Fermentation Of Lignocellulosic Hydrolysates Research Articles

Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses.

The need for sustainable alternatives to oil-based production of biochemicals and biofuels is undisputable. Saccharomyces cerevisiae is the most commonly used industrial fermentation workhorse. The fermentation of lignocellulosic hydrolysates, second-generation biomass unsuited for food and feed, is still hampered by lowered productivities as the raw material is inhibitory for the cells. In order to map the genetic responses of different S. cerevisiae strains, we performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. While the response to inhibitors of S. cerevisiae has been studied earlier, this has in previous studies been done in aerobic conditions. The transcriptomic analysis highlights different evolutionary adaptations among the different S. cerevisiae strains and suggests novel gene targets for improving fermentation performance and robustness.

From straw to salmon: a technical design and energy balance for production of yeast oil for fish feed from wheat straw

BackgroundAquaculture is a major user of plant-derived feed ingredients, such as vegetable oil. Production of vegetable oil and protein is generally more energy-intensive than production of the marine ingredients they replace, so increasing inclusion of vegetable ingredients increases the energy demand of the feed. Microbial oils, such as yeast oil made by fermentation of lignocellulosic hydrolysate, have been proposed as a complement to plant oils, but energy assessments of microbial oil production are needed. This study presents a mass and energy balance for a biorefinery producing yeast oil through conversion of wheat straw hydrolysate, with co-production of biomethane and power.ResultsThe results showed that 1 tonne of yeast oil (37 GJ) would require 9.2 tonnes of straw, 14.7 GJ in fossil primary energy demand, 14.6 GJ of process electricity and 13.3 GJ of process heat, while 21.5 GJ of biomethane (430 kg) and 6 GJ of excess power would be generated simultaneously. By applying economic allocation, the fossil primary energy demand was estimated to 11.9 GJ per tonne oil.ConclusionsFossil primary energy demand for yeast oil in the four scenarios studied was estimated to be 10–38% lower than for the commonly used rapeseed oil and process energy demand could be met by parallel combustion of lignin residues. Therefore, feed oil can be produced from existing non-food biomass without causing agricultural expansion.

Transcriptome profiling brings new insights into the ethanol stress responses of Spathaspora passalidarum.

Spathaspora passalidarum is a xylose-fermenting microorganism promising for the fermentation of lignocellulosic hydrolysates. This yeast is more sensitive to ethanol than Saccharomyces cerevisiae for unclear reasons. An RNA-seq experiment was performed to identify transcriptional changes in S. passalidarum in response to ethanol and gain insights into this phenotype. The results showed the upregulation of genes associated with translation and the downregulation of genes encoding proteins involved in lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways. Our results also revealed that genes encoding heat-shock proteins and involved in antioxidant response were upregulated, whereas the osmotic stress response of S. passalidarum appears impaired under ethanol stress. A pseudohyphal morphology of S. passalidarum colonies was observed in response to ethanol stress, which suggests that ethanol induces a misperception of nitrogen availability in the environment. Changes in the yeast fatty acid profile were observed only after 12 h of ethanol exposure, coinciding with the recovery of the yeast xylose consumption ability. These findings suggest that the lack of fast membrane lipid adjustments, the halt in nutrient absorption and cellular metabolism, and the failure to induce the expression of osmotic stress-responsive genes are the main aspects underlying the low ethanol tolerance of S. passalidarum. KEY POINTS: • Ethanol stress halts Spathaspora passalidarum metabolism and fermentation • Genes encoding nutrient transporters showed downregulation under ethanol stress • Ethanol induces a pseudohyphal cell shape, suggesting a misperception of nutrients.

