal mixing to improve the performance of batch and continuous fermentations with recombinant Escherichia coli

Fermentations with genetically altered bacteria tend to lose plasmids as the fermentation progresses. Methods such as two‐stage cultivation, cell recycle and the addition of antibiotics are commonly used to enhance plasmid stability. Here we examine a different method, the regulation of mixing in the bioreactor. In particular, large bioreactors are considered where uniform mixing is difficult to achieve and the probability of plasmid loss varies with the specific growth rate. For both batch and continuous cultivations of Escherichia coli C600 gal K containing the plasmid pBR Eco gap, it is seen through a model that both modes of operation exhibit high plasmid stability and cell growth when the broth is incompletely mixed, and mixing near and away from the point of inoculation are unequal. Thus, the natural incomplete mixing in large bioreactors may be utilized to improve plasmid stability. A practical method to implement this idea is suggested. Copyright © 2005 Society of Chemical Industry     

基于高密度培养的反复分批发酵法生产丁二酸

引言丁二酸俗称琥珀酸,是三羧酸循环的中间代谢产物和厌氧代谢的终端还原产物,广泛存在于动物、植物及微生物体内。作为重要的C4平台化合物,丁二酸可用于多种大宗化学品以及生物可降解材料的制备。利用微生物发酵生产丁二酸,由于     

Effects of light wavelength and intensity on the production of ethanol by Saccharomyces cerevisiae in batch cultures

BACKGROUND: Photoreceptors have been identified in Saccharomyces cerevisae, however, the influence of light on the performance of ethanol fermentation of S. cerevisiae is not yet clear. The aims of this study are to elucidate the influence of light wavelength and intensity on the growth and ethanol production of S. cerevisiae and to describe a novel two‐stage LED light process to optimize ethanol fermentation. RESULTS: Experimental results indicated that maximum biomass concentration X_max of the batch under red LED light increased monotonically with light intensity, and the optimal specific product yield Y_p/x was 13.2 g g^?1 at 600 lux. Maximum ethanol concentration P_max of the batch under blue LED light increased monotonically with light intensity, and the optimal Y_p/x was 18.4 g g^?1 at 900 lux. A novel two‐stage LED light process achieved maximum P_max, of 98.7 g dm^?3 resulting in 36% improvement compared with that of the batch in the dark. CONCLUSION: The light wavelength and its intensity significantly affected cell growth and ethanol formation of S. cerevisiae. Red LED light (630 nm) stimulated cell growth but slightly inhibited ethanol formation. In contrast, blue LED light (470 nm) significantly inhibited cell growth but stimulated ethanol formation. A novel two‐stage LED light process has been successfully demonstrated to optimize ethanol fermentation of S. cerevisiae. Copyright © 2009 Society of Chemical Industry     

Enhanced Production of Podophyllotoxin by Fed‐batch Cultivation of Podophyllum hexandrum

Podophyllum hexandrum (Indian May Apple) was successfully cultivated in a 3 L stirred tank bioreactor under low shear conditions in batch and fed‐batch modes of operation. A statistically optimized culture medium was used for the batch cultivation of Podophyllum hexandrum. Under optimum culture conditions of P. hexandrum, the batch culture showed a growth‐associated product formation with a maximum biomass of 21.4 g/L dry cell weight (DCW) basis and a podophyllotoxin production of 13.8 mg/L in 26 d. A mathematical model was developed to design the nutrient feeding strategies for a fed‐batch cultivation to prolong the productive log phase of cultivation. The fed‐batch cultivation was able to enhance the biomass and podophyllotoxin accumulation to 48 g/L (DCW basis) and 43.2 mg/L, respectively, in 60 d. The volumetric productivity of podophyllotoxin in fed‐batch cultivation was found to be 0.72 mg/(L. d) as opposed to 0.53 mg/(L. d) in batch cultivation under optimized culture conditions.     

Effect of loading rate on TOC consumption efficiency in a sulfate reducing process: sulfide effect in batch culture

