Industrial Relevant Conditions Research Articles

Directly converting CO2 into a gasoline fuel

The direct production of liquid fuels from CO2 hydrogenation has attracted enormous interest for its significant roles in mitigating CO2 emissions and reducing dependence on petrochemicals. Here we report a highly efficient, stable and multifunctional Na–Fe3O4/HZSM-5 catalyst, which can directly convert CO2 to gasoline-range (C5–C11) hydrocarbons with selectivity up to 78% of all hydrocarbons while only 4% methane at a CO2 conversion of 22% under industrial relevant conditions. It is achieved by a multifunctional catalyst providing three types of active sites (Fe3O4, Fe5C2 and acid sites), which cooperatively catalyse a tandem reaction. More significantly, the appropriate proximity of three types of active sites plays a crucial role in the successive and synergetic catalytic conversion of CO2 to gasoline. The multifunctional catalyst, exhibiting a remarkable stability for 1,000 h on stream, definitely has the potential to be a promising industrial catalyst for CO2 utilization to liquid fuels.

Synergistic Effect of Fe–Co Bimetallic Catalyst on FTS and WGS Activity in the Fischer–Tropsch Process: A Kinetic Study

The kinetics of Fischer–Tropsch (FT) and water–gas shift (WGS) reactions were investigated through detailed experimentation over a laboratory prepared 10Fe/20Co/SiO2 catalyst. An investigation was undertaken to understand the prevailing mechanism of CO activation over a Fe–Co bimetallic catalyst. The mechanisms of CO adsorption and intermediate formation are different over an iron and cobalt catalyst, which finally affect the rate of CO consumption. The mechanism of CO consumption in the FT and WGS reactions over this catalyst has been studied in the light of the synergistic effect due to the presence of both Fe and Co phases. These different phase formations (active sites) were investigated using characterization techniques, namely, XRD, TEM, and SAED. The reaction kinetic study was performed at industrial relevant reaction conditions (T = 473–553 K, P = 1–3 MPa, GHSV = 1800–6600 mL/gcat-h, H2/CO molar ratio = 0.5–2.5) in a continuous fixed bed reactor. The models based on rate of CO consumption were der...

Characterization of MoVTeNbOx Catalysts during Oxidation Reactions Using In Situ/Operando Techniques: A Review

Light alkanes are abundant in shale gas resources. The bulk mixed metal oxide MoVTe(Sb)NbOx catalysts play a very important role in dehydrogenation and selective oxidation reactions of these short hydrocarbons to produce high-value chemicals. This catalyst system mainly consists of M1 and less-active M2 crystalline phases. Due to their ability to directly monitor the catalysts under the relevant industrial conditions, in situ/operando techniques can provide information about the nature of active sites, surface intermediates, and kinetics/mechanisms, and may help with the synthesis of new and better catalysts. Sophisticated catalyst design and understanding is necessary to achieve the desired performance (activity, selectivity, lifetime, etc.) at reasonable reaction conditions (temperature, pressure, etc.). This article critically reviews the progress made in research of these MoVTe(Sb)NbOx catalysts in oxidation reactions mainly through in situ/operando techniques and suggests the future direction needed to realize the industrialization of these catalysts.

Conversion of apple pomace waste to ethanol at industrial relevant conditions.

Apple pomace samples were evaluated for conversion to ethanol at industrial relevant conditions. Biomass degradation efficiency by commercial enzymes was evaluated at 20% solid loading for dilute sulfuric acid, calcium oxide, and autoclave without any chemical (control) apple pomace samples. The control and calcium oxide-pretreated pomace provided similar sugar yields, while dilute sulfuric acid pretreatment resulted in reduced sugar yields. The control and calcium oxide-pretreated pomace hydrolysate were fermented to ethanol using a native Saccharomyces cerevisiae yeast strain, producing 38.8 and 36.9g/L of ethanol, respectively. When control apple pomace sample loading was increased from 20 to 30%, 57.5 and 50.1g/L of glucose and fructose was produced, respectively. Lastly, we found that unhydrolyzed solids (UHS) present during fermentation had little effect on ethanol yield, as 53.6 and 53.8g/L of ethanol were produced with and without UHS, respectively. Overall, ethanol yields were 134g per kg of dry apple pomace. A complete process mass balance for enzyme hydrolysis and ethanol fermentation is provided in this manuscript. These results show that apple pomace is an excellent feedstock for producing ethanol that could be either used as biofuel or as beverage.

