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Tunçer H. Özdamar - One of the best experts on this subject based on the ideXlab platform.
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recombinant protein production in pichia pastoris under glyceraldehyde 3 phosphate dehydrogenase promoter from carbon source metabolism to Bioreactor Operation parameters
Biochemical Engineering Journal, 2015Co-Authors: Pınar Çalık, Hande Gunes, Aslan Massahi, Abdullah Keskin, Sibel Ozturk, Gul H Zerze, Tunçer H. ÖzdamarAbstract:Abstract The yeast Pichia (Komagataella) pastoris has become a potential host system for recombinant protein (r-protein) production. Several strong inducible and constitutive promoters have been identified in P. pastoris which make the host ideal in r-protein expressions. The constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene, PGAP, is promising as its use offers the advantage of associating product quantity to growth which can be optimized by a well-designed feeding strategy so long as the product itself is not toxic to the host. In this article, carbon source metabolism and the central carbon pathways of P. pastoris are reviewed. After comparing PGAP with the available promoters of P. pastoris, the Bioreactor Operation parameters and Bioreactor Operation strategies are analyzed for r-protein production under PGAP by focusing on response of the cells, final product concentration, yield, and productivity. The overview of the subject reveals the requirement for in-depth information on the overall regulation of P. pastoris metabolism that can enable fine-tuning Bioreactor Operation parameters in relation with the physiology of the microorganism in order to develop new strategies for the enhanced r-protein production.
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Microarray Studies in Bacillus subtilis
Biotechnology journal, 2009Co-Authors: Pınar Kocabaş, Pınar Çalık, Güzide Çalık, Tunçer H. ÖzdamarAbstract:This review focuses on the construction of a global, comprehensive understanding of Bacillus subtilis through microarray studies. The microarray studies in B. subtilis were analysed based on the theme of the work, by mentioning the growth media, Bioreactor Operation conditions, RNA isolation method, number of data points analysed in exponential or stationary phases, compared genotypes, induction and repression ratios, investigated gene(s) and their positive and/or negative influences. Based on the theme and scope of the studies, the articles were reviewed under seven thematic sections, i.e., effects of gene deletion(s) or overexpression, effects of overexression of heterologous genes, comparison of global gene expression between aerobic and anaerobic respiration, effects of temperature change, effects of transported molecules, effects of limitations and stress conditions, and other microarray studies in B. subtilis.
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overexpression of a serine alkaline protease gene in bacillus licheniformis and its impact on the metabolic reaction network
Enzyme and Microbial Technology, 2003Co-Authors: Pınar Çalık, Gregory C Tomlin, Stephen G Oliver, Tunçer H. ÖzdamarAbstract:Abstract This work reports on cloning of serine alkaline protease (SAP) encoding gene sub C to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the sub C gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined Bioreactor-Operation conditions. Significant physiological changes were observed in the recombinant B. licheniformis in response to altered Bioreactor-Operation conditions, i.e. initial glucose concentration. The growth kinetics of microbial cells were investigated prior to the investigation of intracellular reactions and rates within the cell; the unstructured substrate inhibition and Monod models were found valid for the wild-type and recombinant B. licheniformis , respectively. Optimum initial glucose concentration for maximum SAP production and the corresponding cultivation time of the recombinant B. licheniformis shifted respectively from C G0 =6 to 8 kg m −3 and from t =43 to 67 h. The maximum SAP activity was obtained as 950 U cm −3 with the recombinant B. licheniformis , which was ca. 2.5-fold higher than that of the wild-type. Carbon fluxes through the central metabolic pathways in the wild-type and recombinant B. licheniformis were calculated, using a mass balance-based mathematical model that contains 105 metabolites and 148 reaction fluxes and the time profiles of glucose, dry cell weight, organic acids, amino acids and SAP obtained in 3.5 dm 3 Bioreactor systems at C G0 =6 kg m −3 for the exponential growth phase and the SAP production phase. The bioreaction network flux analyses were first accomplished by using the theoretical data-based approach; and then by using the theoretical data-based capacity analysis approach. During the SAP synthesis period, the actual fluxes of the glycolysis pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, the amino acids biosynthetic pathways (and, consequently, SAP synthesis) are higher in the recombinant B. licheniformis strain than in the wild-type. Further, the normalised relative flux values of all the pathways, except the glycolysis pathway, change considerably in the recombinant bacteria. The effectiveness factor, defined as the SAP synthesis rate per maximum possible SAP synthesis rate was η =0.20 for the recombinant B. licheniformis . This indicates the possibility of a further increase in SAP production through metabolic engineering, and potential strategies to achieve this are also discussed.
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Bioreactor Operation parameters as tools for metabolic regulations in fermentation processes: influence of pH conditions
Chemical Engineering Science, 2003Co-Authors: Pınar Çalık, Esra Bilir, Güzide Çalık, Tunçer H. ÖzdamarAbstract:The influence of controlled- and uncontrolled-pH conditions together with the initial pH on the product and by-product distributions and oxygen transfer characteristics, whereupon the process rate limitations in relation to the intracellular reaction rates were investigated in serine alkaline protease (SAP) fermentation process by recombinant Bacillus licheniformis carrying pHV1431::subC on a defined medium with the sole carbon source glucose in the pH range of 6.80-7.25 in batch Bioreactors. Although the same amount of cell was produced at all the conditions in each type of Operation, with the increase of initial pH the cell formation rates increased; moreover, uncontrolled-pH Operation was favourable for the cell formation. The SAP synthesis rates and concentrations were higher at uncontrolled-pH Operations. Among the fermentations, pH 0 = 7.10 uncontrolled-pH Operation produced maximum SAP activity that was ca. 900 U cm -3 at t = 21 h. According to the biomass and SAP production profiles, bioprocess was divided into two periods; Period I(0 < t ≤ 10 h) covers the cell growth phase and Period II(10 < t ≤ 24 h) covers the SAP production phase. In Period I in both Operations, while the oxygen uptake rate (OUR) increased with the increase in initial pH the oxygen transfer rate (OTR) decreased. In Period II, among uncontrolled-pH Operations OUR was the lowest at pH = 7.10 while OTR decreased with the increase in initial pH. At the oxygen transfer condition applied, the bioprocess is biochemical reaction limited at all the conditions; nevertheless, with the decrease in initial pH, Damkohler number that is the oxygen transfer limitation decreases. Further, the perturbation effects of initial pH and the Operation conditions on the intracellular reaction rates were calculated for Periods I and II by using the experimental data obtained and, the diversions in the pathways and certain metabolic reactions and potential strategies for improving SAP production are also discussed.
Pınar Çalık - One of the best experts on this subject based on the ideXlab platform.
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Ethanol fed-batch Bioreactor Operation to enhance therapeutic protein production in Pichia pastoris under hybrid-architectured ADH2 promoter
Biochemical Engineering Journal, 2020Co-Authors: Omar Wehbe, Oğuz Ulaş Yaman, Pınar ÇalıkAbstract:ABSTRACT We presented ethanol fed-batch Bioreactor Operations (FBBOs) designed for enhanced recombinant human growth hormone (rhGH) production in Pichia pastoris constructed with novel hybrid-architectured ADH2 promoter, PADH2-Cat8-L2. The parameters in the fed-batch fermentation of ethanol were investigated, and the boundaries of the production domain in terms of the design parameters were determined. Two FBBO design methods were used, and a platform aiming to adjust cellular metabolism for the generation of constant specific growth rate (μ) and substrate concentration (CEtOH) was established. The highest overall productivity (rP,ov) was generated with model-based continuous feed-stream (CFS) design with μpreD = 0.035 h-1 up to tmax ≤ 9 h as rP,ov = 3.0 mg L-1 h-1 and CrhGH = 34 mg L-1. The ethanol-stat FBBO at CEtOH,set = 0.5 g L-1 allowed increased product titer with high productivity and the highest CrhGH =90.1 mg L-1 at tmax = 24 h. The effect of CEtOH on specific rates for cell reactions was represented by the Haldane model in which the cell generation and rhGH production were simulated with low mean relative errors of 6.5 and 16%, respectively. The results indicate the need for an upgraded CFS design model with kinetic expressions involving substrate inhibition. We conclude that a two-step hybrid fed-batch design strategy to be started (i) with the initial-period of the model-based FBBO, in turn, proceeded (ii) with the controlled feeding with ethanol-stat FBBO enables a gradual approach to the hypothetical performance of FBBOs.
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recombinant protein production in pichia pastoris under glyceraldehyde 3 phosphate dehydrogenase promoter from carbon source metabolism to Bioreactor Operation parameters
Biochemical Engineering Journal, 2015Co-Authors: Pınar Çalık, Hande Gunes, Aslan Massahi, Abdullah Keskin, Sibel Ozturk, Gul H Zerze, Tunçer H. ÖzdamarAbstract:Abstract The yeast Pichia (Komagataella) pastoris has become a potential host system for recombinant protein (r-protein) production. Several strong inducible and constitutive promoters have been identified in P. pastoris which make the host ideal in r-protein expressions. The constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene, PGAP, is promising as its use offers the advantage of associating product quantity to growth which can be optimized by a well-designed feeding strategy so long as the product itself is not toxic to the host. In this article, carbon source metabolism and the central carbon pathways of P. pastoris are reviewed. After comparing PGAP with the available promoters of P. pastoris, the Bioreactor Operation parameters and Bioreactor Operation strategies are analyzed for r-protein production under PGAP by focusing on response of the cells, final product concentration, yield, and productivity. The overview of the subject reveals the requirement for in-depth information on the overall regulation of P. pastoris metabolism that can enable fine-tuning Bioreactor Operation parameters in relation with the physiology of the microorganism in order to develop new strategies for the enhanced r-protein production.
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Microarray Studies in Bacillus subtilis
Biotechnology journal, 2009Co-Authors: Pınar Kocabaş, Pınar Çalık, Güzide Çalık, Tunçer H. ÖzdamarAbstract:This review focuses on the construction of a global, comprehensive understanding of Bacillus subtilis through microarray studies. The microarray studies in B. subtilis were analysed based on the theme of the work, by mentioning the growth media, Bioreactor Operation conditions, RNA isolation method, number of data points analysed in exponential or stationary phases, compared genotypes, induction and repression ratios, investigated gene(s) and their positive and/or negative influences. Based on the theme and scope of the studies, the articles were reviewed under seven thematic sections, i.e., effects of gene deletion(s) or overexpression, effects of overexression of heterologous genes, comparison of global gene expression between aerobic and anaerobic respiration, effects of temperature change, effects of transported molecules, effects of limitations and stress conditions, and other microarray studies in B. subtilis.
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overexpression of a serine alkaline protease gene in bacillus licheniformis and its impact on the metabolic reaction network
Enzyme and Microbial Technology, 2003Co-Authors: Pınar Çalık, Gregory C Tomlin, Stephen G Oliver, Tunçer H. ÖzdamarAbstract:Abstract This work reports on cloning of serine alkaline protease (SAP) encoding gene sub C to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the sub C gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined Bioreactor-Operation conditions. Significant physiological changes were observed in the recombinant B. licheniformis in response to altered Bioreactor-Operation conditions, i.e. initial glucose concentration. The growth kinetics of microbial cells were investigated prior to the investigation of intracellular reactions and rates within the cell; the unstructured substrate inhibition and Monod models were found valid for the wild-type and recombinant B. licheniformis , respectively. Optimum initial glucose concentration for maximum SAP production and the corresponding cultivation time of the recombinant B. licheniformis shifted respectively from C G0 =6 to 8 kg m −3 and from t =43 to 67 h. The maximum SAP activity was obtained as 950 U cm −3 with the recombinant B. licheniformis , which was ca. 2.5-fold higher than that of the wild-type. Carbon fluxes through the central metabolic pathways in the wild-type and recombinant B. licheniformis were calculated, using a mass balance-based mathematical model that contains 105 metabolites and 148 reaction fluxes and the time profiles of glucose, dry cell weight, organic acids, amino acids and SAP obtained in 3.5 dm 3 Bioreactor systems at C G0 =6 kg m −3 for the exponential growth phase and the SAP production phase. The bioreaction network flux analyses were first accomplished by using the theoretical data-based approach; and then by using the theoretical data-based capacity analysis approach. During the SAP synthesis period, the actual fluxes of the glycolysis pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, the amino acids biosynthetic pathways (and, consequently, SAP synthesis) are higher in the recombinant B. licheniformis strain than in the wild-type. Further, the normalised relative flux values of all the pathways, except the glycolysis pathway, change considerably in the recombinant bacteria. The effectiveness factor, defined as the SAP synthesis rate per maximum possible SAP synthesis rate was η =0.20 for the recombinant B. licheniformis . This indicates the possibility of a further increase in SAP production through metabolic engineering, and potential strategies to achieve this are also discussed.
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Bioreactor Operation parameters as tools for metabolic regulations in fermentation processes: influence of pH conditions
Chemical Engineering Science, 2003Co-Authors: Pınar Çalık, Esra Bilir, Güzide Çalık, Tunçer H. ÖzdamarAbstract:The influence of controlled- and uncontrolled-pH conditions together with the initial pH on the product and by-product distributions and oxygen transfer characteristics, whereupon the process rate limitations in relation to the intracellular reaction rates were investigated in serine alkaline protease (SAP) fermentation process by recombinant Bacillus licheniformis carrying pHV1431::subC on a defined medium with the sole carbon source glucose in the pH range of 6.80-7.25 in batch Bioreactors. Although the same amount of cell was produced at all the conditions in each type of Operation, with the increase of initial pH the cell formation rates increased; moreover, uncontrolled-pH Operation was favourable for the cell formation. The SAP synthesis rates and concentrations were higher at uncontrolled-pH Operations. Among the fermentations, pH 0 = 7.10 uncontrolled-pH Operation produced maximum SAP activity that was ca. 900 U cm -3 at t = 21 h. According to the biomass and SAP production profiles, bioprocess was divided into two periods; Period I(0 < t ≤ 10 h) covers the cell growth phase and Period II(10 < t ≤ 24 h) covers the SAP production phase. In Period I in both Operations, while the oxygen uptake rate (OUR) increased with the increase in initial pH the oxygen transfer rate (OTR) decreased. In Period II, among uncontrolled-pH Operations OUR was the lowest at pH = 7.10 while OTR decreased with the increase in initial pH. At the oxygen transfer condition applied, the bioprocess is biochemical reaction limited at all the conditions; nevertheless, with the decrease in initial pH, Damkohler number that is the oxygen transfer limitation decreases. Further, the perturbation effects of initial pH and the Operation conditions on the intracellular reaction rates were calculated for Periods I and II by using the experimental data obtained and, the diversions in the pathways and certain metabolic reactions and potential strategies for improving SAP production are also discussed.
Nei Pereira - One of the best experts on this subject based on the ideXlab platform.
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Microbial community dynamics in diesel waste biodegradation using sequencing batch Bioreactor Operation mode (SBR)
African Journal of Biotechnology, 2012Co-Authors: Edelvio De Barros Gomes, Alexandre Soares Rosado, Fl Do Carmo, Anabela Lopes, R.s. Peixoto, Nei PereiraAbstract:The dynamic of molecular microbial community during diesel waste biodegradation was investigated. The waste was treated in Bioreactors operated in sequencing batch Operation mode (SBR) in four cycles of 72 h, using optimized setpoints (pH, initial waste load, C:N ratio, aeration). Optimal conditions allowed the system to reach biodegradation of 53.3, 96.0, 76.2 and 75.0% at the end of cycles one, two three and four, respectively. Oxygen uptake rate (OUR) indicated increases in microbial activity from cycle one to cycle two (124.9 to 252.9 mgO2/L/h) and decreases in cycles three and four (120.4 to 108.8 mgO2/L/h, respectively). Investigations of microbial diversity showed changes in the microbial community members at the end of the cycle one. Significant reductions in the relative ecotoxicity were observed beginning with cycle two, and the reductions extended until the end of process. The SBR Operation mode proved to be an efficient method for treating the diesel waste, and the process allowed for relevant reductions in the hydrocarbon content of the waste along with an increase in its environmental quality. Changes in the microbial members are evidence of the synergistic action of the microbiota in the process. Key words : Microbial diversity, denaturing gradient gel electrophoresis, sequencing batch, biodegradation.
