Methanol Pulses Research Articles

Revealing Main Reaction Paths to Olefins and Aromatics in Methanol-to-Hydrocarbons over H-ZSM-5 by Isotope Labeling

The nature of hydrocarbon pool (HCP) intermediates in the methanol-to-hydrocarbons (MTH) process has been thoroughly investigated, especially for BEA- and CHA-type zeolite catalysts like H-β and H-SAPO-34. Herein, we further reveal the dynamic mechanistic details of the MTH process over the H-ZSM-5 catalyst at 400 °C, based on the dual-cycle mechanism and HCP in this medium-pore zeolite. Application of switching sequences of 13C-labeled and unlabeled methanol pulses over a model H-ZSM-5 catalyst combined with on-line MS analysis and a recently reported technique called “fast scanning-pulse GC analysis” provides a direct and quantitative insight into the MTH reactions under quasi-steady-state conditions. The transient product responses showed the almost instant formation of hydrocarbons upon a small pulse of methanol, followed by secondary formation of light aromatics via HCP decomposition and olefin alkylation–dealkylation, especially in a long catalyst bed when methanol is quickly consumed in the initial reaction zone in the catalyst bed. The isotopic analysis of typical aliphatic C3+ product responses after switching 13C-methanol pulses to the unlabeled methanol pulses showed a fast isotope scrambling in the formation of C3+ species. MS analysis of the light aromatics indicates a complete consecutive but slower isotope incorporation process of 12C into 13C-aromatics. Results provide direct experimental confirmation of the kinetically preferred olefin-based cycle over the aromatic-based cycle. The sequential isotopic incorporation strongly suggests that the paring reaction pathway through aromatic ring contraction and re-expansion steps is operative. In the appearance of aromatics upon pulsing methanol over larger catalyst beds, four processes are directly discerned, involving the displacement of adsorbed species by formed water, isotope incorporation yielding directly labeled and unlabeled products through the paring mechanism and direct aromatization, and HCP conversion through secondary reactions.

Direct discerning reaction pathways in methanol-to-hydrocarbons by transient operation – FASPA

Monitoring complex catalytic pathways under industrially-relevant conditions is one of the key challenges in catalysis chemistry and technology. Herewith we describe a direct technique called ‘fast scanning-pulse analysis’ (FASPA) that allows the direct characterization and detailed kinetic analysis of intimately interweaved catalytic pathways. The power and potential of the FASPA approach are demonstrated with an industrially relevant methanol-to-hydrocarbons (MTH) process over H-ZSM-5 zeolite. This reaction proceeds via a hydrocarbon pool (HCP) mechanism producing olefins and aromatics. The HCP is built-up upon exposure to methanol during the induction period, followed by a transition regime to a quasi steady-state MTH operation. This FASPA technique allows (sub-)second resolution of the full temporal products response upon a methanol pulse providing direct and quantitative insights into the MTH reactions. Globally, two consecutive pathways can be discerned: a very fast primary product formation in the presence of methanol in a narrow active MTH reaction zone, followed by a slower formation of light aromatics, which is closely related to the decomposition and release of HCP species and secondary reactions in absence of methanol in the downstream part of the catalyst bed. The time delay between the appearance of inert tracer and primary products represents the time needed to build-up the HCP in the induction period, where methane is observed prior to other products. The primary products (alkanes, olefins, and light aromatics) are nearly instantaneously formed from the pulsed methanol. These results demonstrate the highly dynamic character of the HCP in the MTH process over H-ZSM-5.

Probing the methanol-assisted autocatalytic formation of methanol over Cu/ZnO/Al2O3 by high-pressure methanol and methyl formate pulses

Using high-pressure methanol and methyl formate pulses as a surface-sensitive operando method for high-pressure methanol synthesis over Cu/ZnO/Al2O3, the recently found autocatalytic pathway was confirmed.