Synthesis of monolithic Al-fiber@HZSM-5 core-shell catalysts for methanol-to-propylene reaction

Monolithic microstructured Al-fiber@HZSM-5 core-shell catalysts have been synthesized by directly growing the shell of HZSM-5 with varied SiO2/Al2O3 molar ratio on the core of sinter-locked aluminum microfibers. Such monolithic catalysts are examined in the methanol-to-propylene reaction, and the best microstructured catalyst of HZSM-5 with SiO2/Al2O3 molar ratio of 200 delivers a C2–C4 selectivity of 62% (~36% to C3) as well as a single-run lifetime of 160h under industrial relevant conditions.

Adiabatic modelling of CO2 capture by amine solvents using membrane contactors

The modelling of CO2 chemical absorption using hollow fiber membrane contactors is addressed. A one-dimensional, multicomponent adiabatic model for the CO2 absorption using an aqueous solution of monoethanolamine is established. The model is validated using both, laboratory and pilot-scale experiments. The simulation results are compared to those from an isothermal model in order to investigate the influence of heat release on contactor performance. When industrial relevant operating conditions are applied, the adiabatic simulations show significant axial temperatures peaks, up to 30°C. Correspondingly, local gas-phase vapor molar fractions values of up to 0.4 are attained. If compared to simulations from an isothermal model, deviations of about 60% were obtained, thus clearly demonstrating the necessity of adiabatic modelling under industrial conditions. Intensification factors comprised of between 2 and 10, for external fiber radiuses in the range of 300–100μm are attained. The mass transfer coefficient is varied from 10−4 to 10−3ms−1 which corresponds to experimentally observed values of microporous membranes that are presumably resistant to liquid breakthrough. However, wetting remains a major problem in microporous as well as composite membranes, as capillary condensation is likely to occur.

Influence of elevated surface texture hydrated titania on Ce-doped Mn/TiO2 catalysts for the low-temperature SCR of NOx under oxygen-rich conditions

In order to boost the low-temperature activity, a series of Mn–Ce/TiO2-X (X=Hk, N1, N2 and N3) were prepared by adopting incipient wetness technique and investigated for the low-temperature selective catalytic reduction (SCR) of NOx with NH3 at industrial relevant conditions. Prior to that, hydrous TiO2-nanosized samples were synthesized by a deposition technique at constant pH kept in the range of 5–8 and constant temperature 30–80°C. Titanium oxide hydrates (N1, N2, and N3) possess high specific surface area as 620m2/g, 457m2/g, 398m2/g, whereas TiO2 (Hk) preserves 309m2/g surface area. In our studies, it was found that the NOx conversion over Mn–Ce/TiO2-Hk with the atomic ratio of Mn/Ce=5.1 was apparently higher compared with that over Mn–Ce(5.1)/TiO2-X (X=N1, N2, and N3). Our activity results showed that 93.0% NOx conversion was obtained over Mn–Ce(5.1)/TiO2-Hk at 100°C at a space velocity of 80,000h−1. Our XRD results suggest that the loading of manganese and ceria onto hydrated titania led to the evolution of diffraction peaks which can be attributed to the formation of crystalline manganese dioxide (MnO2). Among all the catalysts, Mn–Ce/TiO2 N2 showed high intensity diffraction peaks at 2θ=37.2°, which corresponds to the highly crystalline (101) plane of manganese dioxide. Once the catalysts with the best performance were identified, experiments were performed with the aim of optimizing these formulations with respect to the dopant and Mn/Ce atomic ratio. Both the ceria co-doping and Mn/Ce atomic ratios played a key role to achieve high NOx conversions at 100°C. The disappearance or low-temperature shift of ceria reduction peak in H2-TPR indicates the increase of active components’ reduction potential, oxygen vacancies, and the existence of surface-capping oxygen species in Mn–Ce/TiO2 (Hk). The H2-TPR results are in good accordance with our XPS analysis where the relative atomic ratios of Mn4+/Mn3+, Ce3+/Ce4+, and the existence of surface oxygen species greatly enhanced in Mn–Ce/TiO2 (Hk) compared to other catalysts in this work. The relative atomic ratio of Mn4+/Mn3+ (2.19) in Mn–Ce(5.1)/TiO2-Hk calculated from deconvoluted XPS spectra is much higher than that of other catalysts (1.90, 0.89, and 2.03 for N1, N2, and N3, respectively). Moreover, the superior ratio of Ce3+/Ce4+ can generate a charge imbalance, oxygen vacancies, and unsaturated chemical bonds over the catalyst surface to promote the oxidation of NO to NO2. It is highly remarkable to note that the deNOx efficiency of all the prepared catalysts is indeed correlated with the surface concentrations of Ce3+/Ce4+ and Mn4+/Mn3+. NH3-TPD results imply that the co-doping of manganese and ceria onto Hk TiO2 can remarkably improve the acid sites distribution and the concentration of acid sites of the Mn–Ce/TiO2 catalyst. Our investigation results illustrate that the enhancement in reduction potential of active components, broadening of acid sites distribution, and the promotion of Mn4+/Mn3+, Ce3+/Ce4+ ratios including surface labile oxygen and small pore openings seem to be the reason for high deNOx efficiency of Mn–Ce/TiO2 (Hk) at low temperatures.

