Carl Johan Franzén

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.

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

The fermentation performance of nine strains of Saccharomyces cerevisiae in batch and fed-batch cultures in dilute-acid wood hydrolysate

Large differences in colony forming capacity, ethanol production and inhibitor conversion were noted between nine different strains of Saccharomyces cerevisiae in anaerobic batch and fed-batch cultures on dilute acid wood hydrolysate. S. cerevisiae ATCC 96581 was able to metabolize all added glucose and mannose in fed-batch experiments. The choice of production strain will have a significant effect on the performance of a hydrolysate-based ethanol production plant.

Metabolic flux analysis of RQ‐controlled microaerobic ethanol production by Saccharomyces cerevisiae

Microaerobic ethanol production by Saccharomyces cerevisiae CBS 8066 was investigated at different growth rates in respiratory quotient (RQ)‐controlled continuous culture. The RQ was controlled by changing the inlet gas composition by a feedback controller while keeping other parameters constant. The ethanol yield increased slightly from the anaerobic values with decreasing RQ, reaching a broad maximum at RQ 20 to 12. There was little or no glycerol production at RQ values below 17 over a wide range of dilution rates. Metabolic flux analysis revealed that a decrease in the ethanol yield at RQ 6 coincided with the cyclic, oxidative operation of the TCA cycle reactions. The model indicated that respiratory dissimilation of glucose only occurs when the oxygen uptake rate is high enough to completely substitute for glycerol formation. The cytosolic and the mitochondrial NADH balances were important for determining the flux distributions. The smallest deviations between estimated and measured product yields were obtained when the unknown co‐factor requirements of a limited number of biosynthetic reactions were chosen so that the amount of excess NADH formed in biosynthesis was minimized. The biomass yield was positively correlated with the net amount of NADH reoxidized in respiration and glycerol formation, indicating that the turnover of excess NADH from biosynthesis is an important factor influencing the biomass yield under oxygen‐limiting conditions. Copyright

Continuous fermentation of undetoxified dilute acid lignocellulose hydrolysate by Saccharomyces cerevisiae ATCC 96581 using cell recirculation.

Saccharomyces cerevisiae ATCC 96581 was cultivated in a chemostat reactor with undetoxified dilute acid softwood hydrolysate as the only carbon and energy source. The effects of nutrient addition, dilution rate, cell recirculation, and microaerobicity were investigated. Fermentation of unsupplemented dilute acid lignocellulose hydrolysate at D = 0.10 h‐1 in an anaerobic continuous reactor led to washout. Addition of ammonium sulfate or yeast extract was insufficient for obtaining steady state. In contrast, dilute acid lignocellulose hydrolysate supplemented with complete mineral medium, except for the carbon and energy source, was fermentable under anaerobic steady‐state conditions at dilution rates up to 0.14 h‐1. Under these conditions, washout occurred at D = 0.15 h‐1. This was preceded by a drop in fermentative capacity and a very high specific ethanol production rate. Growth at all different dilution rates tested resulted in residual sugar in the chemostat. Cell recirculation (90%), achieved by cross‐flow filtration, increased the sugar conversion rate from 92% to 99% at D = 0.10 h‐1. Nutrient addition clearly improved the long‐term ethanol productivity in the recirculation cultures. Application of microaerobic conditions on the nutrient‐supplemented recirculation cultures resulted in a higher production of biomass, a higher cellular protein content, and improved fermentative capacity, which further improves the robustness of fermentation of undetoxified lignocellulose hydrolysate.

Ethanol production at elevated temperatures using encapsulation of yeast

The ability of macroencapsulated Saccharomyces cerevisiae CBS 8066 to produce ethanol at elevated temperatures was investigated in consecutive batch and continuous cultures. Prior to cultivation yeast was confined inside alginate-chitosan capsules composed of an outer semi-permeable membrane and an inner liquid core. The encapsulated yeast could successfully ferment 30 g/L glucose and produce ethanol at a high yield in five consecutive batches of 12 h duration at 42°C, while freely suspended yeast was completely inactive already in the third batch. A high ethanol production was observed also through the first 48 h at 40°C during continuous cultivation at D=0.2 h(-1) when using encapsulated cells. The ethanol production slowly decreased in the following days at 40°C. The ethanol production was also measured in a continuous cultivation in which the temperature was periodically increased to 42-45°C and lowered to 37°C again in periods of 12h. Our investigation shows that a non-thermotolerant yeast strain improved its heat tolerance upon encapsulation, and could produce ethanol at temperatures as high as 45°C for a short time. The possibility of performing fermentations at higher temperatures would greatly improve the enzymatic hydrolysis in simultaneous saccharification and fermentation (SSF) processes and thereby make the bioethanol production process more economically feasible.

Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae.

ABSTRACT Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.

Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production

ABSTRACT Yeast has long been considered the microorganism of choice for second-generation bioethanol production due to its fermentative capacity and ethanol tolerance. However, tolerance toward inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked to the actual flocculation ability per se. In this study, variants of the flocculation gene FLO1 were transformed into the genome of the nonflocculating laboratory yeast strain Saccharomyces cerevisiae CEN.PK 113-7D. Three mutants with distinct differences in flocculation properties were isolated and characterized. The degree of flocculation and hydrophobicity of the cells were correlated to the length of the gene variant. The effect of different strength of flocculation on the fermentation performance of the strains was studied in defined medium with or without fermentation inhibitors, as well as in media based on dilute acid spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural but not against less convertible inhibitors such as carboxylic acids. During fermentation of dilute acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed significantly faster sugar consumption. The modified strain with the weakest flocculation showed a hexose consumption profile similar to the untransformed strain. These findings may explain why flocculation has evolved as a stress response and can find application in fermentation-based biorefinery processes on lignocellulosic raw materials.

Effects of encapsulation of microorganisms on product formation during microbial fermentations

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.

Current progress in high cell density yeast bioprocesses for bioethanol production

High capital costs and low reaction rates are major challenges for establishment of fermentation‐based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.