Methanol Consumption Research Articles

Scale-up and cost analysis of biodiesel production using liquid-liquid film reactors: Reduction in the methanol consumption and investment cost

Biodiesel is an important renewable fuel industrially produced by transesterification of fats and oils. Conventional process productivity is limited by high residence time in the reaction and separation stages. Liquid-Liquid Film Reactor (LLFR) is a new technology able to overcome this limitation. This work presents the scale-up, cost analysis and process feasibility for the LLFR. The study was made using a complete mathematical model which includes fluid dynamics, kinetics, mass transfer resistance, liquid-liquid equilibria, and cost evaluation. This study recommended the construction of plants with capacities over 5 t/h (about 40,000 t/year) in order to avoid over costs in the process and also recommended the employ of multiple reaction stages to reduce until 32% the global methanol consumption. Results also shows that to get a profit in this biodiesel production, the vegetable oil price must be less than 830 US$/t. Finally, it was found that the use of LLFR technology permit a reduction up to 60% in the required volume when two LLFR stages are employed in comparison to the traditional use of continuous stirred tank reactors (CSTR), this reduction promotes a final cost reduction up to 0.6% per kg of biodiesel.

Development of a New High-Cell Density Fermentation Strategy for Enhanced Production of a Fungus β-Glucosidase in Pichia pastoris.

Traditional diosgenin manufacturing process has led to serious environmental contamination and wastewater. Clean processes are needed that can alternate the diosgenin production. The β-glucosidase FBG1, cloned from Fusarium sp. CPCC 400709, can biotransform trillin and produce diosgenin. In this study, Pichia pastoris production of recombinant FBG1 was implemented to investigate various conventional methanol induction strategies, mainly including DO-stat (constant induction DO), μ-stat (constant exponential feeding rate) and m-stat (constant methanol concentration). The new co-stat strategy combining μ-stat and m-stat strategies was then developed for enhanced FBG1 production during fed-batch high-cell density fermentation on methanol. The fermentation process was characterized with respect to cell growth, methanol consumption, FBG1 production and methanol metabolism. It was found that large amounts of formaldehyde were released by the enhanced dissimilation pathway when the co-stat strategy was implemented, and therefore the energy generation was enhanced because of improved methanol metabolism. Using co-stat feeding, the highest volumetric activity reached ∼89 × 104 U/L, with the maximum specific activity of ∼90 × 102 U/g. After 108 h induction, the highest volumetric production reached ∼403 mg/L, which was ∼91, 154, and 183 mg/L higher than the maximal production obtained at m-stat, μ-stat, and DO-stat strategies, respectively. FBG1 is the first P. pastoris produced recombinant enzyme for diosgenin production through the biotransformation of trillin. Moreover, this newly developed co-stat induction strategy represents the highest expression of FBG1 in P. pastoris, and the strategy can be used to produce FBG1 from similar Pichia strains harboring Fbg1 gene, which lays solid foundation for clean and sustainable production of diosgenin. The current work provides unique information on cell growth, substrate metabolism and protein biosynthesis for enhanced β-glucosidase production using a P. pastoris strain under controlled fermentation conditions. This information may be applicable for expression of similar proteins from P. pastoris strains.

Analysis of heat release characteristics when working on methanol

Existing and currently being upgraded simplified models of the internal combustion engine workflow have very limited capabilities in terms of describing the fuel combustion process. Gorenje it is known that the most important means of studying the combustion in diesel fuel is the analysis of the indicator diagram for heat release. By analyzing the shape of the indicator chart, you can get information about the process of heat release during fuel combustion. The heat release characteristic is a function of the amount of heat released in the cylinder, from the angle of rotation of the crankshaft or time. The heat release characteristics are expressed in different ways: as a dependency of the absolute amount of heat from the current corner p.c.v., the integrated characteristic or the so-called dependence of heat release rate — differential characteristic. In most cases, the characteristics of relative heat release are used, rather than absolute. Vyatka agricultural Academy continues to study the impact of alcohol consumption in diesel engines when they are fed using a dual fuel supply system. The article presents the effect of methanol consumption on the characteristics of heat release.