Probing the Coverage Dependence of Site and Adsorbate Configurational Correlations on (111) Surfaces of Late Transition Metals

Correlations between adsorption energies allow for the prediction of a large set of adsorption energies from a few quantum-chemical calculations, enabling quick evaluations of the catalytic activity of surfaces for multistep reactions. A critical question is whether these correlations—initially established at low coverage on geometrically similar surfaces—are useful at high coverage and in various geometric environments, which are the relevant industrial conditions of real catalysts. To answer this question, we use density functional theory to calculate almost 900 adsorption energies of several atomic adsorbates on late transition metal fcc (111) surfaces at a variety of coverages and configurations. We observe strict requirements of the geometric similarity between adsorption sites for the existence of correlations in adsorption energies. We also find that the introduction of higher coverages generally weakens and changes the correlations. We use our results to assess a simple model constructed from previously discovered scaling relationships and discuss their ramifications on catalyst discovery as well as strategies to incorporate this coverage dependence.

Prediction of the unsteady turbulent flow in an axial compressor stage. Part 1: Comparison of unsteady RANS and LES with experiments

A better understanding of turbulent unsteady flows is a necessary step towards a breakthrough in the design of modern gas turbine components. With the increase in computing power, LES emerges as a promising method to improve both knowledge of complex physics and reliability of flow solver predictions. However, there is still a lack of evidences in the literature that LES is applicable for turbomachinery at conditions relevant to industrial applications. In that context, the objective of the present work is to investigate the capability of LES to predict the turbulent flow in a stage of an axial compressor and compare the results with unsteady RANS data and experiments. The compressor operates at industrial relevant conditions, with Mach and Reynolds numbers equal to M=0.5 and Re=7×105, respectively. This paper presents the numerical method and a comparison of URANS and LES results to experimental data. A particular care is brought to estimate the results sensitivity to grid refinement (LES) and to turbulence and transition modelling (URANS). The comparison to experiments shows that LES better predicts time-dependent quantities than URANS, especially close to the casing. However, both URANS and LES fail to accurately estimate the compressor performance (efficiency and pressure ratio).

In-situ Gas Dosed Powder Diffraction Under Industrially Relevant Conditions

The adsorption of small molecules onto functionalized, high surface area microporous materials is important for the advancement of industrial and environmental processes ranging from catalysis and chemical separations, to CO2 sequestration and energy storage. Over the past several years we have focused our research efforts on understanding the molecular interactions of these small molecules with a variety of microporous materials using in-situ powder diffraction methods to correlate structure with chemical properties. Background will be given on the design of gas dosing apparatus for in-situ diffraction studies at synchrotron X-ray and neutron powder beamlines. The result is that accurate doses can be made per quantity of interest (moles of cations, per unit cell, per pore, etc.), or under high pressures (100 bar), and/or chemical reactions can be followed versus temperature/pressure. Several of our recent investigations of CO2/N2/CH4 sorption in cation-exchange zeolites including Zeolite A (5A) and CHA are presented. While many industrial processes use zeolites to carry out these functions, more emphasis has been placed on metal-organic frameworks (MOFs) on late since their properties can be tuned by varying the synthetic components. A number of studies on an isostructural series, M-MOF-74, have been considered investigating why certain functionalization leads to increased specificity for applications such as CO2, O2, CO, and hydrocarbon separations. The ultimate goal is to use the knowledge gained to improve the design of new MOF materials.

