Maximum Product Concentration Research Articles

Effect of process mode, nitrogen source and temperature on L-malic acid production with Aspergillus oryzae DSM 1863 using acetate as carbon source.

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4–3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.

Anaerobic fermentation for the production of short chain organic acids: Product concentration, yield and productivity in batch experiments at high feed concentration

With the general aim of maximising product concentration, yield and productivity in the anaerobic fermentation (AF) of biomass to make short-chain organic acids (SCOAs) with low-cost and environmentally sustainable processes, this study investigates the effect of high substrate concentration on the batch digestion of model biomass at uncontrolled temperature. Five substrate concentrations, between 24.7 and 394.6 gCOD l−1 (the latter is the highest substrate concentration reported in the literature for these types of studies), were investigated in batch reactors with no pH control. The highest substrate concentration led to a maximum product concentration of 61.5 g l−1, composed mainly of lactic acid (85 wt%). The lowest substrate concentration produced mainly acetic acid (54 wt%). The pH was acidic in all cases but was higher for the most diluted feed (6.2). Similar yields at the end of the experiments, between 15.7% and 22.0% COD COD−1, were observed in all reactors. Over the length of the experiments, generally yields increased and productivities decreased, indicating the need of a compromise between yield and productivity. For the highest substrate concentration, a final productivity of 1.5 g l−1 d−1 was obtained. Experiments with pH adjustment in the range 4.0–6.0 were performed, and results generally showed an increase in product concentration, yield and productivity as the target pH increased, with the highest values of concentration, yield and productivity (163 g l−1, 56% COD COD−1 and 3.9 g l−1 d−1 respectively) obtained at pH 5.5.

Heat transfer evaluation for conventional and extractive ethanol fermentations: Saving cooling water

Ethanol production in Brazil has two major drawbacks, related to the maximum product concentration and control of the process temperature. During the fermentation, yeast metabolism completely ceases when the ethanol concentration reaches 95 g.L−1, while high temperatures increase both the risk of bacterial contamination and the sensitivity of the yeast to ethanol toxicity. In order to address these problems, extractive ethanol fermentation with carbon dioxide stripping offers a way to overcome the toxic effect of ethanol on yeast cell growth, while also avoiding increase of the broth temperature during the fermentation. Although ethanol removal by gas stripping has a positive effect on ethanol volumetric productivity, the application of this technique for economization of cooling water during the ethanol fermentation process requires a technical analysis. Therefore, in the present work, evaluation was made of heat removal from the culture broth during extractive fermentation with ethanol stripping by CO2. First, ethanol fermentations were carried out using a 10-L bench-scale bubble column bioreactor. The mass and energy balances were used to obtain kinetic parameters, with the heat generated during fermentation being close to theoretical values. Extractive batch fermentations were performed using two carbon dioxide specific flow rates (0.5 and 1.0 vvm), resulting in efficient removal of heat from the fermentation broth, as well as reducing cooling water consumption by up to 62.7%. These results were then validated in a pilot-scale (100-L) extractive fed-batch fermentation performed at 1.0 vvm, resulting in a 63.1% reduction of cooling water consumption. In an industrial distillery, this lower consumption of cooling water would allow substantial savings of chemicals needed for cooling water treatment, making the ethanol production process in Brazil more competitive in the near future.

Defatted Chlorella biomass as a renewable carbon source for polyhydroxyalkanoates and carotenoids co-production