Development of highly efficient methanol steam reforming system for hydrogen production and supply for a low temperature proton exchange membrane fuel cell

Methanol steam reforming (MSR) has been regarded as a promising hydrogen supply method for proton exchange membrane fuel cell (PEMFC), while the efficiency for hydrogen production and integration method of MSR with PEMFC are two major challenges for commercial applications. Here, we present a highly efficient MSR system for hydrogen production and supply for low temperature PEMFC (LT-PEMFC). The MSR system has a highly compact microreactor, wherein MSR, methanol combustion, and CO selective methanation reactions occur. The CO selective methanation is used to reduce the content of CO concentration to remit the CO poison, then the reformate of MSR system is mixed with air and supply for the LT-PEMFC. Then, experimental tests are conducted to investigate the effects of operating parameters on hydrogen production. A staged supply strategy is proposed, it enables to startup the system within 11.2 min and with methanol consumption of 34.72 g. Results show that the methanol conversion can reach up to 93.0% and system's energy efficiency of 76.2%. After integration with a LT-PEMFC, a maximum 160 W electricity can be generated. The results obtained in this study demonstrated that the developed MSR system can be used to supply hydrogen for LT-PEMFC and able to power mobile device requiring hundreds of watts power consumption.

Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system

α-Amylase, which was isolated from Geobacillus stearothermophilus SR74, has shown its potential to be used in industrial applications. However, its expression in the Pichia pastoris expression system with the alcohol oxidase 1 promoter (PAOX1) requires high methanol consumption and is time-consuming. This study aimed to express SR74 α-amylase in an alternative yeast system, using Meyerozyma guilliermondii strain SO, which was isolated from a spoiled orange (SO) under the regulation of a formaldehyde dehydrogenase promoter (PFLD). Qualitative screening showed that strain SO possessed a native amylase grown on YPD-starch plate at 30 °C. The recombinant SR74 α-amylase was further quantified and validated using the Western blot test. It was confirmed that SR74 α-amylase was expressed by strain SO extracellularly with a size of 59 kDa. Optimization in a shake flask showed that the recombinant SR74 α-amylase, which was regulated by PFLD, was successfully produced (26 U/mL) without any external inducer in the YPT medium after 24 h of cultivation. In conclusion, strain SO was able to produce SR74 amylase without methanol in one-fifth the fermentation time of P. pastoris. Further optimization of the expression may be done to improve the yield, as this methanol-free host is still underexplored.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Methanol can be effectively removed from air by biofiltration (Shareefdeen et al., 1993; Babbitt et al., 2009 [1,2]). However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms (Negruţa et al., 2010 [3]), and it can be released out of the cell constituting a secondary emission. The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m−3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1. Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment. How to cite: Guerrero K, Arancibia A, Caceres M, et al. Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115. Electron J Biotechnol 2019;40. https://doi.org/10.1016/j.ejbt.2019.04.003.

Used-cooking-oil biodiesel: Life cycle assessment and comparison with first- and third-generation biofuel

The environmental sustainability of second-generation biodiesel (used-cooking-oil) was examined, at industrial-scale, in Greece. The total carbon and environmental footprint per tonne of biodiesel production was ∼0.55t CO2eq (i.e. ∼14g CO2eq/MJ) and 58.37 Pt, respectively. This is ∼40% lower compared to first-generation biodiesel, an order of magnitude lower than the third-generation (microalgae), since the latter is not a fully-fledged technology yet. A threefold reduction in environmental impacts was observed compared to petrodiesel. Environmental hotspots include energy inputs to drive the process, followed by methanol (CH3OH) and potassium methoxide (CH3KO) consumption. Glycerol (C3H8O3) and potassium sulfate (K2SO4), both process co-products, resulted to avoided environmental burdens. Furthermore, used-cooking-oil valorisation for biodiesel production can address water pollution concerns from its disposal to the sewage system. The total distance and means of transport were found to influence the system’s environmental sustainability. Strong incentives for used-cooking-oil recycling, widespread collection systems, and biodiesel supply chain optimization are still pending in Greece, Europe, and further afield. Given its overall low environmental footprint and capability to be produced at commercial scales, the second-generation biodiesel, which currently represents 15% of the biodiesel market in Greece, could act as a stepping-stone in decarbonizing Europe’s transport sector and improving supply and energy security.

Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.