Mechanical reliability of geometrically imperfect tubular oxygen transport membranes

Mixed ionic and electronic conductors have potential applications as oxygen transport membranes. Realization of the technology is challenged by mechanical reliability of the components which are subjected to stresses arising from oxygen stoichiometry gradients and external overpressure during operation. This paper investigates numerically the failure risk of tubular oxygen transport membranes under industrial operating conditions using finite element modeling and Weibull strength analysis. The effects of component manufacturing defects on fracture probability are elucidated by explicit modeling of imperfections in the tubular membrane geometry. A supported membrane made of dense and porous Zr-doped-BSCF is studied as an illustrative example. It is shown that stresses induced by oxygen stoichiometry gradients relax over time due to creep and external pressure is the dominating source of stress in the long term. Therefore, creep has no adverse effect for geometrically perfect membranes. For geometrically imperfect membranes, curl and eccentricity are found to have insignificant influence on fracture risk while ovality is identified as the most critical geometric imperfection. Under the influence of external pressure, ovality may lead to dramatic stress increase and flattening of oval cross sections. Oval membranes can fail in the long term even though the instantaneous fracture risk is tolerable. Based on industrial relevant conditions, the requirements to the material creep rate and component quality (in terms of specification of tolerable deviation from perfect tubular shape) that allows fail-safe operation are deduced.

Catalytic upgrading of crude tall oil into a paraffin-rich liquid

Catalytic upgrading via hydrotreatment is an essen- tial method to produce paraffin-range renewable feedstocks from bio-derived oils. This research approach studies the catalytic hydrotreating of crude tall oil (CTO) over a sulfided NiMo catalyst under industrial-relevant conditions in order to produce hydrocarbon fraction, a steam cracker feed with maximum amount of paraffins and high degree of deoxygen- ation. Initial activity of the catalyst (6 h of time on stream) has been investigated, and we report that hydrotreating experiments at most favorable tested conditions (weight hourly space velocity, WHSV=1 h �1 and T=350-400 °C) produces hydrocarbon fraction with 45 wt% of paraffins (n-alkanes+i-alkanes). The highest degree of deoxygen- ation (91 %) was achieved at WHSV=1 h �1 and T=350 °C. Furthermore, the potential of hydrotreated CTO for steam cracking applications has been assessed in this study in view of the achieved product distribution and degree of deoxygen- ation from the hydrotreating of CTO.

Washcoating and chemical testing of a commercial Cu/ZnO/Al2O3 catalyst for the methanol synthesis over copper open-cell foams

We herein present the preparation of washcoated copper open-cell foam prototypes by dip-blowing a ball-milled aqueous slurry made of commercial powdered Cu/ZnO/Al2O3 methanol catalyst, deionized water and methylhydroxyethylcellulose. We then show the results of the chemical testing of such prototypes in the low temperature-low pressure methanol synthesis at relevant industrial conditions (i.e. T=505K, P=50 bara, H2/CO/CO2/CH4/N2 feed molar ratio=73.2/8.3/2.6/5.1/11.2). Three levels of temperature (485, 505 and 525K) were investigated, keeping constant all the other operating conditions. The performances were compared to those of the original powder.We found that washcoated copper foams coated with a 75μm thick catalytic layer were active in the methanol synthesis, but showed lower COx conversion and MeOH productivity than the original powder. Slurried powders (i.e. slurry dried and calcined) showed the same chemical activity as the washcoated foams, therefore ruling out any effect related to the deposition step and singling out the slurry preparation and/or calcination procedure as responsible for a change in the catalytic performances.We also tested the same catalytic systems in the reverse-water–gas-shift (RWGS) reaction at P=1 bara, T=505K and with H2/CO2=20. The campaign of low pressure tests confirmed the lower activity of slurried powders with respect to the original catalytic powder and leads us to the conclusion that the RWGS reaction can be considered as a representative probe reaction for ranking the intrinsic activity of Cu/ZnO/Al2O3 methanol catalysts without requiring testing under the high pressures typical of the commercial methanol synthesis process.