Defatted Chlorella biomass was investigated as an unconventional and promising carbon source for the co-production of polyhydroxyalkanoates and cartenoids via batch cultivation of Paracoccus sp. LL1. 10% (w/v) of algal biomass was subjected to acid hydrolysis using different concentrations (0.1, 0.2, 0.3, and 0.5 N) of either sulfuric or hydrochloric acid to yield fermentable sugars. Regardless of the acid type, reducing sugar content significantly increased with an increasing acid concentration, reaching a maximum yield of 52 and 46 g/L respectively for hydrochloric and sulfuric acid catalyzed hydrolysis at the highest acid concentrations (p < 0.05). Shake flask fermentation process showed the ability of Paracoccus sp. LL1 to convert fermentable sugars from 0.3 N hydrochloric acid catalyzed hydrolysis into polyhydroxyalkanoates and carotenoids with a maximum product concentration of 1.48 g/L (i.e., 37.4% of dry cell weight) and 6.08 mg/L, respectively. Moreover, 144 and 92% increases in polyhydroxyalkanoates and carotenoids production (i.e., 3.62 g/L polyhydroxyalkanoates and 11.7 mg/L carotenoids) was recorded upon upscaling the biotransformation of defatted Chlorella biomass hydrolysate in 5 L fermenter by Paracoccus sp. LL1. Notably, these results corresponded to 92 and 54% greater production of polyhydroxyalkanoates and carotenoids than those observed in fermenter operated under identical conditions with glucose as a carbon source. Subsequent analyses of gas chromatography, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance spectroscopy further identified the accumulated polyhydroxyalkanoate as a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate with a 3-hydroxyvalerate fraction of 6.22 mol%.

Perfusion CHO cell culture applied to lower aggregation and increase volumetric productivity for a bispecific recombinant protein

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an “apparent” 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

Optimization of limonene biotransformation to limonene-1,2-diol by Colletotrichum nymphaeae CBMAI 0864

Abstract This study aimed to optimize the conditions of the biotransformation of limonene to limonene-1,2-diol by C. nymphaeae. In the initial modeling, in shake flasks, the use of 13.2 g.L−1 biomass, 27 °C, 250 rpm, and pH of 6.0 could maximize the production of limonene-1,2-diol (6.75 g.L−1). Subsequently, optimal conditions were transposed to a bioreactor, where agitation and aeration were also evaluated. A minimum 60% of dissolved oxygen was defined for this process. The limonene volatilization could be reduced almost three times when a larger condenser is used. Finally, when the bioreactor was operated at 27 °C, 300 rpm, 1 vvm, and with 13.2 g.L−1 biomass, limonene-1,2-diol production reached 7.1, 7.8, and 5.6 g.L−1 after 72 h when using 20 g.L−1 of R-(+)-, S-(−)-limonene or citrus terpene as substrate, respectively. This is approximately the same maximum product concentration found for the shake flasks, but it represents an almost three times higher productivity.

Application of a thermostable Baeyer-Villiger monooxygenase for the synthesis of branched polyester precursors.

BACKGROUNDIt is widely accepted that the poor thermostability of Baeyer–Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5‐trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ϵ‐caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi‐enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co‐factor regeneration.RESULTSUsing reaction engineering, the reaction rate and product formation of the regio‐isomeric branched lactones were improved and the use of co‐solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water‐immiscible organic solvent. A maximum volumetric productivity, or space–time‐yield, of 1.20 g L‐1 h‐1 was achieved with continuous feeding of substrate using methanol as co‐solvent, while a maximum product concentration of 11.6 g L‐1 was achieved with toluene acting as a second phase and substrate reservoir.CONCLUSIONThese improvements in key process metrics therefore demonstrate progress towards the up‐scaled Baeyer–Villiger monooxygenase‐biocatalyzed synthesis of the target building blocks for polymer application. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A mobile-mobile transport model for simulating reactive transport in connected heterogeneous fields

Mobile-immobile transport models can be effective in reproducing heavily tailed breakthrough curves of concentration. However, such models may not adequately describe transport along multiple flow paths with intermediate velocity contrasts in connected fields. We propose using the mobile-mobile model for simulating subsurface flow and associated mixing-controlled reactive transport in connected fields. This model includes two local concentrations, one in the fast- and the other in the slow-flow domain, which predict both the concentration mean and variance. The normalized total concentration variance within the flux is found to be a non-monotonic function of the discharge ratio with a maximum concentration variance at intermediate values of the discharge ratio. We test the mobile-mobile model for mixing-controlled reactive transport with an instantaneous, irreversible bimolecular reaction in structured and connected random heterogeneous domains, and compare the performance of the mobile-mobile to the mobile-immobile model. The results indicate that the mobile-mobile model generally predicts the concentration breakthrough curves (BTCs) of the reactive compound better. Particularly, for cases of an elliptical inclusion with intermediate hydraulic-conductivity contrasts, where the travel-time distribution shows bimodal behavior, the prediction of both the BTCs and maximum product concentration is significantly improved. Our results exemplify that the conceptual model of two mobile domains with diffusive mass transfer in between is in general good for predicting mixing-controlled reactive transport, and particularly so in cases where the transfer in the low-conductivity zones is by slow advection rather than diffusion.

Acidogenic fermentation of vegetable and salad waste for chemicals production: Effect of pH buffer and retention time

This study investigated the effect of pH buffer and solids retention time (SRT) on the anaerobic fermentation of vegetable and salad waste (VSW). Experiments were carried out in batch and semi-continuous reactors at 35°C. In the batch experiments, the effect of pH buffer on the production of volatile fatty acids (VFAs) and ethanol was investigated. Acetate and butyrate were the main fermentation products. The maximum total product concentration was 43.3 and 18.5gCODl−1 in the buffered and unbuffered batch reactors resulting in a yield of 62 and 27% (CODtotalproduct/CODfeed) respectively. Volatile suspended solids (VSS) removal was higher in the buffered semi-continuous reactor (57%) compared to the unbuffered-acidic reactor (39%), but similar yields (15%, CODtotalproduct/CODfeed) were observed because biogas production was stimulated in the buffered reactor. The effect of SRT on the VSS removal and product distribution in unbuffered systems was investigated at 10, 20 and 30days SRT. The VSS removal increased as the SRT increased, ranging between 18.2–49.1%, likewise the total product concentration, 9.1–19.4gCODl−1, and product yield, 7–24% (CODtotalproduct/CODfeed). Acetate and butyrate were the predominent fermentation products at all conditions followed by caproate although caproate to this was only detected at 20 and 30days SRT. Total COD removal ranged between 15.2 and 35.1% with the highest removal observed at 30days SRT.

Definition of optimum basic nutrients ratio in media for bioethanol production with immobilised yeast cells

The production of bioethanol as an alternative to fossil fuel has been increasing owing to growing biofuel demand. Combination of yeast immobilisation and media optimisation has become a popular technique for the improvement of bioethanol technology. The aim of this study was the optimisation, by response surface methodology, of sodium alginate concentration and nitrogen and phosphorus content in media for bioethanol production using immobilised Saccharomyces cerevisiae. Ratio of carbon, nitrogen and phosphorus (C : N : P) in media was calculated based on optimum factors values and initial glucose content. The developed model predicts that maximum product concentration, minimum 'eluted' yeast cells number and minimum nutrients content are achieved when the sodium alginate concentration is 71.99 g/L and C : N : P ratio is 50 : 3.5 : 1.3. The obtained results can be used for techno-economic analysis of the process to select optimum fermentation conditions for industrial application.

Definition of optimum basic nutrients ratio in media for bioethanol production with immobilised yeast cells

The production of bioethanol as an alternative to fossil fuel has been increasing owing to growing biofuel demand. Combination of yeast immobilisation and media optimisation has become a popular technique for the improvement of bioethanol technology. The aim of this study was the optimisation, by response surface methodology, of sodium alginate concentration and nitrogen and phosphorus content in media for bioethanol production using immobilised Saccharomyces cerevisiae. Ratio of carbon, nitrogen and phosphorus (C : N : P) in media was calculated based on optimum factors values and initial glucose content. The developed model predicts that maximum product concentration, minimum 'eluted' yeast cells number and minimum nutrients content are achieved when the sodium alginate concentration is 71.99 g/L and C : N : P ratio is 50 : 3.5 : 1.3. The obtained results can be used for techno-economic analysis of the process to select optimum fermentation conditions for industrial application.

Continuous bioproduction of 1,3‐propanediol from biodiesel raw glycerol: Operation with free and immobilized cells of Clostridium butyricum DSM 10702

AbstractThis work investigates the bioproduction of 1,3‐propanediol (1,3‐PD) from biodiesel derived raw glycerol with free and immobilized cells of Clostridium butyricum DSMZ 10702. The cultures were carried out under continuous conditions in a stirred tank bioreactor with suspended cells and a fluidized‐bed bioreactor with immobilized cells. The microorganisms were immobilized by entrapment in calcium alginate gel beads. The effect of dilution rate and glycerol concentration in the feed on 1,3‐PD production was investigated for both bioreactor configurations. Results showed that 1,3‐PD concentration and glycerol uptake decreased when dilution rate increased. Maximum product concentration was similar in both bioreactors, 24.3 g/L and 20.5 g/L for suspended cells and Ca‐alginate immobilized cells, respectively, for a glycerol feed concentration of 50 g/L and a dilution rate of 0.05 h−1. The higher productivities were observed for a dilution rate of 0.15 h−1 for the suspended culture bioreactor and 0.2 h−1 for the immobilized cell bioreactor. Although maximum productivity was similar in both systems, 2.8 g 1,3‐PD/(L · h), cell immobilization was shown to be more resistant to washout at higher dilution rates.