Synthesis of biodiesel from waste cooking oil using a pulsed high-intensity ultrasound method

ABSTRACT Biodiesel is widely recognized as an environmentally friendly biofuel. The key to the industrial production of biodiesel is the cost of components, especially that of the feedstock. In this study, a green pulsed ultrasound-assisted synthesis method for biodiesel production is reported. The biodiesel was prepared from waste cooking oil by transesterification in the presence of KOH, a homogeneous catalyst, under pulsed high-intensity ultrasonic treatment. The effects of various experimental parameters such as catalyst type, catalyst dosage, molar ratio of oil to methanol, temperature, ultrasonic power, and ultrasonic pulse mode were investigated. The results demonstrated that KOH had high catalytic performance compared to a heterogeneous K3PO4 catalyst for the transesterification of waste cooking oil. The yield of biodiesel reached up to 91.5% in 120 min of reaction time under the most optimal reaction conditions. The reaction conditions included a catalyst dose of 3% KOH, oil to methanol molar ratio of 1:6, temperature of 55°C, ultrasonic intensity of 260 W, and pulse mode of 9 s on/1 s off. Kinetic studies were conducted to obtain the rate constants at various reaction temperatures. The kinetic rate constant was observed to increase with increasing temperatures, and the activation energy for the ultrasonic method was calculated to be 35.9 kJ/mol. Hence, this green method shows great potential for applications in biodiesel production.

Increased glycosylated l-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches

Currently, only the biopharmaceutical l-asparaginase (ASNase), a chemotherapy agent, from bacteria is approved for clinical use. Although it is efficient, it causes severe side effects due to its origin. Therefore, new sources are investigated to reduce immunogenicity. Moreover, it is urgent to enhance protein stability, folding and kinetics with post-translational modifications, such as glycosylation. Aiming at these attributes, this study focused on the production of a glycosylated l-asparaginase of Dickeya chrysanthemi expressed by Pichia pastoris Glycoswitch®. Producer strains of Pichia, one with auxotrophy for histidine and another prototrophic were compared by using a complex medium in flasks and a synthetic medium in a bioreactor. The prototrophic strain showed higher productivity than the auxotrophic in both scales. Following, two induction strategies with the prototrophic strain were executed: methanol pulses and a DO-stat. The latter resulted in 2-fold more maximum volumetric activity. Lastly, the glycosylation analysis of the final product showed that the site Asn170, which is associated with allergies in patients, was glycosylated, thus, reducing potential immunogenic effects. Therefore, this study showed that the prototrophic strain was the most suitable l-asparaginase producer and that methanol–oxygen control in bioreactor enhanced the production of a glycosylated ASNase.

Influence of the Reaction Temperature on the Nature of the Active and Deactivating Species During Methanol-to-Olefins Conversion over H-SAPO-34.

The selectivity toward lower olefins during the methanol-to-olefins conversion over H-SAPO-34 at reaction temperatures between 573 and 773 K has been studied with a combination of operando UV–vis diffuse reflectance spectroscopy and online gas chromatography. It was found that the selectivity toward propylene increases in the temperature range of 573–623 K, while it decreases in the temperature range of 623–773 K. The high degree of incorporation of olefins, mainly propylene, into the hydrocarbon pool affects the product selectivity at lower reaction temperatures. The nature and dynamics of the active and deactivating hydrocarbon species with increasing reaction temperature were revealed by a non-negative matrix factorization of the time-resolved operando UV–vis diffuse reflectance spectra. The active hydrocarbon pool species consist of mainly highly methylated benzene carbocations at temperatures between 573 and 598 K, of both highly methylated benzene carbocations and methylated naphthalene carbocations at 623 K, and of only methylated naphthalene carbocations at temperatures between 673 and 773 K. The operando spectroscopy results suggest that the nature of the active species also influences the olefin selectivity. In fact, monoenylic and highly methylated benzene carbocations are more selective to the formation of propylene, whereas the formation of the group of low methylated benzene carbocations and methylated naphthalene carbocations at higher reaction temperatures (i.e., 673 and 773 K) favors the formation of ethylene. At reaction temperatures between 573 and 623 K, catalyst deactivation is caused by the gradual filling of the micropores with methylated naphthalene carbocations, while between 623 and 773 K the formation of neutral poly aromatics and phenanthrene/anthracene carbocations are mainly responsible for catalyst deactivation, their respective contribution increasing with increasing reaction temperature. Methanol pulse experiments at different temperatures demonstrate the dynamics between methylated benzene and methylated naphthalene carbocations. It was found that methylated naphthalene carbocations species are deactivating and block the micropores at low reaction temperatures, while acting as the active species at higher reaction temperatures, although they give rise to the formation of extended hydrocarbon deposits.

Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations

Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.