Simulation and economic assessment of large-scale enzymatic N-acetyllactosamine manufacture

N-acetyllactosamine (LacNAc) is an important lactose-derived molecule which can act as an effective prebiotic. In this study a process for the enzymatic synthesis and downstream purification of LacNAc was designed based on the use of thermostable β-galactosidases from Bacillus circulans (BgaD-D), Thermus thermophilus HB27 or Pyrococcus furiosus (CelB) respectively. Four configurations for the purification stage were simulated; anion-exchange chromatography, an activated charcoal-Celite column, N-acetylglucosamine (GlcNAc) crystallization and an activated charcoal-Celite column, as well as selective crystallization. While the enzyme CelB has greater stability at higher temperatures, this enzyme gives a lower LacNAc yield, leading to significant capital investment. For the design based on the BgaD-D biocatalyst and anion exchange chromatography, recovery of GlcNAc improved the project profitability when the GlcNAc price was greater than $10 per kg. GlcNAc was the main contributor to the raw material costs for most processes, although methanol contributed 72% of these costs for the process based on an activated charcoal column. The use of a crystallizer for GlcNAc separation before this column, reduced this methanol consumption by 73%. The use of selective crystallization proved the best approach, reducing the minimum LacNAc sales price to $2 per gram. The plant was more economic when the acceptor to donor ratio was reduced from 10 to 4 and the lactose concentration increased from 50 mM to 550 mM.

Continuous fermentation of recombinant Pichia pastoris Mut+ producing HBsAg: Optimizing dilution rate and determining strain-specific parameters

There is a growing interest in the biopharmaceutical industry to enhance production efficiency via shifting batch to continuous manufacturing. In the previous study, we demonstrated that under identical operation condition, continuous fermentation of recombinant Pichia pastoris (P. pastoris) Mut+ producing intracellular hepatitis B surface antigen (HBsAg) has considerably higher efficiency compared to the conventional fed-batch process. In the current work, by examining various dilution rates, we further optimized the process efficiency, and also determined some strain-specific parameters. According to the results, continuous operation at the dilution rate of 0.015 1/h demonstrated the highest performance compared to other dilution rates of 0.009, 0.02, 0.25, 0.03 and 0.0417 1/h. In the optimum dilution rate, the average HBsAg titer, yield, specific and volumetric productivity were 283.9mgHBsAg/L, 0.71mgHBsAg/g MeOH, 0.0097mgHBsAg/g wet cell weight (WCW)/h and 4.26mgHBsAg/L/h, respectively. Compared to the continuous fermentation in the previous study, the volumetric and specific productivities were improved by the factors of 2.0 and 2.5, respectively. Following Herbert-Pirt linear relation between specific growth rate and methanol consumption rate, the obtained biomass yield and maintenance coefficient in the continuous fermentation were 1.22 (g WCW/g MeOH) and 0.008 (g MeOH/g WCW/h), respectively. The consistency in the process condition and efficiency without any genetic instability and contamination in the one-month operation- in each fermentation run- suggests that the studied recombinant strain could be one of the potential candidates for continuous biomanufacturing of hepatitis B vaccine.

The optimization performance of cross‐linked sodium alginate polymer electrolyte bio‐membranes in passive direct methanol/ethanol fuel cells

Consumption of methanol and ethanol as a fuel in the passive direct fuel cells technologies is suitable and more useful for the portable application compared with hydrogen as a preliminary fuel due to the ease of management, including design of cell, transportation, and storage. However, the cost production of commercial membrane is still far from the acceptable commercialization stage. Based to our previous works, the low cost of cross-linked sodium alginate (SA) polymer electrolyte bio-membrane shown the virtuous chemical, mechanical, and thermal characterization as polymer electrolyte membrane in the direct methanol fuel cells (DMFCs). This study will further the investigation of cross-linked SA polymer electrolyte bio-membrane performance in the passive DMFCs and the passive direct ethanol fuel cells (DEFCs). The experimental study investigates the influence of the membrane thickness, loading of catalysts, temperature, type of fuel, and fuel concentration in order to achieve the optimal working operation performances. The passive DMFCs is improved from 1.45 up to 13.5 mW cm−2 for the maximum peak of power density, which is obtained by using 0.16 mm as an optimum thick of SA bio-membrane that shown the highest selectivity 6.31 104 S s cm−3, 4 mg cm−2 of Pt-Ru as an optimum of anode catalyst loading, 2 mg cm−2 of Pt at the cathode, 2M of methanol as an optimum fuel concentration, and an optimum temperature at 90°C. Under the same conditions of cells, the passive DEFCs are shown to be 10.2 mW cm−2 in the maximum peak of power density with 2M ethanol. Based on our knowledge, this is the first work that reports the optimization works of performance SA-based membrane in the passive DMFCs via experimental studies of single cells and the primary performance of passive DEFCs using the SA-based membrane as polymer electrolyte membrane.

