Methanol Feed Rate Research Articles

A three-dimensional simulation of a mid-and-low temperature solar receiver/reactor for hydrogen production

The solar receiver/reactor is a key component that influences the conversion efficiency in the solar thermochemical process. A thermochemical solar reactor/receiver consisting of a porous Cu/ZnO/Al2O3 catalyst bed is studied in this paper. A three-dimensional thermochemical coupling model that incorporates the fluid flowing through the porous catalyst bed and energy conservation equations coupling the radiation/convection/conduction heat transfer with the reaction kinetics is proposed to investigate the performances of the receiver/reactor. The factors of influencing the hydrogen production and the temperature distribution, including the mole ratio of water/methanol, the solar radiation and the inlet temperature, are numerically investigated. Numerical simulation results indicate that the deactivation of the catalyst may appear near the receiver/reactor tube wall. The methanol conversion decreases with the increase of the methanol feeding rate, and the low inlet methanol feeding rate should be avoided for the protection of the catalyst bed. A new solar receiver/reactor is proposed by changing the aperture width along the flow direction to make the concentrated solar energy level match the chemical reaction. Compared with traditional solar receivers/reactors, the thermochemical efficiency can be increased by 3% points. The research findings will pave the way for the future development of the mid-and-low temperature solar receiver/reactor.

Methanol catalytic micro-combustor with pervaporation-based methanol supply system

A catalytic micro-combustor using a pervaporation membrane as the passive methanol vaporizer is developed for direct methanol fuel cell (DMFC) applications, which is able to realize self-starting in an ambient environment. The methanol feed rate and the membrane diffusion coefficient related to the permeation flux are experimentally evaluated. Results indicate that the air feed rate (AFR) has positive effects on enhancing the methanol production and also increasing the catalyst temperature. A higher AFR promotes vapor generation and improves the combustion rate, thereby facilitating a higher start-up performance of the combustor. The infrared thermal analysis verifies the positive effect of increasing the AFR on the status of heat production and distribution. Results also validate the feasibility of changing the operating orientation of the combustor, which illuminates its potential use for portable applications. The online fuel cell tests validate that this combustor is viable to be used as a heat generator for a vapor-feed DMFC.

Enhancement of thermostable β-glucosidase production in a slow methanol utilization strain of Pichia pastoris by optimization of the specific methanol supply rate

Control of the methanol supply during the production stage is a crucial parameter for maintaining cell growth and enhancing recombinant protein production by Pichia pastoris. In this study, optimization of the initial specific methanol supply rate was explored for high cell density fed-batch cultivation of recombinant fungal β- glucosidase in a slow methanol utilization strain (MutS), P. pastoris KM71. By varying the methanol feed rates for initiating the induction at a cell concentration of 60 g/L, the optimum initial specific methanol supply rate was determined to be 30.34 ± 0.34 mg/g·h. A methanol feed rate was proposed according to this optimum parameter and applied for the production of recombinant β-glucosidase at the higher cell concentrations of 80 and 100 g/L. The highest recombinant β-glucosidase activity obtained was 2851.7 ± 14.6 U/mL, which was four times higher than that obtained with the reference condition (40 g/L initial cell concentration). The success of this approach suggests that the strategy of optimizing the initial specific methanol supply rate could be adopted and applied for the production of other recombinant proteins in P. pastoris employing a methanol inducible system.

Cogeled copper–silica aerogel as a catalyst in hydrogen production from methanol steam reforming

In this work, the copper–silica aerogel catalyst was prepared by cogelation of copper and silica precursors in a sol–gel process followed by chemical surface modification and ambient pressure drying. The results of characterizations by XRD, BET, FE-SEM, TEM, FTIR and TPR showed the prepared catalysts had high surface area and pore size distribution in the range of 1–10 nm. Also, uniform nanostructural morphology with particle size of 5–20 nm was observed. The copper loading and calcination temperature influenced the morphology and texture of the catalyst and the nature of copper species. So, copper existed within the silica matrix as highly dispersed isolated copper ions or CuO crystallite clusters which have good interaction with the matrix. Catalytic performance for hydrogen production from methanol reforming was evaluated at 300 °C in the methanol feed rate of 12–65 mmol g−1 h−1. The catalyst activity was increased by increasing the copper loading and calcination temperature. In comparison with CuO/ZnO/Al2O3 commercial catalyst which showed 6–11% CO selectivity, no CO formation was detected using the prepared Cu/SiO2 aerogel catalysts.

Continuous supercritical decrosslinking extrusion process for recycling of crosslinked polyethylene waste

ABSTRACTThis work demonstrates that extrusion process can be used as a continuous reactor for the recycling of crosslinked polyethylene (XLPE) via supercritical methanol decrosslinking reaction. The multistage single screw extruder (L/D = 36, D = 40 mm) with methanol injection pump was customized for the continuous supercritical decrosslinking reaction. Reaction temperature was ranged from 360°C to 390°C. The amount of methanol was varied from 0 to 7 mL/min. The extruder provided the crosslinked polymers with the supercritical conditions of methanol during continuous process. The gel content of XLPE decreased with the increase in the reaction temperature and methanol content. Although XLPE experienced supercritical methanol for less than 2 min retention time in the continuous supercritical extruder, it was completely decrosslinked above 390°C at the methanol feeding rate of 7 mL/min. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41442.

Identification of critical parameters in liquid enzyme-catalyzed biodiesel production.

Callera™ Trans L, a liquid formulation of Thermomyces lanuginosus lipase, has recently shown great promise as a cost-efficient catalyst for methanolysis of triglyceride substrates, specifically in the BioFAME process. However, identifying the right combination of temperature and concentrations of catalyst, water and methanol to realize the full potential of the reaction system has remained a challenge. This study presents an investigation of the impact of temperature, enzyme and water concentration on the reaction, as well as the effect of methanol feed rate for the conversion of rapeseed oil in a fed-batch reaction system. It was observed that the reaction can be divided into two distinct parts. The first part of the reaction, during which primarily tri- and diglycerides are converted, proceeded at a high rate and thus required a high rate of methanol supply. The second part of the reaction, where the remaining di- and monoglycerides are converted, proceeded at a much lower rate. Consequently, it is necessary to reduce the methanol feed rate during the latter part of the reaction to avoid inhibition or even inactivation of the enzyme. Since the second part of the reaction occupied most of the 24-h reaction time, it was concluded that this is the part of the process where further development efforts should be targeted. This point was demonstrated by partially substituting the catalyst with a lipase with a different specificity, which enhanced the performance during the second phase of the reaction.

Fast development of Pichia pastoris GS115 Mut+ cultures employing batch-to-batch control and hybrid semi-parametric modeling

In this paper, we implemented a model-based optimization platform for fast development of Pichia pastoris cultures employing batch-to-batch control and hybrid semi-parametric modeling. We illustrate the methodology with a P. pastoris GS115 strain expressing a single-chain antibody fragment (scFv) by determining the optimal time profiles of temperature, pH, glycerol feeding and methanol feeding that maximize the endpoint scFv titer. The first hybrid model was identified from data of six exploratory experiments carried out in a pilot 50-L reactor. This model was subsequently used to maximize the final scFv titer of the proceeding batch employing a dynamic optimization program. Thereupon, the optimized time profiles of control variables were implemented in the pilot reactor and the resulting new data set was used to re-identify the hybrid model and to re-optimize the next batch. The iterative batch-to-batch optimization was stopped after 4 complete optimized batches with the final scFv titer stabilizing at 49.5mg/L. In relation to the baseline batch (executed according to the Pichia fermentation guidelines by Invitrogen) a more than fourfold increase in scFv titer was achieved. The biomass concentration at induction and the methanol feeding rate profile were found to be the most critical control degrees of freedom to maximize scFv titer.

Exergy and environmental impact assessment of solar photoreactors for catalytic hydrogen production

In this paper, a new photo-catalytic energy conversion system is analyzed for continuous production of hydrogen at a pilot-plant scale. Two methods of photo-catalytic water splitting and solar methanol steam reforming are investigated as two potential solar-based methods of catalytic hydrogen production. The exergy efficiency, exergy destruction, environmental impact and sustainability index are investigated for these systems, as well as exergoenvironmental analyses. A Compound Parabolic Concentrator (CPC) is presented for the sunlight-driven hydrogen production system. This study shows that an optimum water flow rate exists, where the exergy efficiency of the photo-catalytic hydrogen production is maximized. The amount of CO2 emissions that are reduced by this process increases at higher flow rates. The light intensity is one of the key parameters in design optimization of the photo-reactors, in conjunction with light absorptivity of the catalyst. The results show that a trade-off exists in terms of exergy efficiency improvement and CO2 emissions of the photo catalytic hydrogen production system. The optimal working condition of solar methanol reforming to satisfy the exergy–environmental considerations is found to be light intensity range of 530Wm−2<J<600Wm−2 and water–methanol mole ratio of 1.5–2. An optimum methanol feeding rate associated with CO2 emissions of methanol steam reforming is determined in order to establish the required solar flux for photo-catalytic conversion of methanol to hydrogen.

Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.

A single step non-catalytic esterification of palm fatty acid distillate (PFAD) for biodiesel production

In this work, the single step method for non-catalytic esterification of palm fatty acid distillate (PFAD), which is readily applicable to actual production of biodiesel, was investigated. In this method, the esterification reaction is accomplished in a single step by ensuring water-free reaction conditions and the acid value is reduced to below 0.5 (mgKOH/g) which has not been possible in previous methods. The reaction was completed (<0.5mgKOH/g) within 180min at relatively high temperature (>250°C) enough to be above boiling point of water and at moderate pressure (0.85–1.20MPa) without any catalyst. The effects of temperature, methanol feed rate and pressure on a semi-batch reaction were investigated and the optimal values of these variables were found (temperature: 290°C, pressure: 0.85MPa, feed rate: 2.4g/min). The acid value was reduced from 191.4 to 0.36 (mgKOH/g) just in 180min at these conditions. From the kinetic study on non-catalytic esterification of PFAD, it was found that the activation energy is 17.74kJ/mol and the frequency factor is 2.12min−1.

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9gL(-1) vs. 1.2gL(-1)), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.

Dynamic flux balance analysis for pharmaceutical protein production by Pichia pastoris: Human growth hormone

The influence of methanol feeding rate on intracellular reaction network of recombinant human growth hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates, namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04h−1 (MS-0.04) where Period-I (33≤t<42h) includes the early exponential growth phase; Period-II (42≤t<48h) is the exponential growth phase where the specific cell growth rate decreases; Period-III (48≤t≤51h) is the exponential growth phase where rhGH concentration was the highest; and Period-IV (t>51h) is the diminution phase for rhGH and cell synthesis. In Period-I, almost all of the formaldehyde entered the assimilatory pathway, at MS-0.02 and MS-0.03, whereas, at MS-0.04 high methanol feeding rate resulted in an adaptation problem. In Period-III, only at MS-0.02 co-carbon source sorbitol uptake-flux was active showing that sorbitol uptake does not affected from the predetermined feeding rate of methanol at μ0>0.02h−1. The biomass synthesis flux value was the highest in Period-I, -II and -III, respectively at MS-0.03 & MS-0.04, MS-0.04 and MS-0.02; whereas, rhGH flux was the highest in Period-I, -II, and -III, respectively at MS-0.03, MS-0.02 and MS-0.03. Based on the fluxes, Period-I should start with MS-0.03 methanol feeding rate and starting from the middle of Period-II methanol feeding rate should be shifted to MS-0.02.

Antibody expression kinetics in glycoengineered Pichia pastoris

Growth of the antibody market has fueled the development of alternative expression systems such as glycoengineered yeast. Although intact antibody expression levels in excess of 1 g L(-1) have been demonstrated in glycoengineered yeast, this is still significantly below the titers reported for antibody fragments in fungal expression systems. This study presents a simplified approach to estimate antibody secretion kinetics and oxygen uptake rate requirements as a function of growth-rate controlled by a limiting methanol feed rate in glycoengineered Pichia pastoris. The yield of biomass from methanol and the specific oxygen requirements predicted in this study compare well with values reported in the literature for wild-type P. pastoris, indicating the intrinsic nature of these yields independent of glycoengineering or the heterologous protein expressed. Specific productivity was found to be a non-linear function of specific growth rate. Based on comparison with relationships between specific growth rate and specific productivity reported in the literature this correlation seems empirical in nature and cannot be established a priori. These correlations were then used in a simple mass balance based model to predict the cultivation performance of carbon limited cultivations under oxygen transfer limited conditions to indicate the usefulness of this approach to predict large scale performance and aid in process development.

Fermentation and oxygen transfer characteristics in recombinant human growth hormone production byPichia pastorisin sorbitol batch and methanol fed-batch operation

BACKGROUND: The influence of methanol feed rate on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut+ in medium containing sorbitol was investigated at three different specific growth rates (µ), namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04 (MS-0.04). RESULTS: Increasing methanol feed rate above MS-0.03 did not affect sorbitol consumption, showing that µ = 0.03 h−1 is a threshold limiting value, above which sorbitol utilization became independent of methanol feed rate. Moreover, when sorbitol was consumed, no further cell growth was observed. Increase in methanol feed rate triggered cell synthesis and the highest cell concentration was obtained at MS-0.04 as 48 g L−1 (t = 18 h); whereas, the highest rhGH production, 270 mg L−1, was obtained at MS-0.03 as a consequence of lower extracellular protease production and higher AOX activity (41 U g−1 CDW). Oxygen uptake rate increased with increasing µ, having the maximum value, 76.6 mmol m−3 s−1, at MS-0.04. KLa had a tendency to increase with µ, having a maximum value of 0.15 s−1 at MS-0.04 (t = 15 h). CONCLUSION: By considering rhGH concentration and oxygen transfer characteristics, the bioprocess can be improved by a two-stage feeding strategy, operating at MS-0.04 at the beginning of fermentation, and thereafter shifting to MS-0.03. Copyright © 2009 Society of Chemical Industry

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut(+)) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry-based model containing 102 metabolites and 141 reaction fluxes. Four fed-batch operations with (MS-) and without (M-) sorbitol were performed at three different constant specific growth rates (h(-1)), and denoted as M-0.03, MS-0.02, MS-0.03, and MS-0.04. Considering the methanol consumption pathway, the M-0.03 and MS-0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS-0.03 to MS-0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher micro(0). Comparing M-0.03 and MS-0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4-fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS-0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS-0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism.

A structured kinetic model for recombinant protein production by Mut+ strain of Pichia pastoris

A structured kinetic biochemical model for Pichia pastoris growth and recombinant protein production, able to predict r-protein production and optimize the feeding strategy is reported. Pichia pastoris was divided into three compartments, the recombinant product human erythropoietin (rHuEPO) was considered in one compartment, alcohol oxidase and extracellular protease enzymes were assembled in the enzymatic compartment, and rest of the cell components (including intracellular proteases) were gathered in the cellular compartment. After parameter identification by the simplex method, sensitivity analysis was carried out to confirm that the global optimum values were reached for the parameters of the model. The model successfully simulated the dynamic changes and the resulting rHuEPO production caused by variations in methanol feeding rate, even in the lag and the stationary phases. The model successfully predicted the optimal feeding strategies and has great potential for use in process simulation and control of glycosylated recombinant protein production by Pichia pastoris cells.

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch-wise sorbitol addition as a co-substrate at the induction phase of methanol fed-batch fermentation by Pichia pastoris (Mut(+)) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch-wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag-phase for the cells and reaching 'high cell density production' at t = 24 h of the process (C(X) = 70 g CDW/l); (b) achieving 1.8-fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2-fold; (d) eliminating the lactic acid build-up period; (e) lowering the oxygen uptake rate two-fold; and (f) obtaining 1.4-fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut(+) strains of P. pastoris for the production of any bio-product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris.

Preparation, characterization, and catalytic activity of H5PMo10V2O40 immobilized on nitrogen-containing mesoporous carbon (PMo10V2/N-MC) for selective conversion of methanol to dimethoxymethane

Nitrogen-containing mesoporous carbon (N-MC) was synthesized by a templating method using SBA-15 and polypyrrole as a templating agent and a carbon precursor, respectively. The N-MC was then modified to have a positive charge, and thus, to provide a site for the immobilization of [PMo10V2O40]5−. By taking advantage of the overall negative charge of [PMo10V2O40]5−, H5PMo10V2O40 (PMo10V2) catalyst was chemically immobilized on the N-MC support as a charge-matching component. Characterization results showed that nitrogen in the N-MC played an important role in forming a nitrogen-derived functional group (amine group), and PMo10V2 catalyst was finely and chemically immobilized on the nitrogen-derived functional group of N-MC support. In the vapor-phase selective conversion of methanol, the PMo10V2/N-MC catalyst showed a higher conversion of methanol than the bulk PMo10V2 catalyst. Furthermore, the PMo10V2/N-MC catalyst showed a higher selectivity for dimethoxymethane (a product formed by bifunctional oxidation-acid-acid catalysis) and a higher selectivity for methylformate (a product formed by bifunctional oxidation-acid-oxidation catalysis) than the PMo10V2 catalyst. Reaction pathway for selective conversion of methanol to dimethoxymethane over PMo10V2/N-MC catalyst could be controlled by changing the methanol feed rate.

Prevention of human granulocyte colony‐stimulating factor protein aggregation in recombinant Pichia pastoris fed‐batch fermentation using additives

rhG-CSF (recombinant human granulocyte colony-stimulating factor) was expressed in the yeast Pichia pastoris under the control of the AOX1 (alcohol oxidase 1) promoter. The production of rhG-CSF was induced by switching from growth on glycerol to growth on methanol. In the induction phase, the methanol feed rate had a significant effect on the specific expression rate of rhG-CSF. A constant feed rate of 16 ml.h(-1).l(-1) was found to be optimal for a high specific expression rate of rhG-CSF (0.058 mg(-1).h(-1).g DCW(-1); DCW is dry cell weight). Under this condition, a maximum concentration of 300 mg/l of rhG-CSF and the expression yield of 0.6 mg of rhG-CSF/g of methanol were attained. However, the secreted rhG-CSF was shown to exist as aggregates in the culture broth, owing to hydrophobic interactions. To prevent undesirable protein aggregation, the presence of additional additives in the P. pastoris culture medium was investigated. Among seven additives tested, Tween 20, Tween 80 and betaine exhibited the best results in respect of preventing the formation of rhG-CSF protein aggregates.

Production of monoclonal antibodies by glycoengineered Pichia pastoris

The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l −1 of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man 5GlcNAc 2. N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5–7.5), temperature (16–24 °C), dissolved oxygen concentration (0.85–3.40 mg l −1) and specific methanol feed rate (9–19 mg g −1 h −1) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.