ΒackgroundThe methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.ResultsA fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.ConclusionsCo-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

A fast approach to determine a fed batch feeding profile for recombinant Pichia pastoris strains

BackgroundThe microorganism Pichia pastoris is a commonly used microbial host for the expression of recombinant proteins in biotechnology and biopharmaceutical industry. To speed up process development, a fast methodology to determine strain characteristic parameters, which are needed to subsequently set up fed batch feeding profiles, is required.ResultsHere, we show the general applicability of a novel approach to quantify a certain minimal set of bioprocess-relevant parameters, i.e. the adaptation time of the culture to methanol, the specific substrate uptake rate during the adaptation phase and the maximum specific substrate uptake rate, based on fast and easy-to-do batch cultivations with repeated methanol pulses in a batch culture. A detailed analysis of the adaptation of different P. pastoris strains to methanol was conducted and revealed that each strain showed very different characteristics during adaptation, illustrating the need of individual screenings for an optimal parameter definition during this phase. Based on the results obtained in batch cultivations, dynamic feeding profiles based on the specific substrate uptake rate were employed for different P. pastoris strains. In these experiments the maximum specific substrate uptake rate, which had been defined in batch experiments, also represented the upper limit of methanol uptake, underlining the validity of the determined process-relevant parameters and the overall experimental strategy.ConclusionIn this study, we show that a fast approach to determine a minimal set of strain characteristic parameters based on easy-to-do batch cultivations with methanol pulses is generally applicable for different P. pastoris strains and that dynamic fed batch strategies can be designed on the specific substrate uptake rate without running the risk of methanol accumulation.

A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

BackgroundPichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets.ResultsHere, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for qs were increased stepwise until a qs max of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1.ConclusionsOur strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.

Comprehensive characterization of recombinant Pichia pastoris using dynamic process conditions and process analytical technology

HRP_ FB I HRP_FB II HRP_B II Fig 3. Induction by methanol pulses in a batch culture. Cells were grown using D-glucose as C-source. When the substrate was exhausted, induction of protein expression was carried out by successive pulses of methanol: 1 adaptation pulse with 0,5 % methanol (in gray), successive pulses with 1 % methanol (in orange) and 1 pulse with 2 % methanol (not shown). The on-line calculated carbon dioxide evolution rate (CER) and the oxygen uptake rate (OUR) are shown. These calculations were performed by a metabolic balance analyzer implemented on-line. HRP activity was detected both in the absence and in the presence of D-Ala with slightly better results in the latter case, therefore D-Ala was added to the fed-batch cultures.

Overexpression of the Neocallimastix frontalis xylanase gene in the methylotrophic yeasts Pichia pastoris and Pichia methanolica

The xylanase gene from the rumen fungus Neocallimastix frontalis was expressed in Pichia pastoris and Pichia methanolica. Using a complex medium with medium replacement before induction and the maintenance of the methanol induction level at 0.5%, P. pastoris was able to produce about 5400 U/ml of xylanase after 10 days of induction. With P. methanolica, on the other hand, about 6200 U/ml of xylanase was reached after 10 days of induction using synthetic medium as first culture medium and then direct induction by continuous methanol feed at 1.8 ml l −1 h −1. In general, the advantages of using P. methanolica to produce the xylanase included higher protein production, the lack of medium replacement, and an ease of scale up. However, because the P. pastoris culture supernatant contained fewer secreted non-target proteins compared to P. methanolica, xylanase purification would be easier with the P. pastoris system. In addition, experiments involving methanol pulses suggested that a relationship exists among base feeding, methanol consumption and xylanase activity.

Ultralow drift in organic thin-film transistor chemical sensors by pulsed gating

A pulsed gating method has been developed to enhance the baseline stability of organic thin-film transistor (OTFT) chemical sensors. Trap states in the organic films are the major source of the OTFTs baseline drift under static gate bias, which is identified as the bias stress effect (BSE). BSE typically reduces the baseline current by 60% over 20h in phthalocyanine based OTFT sensors. The baseline drift has been reduced below 1% over 20h in the absence of the analyte using the pulsed gating method. With pulsed gating, the baseline drift on exposure to 15 methanol pulses is less than 0.09%∕h, and the response to this analyte is fully recoverable. Similar ultralow drift results were obtained for methanol sensing on three different phthalocyanine OTFTs. Combining the pulsed gating with low duty cycle analyte pulses, this method is also applicable to obtain ultralow drift (0.04%∕h) even for low vapor pressure analytes such as organophosphonate nerve agent simulants.