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

AbstractBACKGROUNDProcess variability in bioprocess systems involving genetically engineered strains is a common bottleneck and a real‐time insight of the on‐going process is crucial to achieve the desired product and its quality. In this study, a process analytical technology (PAT) platform was developed for the monitoring and control of specific growth rate during methanol induction phase (μmet) of glycoengineered Pichia pastoris fermentation for human interferon alpha 2b (huIFNα2b) production.RESULTSThe PAT guided approach involves real‐time monitoring of capacitance (ΔC) facilitating online estimation of specific growth rate (μest), which serves as a process input during controller application. Fed‐batch experiments using pulsed‐feeding of methanol at different dosage (20 g and 30 g) did not significantly influence μmet. Exponential methanol feeding was achieved using a modified proportional, integral and derivative (PID) controller for different predefined specific growth rate set point (μsp) values, namely 0.015, 0.03, 0.04 and 0.06 h−1. Exponential feeding strategy during the induction phase resulted in two crucial outcomes: (i) controlled methanol feeding rate (regulated by the developed PID controller) balanced the methanol consumption rate (qs, met) of the P. pastoris; (ii) significant improvement in huIFNα2b titer (1483 mg L−1) and specific productivity(>0.4 mg g−1. h) was achieved by the robust control of μmet at optimal (0.04 h−1) value. The purified huIFNα2b was found to exhibit antiproliferative effect against human breast cancer cell lines.CONCLUSIONSEfficient control of μmet at a very low narrow range was achieved with a long‐term controller stability (>10 h) and the highest titer reported to date for huIFNα2b (at optimal μmet) in the yeast expression platform. © 2019 Society of Chemical Industry

Semi-preparative separation of dihydromyricetin enantiomers by supercritical fluid chromatography and determination of anti-inflammatory activities

Dihydromyricetin, extracted from Ampelopsis grossedentata, has been widely used as one of Chinese health products in recent years. However, limited chiral separation method hinders the studies of pharmacological and pharmacokinetic activity differences of (+)-dihydromyricetin, (−)-dihydromyricetin, and (±)-dihydromyricetin. Herein, we developed a supercritical fluid chromatography approach for chiral separation of dihydromyricetin. Firstly, effects of chiral stationary phase, co-solvent, and flow rate of mobile phase have been investigated in detail. The resolution of 5.11 was achieved for dihydromyricetin enantiomers on amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase with the CO2-methanol mixture (60:40, v/v). With respect to the enantiomeric purity, production rate and solvent consumption of 15 stacked injections, sample loading for semi-preparative separation of dihydromyricetin was optimized in three given equivalents set by volume overloading. Along with increase of sample loading per injection from 40 mg to 120 mg, the productivity of dihydromyricetin increased from 0.07 g (racemate)/g (chiral stationary phase) /24 h to 0.27 g (racemate) /g (chiral stationary phase)/24 h, and the consumption of methanol significantly reduced from 5.86 L/g (racemate) to 1.76 L/g (racemate). Moreover, (−)-dihydromyricetin exhibited better anti-inflammatory activity in TLR 2-related Raw 264.7 cells than (+)-dihydromyricetin and (±)-dihydromyricetin.

Application of a mid-/low-temperature solar thermochemical technology in the distributed energy system with cooling, heating and power production

A new solar-assisted cooling, heating and power (CCHP) system is developed for improving the energy conversion efficiency in this work. Solar thermal energy (250–350 °C) collected by a parabolic trough solar collector is used to drive the thermochemical reaction of methanol decomposition, then the generated solar fuel in the form of syngas is fed into an internal combustion engine (ICE) to generate electricity. The high-temperature exhaust gas released by the ICE is used to produce cooling and heating energies via a double-effect LiBr-H2O absorption refrigerator and a heat exchanger. Solar energy is converted to chemical energy in the form of syngas, the distinct advantages of energy cascade utilization and thermochemical storage can be realized. Numerical simulation results show that in the proposed CCHP system the energy and exergy efficiencies at the designate condition are 82.0% and 58.72%, respectively. The off-design performances are investigated by deploying the proposed CCHP system to a shopping center building, the annual efficiency and the solar fraction reach 53.6% and 9.81%, respectively. In comparison with the reference solar-assisted CCHP system, the annual methanol consumption of the proposed CCHP system decreases to 874.31 tons with the fuel saving ratio of 4.56% and less CO2 emission. This novel solar CCHP system presents a promising cost-effective performance and the system life cycle cost saving ratio reaches 2.84%. The research findings provide an alternative route towards efficiently utilizing solar energy.

