Hybrid SSF/SHF Processing of SO2 Pretreated Wheat Straw-Tuning Co-fermentation by Yeast Inoculum Size and Hydrolysis Time.

Wheat straw is one of the main agricultural residues of interest for bioethanol production. This work examines conversion of steam-pretreated wheat straw (using SO2 as a catalyst) in a hybrid process consisting of a short enzymatic prehydrolysis step and a subsequent simultaneous saccharification and fermentation (SSF) step with a xylose-fermenting strain of Saccharomyces cerevisiae. A successful process requires a balanced design of reaction time and temperature in the prehydrolysis step and yeast inoculum size and temperature in the SSF step. The pretreated material obtained after steam pretreatment at 210 °C for 5 min using 2.5 % SO2 (based on moisture content) showed a very good enzymatic digestibility at 45 °C but clearly lower at 30 °C. Furthermore, the pretreatment liquid was found to be rather inhibitory to the yeast, partly due to a furfural content of more than 3 g/L. The effect of varying the yeast inoculum size in this medium was assessed, and at a yeast inoculum size of 4 g/L, a complete conversion of glucose and a 90 % conversion of xylose were obtained within 50 h. An ethanol yield (based on the glucan and xylan in the pretreated material) of 0.39 g/g was achieved for a process with this yeast inoculum size in a hybrid process (10 % water-insoluble solid (WIS)) with 4 h prehydrolysis time and a total process time of 96 h. The obtained xylose conversion was 95 %. A longer prehydrolysis time or a lower yeast inoculum size resulted in incomplete xylose conversion.

An approach for manganese biomonitoring using a manganese carrier switch in serum from transferrin to citrate at slightly elevated manganese concentration.

After high-dose-short-term exposure (usually from occupational exposure) and even more under low-dose long term exposure (mainly environmental) manganese (Mn) biomonitoring is still problematic since these exposure scenarios are not necessarily reflected by a significant increase of total Mn in blood or serum. Usually, Mn concentrations of exposed and unexposed persons overlap and individual differentiation is often not possible. In this paper Mn speciation on a large sample size (n=180) was used in order to be able to differentiate between highly Mn-exposed or low or unexposed individuals at low total Mn concentration in serum (Mn(S)). The whole sample set consisted of three subsets from Munich, Emilia Romagna region in Italy and from Sweden. It turned out that also at low total Mn(S) concentrations a change in major Mn carriers in serum takes place from Mn-transferrin (Mn-Tf(S)) towards Mn-citrate (Mn-Cit(S)) with high statistical significance (p<0.000002). This carrier switch from Mn-Tf(S) to Mn-Cit(S) was observed between Mn(S) concentrations of 1.5µg/L to ca. 1.7µg/L. Parallel to this carrier change, for sample donors from Munich where serum and cerebrospinal fluid were available, the concentration of Mn beyond neural barriers - analysed as Mn in cerebrospinal fluid (Mn(C)) - positively correlates to Mn-Cit(S) when Mn(S) concentration was above 1.7µg/L. The correlation between Mn-Cit(S) and Mn(C) reflects the facilitated Mn transport through neural barrier by means of Mn-citrate. Regional differences in switch points from Mn-Tf(S) to Mn-Cit(S) were observed for the three sample subsets. It is currently unknown whether these differences are due to differences in location, occupation, health status or other aspects. Based on our results, Mn-Cit(S) determination was considered as a potential means for estimating the Mn load in brain and CSF, i.e., it could be used as a biomarker for Mn beyond neural barrier. For a simpler Mn-Cit(S) determination than size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS), ultrafiltration (UF) of serum samples was tested for suitability, the latter possibly being a preferred choice for routine occupational medicine laboratories. Our results revealed that UF could be an alternative if methodical prerequisites and limitations are carefully considered. These prerequisites were determined to be a thorough cleaning procedure at a minimum Mn(S) concentration >1.5µg/L, as at lower concentrations a wide scattering of the measured concentrations in comparison to the standardized SEC-ICP-MS results were observed.

