Claes Niklasson

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae

Abstract 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 · 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.

Acetic acid-friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae?

The permissible region of growth of Saccharomyces cerevisiae on glucose under anaerobic conditions was determined as a function of both pH and the concentration of added acetic acid to the medium. In the absence of acetic acid, growth was possible at a pH as low as 2.5, whereas a total acetic acid addition of 10 gl−1 increased the minimum allowable pH for growth to 4.5. The results showed that the concentration of the undissociated form of acetic acid should not exceed 5 gl−1 in the medium for growth to occur. The addition of acetic acid had a profound effect on growth energetics, thereby leading to an increased ethanol yield on glucose. At a concentration of 3.3 gl−1 of undissociated acetic acid, the ethanol yield was 20% higher than without added acetic acid. Furthermore, the biomass and glycerol yields decreased by 45 and 33%, respectively.

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.

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

Abstract 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 μg/l pantothenate showed no significant differences with respect to growth rates and product yields. However, with an initial pantothenate concentration of 30 μg/l, the average glucose consumption rate was 50% lower than in rich medium and, at even lower concentrations 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 μg/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 μg/l) also.

Production of biofuels, limonene and pectin from citrus wastes.

Production of ethanol, biogas, pectin and limonene from citrus wastes (CWs) by an integrated process was investigated. CWs were hydrolyzed by dilute-acid process in a pilot plant reactor equipped with an explosive drainage. Hydrolysis variables including temperature and residence time were optimized by applying a central composite rotatable experimental design (CCRD). The best sugar yield (0.41g/g of the total dry CWs) was obtained by dilute-acid hydrolysis at 150 degrees C and 6min residence time. At this condition, high solubilization of pectin present in the CWs was obtained, and 77.6% of total pectin content of CWs could be recovered by solvent recovery. Degree of esterification and ash content of produced pectin were 63.7% and 4.23%, respectively. In addition, the limonene of the CWs was effectively removed through flashing of the hydrolyzates into an expansion tank. The sugars present in the hydrolyzates were converted to ethanol using bakers yeast, while an ethanol yield of 0.43g/g of the fermentable sugars was obtained. Then, the stillage and the remaining solid materials of the hydrolyzed CWs were anaerobically digested to obtain biogas. In summary, one ton of CWs with 20% dry weight resulted in 39.64l ethanol, 45m(3) methane, 8.9l limonene, and 38.8kg pectin.

Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats,

ABSTRACT Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

Process design and economic analysis of a citrus waste biorefinery with biofuels and limonene as products

Process design and economic analysis of a biorefinery for the treatment of citrus wastes (CW) at different capacities was carried out. The CW is hydrolyzed using dilute sulfuric acid and then further processed to produce limonene, ethanol and biogas. The total cost of ethanol for base case process with 100,000 tons/year CW capacity was calculated as 0.91 USD/L, assuming 10 USD/ton handling and transportation cost of CW to the plant. However, this price is sensitive to the plant capacity. With constant price of methane and limonene, changing the plant capacity from 25,000 to 400,000 tons CW per year results in reducing ethanol costs from 2.55 to 0.46 USD/L in an economically feasible process. In addition, the ethanol production cost is sensitive to the transportation cost of CW. Increasing this cost from 10 to 30 USD/ton for the base case results in increasing the ethanol costs from 0.91 to 1.42 USD/L.

Conversion of dilute-acid hydrolyzates of spruce and birch to ethanol by fed-batch fermentation

Fermentation techniques for conversion of dilute acid hydrolyzates were examined. Batch and fed-batch fermentations of hydrolyzates from spruce and birch woods were made in a lab-scale (3.31) anaerobic bioreactor using the yeast Saccharomyces cerevisiae. The spruce and birch hydrolyzates contained high initial concentrations of furfural (2.2 and 5.7 g/l) and 5-hydroxymethylfurfural (HMF, 7.3 and 2.4 g/l), and were found to be strongly inhibiting to the yeast strain used in this study. Fermentation of the hydrolyzates was not possible using a batch mode of operation. However, using a fed-batch technique with a suitably adjusted feed rate, it was found possible to completely ferment the glucose and mannose sugars in both hydrolyzates. Most of the furfural (90%), and part of the HMF (40–70%), present in the hydrolyzates was converted during the fed-batch operation. It is suggested that the success of the fed-batch operation is related to the conversion of furfural and HMF.

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.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD+‐dependent glycerol‐3‐phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol‐3‐phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright