Glycerol Feeding Research Articles

An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures.

BackgroundMembrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson’s disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN).ResultsThe optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks.ConclusionsThe ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure highlights the potential role of chemical chaperones such as DMSO in improving yields of recombinant membrane proteins with a different topology than G-coupled receptors. Finally, the proposed ANN shows that the manipulation of classic fermentation parameters coupled with the addition of specific molecules can open and reinforce new perspectives in the optimization of P. pastoris bioprocesses for membrane proteins biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0304-7) contains supplementary material, which is available to authorized users.

Enhancing allyl alcohol selectivity in the catalytic conversion of glycerol; influence of product distribution on the subsequent epoxidation step

AbstractConversion of glycerol to allyl alcohol in a flow reactor and subsequent conversion to glycidol in a batch reactor are discussed in this paper. To increase reaction selectivity in the glycerol to allyl alcohol reaction, the catalyst was modified with alkali metal salts. The treatment of the catalyst (Fe/Al2O3) with potassium, caesium and rubidium salts enhanced the allyl alcohol yield when a 35 wt% glycerol solution was used as feed. Furthermore, the addition of organic reductants or hydrogen to the glycerol feed increased the allyl alcohol yield. Optimisation of allyl alcohol selectivity was achieved by a combination of alkali metal cation modifiers and the addition of reductants. The influence of acid, base and redox properties on selectivity is presented, focusing on the effect of these properties on product distribution. The effect of by‐products from the conversion of glycerol to allyl alcohol on the epoxidation reaction was examined to identify the ideal reaction conditions for maximising the glycidol yield. Epoxidation of allyl alcohol by 30 wt% hydrogen peroxide was conducted in a batch reactor at 333K using titanium silicate‐1 catalyst. Conversion of allyl alcohol was high for all conditions; however, hydrolysis of glycidol to glycerol reduces selectivity. © 2015 Curtin University of Technology and John Wiley & Sons, Ltd.

Effect of Ethanol Accumulation on Porcine Interferon-α Production by Pichia pastoris and Activities of Key Enzymes in Carbon Metabolism.

In production of porcine interferon α (pIFN-α) by Pichia pastoris, improper glycerol feeding strategy leads to ethanol accumulation in the last stage of growth phase. In the present study, taking two runs with low ethanol accumulation under 2 g/L as control group, effects of long-term (>4 h) and instantaneous high ethanol concentration (>10 g/L) on pIFN-α production, and activities of key enzymes in carbon metabolism were discussed. As a result, compared with control group, pIFN-α expression level was decreased about 4~12 folds under long-term high ethanol concentration, from the level above 3 g/L to the level under 1 g/L; pIFN-α expression level was decreased about 8 folds under instantaneous high ethanol concentration, reaching to the low level of 0.42 g/L. The low production of pIFN-α was caused by the severe inhibitory effect of ethanol on these enzymes.

Hydrogen production from supercritical water reforming of glycerol over Ni/Al2O3–SiO2 catalyst

Hydrogen production from the supercritical water reforming of glycerol was studied in a tubular fixed-bed reactor by using a Ni-based catalyst supported on Al2O3 and SiO2. Tests were carried out at a pressure of 240 bar, temperatures of 500–800 °C, glycerol feed concentrations of 5–30 wt.%, and weight hourly space velocity from 1.25 to 22.5 gGly h−1 gCat−1 (residence time from 1.6 to 4.8 s through the bed). The dry gas is mainly composed of H2, CO2, CO, CH4. The results showed that the glycerol conversion was almost complete, except at the highest glycerol feed concentration and lowest temperature. Hydrogen yields were very close to those values predicted by equilibrium at a short residence time. Nickel on catalyst was completely reduced, and structured carbon nanotubes were encountered at glycerol concentrations higher than 20 wt.%. This study illustrates that the reforming of glycerol using supercritical water over a Ni catalyst makes it possible to reduce the reforming temperature needed when no catalyst is used (from 800 °C to 600 °C), achieving a high-yield hydrogen production, very close to equilibrium, and requiring less energy.

Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut(+) phenotype in continuous culture.

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol-glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol-glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04-0.06 h(-1) , while ROL yield decreased along the whole dilution rate range evaluated (0.03-0.1 h(-1) ). Compared to production level achieved with methanol-only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g-biomass(-1) h(-1) ), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut(+) phenotype for heterologous protein production.

Supported silicotungstic acid on zirconia catalyst for gas phase dehydration of glycerol to acrolein

The gas phase dehydration of glycerol to acrolein over a series of supported silicotungstic acid (HSiW) on zirconia (10HZ, 20HZ, 30HZ and 40HZ) has been investigated. The catalysts were characterized by temperature programmed desorption, nitrogen adsorption–desorption, thermogravimetric analysis, Fourier transform infrared spectroscopy, X-ray diffraction, field-emission scanning electron microscopy and energy dispersive X-ray techniques. The large pore diameters (>19nm) of the prepared catalysts alleviated the coke deposition effect. Also, the specific surface area and acidity of the samples surged from 18 to 22m2/g and 0.38 to 1.24mmol/gcat, respectively by varying HSiW loadings from 10 to 40wt% on zirconia. The highest acrolein yield achieved was 63.75% at 92% glycerol conversion over 30HZ catalyst for 10wt% glycerol feed concentration and 300°C reaction temperature in 3h. The combined physico-chemical characteristics of 30HZ made it more superior compared with other samples in the current study.

DESIGN AND PERFORMANCE EVALUATION OF A PLANT FOR GLYCEROL CONVERSION TO ACROLEIN

The development of biodiesel industry in the recent past brought into discussion the valorization of glycerol as the byproduct of this technology. Dehydration of glycerol leads to acrolein, mainly used as a raw material in the production of acrylic acid and its esters. Several research efforts dealing with the synthesis of acrolein from glycerol are reported in the literature. However, they are limited to catalyst testing on laboratory scale, no attention being paid to the process feasibility at an industrial scale. The goal of this paper is to fill this gap by presenting an integrated design study of the glycerol dehydration process. A simplified kinetic model was developed based on published data, this one being able to predict with sufficient accuracy the rate of the main reaction and the formation of relevant by-products (carbonyl compounds, hydrocarbons, carbon monoxide and coke). A reaction system was designed, similar to the reactor-regenerator unit usually used in hydrocarbon catalytic cracking (FCC). Moreover, the operating conditions were determined in such a way to maximize the selectivity and ensure the autothermal operation. The reactor effluent is sent to a separation section consisting mainly in distillation units. Due to formation of a low-boiling azeotrope, extractive distillation is employed for separating the acrolein-water mixture, using a part of the fresh glycerol feed as solvent. For a set of typical operating conditions, the separation section was designed in Aspen Plus environment. An economic evaluation study was performed using Aspen Economic Evaluation module, following which an acrolein price of 1.13 EUR/kg was determined. By comparing this value with the market one of 3.3 EUR/kg, the feasibility of the proposed process from the economical point of view will be emphasized.

Optimal dilution strategy for a microbial continuous culture based on the biological robustness

A robust optimal parameter selection model is proposed to formulate the microbial continuous culture process of glycerol bio-dissimilation to 1,3-propanediol (1,3-PD), in which the dilution rate and the glycerol concentration in feed medium are taken as the optimization variables. In consideration of the uncertain factors that some system parameters may be changed with the change of the optimization variables, we establish a novelty mathematical model which is represented by an eight-dimensional nonlinear dynamical system with the unknown parameters. On the basis of biological robustness, we give a quantitative definition of robustness index. The concentration of 1,3-PD at the approximately steady-state time together with the robustness index are taken as the cost functional in consideration of uncertain metabolic mechanisms. A parallel particle swarm optimization -- optimal parameter selection algorithm (PPSO-OPSA) is constructed to find the optimal dilution rate and the feeding glycerol concentration. Numerical results show that, by employing the obtained optimal input strategy, not only the concentration of 1,3-PD at the approximately steady-state time can be increased considerably compared with the previous experimental results, but also the obtained optimal parameters are robust for the dynamical system.