Quantitative understanding of the impact of stress factors on xylose fermentation at different high solid biomass loads

During lignocellulosic fermentation, cells are challenged by multiple stresses, leading to decreased performances, especially xylose conversion. Quantitatively understanding stress factors in alcoholic fermentation on xylose fermentation is prerequisite for designing and/or modifying lignocellulosic ethanol fermentation. Nonetheless, most studies only focus on just one type of stress, even only one target chemical compound, making it difficult to comprehensively understand the whole stress factors on xylose fermentation. In this study, stress factors in alcoholic fermentation of lignocellulosic hydrolysate were divided into five categories: inhibitors, fermentation metabolites, solid biomass residues, metal ions, and ethanol. Especially, extracted inhibitors, instead of target known inhibitors, were used for studying their effects on fermentation. The effects of each stress on xylose utilization were detailed investigated and compared. Principal component analysis was then performed for comprehensively analyzing effects of the above-mentioned five stresses on xylose fermentation. Results showed that high dose of ethanol is the most significant inhibition factor for xylose fermentation, followed by inhibitors and fermentation metabolites. Metal ions and solid biomass residues in hydrolysate have small effects on xylose fermentation. This study will provide fundamental theoretical support for designing efficient xylose conversion processes and constructing robust xylose-utilizing S. cerevisiae.

Dark Fermentation of Arundo donax: Characterization of the Anaerobic Microbial Consortium

The dark fermentation of lignocellulose hydrolysates is a promising process for the production of hydrogen from renewable sources. Nevertheless, hydrogen yields are often lower than those obtained from other carbohydrate sources due to the presence of microbial growth inhibitors in lignocellulose hydrolysates. In this study, a microbial consortium for the production of hydrogen by dark fermentation has been obtained from a wild methanogenic sludge by means of thermal treatments. The consortium has been initially acclimated to a glucose-based medium and then used as inoculum for the fermentation of Arundo donax hydrolysates. Hydrogen yields obtained from fermentation of A. donax hydrolysates were lower than those obtained from glucose fermentation using the same inoculum (0.30 ± 0.05 versus 1.11 ± 0.06 mol of H2 per mol of glucose equivalents). The hydrogen-producing bacteria belonged mainly to the Enterobacteriaceae family in cultures growing on glucose and to Clostridium in those growing on A. donax hydrolysate. In the latter cultures, Lactobacillus outcompeted Enterobacteriaceae, although Clostridium also increased. Lactobacillus outgrowth could account for the lower yields observed in cultures growing on A. donax hydrolysate.

Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate

Economically feasible bioethanol process from lignocellulose requires efficient fermentation by yeast of all sugars present in the hydrolysate. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth, and fermentation rate, and in addition damage cellular structures. In order to evaluate the capability of S. cerevisiae to adapt and respond to lignocellulosic hydrolysates, the physiological effect of cultivating yeast in the spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome-wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. The adaptation was shown to involve the induction of NADPH-dependent aldehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. These properties were correlated to the expression of several genes encoding oxidoreductases, notably AAD4, ADH6, OYE2/3, and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1, or ATR1. A large set of genes were found to be associated with transcription factors (TFs) involved in stress response (Msn2p, Msn4p, Yap1p) but also cell growth and division (Gcr4p, Ste12p, Sok2p), and these TFs were most likely controlling the response at the post-transcriptional level.

Integrated production of ethanol and xylitol from Brassica juncea using Candida sojae JCM 1644

The present study demonstrates a novel strategy involving two-step fermentation of lignocellulosic hydrolysate for the integrated production of ethanol and xylitol using a newly isolated yeast strain, Candida sojae JCM 1644. The isolated strain was characterised by its carbohydrate assimilation efficiency and tolerance towards inhibitors generated during pretreatment and fermentation of lignocellulosic biomass. In brief, the study comprised alkali treatment of Brassica juncea followed by its saccharification with cellulase consortia. An isolated strain was used for the co-production of xylitol and ethanol from sugar hydrolysate, and several parameters were systematically optimised for maximum co-production of ethanol and xylitol. Out of total glucose (149.72 g/L) and xylose (84.21 g/L) present in biomass hydrolysate, a product yield of 0.45 g/g (ethanol) and 0.62 g/g (xylitol) was achieved for a two-step fermentation process, which was 15.57% and 11.78% higher than the yield achieved for ethanol and xylitol, respectively, in a one-step fermentation process.