Production of 8-Hydroxygenistein through Biotransformation by Using Aspergillus oryzae

8-Hydroxygenistein is one of the orthohydroxygenisteins, which are derivatives of genistein. Although 8-hydroxygenistein is generally produced through the biotransformation of soya isoflavones by using specific microorganisms, the yield of produced 8-hydroxygenistein is low. In this study, we optimized the cultivation medium for 8-hydroxygenistein production. The results of the central composite design indicated that the optimal nutrient concentrations were 35 g/L of glucose, 22 g/L of malt extract, and 4.75 g/L of soya peptone. The maximum product concentration of 8-hydroxygenistein obtained was 77.76 mg/L. Based on literature review, this concentration of 8-hydroxygenistein is the highest yet reported. The effects of operating conditions on the concentration of 8-hydroxygenistein were also determined using a fermenter. After investigating the effects of the agitation speed and aeration rate, a final product concentration of 83.01 mg/L was obtained. Thus, large-scale biotransformation of genistein to 8-hydroxygenistein can be performed using Aspergillus oryzae.

Bioprocess Development for Chlorella vulgaris Cultivation and Biosynthesis of Anti-phytopathogens Silver Nanoparticles

Bioprocess Development for Chlorella vulgaris Cultivation and Biosynthesis of Anti-phytopathogens Silver Nanoparticles In this study, growth media formulations and environmental conditions were varied to optimize the growth media composition and conditions for maximized algal biomass production. The production both of Chlorella vulgaris in different photobioreactor configurations (250 ml flask, 5L flask and 7.5L stirred tank reactor) was investigated, using different organic and inorganic media formulations. The effect of initial cell concentrations, agitation speed, airflow rate and photoperiodic time were studied using inexpensive poultry manure medium, the results indicate that 0.5 × 107 (n/ml), 200 rpm, 1 bar and 16:8hr respectively which better for biomass productivity at growth periods. The highest overall specific growth rate (μ) and doubling time (td) were found when growing C. vulgaris in batch operation in a 7.5 L stirred tank bioreactor, μ=0.08 d-1 and 3.5 days respectively which also reached the maximum biomass concentration of 0.59 g/L. Biosynthesized silver nanoparticles (AgNPs) are being widely using in the field of agriculture. Biosynthesis of silver nanoparticles was carried out by using the algal cells for the reduction of aqueous silver ions in short period. A Plackett-Burman experimental design was applied to screen seven parameters for biosynthesis of AgNPs, the most important parameters were pH, temperature and container head space; the results indicate that pH 6, 40ºC and 75% container head space which better for biosynthesis of AgNPs which also reached the maximum final product concentration of 5.59 mg/L. The AgNP formation was confirmed with the help of SEM, XRD and UV-Vis spectroscopy. These AgNPs were tested for antibacterial and antifungal activities. The tested strains are; Erwinia carotovora and Alternaria alternata were used. The results indicate that the AgNPs may have an important advantage over conventional antibiotics.

Effect of Fuel Inject Angle on the Thermal Behavior of a 2D Axisymmetric Non-Premixed Methane–Air Flame in Vertical Cylinder Filled by Porous Media