Dissimilation of [(13)C]methanol by continuous cultures of Bacillus methanolicus MGA3 at 50 degrees C studied by (13)C NMR and isotope-ratio mass spectrometry.

Using a continuous culture of Bacillus methanolicus MGA3 limited by 100 mM methanol in the feed and growing at a dilution rate D=0.25 h(-1), transients in dissolved methanol were studied to determine the effects of methanol toxicity and the pathway of methanol dissimilation to CO(2). Steady-state cultures were disturbed by pulses of methanol resulting in a rapid change in concentration of 6.4-12.8 mM. B. methanolicus MGA3 responded to a sudden increase in available methanol by a transient decline in the biomass concentration in the reactor. In most cases the culture returned to steady state between 4 and 12 h after pulse addition. However, at a methanol pulse of 12.8 mM, complete biomass washout occurred and the culture did not return to steady state. Integrating the response curves of the dry biomass concentration over a 12 h time period showed that a methanol pulse can cause an average transient decline in the biomass yield of up to 22%. (13)C NMR experiments using labelled methanol indicated that the transient partial or complete biomass washout was probably caused by toxic accumulation of formaldehyde in the culture. These experiments also showed accumulation of formate, indicating that B. methanolicus possesses formaldehyde dehydrogenase and formate dehydrogenase activity resulting in a methanol dissimilation pathway via formate to CO(2). Studies using isotope-ratio mass spectrometry provided further evidence of a methanol dissimilation pathway via formate. B. methanolicus MGA3, growing continuously under methanol limitation, consumed added formate at a rate of approximately 0.85 mmol l(-1) h(-1). Furthermore, significant accumulation of (13)CO(2) in the reactor exhaust gas was measured in response to a pulse addition of [(13)C]formic acid to the bioreactor. This indicates that B. methanolicus dissimilates methanol carbon to CO(2) in order to detoxify formaldehyde by both a linear pathway to formate and a cyclic mechanism as part of the RuMP pathway.

Mechanistic Studies on the Oxidative Dehydrogenation of Methanol over Polycrystalline Silver Using the Temporal-Analysis-of-Products Approach

The temporal-analysis-of-products approach was used to study the mechanism of the oxidative dehydrogenation of methanol over an unsupported polycrystalline silver catalyst. Pulse experiments revealed a distinct influence of the state of the silver surface on the adsorption of oxygen and on reactivity toward methanol. The surface-embedded oxygen species O γ, which is stable at high temperatures, was found to provide a highly selective reaction pathway for the oxidation of methanol to formaldehyde above 550 K. A comparison of the pulse responses of formaldehyde, water, and hydrogen to methanol pulses showed that water is a primary gas-phase product and that only small amounts of hydrogen are formed. Furthermore, it was found that the O γ species is not active in the oxidation of hydrogen or carbon monoxide. In contrast, the much more active adsorbed oxygen species O α was observed to be involved in the nonselective deep oxidation of methanol at lower temperatures, yielding carbon dioxide at high coverages. However, at low coverages O α also causes the selective formation of formaldehyde.

Cosolvent-Water Displacement in One-Dimensional Soil Column

A one-dimensional flow and transport model with dynamic fluid density and viscosity terms is proposed for modeling cosolvent flushing in a water-saturated porous medium. Given knowledge of the density and viscosity functions for cosolvent-water mixtures, the model is controlled by two flow parameters (specific storage and permeability) and one transport parameter (dispersivity). Sensitivity analysis demonstrates that the model solution is relatively insensitive to the flow parameters for the imposed constant head, constant flow conditions. The dynamic density and viscosity model is tested against the conventional transport model in simulating breakthrough data from soil column experiments in which water is displaced by pure methanol pulses (or slugs). Methanol slug breakthrough behavior is first predicted using independent parameter estimates (dispersivity obtained from tracer tests), then the dispersivity was adjusted to obtain optimal fits. The dynamic model provided slightly better predictions than the conventional transport model but failed to accurately reproduce methanol breakthrough behavior. Irregularities in observed slug breakthrough curves suggest that frontal instabilities may have been the cause of the discrepancy between the model and observations. Cosolvent overriding may have also contributed to the discrepancy in the horizontal displacement case.

Physiological responses of a methylotrophic bacterium after sudden shifts from C‐limited chemostat to C‐excess batch growth conditions

The response of steady‐state continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady‐state cultures was low (D= 0.046 h−1), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol l−1 to 280 mmol l−1, uncoupled growth occurred and the molar cell yield decreased. Conversely, steady‐state cultures at high growth rate (D= 0.2 h−1) showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1−1). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1−1 Slow‐growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast‐growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate.It is concluded that slow‐growing cultures are more stable than fast‐growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow‐growing and fast‐growing cultures.

The process utility of thermotolerant methylotrophic bacteria: II. An evaluation of transient responses

In Part II, the process utility of a thermotolerant methylotrophic bacterium is evaluated with respect to its dynamic response when various substrate or nutrient pulses are imposed on it while growing under steady-state conditions in a chemostat. The pulses investigated were methanol pulses on methanol-, methanol/formaldehyde-, and dual methanol/ammonia-limited cultures and an ammonia pulse on a severely nitrogen (ammonia)-limited culture. The results obtained, although exemplifying the complex biochemistry of such bacteria, clearly demonstrate the bacteriums flexibility and versatility in handling process transients. Its lack of fastidiousness in unsteady state continuous culture make it a most promising candidate for inclusion in process cultures for elevated temperature industrial wastewater treatment.

Utilization of methanol by a catalase-negative mutant of Hansenula polymorpha

In methanol-utilizing yeasts, catalase is an essential enzyme for the destruction of hydrogen peroxide generated by methanol oxidase (E.C. 1.1.3.13). It was found however that a catalase-negative mutant of Hansenula polymorpha is able to consume methanol in the presence of glucose in continuous cultures. At a dilution rate of 0.1 h-1, stable continuous cultures could be obtained during growth on methanol/glucose mixtures with a molar ratio of methanol/glucose between 0 to 2.4. In these cultures methanol oxidase was induced up to a level of 40% of that obtained in the wild-type strain. The hydrogen peroxide-decomposition activity of the mutant was studied in more detail by pulsing methanol to samples of steady-state cultures. Only after the addition of excess methanol the hydrogen peroxide-decomposing system became saturated, and the cells excreted hydrogen peroxide. This was accompanied by excretion of formaldehyde and a rapid loss of viability. The presence of extracellular catalase during a methanol pulse prevented the loss of viability. The nature of the alternative hydrogen peroxide-decomposing enzyme system remains to be elucidated. Its capacity strongly depended on the cultivation conditions and pretreatment of the cells. Cells grown on formaldehyde/glucose mixtures showed a lower methanol tolerance than those grown on the methanol/glucose mixtures. Freeze-drying of cells drastically enhanced the excretion of hydrogen peroxide, probably as a result of an inactivation of the decomposing system.

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. I. Fast transients induced by methanol pulses and methanol accumulation

The dynamic behavior of the Ribulose Monophosphate-type Methylomonas L3 in continuous cultures was studied, using methanol pulses to induce fast transients in steady-state cultures of single (methanol) and mixed (methanol plus formaldehyde) substrates. In several experiments, the methanol-uptake rate (MUR) profiles displayed negative MUR values for a time period following the methanol pulse, and significant amounts of methanol disappeared immediately following the pulse. These phenomena suggested the accumulation of methanol in the cells upon pulsing, apparently due to an active transport system. Accordingly, and in order to estimate the potential of the transport system for methanol accumulation, accumulation profiles were calculated for several pulse experiments. The calculations are based on a methanol balance and experimentally determined values of the cell volume and the true transient biomass yields. It is calculated that methanol accumulates up to 200-fold to very high intracellular concentrations. The accumulation is calculated to be much higher in single- (methanol) substrate cultures of low dilution rate than in cultures of high dilution rate or of mixed substrates. The specific growth rate immediately following the methanol pulse decreased in single-substrate cultures and increased in mixed-substrate ones. The biomass yield decreased after the methanol addition in all experiments; however, the drop was less severe in the mixed-substrate experiments. It is also suggested that formaldehyde as a methanol cosubstrate may be an effective means of providing more stable biomass yields and growth rates in reactors with imperfect mixing, and of protecting the reactor against accidentally induced methanol accumulation.