Glucose/Xylose Co-Fermenting Saccharomyces cerevisiae Increases the Production of Acetyl-CoA Derived n-Butanol From Lignocellulosic Biomass.

Efficient xylose catabolism in engineered Saccharomyces cerevisiae enables more economical lignocellulosic biorefinery with improved production yields per unit of biomass. Yet, the product profile of glucose/xylose co-fermenting S. cerevisiae is mainly limited to bioethanol and a few other chemicals. Here, we introduced an n-butanol-biosynthesis pathway into a glucose/xylose co-fermenting S. cerevisiae strain (XUSEA) to evaluate its potential on the production of acetyl-CoA derived products. Higher n-butanol production of glucose/xylose co-fermenting strain was explained by the transcriptomic landscape, which revealed strongly increased acetyl-CoA and NADPH pools when compared to a glucose fermenting wild-type strain. The acetate supplementation expected to support acetyl-CoA pool further increased n-butanol production, which was also validated during the fermentation of lignocellulosic hydrolysates containing acetate. Our findings imply the feasibility of lignocellulosic biorefinery for producing fuels and chemicals derived from a key intermediate of acetyl-CoA through glucose/xylose co-fermentation.

Conversion of corn fiber into fuel ethanol

Corn fiber due to its chemical composition (up to 20% starch, 50 - 60% non-starch polysaccharides) and availability has potential to serve as a substrate for manufacture of various products, including fuel ethanol. This paper deals with assessment of fiber-to-ethanol conversion. The water/dry fiber ratio in suspensions was 10/1. Enzyme liquefaction and saccharification of residual starch in corn fiber was carried out in two steps with thermostable α-amylase (20 min, 120°C) and mixture of pullulanase and glucomalyse (24 hours, 60°C). Procedures resulted in release of 57.7±1.6 mg of glucose per gram of dry fiber basis. It responds to the dextrose equivalent expression to 96.7±2.2%. By fermentation of the starch hydrolysates by yeasts Saccharomyces cerevisiae CCY-11-3 (5% v/v inoculum, 28°C, 72 hours) 0.48 g of ethanol per gram of glucose in hydrolysates was obtained. The solids after starch hydrolysis were separated by filtration and processed by acid pretreatment (0.1 g of conc. HCl/g of biomass/5 ml of water, 120°C, 20 min) with subsequent enzyme hydrolysis (24 hours, 60°C) by the multienzyme preparations containing cellulases and hemicellulases. Overall yield of reducing sugars after these two steps was
 740.7±3.9 mg/gram of dry corn fiber basis. Fermentation of lignocellulosic hydrolysates by yeasts Pichia stipitis CCY-39-50-1 and Candida shehatea CCY-29-68-4 (in both cases 5% v/v inoculum, 28°C, 72 hours) resulted in 0.38 and 0.12 g of ethanol per gram of reducing sugars. The results indicate that applied pretreatment methods and used microorganisms are able to produce ethanol from corn fiber.

Enhanced production and in-situ removal of butanol during the fermentation of lignocellulosic hydrolysate of pineapple leaves

The pineapple leaves waste was exploited as a potential biomass for butanol production. The cocktail of lignocellulolytic enzymes secreted by Sphingobacterium sp. ksn- 11 employed for the saccharification of steam pretreated pineapple leaves (100 g) which produced fermentable sugars (42.89 g) and polyphenols in the hydrolysate. The polyphenols were removed selectively by using residual cornhusk bed from the hydrolysate and subjected to fermentation by Clostridium acetobutylicum NCIM 2337. The butanol tolerance and production capacity of Clostridium acetobutylicum NCIM 2337 were enhanced by supplementing histidine and proline (0.5 %) to the fermentation medium by 1.8-fold and observed increase in butanol production (4.63 g/L). In order to minimize the sub-inhibitory effect of butanol, in-situ product recovery was carried out by the addition of Dowex L-493 resin which enhanced the butanol production (6.73 g/L) by 2.6-fold and achieved maximum yield of 0.16 g/g. Thus, the one pot step optimized process for butanol production was achieved.

Co-cultivation of a novel Fusarium striatum strain and a xylose consuming Saccharomyces cerevisiae yields an efficient process for simultaneous detoxification and fermentation of lignocellulosic hydrolysates

Furfural (FF) and 5-hydroxymethylfurfural (HMF) are furan derivatives commonly generated during the pretreatment of lignocellulosic biomass and often considered among the most inhibitory compounds towards the sugar fermenting strains due to their acute toxicity and high concentrations. The present study describes the simultaneous detoxification and fermentation of lignocellulosic hydrolysates containing high concentrations of FF and HMF by a co-culture of a novel Fusarium striatum strain and a xylose consuming Saccharomyces cerevisiae strain. The process demonstrates a superior performance than those previously described in the literature, as FF and HMF were efficiently transformed into their less toxic added-value alcohol derivatives by F. striatum with high yields (99% and 86%, respectively) and the higher detoxification rates reported (0.56 g/L/h and 0.13 g/L/h, respectively). There was no sugar consumption by the filamentous fungus during the detoxification process, rendering it available for ethanol fermentation by S. cerevisiae, which started immediately after the detoxification of the inhibitors. Ethanol productivities were significantly higher when increasing the inoculum size of F. striatum, confirming its potential for the detoxification of the lignocellulosic hydrolysate. High ethanol yields (0.4 g/g) and productivities (0.46 g/L/h) were obtained in a bench-scale bioreactor (1.5 L) in the presence of 3.5 g/L HMF and 2.5 g/L FF, a concentration of furan derivatives that completely inhibited the fermentation process in the absence of F. striatum. The presented process allows access to lignocellulosic materials and pretreatment methods that result in high concentrations of FF and HMF that are currently not feasible, representing a significant advance for the lignocellulosic ethanol industry.

Yeast immobilization systems for second‐generation ethanol production: actual trends and future perspectives

AbstractYeast immobilization with low‐cost carrier materials is a suitable strategy to optimize the fermentation of lignocellulosic hydrolysates for the production of second‐generation (2G) ethanol. It is defined as the physical confinement of intact cells to a certain region of space (the carrier) with the preservation of their biological activity. This technological approach facilitates promising strategies for second‐generation bioethanol production due to the enhancement of the fermentation performance that is expected to be achieved. Using immobilized cells, the resistance to inhibitors contained in the hydrolysates and the co‐utilization of sugars are improved, along with facilitating separation operations and the reuse of yeast in new production cycles. Until now, the most common immobilization technology used calcium alginate as a yeast carrier but other supports such as biochar or multispecies biofilm membranes have emerged as interesting alternatives. This review compiles updated information about cell carriers and yeast‐cell requirements for immobilization, and the benefits and drawbacks of different immobilization systems for second‐generation bioethanol production are investigated and compared.

Xylitol production from non-detoxified Napiergrass hydrolysate using a recombinant flocculating yeast strain

Xylose derived from lignocellulose can be utilized to produce ethanol and other high-value chemicals, such as xylitol. The xylitol production through fermentation of lignocellulosic hydrolysate by microorganisms offers advantages of high product yield, high selectivity, and efficacy in mild conditions. In this study, non-detoxified hemicellulose hydrolysate from napiergrass was used for xylitol production by a recombinant flocculating strain of Saccharomyces cerevisiae. An optimization study was conducted with the strain at 35 °C. A promising xylitol yield of 0.96 g/g xylose with no addition of glucose required during the fermentation process, which suggests an extensive potential improvement for the economics of lignocellulosic xylitol production.

Origin, Impact and Control of Lignocellulosic Inhibitors in Bioethanol Production—A Review

Bioethanol production from lignocellulosic biomass is still struggling with many obstacles. One of them is lignocellulosic inhibitors. The aim of this review is to discuss the most known inhibitors. Additionally, the review addresses different detoxification methods to degrade or to remove inhibitors from lignocellulosic hydrolysates. Inhibitors are formed during the pretreatment of biomass. They derive from the structural polymers-cellulose, hemicellulose and lignin. The formation of inhibitors depends on the pretreatment conditions. Inhibitors can have a negative influence on both the enzymatic hydrolysis and fermentation of lignocellulosic hydrolysates. The inhibition mechanisms can be, for example, deactivation of enzymes or impairment of vital cell structures. The toxicity of each inhibitor depends on its chemical and physical properties. To decrease the negative effects of inhibitors, different detoxification methods have been researched. Those methods focus on the chemical modification of inhibitors into less toxic forms or on the separation of inhibitors from lignocellulosic hydrolysates. Each detoxification method has its limitations on the removal of certain inhibitors. To choose a suitable detoxification method, a deep molecular understanding of the inhibition mechanism and the inhibitor formation is necessary.

Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern. A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion. This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways.

Enhanced biohydrogen production from cotton stalk hydrolysate of Enterobacter cloacae WL1318 by overexpression of the formate hydrogen lyase activator gene

BackgroundBiohydrogen production from lignocellulose has become an important hydrogen production method due to its diversity, renewability, and cheapness. Overexpression of the formate hydrogen lyase activator (fhlA) gene is a promising tactic for enhancement of hydrogen production in facultative anaerobic Enterobacter. As a species of Enterobacter, Enterobacter cloacae was reported as a highly efficient hydrogen-producing bacterium. However, little work has been reported in terms of cloning and expressing the fhlA gene in E. cloacae for lignocellulose-based hydrogen production.ResultsIn this study, the formate hydrogen lyase activator (fhlA) gene was cloned and overexpressed in Enterobacter cloacae WL1318. We found that the recombinant strain significantly enhanced cumulative hydrogen production by 188% following fermentation of cotton stalk hydrolysate for 24 h, and maintained improved production above 30% throughout the fermentation process compared to the wild strain. Accordingly, overexpression of the fhlA gene resulted in an enhanced hydrogen production potential (P) and maximum hydrogen production rate (Rm), as well as a shortened lag phase time (λ) for the recombinant strain. Additionally, the recombinant strain also displayed improved glucose (12%) and xylose (3.4%) consumption and hydrogen yield Y(H2/S) (37.0%) compared to the wild strain. Moreover, the metabolites and specific enzyme profiles demonstrated that reduced flux in the competitive branch, including succinic, acetic, and lactic acids, and ethanol generation, coupled with increased flux in the pyruvate node and formate splitting branch, benefited hydrogen synthesis.ConclusionsThe results conclusively prove that overexpression of fhlA gene in E. cloacae WL1318 can effectively enhance the hydrogen production from cotton stalk hydrolysate, and reduce the metabolic flux in the competitive branch. It is the first attempt to engineer the fhlA gene in the hydrogen-producing bacterium E. cloacae. This work provides a highly efficient engineered bacterium for biohydrogen production from fermentation of lignocellulosic hydrolysate in the future.

Microwave-assisted dilute acid pretreatment in bioethanol production from wheat and rye stillages

The main problem in the production of cellulosic ethanol is to ensure an efficient and cost-effective raw material pretreatment, guaranteeing a high degree of lignocellulose decomposition. Microwave radiation can be an alternative to conventional biomass heating, which in association with the use of dilute acid and a cellulolytic enzyme ensures a high degree of cellulose degradation. We evaluated the effectiveness of microwave dilute acid pretreatment of wheat and rye stillage in terms of the amount of sugars released, formation of fermentation inhibitors and their removal, and fermentation efficiency. Regardless of the type of stillage used, the highest glucose concentration after pretreatment (above 156 mg g−1 of DW) and the highest yield of cellulose hydrolysis after 24 h of the process (over 75%) were obtained using microwave power of 300 W, 15 min, 54 PSI. Fermentation of lignocellulosic hydrolysates obtained under the above-mentioned conditions ensured high ethanol concentrations of up to 20 g L−1 with full attenuation after just 48 h. Too intensive microwave treatment (152 PSI, 10 min) increased the concentration of sugar dehydration products which inhibited yeast fermentation activity and led to a lower ethanol yield. Detoxification with activated carbon reduced the toxic stress caused by the high concentration of HMF in hydrolysates. The application of microwaves is an interesting alternative to conventional barothermal methods. The possibility of an effective use of waste biomass of wheat/rye stillage for the production of cellulosic ethanol creates a prospect of integrating first and second generation ethanol production within one technological process.