The present manuscript is dedicated to the study of non-premixed combustion within a vertical cylinder filled by inert porous media using a turbulence model. A study of the effect of inlet gas angle on methane/air combustion is performed. Governing equations of Navier-Stokes, energy and the chemical species transport equations in a porous media with local thermal nonequilibrium assumption between the solid and fluid are solved. Distinct energy equations are considered for the porous burner and the fuel in it. Inlet velocity and excess air-to-fuel ratio are varied in order to analyze their effects on temperature, turbulent kinetic energy distribution and flame front location. The temperature field and species concentrations are presented in the premixed methane air combustion. The results show that an increase in angle of inlet gas enhances the mixing rate, the peak temperature, and water and carbon dioxide volume fraction inside the middle region of the chamber. The locations of the maximum temperature and product concentration shift closer to the combustor inlet with an increase in inlet angle. Furthermore, the temperature in porous burners is lower than free flame burners, and lower NOx and CO pollutant are achieved. KeywordsMethane/Air Flame; Turbulent Model; Numerical Simulations; Non-Premixed; Porous Media

Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture

In an effort to reduce the accumulation of ammonia in culture medium, three different TCA cycle intermediates, (alpha-ketoglutarate (α-KG), citric acid and succinic acid) along with glutamic acid for a comparison, were examined as a substitute for glutamine with rCHO cell line producing a Fc-fusion glycoprotein. Among them, α-KG showed the best production performance. When cells were cultivated with 4mM α-KG, the final ammonia concentration did not exceed 3mM, which is less than one fourth of that with 4mM glutamine. The replacement of glutamine increased the lag phase and reduced cell growth. However, it increased the specific productivity by 2.7-fold, resulting in a 1.3-fold increase in the maximum product concentration. Furthermore, the sialic acid content of the Fc-fusion protein with 4mM α-KG was higher than that with 4mM glutamine in all cultures, most likely due to the lower ammonia concentration. The results of Western blotting and activity assays of intracellular α-2,3-sialyltransferase and extracellular sialidases are in good agreement with tests done to assess the sialic acid content of the Fc-fusion protein. Taken together, the data obtained here demonstrate that α-KG is a potential substitute for glutamine for improved glycoprotein production in rCHO cells.

Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation

This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

Using Molecular Markers to Characterize Productivity in Chinese Hamster Ovary Cell Lines

Selection of high producing cell lines to produce maximum product concentration is a challenging and time consuming task for the biopharmaceutical industry. The identification of early markers to predict high productivity will significantly reduce the time required for new cell line development. This study identifies candidate determinants of high productivity by profiling the molecular and morphological characteristics of a panel of six Chinese Hamster Ovary (CHO) stable cell lines with varying recombinant monoclonal antibody productivity levels ranging between 2 and 50 pg/cell/day. We examined the correlation between molecular parameters and specific productivity (qp) throughout the growth phase of batch cultures. Results were statistically analyzed using Pearson correlation coefficient. Our study revealed that, overall, heavy chain (HC) mRNA had the strongest association with qp followed by light chain (LC) mRNA, HC intracellular polypeptides, and intracellular antibodies. A significant correlation was also obtained between qp and the following molecular markers: growth rate, biomass, endoplasmic reticulum, and LC polypeptides. However, in these cases, the correlation was not observed at all-time points throughout the growth phase. The repeated sampling throughout culture duration had enabled more accurate predictions of productivity in comparison to performing a single-point measurement. Since the correlation varied from day to day during batch cultivation, single-point measurement was of limited use in making a reliable prediction.

In-situ Product Recovery as a Strategy to Increase Product Yield and Mitigate Product Toxicity

Product inhibition is often the cause limiting the maximum product concentration attainable in fermentation. This study showed the product yield of p-cresol could be improved by in-situ product recovery (ISPR). Escherichia coli transformed with the hpd BCA operon from Clostridium difficile was shown in this study to express phydroxyphenylacetate decarboxylase which converted p-hydroxyphenylacetate into p-cresol under anaerobic fermentation. Toxicity of p-cresol found at a concentration as low as 5 mM in a broth spiked with p-cresol was shown to have limited the maximum product concentration at 1 ± 0.1 mM after 30 hours of batch fermentation. Product yield was however shown to increase by 51% when activated carbon was used to remove p-cresol in-situ production. The activated carbon concentrated p-cresol on the solid adsorbent which was subsequently separated by sedimentation and p-cresol recovered by ultrasonic-assisted solvent extraction. Desorption of p-cresol from the spent activated carbon allowed the adsorbent to be regenerated for further product recovery. The ISPR strategy reported here was shown to improve the yield of a toxic product, was sustainable, and when adapted to a continuous process would increase productivity.

Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system

The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.