Anaerobic codigestion of sewage sludge and glycerol, focusing on process kinetics, microbial dynamics and sludge dewaterability

Anaerobic codigestion (AcoD) is a proven option to significantly boost biogas production while utilizing existing digesters and infrastructure. The aim of the present research was to conduct an exhaustive study regarding anaerobic codigestion of mixed sewage sludge and crude glycerol considering impacts on organic load, hydraulic load, process performance and microbial community. The methane potential of crude glycerol varied from 370 mL CH4·g−1 VS to 483 mL CH4·g−1 VS for different samples tested. The half maximal inhibitory concentration of crude glycerol was 1.01 g VS L−1, and the primary mechanism of inhibition was through overload from rapid fermentation rather than the presence of toxic compounds in the crude glycerol. In continuous operation over 200 days, feeding glycerol at up to 2% v/v, increased organic load by up to 70% and resulted in a 50% increase in methane production. Glycerol dosing resulted in no change in apparent dewaterability, with both codigestion and control reactors returning values of 22%–24%. Members of the phylum Thermotogae emerged as a niche population during AcoD of sewage sludge and glycerol; however there was no gross change in microbial community structure and only minimal changes in diversity. AcoD did not result in synergisms between sewage sludge and crude glycerol. Actually, at dose rate up to 2% v/v glycerol dosing is still an effective strategy to increase the organic loading rate of continuous anaerobic digesters with minimal impact of the hydraulic retention time. Nonetheless, the dose rate must be managed to: (i) prevent process inhibition and (ii) ensure sufficient degradation time to produce a stable biosolids product.

Syngas methanation from the supercritical water reforming of glycerol

An overall heat-integrated process of SCW (supercritical water) reforming of glycerol for methanation of the syngas obtained and power generation is proposed and analyzed. Methanation is the methane synthesis from the hydrogenation of CO and CO2. The SCW reforming is performed at 240 bars. Reforming temperatures from 700 °C to 900 °C and glycerol feed concentrations between 25 wt.% and 50 wt.%, needed to reach an energy self-sufficient process, are studied. For methanation, three adiabatic, fixed-bed reactors are connected in series with intermediate gas cooling, operating at 30 bars. The exit temperatures of these reactors range from 600 °C to 300 °C, respectively. The feed for the methanation section is previously conditioned by a Pressure Swing Adsorption unit to achieve a stoichiometric number of 3. The recommended operating conditions are a reforming temperature of 800 °C and a glycerol concentration of 33 wt.% to obtain 0.166 kg CH4/kg glycerol, 0.433 kWe/kg glycerol and an overall energy efficiency of 61.6%, which may increase up to 76.1% if the hot water leaving the process at 90 °C is considered (cogeneration water). The results of this process were compared to those of the methanol synthesis, previously published, resulting in a better performance, because the carbon proportion converted into methane is higher than into methanol from SCW reforming of glycerol, and the higher specific overall value for the methane production, which considers the price of the product and the electricity jointly.

Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks

Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60–70h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.

Process optimization of high-level extracellular production of alkaline pectate lyase in recombinant Escherichia coli BL21 (DE3)

Eco-effective high-level production of alkaline pectate lyase (PL) is a necessary prerequisite for the application of this enzyme in large-scale industries, such as bioscouring. For this purpose, in this work, a multi-step glycerol feeding strategy based on specific growth rate for high cell density cultivation of recombinant Escherichia coli was first established, which could effectively control cell growth and acetate yield. Next, the effects of induction time, temperature, and inducer concentration on cell growth and extracellular secretory production of recombinant PL were investigated. When induced by IPTG (using either low IPTG concentration at 25°C or high IPTG concentration at 30°C), two different extracellular secretion strategies were found to be appropriate for extracellular PL production. On the other hand, when cultivations were induced by mild continuous lactose feeding strategy, the extracellular and total PL activities reached 4478 and 5337U/mL, respectively, representing the highest PL yield reported to date.

Catalytic reforming of glycerol in supercritical water with nickel-based catalysts

The catalytic performance of nickel catalysts supported on La2O3, α-Al2O3, γ-Al2O3, ZrO2, and YSZ for supercritical water reforming of glycerol was investigated. Experiments were conducted in a tubular reactor made of Inconel-625 with the temperature range of 723–848 K under a pressure of 25 MPa. Carbon formation causing operation failure was observed for α-Al2O3, γ-Al2O3 and ZrO2 at temperatures higher than 748, 798 and 823 K, respectively. Ni/La2O3 exhibited the highest H2 yield where almost complete conversion was obtained at 798 K. Moderate space velocities (WHSV = 6.45 h−1) and glycerol feed concentration (5wt.%) favor high hydrogen selectivity and yield. Methanation is favored at a low WHSV or high glycerol feed concentration, resulting in a lower H2 yield. Increasing Ni loading on the Ni/La2O3 catalyst strongly promoted the reforming, water–gas shift, and methanation reactions, which contributed significantly to the product species distribution.