Bio-ethanol--the fuel of tomorrow from the residues of today.

The increased concern for the security of the oil supply and the negative impact of fossil fuels on the environment, particularly greenhouse gas emissions, has put pressure on society to find renewable fuel alternatives. The most common renewable fuel today is ethanol produced from sugar or grain (starch); however, this raw material base will not be sufficient. Consequently, future large-scale use of ethanol will most certainly have to be based on production from lignocellulosic materials. This review gives an overview of the new technologies required and the advances achieved in recent years to bring lignocellulosic ethanol towards industrial production. One of the major challenges is to optimize the integration of process engineering, fermentation technology, enzyme engineering and metabolic engineering.

The YIG1 (YPL201c) encoded protein is involved in regulating anaerobic glycerol metabolism in Saccharomyces cerevisiae.

Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD(+) from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (Ypl201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level.

Thoracic size-selective sampling of fibres: performance of four types of thoracic sampler in laboratory tests.

The counting of fibres on membrane filters could be facilitated by using size-selective samplers to exclude coarse particulate and fibres that impede fibre counting. Furthermore, the use of thoracic size selection would also remove the present requirement to discriminate fibres by diameter during counting. However, before thoracic samplers become acceptable for sampling fibres, their performance with fibres needs to be determined. This study examines the performance of four thoracic samplers: the GK2.69 cyclone, a Modified SIMPEDS cyclone, the CATHIA sampler (inertial separation) and the IOM thoracic sampler (porous foam pre-selector). The uniformity of sample deposit on the filter samples, which is important when counts are taken on random fields, was examined with two sizes of spherical particles (1 and 10 microm) and a glass fibre aerosol with fibres spanning the aerodynamic size range of the thoracic convention. Counts by optical microscopy examined fields on a set scanning pattern. Hotspots of deposition were detected for one of the thoracic samplers (Modified SIMPEDS with the 10 microm particles and the fibres). These hotspots were attributed to the inertial flow pattern near the port from the cyclone pre-separator. For the other three thoracic samplers, the distribution was similar to that on a cowled sampler, the current standard sampler for fibres. Aerodynamic selection was examined by comparing fibre concentration on thoracic samples with those measured on semi-isokinetic samples, using fibre size (and hence calculated aerodynamic diameter) and number data obtained by scanning electron microscope evaluation in four laboratories. The size-selection characteristics of three thoracic samplers (GK2.69, Modified SIMPEDS and CATHIA) appeared very similar to the thoracic convention; there was a slight oversampling (relative to the convention) for d(ae) < 7 microm, but that would not be disadvantageous for comparability with the cowled sampler. Only the IOM thoracic sampler tended to undersample the fibres relative to the thoracic convention. With the data divided into four classes based on fibre length, the size-selection characteristics appeared to be unaffected by fibre length for GK2.69, Modified SIMPEDS and CATHIA. Only the IOM thoracic sampler (with the foam selector) showed slightly lower selection for longer length classes of fibres. These results indicate that the tested samplers follow the thoracic sampling convention for fibres, and may be used to improve the quality and reliability of samples that are taken when there is likely to be significant background dust.

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae.

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).

Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae.

Furfural is an important inhibitor of yeast metabolism in lignocellulose-derived substrates. The effect of furfural on the physiology of Saccharomyces cerevisiae CBS 8066 was investigated using anaerobic continuous cultivations. Experiments were performed with furfural in the feed medium (up to 8.3 g/L) using three different dilution rates (0.095, 0.190, and 0.315 h(-1)). The measured concentration of furfural was low (< 0.1 g/L) at all steady states obtained. However, it was not possible to achieve a steady state at a specific conversion rate of furfural, q(f), higher than approximately 0.15 g/g.h. An increased furfural concentration in the feed caused a decrease in the steady-state glycerol yield. This agreed well with the decreased need for glycerol production as a way to regenerate NAD+, i.e., to function as a redox sink because furfural was reduced to furfuryl alcohol. Transient experiments were also performed by pulse addition of furfural directly into the fermentor. In contrast to the situation at steady-state conditions, both glycerol and furfuryl alcohol yields increased after pulse addition of furfural to the culture. Furthermore, the maximum specific conversion rate of furfural (0.6 g/g.h) in dynamic experiments was significantly higher than what was attainable in the chemostat experiments. The dynamic furfural conversion could be described by the use of a simple Michaelis-Menten-type kinetic model. Also furfural conversion under steady-state conditions could be explained by a Michaelis-Menten-type kinetic model, but with a higher affinity and a lower maximum conversion rate. This indicated the presence of an additional component with a higher affinity, but lower maximum capacity, either in the transport system or in the conversion system of furfural.

Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol.

Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.

[Functionality of inhalation aerosols is inadequate or lost at low temperature]. / Inhalationsaerosolers funktion försämras eller uteblir i kyla.

Correct and safe performance of metered-dose inhalers are necessary for efficient treatment of asthma under all climatic conditions. The spray mechanism malfunctioned when canister temperature was below -15 degrees C. The range and propagation of the spray plume at canister temperatures below 10 degrees C deviated clearly from those at 20 degrees C. Also the composition and particle concentration of the spray plume at low temperatures differed from those at more normal temperatures. Information must be provided to the effect that a canister must not be cold when used.

Weighing imprecision and handleability of the sampling cassettes of the IOM sampler for inhalable dust.

The weight stability of the sampling cassette of the IOM sampler for inhalable dust was tested in several weighing experiments. The results show that the reliability of repeated weighings was good, but the absorption of water vapour was slow and varied considerably among cassette specimen. The exponential time constant for water absorption was approximately 4 days, and 15-20 days were needed to obtain weight stability. With the help of cassette blanks the imprecision in dust weight could be held below 0.05 mg, if the cassettes were allowed one week's storage in the weighing room before weighing, both before and after sampling. The IOM sampling cassettes seem to consist of a few subsets, each with identical relative weight increase in a weighing room. To keep the variability low it is important that both the blanks and the cassettes used for sampling come from the same subset. Experiments indicate that the conducting plastic of the IOM sampling cassette may be replaced with another kind of plastic with similar electrical conductivity, but whose humidity absorption is 30 times lower. A lid, which is weighed with the cassette, was designed so that the potential dust loss from the cassette proper to the commercial transport clip was eliminated. A flow adapter, which simplifies the measurement of the air flow during personal sampling, was designed.

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae.

The physiological effects of 5-hydroxymethylfurfural (HMF) on Saccharomyces cerevisiae CBS 8066 in the presence and absence of furfural were studied. Experiments were carried out by pulse addition of HMF (2-4 g/l) as well as HMF (2 g/l) together with furfural (2 g/l) to batch cultivations of S. cerevisiae. Synthetic medium with glucose (50 g/l) as carbon and energy source was used. Addition of 4 g/l of HMF caused a decrease (approx. 32%) in the carbon dioxide evolution rate. Furthermore, the HMF was found to be taken up and converted by the yeast with a specific uptake rate of 0.14 (+/-0.03) g/g x h during both aerobic and anaerobic conditions, and the main conversion product was found to be 5-hydroxymethylfurfuryl alcohol. A previously unreported compound was found and characterized by mass spectrometry. It is suggested that the compound is formed from pyruvate and HMF in a reaction possibly catalysed by pyruvate decarboxylase. When HMF was added together with furfural, very little conversion of HMF took place until all of the furfural had been converted. Furthermore, the conversion rates of both furfural and HMF were lower than when added separately and growth was completely inhibited as long as both furfural and HMF were present in the medium.

On-line control of fed-batch fermentation of dilute-acid hydrolyzates.

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).

Personal sampling in parallel with open-face filter cassettes and IOM samplers for inhalable dust--implications for occupational exposure limits.