BACKGROUND: The sulfate reducing process (SRP) was analyzed in order to identify factors that diminish the effectiveness of the SRP during wastewater treatment. The effect of different sulfate loading rates (SLR, 290 to 981 mg SO₄‐S L^?1d^?1) and lactate at a stoichiometric C/S ratio of 0.75 on SRP was studied in an upflow anaerobic sludge blanket (UASB) reactor. The effect of sulfide concentration (0 to 200 mg sulfide‐S L^?1) on SRP in batch culture was evaluated. RESULTS: When the SLR was increased, the total organic carbon (TOC) and sulfate consumption efficiencies decreased from 93% ± 3 to 66% ± 2 and 60% ± 5 to 45% ± 4, respectively. Acetate and propionate were accumulated. Microbial analysis showed the presence of microorganisms related with the SRP, fermentation and methanogenesis. In batch culture, when lactate and sulfate were present, SRP and fermentation were observed. When sulfide was added only SRP was observed. At concentrations higher than 150 mg sulfide‐S L^?1 the efficiencies, yields and specific consumption rates (q) decreased. CONCLUSION: Based on the sulfide‐S/volatile suspended solid ratio, it was found that the decrease in efficiency and accumulation of acetate and propionate in the UASB reactor was not related to sulfide inhibition but to the q of acetate and propionate, which were up to 11 times lower than lactate. Copyright © 2008 Society of Chemical Industry     

Assessment of in situ butanol recovery by vacuum during acetone butanol ethanol (ABE) fermentation

BACKGROUND: Butanol fermentation is product limiting owing to butanol toxicity to microbial cells. Butanol (boiling point: 118 °C) boils at a higher temperature than water (boiling point: 100 °C) and application of vacuum technology to integrated acetone–butanol–ethanol (ABE) fermentation and recovery may have been ignored because of direct comparison of boiling points of water and butanol. This research investigated simultaneous ABE fermentation using Clostridium beijerinckii 8052 and in situ butanol recovery by vacuum. To facilitate ABE mass transfer and recovery at fermentation temperature, batch fermentation was conducted in triplicate at 35 °C in a 14 L bioreactor connected in series with a condensation system and vacuum pump. RESULTS: Concentration of ABE in the recovered stream was greater than that in the fermentation broth (from 15.7 g L^?1 up to 33 g L^?1). Integration of the vacuum with the bioreactor resulted in enhanced ABE productivity by 100% and complete utilization of glucose as opposed to a significant amount of residual glucose in the control batch fermentation. CONCLUSION: This research demonstrated that vacuum fermentation technology can be used for in situ butanol recovery during ABE fermentation and that C. beijerinckii 8052 can tolerate vacuum conditions, with no negative effect on cell growth and ABE production. Copyright © 2011 Society of Chemical Industry     

Production of GST–SOD fusion protein by recombinant E coli XL1 Blue

A recombinant plasmid was constructed by inserting a DNA fragment with the coding region of Cu/Zn–superoxide dismutase (Cu/Zn–SOD) cDNA from sweet potato, Ipomoea batatas (l) Lam cv Tainong 57, into the 3′ end of the open reading frame of the glutathione S‐transferase (GST) gene in an expression vector, pGEX‐2T. The constructed plasmid was transformed into E coli XL1 Blue. Fusion proteins of Cu/Zn–SOD and GST (GST–SOD) were produced from the recombinant E coli. About 6 mg of GST–SOD fusion proteins could be obtained from 1 dm³ of cultural broth after induction with 0.075 mmol dm⁻³ Isopropyl‐β‐D ‐thiogalactoside (IPTG). Lactose was not an efficient inducer. High cell density culture was performed by fed‐batch fermentation using a glucose analyzer to control glucose concentration at 1 g dm⁻³. The cell density of the fed‐batch culture reached an OD₆₀₀ of 30, the total amount of GST–SOD fusion protein was 100 mg dm⁻³ which is about 14 times more than that of the batch culture. Most of the fusion proteins were shown to be in an active monomeric form, and the molecular weight was estimated to be 45 kDa by SDS–PAGE and 47 kDa by gel filtration. The specific activity of the purified fusion proteins was about 1200 mg⁻¹ and equal to 3200 unit per mg of SOD domain only. © 2000 Society of Chemical Industry     

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xyl_A) was recombined with pRSETA vector, and the pRSETA::xyl_A obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L^?1 (20.6 U mg^?1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h^?1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h^?1 operation as 9.6 g L^?1 at t = 32 h. The highest oxygen uptake was 4.57 mol m^?3 s^?1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry     

Nonlinear Predictive Control of Fed‐Batch Cultures of Escherichia coli

A strategy for controlling a fed‐batch Escherichia coli culture is described to maintain the culture at the boundary between oxidative and oxido‐fermentative regimes. A nonlinear predictive controller is designed to regulate the acetate concentration, constraining the feed rate to follow an optimal reference profile which maximizes the biomass growth. For the sake of simplicity and efficiency, the original problem is converted into an unconstrained nonlinear programming problem, solved by control vector parameterization techniques. The robustness of the structure is further improved by explicitly including the difference between system and model prediction. A robustness study based on a Monte Carlo approach is used to evaluate the performance of the proposed controller. This control law is finally compared to the generic model control strategy.