Improved Design and Control of Triacetin Reactive Distillation Process for the Utilization of Glycerol

Glycerol utilization is an important research topic because of recent surging biodiesel production through transesterification of vegetable oils and animal fats. One of the valuable products from glycerol is gained through esterification with acetic acid to produce triacetin. Hasabnis and Mahajani1 proposed an entrainer-based reactive column configuration with stoichiometric feed ratio to obtain high selectivity and conversion. This paper corrects the kinetic parameters in Hasabnis and Mahajani1 to better describe this reaction system. A more effective reactive entrainer for this reactive distillation system is also proposed to better carry water to the top of the column. The other modification is to assume feed compositions of the glycerol and acetic acid feed streams containing some water as impurity as opposed to idealistic pure feed assumptions. The operation and control of this system are also investigated. The proposed tray-temperature control strategy is able to maintain product purity despite disturbances from throughput and feed composition changes.

Energy and exergy analysis as tools for optimization of hydrogen production by glycerol autothermal reforming

In this work, various assessment tools were comprehensively applied to investigate hydrogen production via glycerol autothermal reforming. These tools are used to study the chemical reactions, design and simulate the entire hydrogen production process, investigate the energetic and exergetic performances of the processes and perform parametric analyses (using intuitive and design of experiment-based methods).Investigating the chemical reactions of autothermal reforming (ATR) of glycerol reveals that the optimal conditions, based on maximizing the hydrogen production, minimizing the methane and carbon monoxide contents and eliminating coke formation at thermoneutral conditions, can be achieved at a water–glycerol feed ratio (WGFR), reforming temperature (T) and oxygen–glycerol feed ratio (OGFR) of 9, 900 K and 0.35, respectively. The energetic study of the resulting process indicates that approximately two-thirds of the energy fed to the process is recovered in the useful product (H2) and that the remaining incoming process energy is exhausted in the off-gas. The exergetic investigation reveals that the exergetic efficiency of the ATR process is 57% and that 152 kJ are destroyed to generate 1 mol of hydrogen. The process operating conditions recommended by the chemical reaction investigation suffers from low performance because energetic and exergetic efficiencies are comparatively lower than values previously reported in literature for other reformates. The parametric investigation indicates that more accurate conditions are needed to convert glycerol–hydrogen. These conditions ensure the lowest consumption of energy to generate a given amount of hydrogen. This paper recommends WGFR = 5.5, T = 900 K and OGFR = 0.96 as the optimum conditions for the entire glycerol-to-hydrogen process. For this configuration, the thermal and exergetic efficiencies are 78.7% and 67.8%, respectively.

Aromatization of glycerol/alcohol mixtures over zeolite H-ZSM-5

The aromatization of glycerol is conducted over zeolite H-ZSM-5 for glycerol valorization. Due to its high viscosity, glycerol is diluted with water, methanol, ethanol, isopropanol or isobutanol. With the aqueous glycerol feed, the total aromatics yield is low and catalyst deactivation is fast. The mixture of glycerol with each alcohol is thus used as a feed, resulting in higher aromatics yields. Furthermore, the carbon fraction of aromatic products depends upon the alcohol mixed with glycerol. In case of the glycerol/methanol feed, the formation of trimethylbenzenes along with xylenes is pronounced, since both reactant constituents can be transformed into heavy C9 aromatics and then dealkylated to xylenes (major) and toluene (minor). In contrast, the use of higher alcohols for glycerol dilution leads to the preferential production of ethylbenzene and ethylmethylbenzenes, though xylenes and trimethylbenzenes are produced, due to alkylation of ethylene formed via dehydration (ethanol) or dehydration/cracking (isopropanol and isobutanol).