Process Integration and Feedstock Optimisation of a Two-Step Biodiesel Production Process from Jatropha Curcas Using Aspen Plus

Abstract Jatropha curcas oil (JCO) has been recognized as a viable non-edible feedstock for biodiesel production with the focus of achieving lesser reliance on fossil fuels. The aim of this work is to integrate and simulate the production of biodiesel from Jatropha curcas oil by a two-step process; a hydrolysis step and a trans-esterification step. The challenge is then to optimise the feedstock ratios to obtain the minimal water and methanol consumption to give optimal biodiesel yield. For this purpose, steady-state simulation model of a two-step production process of biodiesel from Jatropha curcas oil was prepared using ASPEN Plus V8.8. The response surface methodology (RSM) based on a central composite design (CCD) was used to design optimisation experiments for the research work. From the ANOVA, methanol/oil ratio of the trans-esterification step was found to have a significant effect on the biodiesel yield compared to the water/oil ratio of the hydrolysis step. The linear model developed was shown to be a good predictor of feedstock ratios for biodiesel yield. The surface plot revealed that both feedstock ratios do not show a significant combinatorial effect on each other. Numerical optimisation gave the optimum values of the feedstock ratios as a methanol/oil ratio of 2.667 and a water/oil ratio of 1. The optimisation results also indicated a predicted optimum biodiesel yield of 10.0938 kg/hr.

Simulation-Based Multiobjective Optimization of the Product Separation Process within an MTP Plant

This work proposes a simulation-based multi-objective optimization model for the operation of the product separation process in a methanol to propylene (MTP) plant, striving to enhance the system energy utilization eciency. The formation of byproducts including an oxygenated hydrocarbon, dimethyl ether (DME), makes the product rening process highly energy-intensive, not only because that the various hydrocarbon byproducts require a long train of distillation columns but also owing to the fact that DME forms azeotrope with product propylene. Furthermore, the formation of oxygenated DME byproduct varies with the catalyst activity, which makes the system more complex. The contribution of this paper is twofold. First, it proposes a novel way of DME removal with responsive consumption of extractant methanol according to the DME concentration during one production period. In this way, the energy consumption of the condenser and reboiler of the methanol recovery unit can be expected to decrease by 61.5% and 37.6...

IMPROVMENT THE MATHEMATICAL MODEL FOR THE HYDRATES FORMATION CALCULATING IN THE GAS GATHERING NETWORK

Background Most gas fields are characterized by complex and branched systems for collecting and inter-field gas transportation. It should be noted that the main characteristics of gas fields are well cluster location and an extensive plume system from wells to the integrated gas treatment unit (GTU) for transportation, as well as the complex network structure of the inter-field collector. Such systems form a complex hierarchical structure, in which field information has a high degree of inaccuracy, and often there are a large number of errors in the measurements of technological parameters, such as measurements of flow, pressure and temperature. In some nodes of the gas gathering network, these data are not displayed at all or the corresponding parameters are not measured due to the lack of instrumentation. All this leads to increased consumption of chemical reagent methanol. Aims and Objectives To improve gas collection systems and reduce the consumption of chemical reagents during operation, it is necessary to analyze existing mathematical models and, based on this analysis, create a more sophisticated mathematical model of calculation that takes into account the optimal number of factors affecting the formation of hydrates in field systems. Especially this problem is relevant in the conditions of operation of wells at negative ambient temperatures, that is, in the winter period or for the fields of the Far North. Results A completely new mathematical model has been developed that allows to diagnose the formation of hydrates in the plumes of the gas gathering network prior to the integrated gas treatment unit. A formula is proposed for calculating the temperature in gas pipelines, which reflects two independent (physically meaningful) parameters that take into account the presence of a thermal boundary layer and the temperature variation along the length of the pipeline. A flowchart of the proposed mathematical model has been developed, which can be easily realized and obtained a system for diagnosing and preventing the formation of hydrates for the control units of the GTU.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris.

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible PAOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a PAOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01hr-1 ) and higher consumption of methanol (2.25 vs. 1.81mmol/gDCW .hr) and oxygen (2.2 vs. 0.66mmol/gDCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3gDCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Methanol can be effectively removed from air by biofiltration. However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms, and it can be released out of the cell constituting a secondary emission.

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m−3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.