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Biotreatment of diesel waste by sequencing batch Bioreactor Operation mode (SBR).
International Biodeterioration & Biodegradation, 2010Co-Authors: Edelvio De Barros Gomes, Ricardo F. Silva, Alexandre Soares Rosado, Nei PereiraAbstract:Abstract The performance of a stirred tank Bioreactor operated in sequencing batch Operation mode (SBR) to treat diesel waste from storage tanks, was investigated. The SBR run in four cycles of 72 h, using optimized setpoints for pH, initial waste load, C:N ratio and agitation speed. Optimal conditions allowed the system to reach biodegradation percentages of 53.3, 96.0, 76.2 and 75.0% at the end of cycles one, two three and four, respectively. Investigations of microbial diversity showed changes in the microbial community members at the end of the cycle one. Significant reductions in the relative ecotoxicity were observed beginning with cycle two, and the reductions extended until the end of process. The SBR Operation mode proved to be an efficient method for treating the diesel waste, allowing relevant reductions in the hydrocarbon content of the waste along with an increase in its environmental quality.
Anna Trusek-holownia - One of the best experts on this subject based on the ideXlab platform.
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Two-step treatment of harmful industrial wastewater: an analysis of microbial reactor with integrated membrane retention for benzene and toluene removal
Polish Journal of Chemical Technology, 2015Co-Authors: Anna Trusek-holownia, Andrzej NoworytaAbstract:Abstract Standards for highly toxic and carcinogenic pollutants impose strict guidelines, requiring values close to zero, regarding the degradation of such pollutants in industrial streams. In many cases, classic bioremoval processes fail. Therefore, we proposed a stream leaving the microbial membrane Bioreactor (MBR) that is directed to an additional membrane separation mode (NF/RO). Under certain conditions, the integrated process not only benefits the environment but may also increase the profitability of the Bioreactor Operation. An appropriate model was developed and tested in which the bioremoval of benzene and toluene by Pseudomonas fluorescens was used as an example. This paper presents equations for selecting the Operation parameters of the integrated system to achieve the expected degree of industrial wastewater purification.
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Wastewater treatment in a microbial membrane Bioreactor — a model of the process
Desalination, 2008Co-Authors: Anna Trusek-holowniaAbstract:Abstract A model of microbial membrane Bioreactor with convective flow through the membrane is presented in the paper. Permeability coefficients hi in reference to microbial cells, substrates and possible reaction products as well as intensification coefficient Ψ which is a hydrodynamic parameter very significant for this Bioreactor type, were introduced. The latter coefficient is used to control the Bioreactor Operation. Advantages of the membrane Bioreactor over the classical flow Bioreactor are presented. A reduction of residence time, with other parameters kept constant, or an increase of the conversion degree which enables the growth of feed stream, is reached in the range of coefficient 1 Due to high efficiency of the membrane Bioreactor operating in a broad range of inlet concentrations and at low outlet concentration, this type of a microbial Bioreactor is particularly recommended in industrial wastewater biodegradation.
Saravanamuth Vigneswaran - One of the best experts on this subject based on the ideXlab platform.
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quantification of air stripping and biodegradation of organic removal in acrylonitrile butadiene styrene abs industry wastewater during submerged membrane Bioreactor Operation
Desalination, 2006Co-Authors: Chiayuan Chang, Jingsong Chang, Yaowen Lin, Laszlo Erdei, Saravanamuth VigneswaranAbstract:With the rapid development of the petrochemical industry in Taiwan, a significant amount of nitrogenous organic wastewater is generated. Some of this wastewater contains volatile residual monomers, such as acrylonitrile-butadienestyrene (ABS) manufacturing wastewater. Because of the high volatility of the acrylonitrile monomer, this chemical will be partly removed from the aeration system by the air stripping effect. An empirical model was developed for evaluating BOD removal by air stripping and biodegradation when the wastewater was treated in an aerobic submerged membrane Bioreactor (ASMBR). The results showed that the removal of BOD from ABS wastewater by air stripping was in the range of 2–10%. These results also showed that the estimation of contribution of air stripping and biodegradation in the BOD removal in the MBR is very important in the optimum design of the MBR for ABS wastewater.