Impact of glycerol and nitrogen concentration on Enterobacter A47 growth and exopolysaccharide production

Enterobacter A47 produces a fucose-containing exopolysaccharide (EPS) by cultivation in mineral medium supplemented with glycerol. EPS synthesis by Enterobacter A47 was shown to be influenced by both the initial glycerol and nitrogen concentrations and by the nutrients’ feeding rate during the fed-batch phase. Initial nitrogen concentrations above 1.05g/L were detrimental for EPS synthesis: the productivity was reduced to 0.35–0.62g/Ld (compared to 1.89–2.04g/Ld under lower nitrogen concentrations) and the polymer had lower fucose content (14–17%mol, compared to 36–38%mol under lower nitrogen concentrations). On the other hand, EPS productivity was improved to 5.66g/Ld by increasing the glycerol and nitrogen feeding rates during the fed-batch phase. However, the EPS thus obtained had lower fucose (26%mol) and higher galactose (34%mol) contents, as well as lower average molecular weight (7.2×105). The ability of Enterobacter A47 to synthesize EPS with different physico-chemical characteristics may be useful for the generation of biopolymers with distinct functional properties suitable for different applications.

Recovery of hydrochloric acid and glycerol from aqueous solutions in chloralkali and chemical process industries by membrane distillation technique

Membrane distillation (MD) is a rapidly advancing process for separation of azeotropic and close boiling liquid mixtures besides dewatering of high boiling solvents. Recovery of hydrochloric acid (HCl) from a chloralkali industrial effluent and dehydration of glycerol/water mixture was performed using MD technique. HCl was recovered using chemically resistant polytetrafluoroethylene (PTFE) membrane of 0.22μm pore size and 78% porosity. The effluent feed contained 32.8wt.% of aqueous HCl with color forming Fe compounds and heavy hydrocarbon (C9–C14) impurities that gave an oily appearance. Permeate obtained was colorless aqueous HCl (33wt.%) at a high flux with negligible impurity levels. An increase in permeate pressure from 5 to 15mmHg resulted in a gradual reduction in flux from a high value of 8.57kg/m2/h to a moderate 1.02kg/m2/h at ambient temperature of 28°C. Effect of feed composition in terms of acid and inorganic salt contents besides feed temperature (25–60°C) on flux and separation efficiency was demonstrated at a constant downstream vacuum of 8–10mmHg. Dehydration of glycerol was performed using novel indigenously synthesized ultraporous hydrophobic polystyrene (PS) membrane of 0.72μm pore size. Permeate was found to contain pure water due to the low vapor pressure and larger molecular size of glycerol which cannot penetrate PS as it does not get wetted by water. This indicated a selectivity of infinity (∞) which is associated with a reasonable water flux in the range 0.56–0.02kg/m2/h at a vacuum of 5mmHg for feed glycerol concentration varying from 10 to 90wt.%. PS membrane was characterized by SEM, FTIR, XRD and TGA to assess surface and cross-sectional morphologies, structural elucidation, crystallinity and thermal stability of the membrane, respectively. A detailed economic estimation of HCl recovery for a feed effluent capacity of 2m3/h is presented. The study showed that commercial grade HCl and glycerol could be recovered from aqueous streams at a reasonable price by employing MD technique.

Fabrication of Reversible Phase Transition Polymer Gels toward Metal Ion Sensing

We report the synthesis of a new type of triple stimuli-responsive, i.e., thermo-, pH-, and metal ion-responsive copolymers based on poly(N-vinylimidazole-co-methacrylic acid) (poly(VI-co-MAA)) and their application as metal ion sensors. The copolymers exhibit reversible sol–gel phase transition behavior in aqueous media. The sol-to-gel transition temperature (Tsol–gel) can be shifted in the range of 20–80 °C, by varying monomer ratio and feeding glycerol content, by adjusting the copolymer concentration in aqueous solution, by tuning the pH of the solution, or by adding various divalent metal ions. Metal ion sensors were designed upon an inverse opal photonic film loaded with aqueous solution of poly(VI-co-MAA), which allows the easy reorganization of various divalent metal ions by combining the diversity of Tsol–gel of the copolymers on different metal ions and flexible reflection spectra of the film. In addition, a fluorescent reversible sol–gel transition system was established by in situ generation of nanocrystals in the copolymer matrix. These extensions may provide the multiresponsive copolymers great flexibility for applications in biomedical, optical, and sensory fields.

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014