Parallel personal sampling was carried out with the open-face filter cassette and the IOM sampler for inhalable dust for nine types of organic dust. Parallel samples numbering 749 were obtained from 152 plants. Extremely large values and outliers were disregarded, and the remaining data for each type of dust were divided into subsets according to type of product or work task, and analyzed with the aid of linear regression. The coefficient of regression for each subset ranged between 0.2 and 0.7. Hypothetical occupational exposure limits (OELs) for inhalable dust were calculated based on the linear relation obtained between the dust concentrations measured with the open-face filter cassette and the IOM sampler. The fraction of person days with time-weighted average (TWA) concentrations exceeding the calculated hypothetical OELs for inhalable dust was obtained from the distribution of measured TWA inhalable dust concentrations. Based on the results of this study and the difference in sampling efficiency for large particles between the two samplers, it was concluded that the numerical value of the OEL for inhalable dust may be set at approximately twice the numerical value of the corresponding limit value for "total dust." Additional consideration of recently discovered health effects, and technical and economical factors may result in other numerical values of future OELs for inhalable dust.

Inhibition effects of furfural on aerobic batch cultivation of Saccharomyces cerevisiae growing on ethanol and/or acetic acid.

Physiological effects of furfural on Saccharomyces cerevisiae growing on ethanol (15 g.l(-1)) or acetate (20 g.l(-1)) as the carbon and energy source were investigated. Furfural (4 g.l(-1)), which was added during the exponential growth phase in batch cultures, was found to strongly inhibit cell growth on both carbon sources. No biomass formation occurred in the presence of furfural. However, furfural was in both cases converted to furfuryl alcohol and furoic acid, and growth resumed after complete conversion of furfural. During growth on ethanol, a rapid initial conversion of furfural to furfuryl alcohol was observed during the first few minutes after the addition of furfural, after which the conversion rate decreased to approximately 0.15 g.g(-1).h(-1) for the remaining conversion time. Acetaldehyde accumulated in the medium during the first few hours of conversion. Interestingly, addition of acetate after furfural addition resulted in an increased conversion rate of furfural and a higher carbon dioxide evolution rate, but no growth was observed until after complete conversion of furfural. Furfural addition to cells growing on acetate as the sole carbon source induced no formation of acetaldehyde, and the furfural conversion rate was lower than that on ethanol. The relationship between inhibition effects of furfural and NADH consumption is discussed.

Conversion of furfural in aerobic and anaerobic batch fermentation of glucose by Saccharomyces cerevisiae.

The effect of furfural on aerobic and anaerobic batch cultures of Saccharomyces cerevisiae CBS 8066 growing on glucose was investigated. Furfural was found to decrease both the specific growth rate and ethanol production rate after pulse additions in both anaerobic and aerobic batch cultures. The specific growth rate remained low until the furfural had been completely consumed, and then increased somewhat, but not to the initial value. The CO(2) evolution rate decreased to about 35% of the value before the addition of 4 g x l(-1) furfural, in both aerobic and anaerobic fermentations. The decrease of the CO(2) evolution rate was rapid at first, and then a more gradual decrease was observed. The furfural was converted mainly to furfuryl alcohol, with a specific conversion rate of 0.6 (+/-0.03) g (furfural) x g(-1) (biomass) x h(-1) by exponentially growing cells. However, the conversion rate of furfural by cells in the stationary phase was much lower. A previously unidentified compound was detected during the conversion of furfural. This compound was characterized by mass spectrometry and it is suggested that it is formed from furfural and pyruvate.

A new whole-body exposure chamber for human skin and lung challenge experiments--the generation of wheat flour aerosols.

A new whole-body exposure chamber for human skin and lung challenge offers possibilities for experimental exposure challenges carried out in clinical practice, for exposure of patients, in research and for investigations of the effects of exposure on the skin and in the respiratory tract. The chamber system can be used for both aerosols and gases. Dynamically controlled, the chamber is relatively easy to operate and to clean. Air exchange rates can be varied between 6-12/h. Initial studies with wheat flour have been carried out. The homogeneity and stability of the wheat flour aerosol concentration (the spatial and the temporal variation) inside the chamber can be kept at acceptable levels.