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

AbstractLignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.

ALKALI PRE-TREATMENT AND ENZYMATIC HYDROLYSIS OF Arundo donax FOR SINGLE CELL OIL PRODUCTION

Microbial oil obtainable from the fermentation of lignocellulose hydrolysates represents a promising and sustainable alternative to first generation biodiesel. Among the lignocellulosic crops, giant reed (Arundo donax) is attracting interest due to the impressive biomass yield and the low input requirement. However, a delignification step is needed to facilitate the fermentable sugar release from the lignocellulosic matrix, paying attention to the production of growth inhibitors that represent a bottleneck in the development of microbial oil production. The aim of this study was to evaluate the suitability of an enzymatic hydrolysate of alkali pre-treated A. donax biomass, as a substrate for the growth of the oleaginous yeast Lipomyces starkeyi. Specific attention was also paid to possible inhibitory effects of compounds generated by the alkaline pre-treatment (10% slurry, NaOH 0-1.5% w/w, 120 C, 20 min). Increasing NaOH levels enhanced the release of phenolic compounds and increased the fermentable sugar yield after enzymatic hydrolysis of the washed pre-treated fiber. Saccharification yields reached a plateau in correspondence to NaOH 1.2% dose, which gave 407 mg of sugars per g of dry biomass. A medium containing 30 g/L of reducing sugars from the hydrolysate resulted suitable for the growth of L. starkeyi and for lipid accumulation, achieving 12.2 g/L of dry cell biomass with 43% w/w of total lipids. The pre-treatment produced soluble inhibitors that affected moderately the yeast growth in an initial phase, followed by a recovery. Thus, extensive washing of the fiber could be avoided, while a thorough filtration after the pre-treatment would be recommended.

Changes in lipid metabolism convey acid tolerance in Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae plays an essential role in the fermentation of lignocellulosic hydrolysates. Weak organic acids in lignocellulosic hydrolysate can hamper the use of this renewable resource for fuel and chemical production. Plasma-membrane remodeling has recently been found to be involved in acquiring tolerance to organic acids, but the mechanisms responsible remain largely unknown. Therefore, it is essential to understand the underlying mechanisms of acid tolerance of S. cerevisiae for developing robust industrial strains.ResultsWe have performed a comparative analysis of lipids and fatty acids in S. cerevisiae grown in the presence of four different weak acids. The general response of the yeast to acid stress was found to be the accumulation of triacylglycerols and the degradation of steryl esters. In addition, a decrease in phosphatidic acid, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine, and an increase in phosphatidylinositol were observed. Loss of cardiolipin in the mitochondria membrane may be responsible for the dysfunction of mitochondria and the dramatic decrease in the rate of respiration of S. cerevisiae under acid stress. Interestingly, the accumulation of ergosterol was found to be a protective mechanism of yeast exposed to organic acids, and the ERG1 gene in ergosterol biosynthesis played a key in ergosterol-mediated acid tolerance, as perturbing the expression of this gene caused rapid loss of viability. Interestingly, overexpressing OLE1 resulted in the increased levels of oleic acid (18:1n-9) and an increase in the unsaturation index of fatty acids in the plasma membrane, resulting in higher tolerance to acetic, formic and levulinic acid, while this change was found to be detrimental to cells exposed to lipophilic cinnamic acid.ConclusionsComparison of lipid profiles revealed different remodeling of lipids, FAs and the unsaturation index of the FAs in the cell membrane in response of S. cerevisiae to acetic, formic, levulinic and cinnamic acid, depending on the properties of the acid. In future work, it will be necessary to combine lipidome and transcriptome analysis to gain a better understanding of the underlying regulation network and interactions between central carbon metabolism (e.g., glycolysis, TCA cycle) and lipid biosynthesis.