Comparison of global models of sub-bituminous coal devolatilization by means of thermogravimetric analysis

Coal gasification and combustion are strongly dependent on devolatilization step. Aim of this work is to obtain the parameters of global kinetics of devolatilization of a sub-bituminous coal with high sulfur content. The kinetic parameters are obtained by means of TG experimental data, and applying different approaches to extrapolate the data to industrial relevant conditions. The simpler method is a model-free one which supposes a single step process whose Arrhenius kinetic parameters (A and E a) have to be determined. Another common approach is the distributed activation energy model (DAEM) which assumes a series of first order parallel reactions occurring and sharing the same pre-exponential factor, A, with a continuous distribution of the activation energy. For the fitting of the experimental data, a numerical solution to DAEM and two approximate methods have been evaluated. Moreover, the results of these kinetic methods based on empirical approaches were compared with simulated data obtained using a more complex model based on percolation theory with cross-linking mechanism and vapor–liquid equilibrium (chemical percolation devolatilization, CPD model), which allows to simulate the coal pyrolysis from volatile yield data.

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae

BackgroundTo effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (mATP). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Yethanol = 0.35 g/g) but slow (qethanol = 0.05 ± 0.01 g/gBM/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway.ResultsBP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h-1 but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h-1; qethanol = 0.28 ± 0.04 g/gBM/h), efficient (Yethanol = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO2/HCO3-, was accompanied by a 2.3-fold increase of mATP while YATP (= 87 ± 7 mmolATP/gBM) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical.ConclusionsIn just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that mATP was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: Potential and temperature dependency at high solid processes

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling.

MFC-structured biodegradable poly(l-lactide)/poly(butylene adipate-co-terephatalate) blends with improved mechanical and barrier properties

Both polylactide (PLA) and poly(butylene adipate-co-terephthalate) (PBAT) are biodegradable polymers. They are thermoplastics which can be processed using conventional polymer processing methods. In this study, microfibrillar-reinforced composites (MFC) based on PLA/PBAT (PLA/Ecoflex®) blends in different weight ratios were prepared under industry-relevant conditions by melt extrusion followed by continuous cold drawing of the extrudates. Strip-like specimens (films) and plates (laminates) of the drawn blends were prepared by compression molding (CM) at processing temperature above the melting temperature (T m) of PBAT, but below T m of PLA. SEM and WAXS observations show that the extruded blend components are isotropic, but become highly oriented after drawing, and they are converted into MFC-structured polymer–polymer composites after CM. An effect of PLA microfibrils on the non-isothermal crystallization of the Ecoflex during cooling from the melt, associated with the formation of crystalline regions of the matrix around the fibrils, was observed. Depending on the blend composition, the compression-molded samples possess a 3- to 7-time higher tensile strength as well as a 15–30 higher modulus than the neat Ecoflex. In addition, the MFC-structured plates exhibited superior barrier properties compared to the neat Ecoflex, e.g., the oxygen permeability decreased by up to 5 times.

Superior activity of rutile-supported ruthenium nanoparticles for HCl oxidation

The activity of supported Ru-based catalysts in the oxidation of HCl to Cl2 is shown to be strongly affected by active phase–support interactions and by the way of incorporation of the Ru species. Depositing colloidally prepared Ru nanoparticles on a rutile carrier results in catalysts attaining very high and stable HCl conversion levels under industrially-relevant conditions using ruthenium loadings as low as 0.16 wt%.

Oxidative dehydrogenation of ethylbenzene to styrene over alumina: effect of calcination

Commercially available γ-Al2O3 was calcined at temperatures between 500 and 1200 °C and tested for its performance in the oxidative ethylbenzene dehydrogenation (ODH) over a wide range of industrially-relevant conditions. The original γ-Al2O3, as well as η- and α-Al2O3, were tested. A calcination temperature around 1000/1050 °C turned out to be optimal for the ODH performance. Upon calcination the number of acid sites (from 637 to 436 μmol g−1) and surface area (from 272 to 119 m2 g−1) decrease, whereas the acid site density increases (from 1.4 to 2.4 sites per nm2). Less coke, being the active catalyst, is formed during ODH on the Al-1000 sample compared to γ-Al2O3 (30.8 wt% vs. 21.6 wt%), but the coke surface coverage increases. Compared with γ-Al2O3, the EB conversion increased from 36% to 42% and the ST selectivity increased from 83% to 87%. For an optimal ST selectivity the catalyst should contain enough coke to attain full conversion of the limiting reactant oxygen. The reactivity of the coke is changed due to the higher density and strength of the Lewis acid sites that are formed by the high temperature calcination. The Al-1000 sample and all other investigated catalysts lost ODH activity with time on stream. The loss of selectivity towards more COX formation is directly correlated with the amount of coke.

Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

BackgroundPichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.ResultsThe shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation.ConclusionsIn the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.