A collaborative European study of personal inhalable aerosol sampler performance.

Following the adoption of new international sampling conventions for inhalable, thoracic and respirable aerosol fractions, a working group of Comité Européen de Normalisation (CEN) drafted a standard for the performance of workplace aerosol sampling instruments. The present study was set up to verify the experimental, statistical and mathematical procedures recommended in the draft performance standard and to check that they could be applied to inhalable aerosol samplers. This was achieved by applying the tests to eight types of personal inhalable aerosol sampler commonly used for workplace monitoring throughout Europe. The study led to recommendations for revising the CEN draft standard, in order to simplify the tests and reduce their cost. However, some further work will be needed to develop simpler test facilities and methods. Several of the samplers tested were found to perform adequately with respect to the inhalable sampling convention, at least over a limited range of typical workplace conditions. In general the samplers were found to perform best in low external wind speeds, which are the test conditions thought to be closest to those normally found in indoor workplaces. The practical implementation of the CEN aerosol sampling conventions requires decisions on which sampling instruments to use, estimation of the likely impact that changing sampling methods could have on apparent exposures, and adjustment where necessary of exposure limit values. The sampler performance data obtained in this project were affected by large experimental errors, but are nevertheless a useful input to decisions on how to incorporate the CEN inhalable sampling convention into regulation, guidance and occupational hygiene practice.

Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae, in which one or both of the genes encoding the two isoforms of NAD-dependent glycerol-3-phosphate dehydrogenase had been deleted, were studied in aerobic batch cultures and in aerobic-anaerobic step change experiments. The respirofermentative growth rates under aerobic conditions with semisynthetic medium (20 g of glucose per liter) of two single mutants, gpd1 delta and gpd2 delta, and the parental strain (mu = 0.5 h-1) were almost identical, whereas the growth rate of a double mutant, gpd1 delta gpd2 delta, was approximately half that of the parental strain. Upon a step change from aerobic to anaerobic conditions in the exponential growth phase, the specific carbon dioxide evolution rates (CER) of the wild-type strain and the gpd1 delta strain were almost unchanged. The gpd2 delta mutant showed an immediate, large (> 50%) decrease in CER upon a change to anaerobic conditions. However, after about 45 min the CER increased again, although not to the same level as under aerobic conditions. The gpd1 delta gpd2 delta mutant showed a drastic fermentation rate decrease upon a transition to anaerobic conditions. However, the CER values increased to and even exceeded the aerobic levels after the addition of acetoin. High-pressure liquid chromatographic analyses demonstrated that the added acetoin served as an acceptor of reducing equivalents by being reduced to butanediol. The results clearly show the necessity of glycerol formation as a redox sink for S. cerevisiae under anaerobic conditions.

A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene.

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.

The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae.

Physiological effects of deficiency of pantothenate, a necessary precursor in the synthesis of coenzyme A, were studied using the yeast strain Saccharomyces cerevisiae CBS 8066. Cells were grown on defined media in anaerobic batch cultures with glucose (50 g/l) as the carbon and energy source. Batch cultures containing more than 60 micrograms/l pantothenate showed no significant differences with respect to growth rates and product yields. However, with an initial pantothenate concentration at 30 micrograms/l, the average glucose consumption rate was 50% lower than in rich medium and, at even lower concentration of pantothenate, the culture did not consume all the glucose in the medium. Furthermore, pantothenate deficiency caused the acetate and pyruvate yields to increase and the biomass yield to decrease, compared to the yields in pantothenate-rich medium. The increased acetate formation could be counteracted by initial addition of acetate to the medium, and thereby the glycerol yield could be decreased. An initial addition of acetate of 1.6 g/l to pantothenate-deficient medium (30 micrograms/l) caused a 35% decrease in glycerol yield and a 6% increase in ethanol yield. Furthermore, the time required for complete conversion of the glucose decreased by 40%. Acetate addition affected the acetate and glycerol yields in a similar way in pantothenate-rich medium (1000 micrograms/l) also.