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Short-chain fatty acids (SCFAs) are considered building blocks for bioproducts in the so-called carboxylate platform. These compounds can be sustainably produced via anaerobic fermentation (AF) of organic substrates, such as microalgae. However, SCFAs bioconversion efficiency is hampered by the hard cell wall of some microalgae. In this study, one thermal and two enzymatic pretreatments (carbohydrases and proteases) were employed to enhance Chlorella vulgaris biomass solubilization prior to AF. Pretreated and non-pretreated microalgae were assessed in continuous stirred tank reactors (CSTRs) for SCFAs production. Aiming to understand microorganisms' roles in AF depending on the employed substrate, not only bioconversion yields into SCFAs were evaluated but microbial communities were thoroughly characterized. Proteins were responsible for the inherent limitation of raw biomass conversion into SCFAs. Indeed, the proteolytic pretreatment resulted in the highest bioconversion (33.4% SCFAs-COD/CODin), displaying a 4-fold enhancement compared with raw biomass. Population dynamics revealed a microbial biodiversity loss along the AF regardless of the applied pretreatment, evidencing that the imposed operational conditions specialized the microbial community. In fact, a reduced abundance in Euryarchaeota phylum explained the low methanogenic activity, implying SCFAs accumulation. The bacterial community developed in the reactors fed with pretreated microalgae exhibited high acidogenic activities, being dominated by Firmicutes and Bacteroidetes. Firmicutes was by far the dominant phylum when using protease (65% relative abundance) while Bacteroidetes was prevailing in the reactor fed with carbohydrase-pretreated microalgae biomass (40% relative abundance). This fact indicated that the applied pretreatment and macromolecule solubilization have a strong effect on microbial distribution and therefore in SCFAs bioconversion yields.

Short-chain fatty acids (SCFAs) are considered building blocks for bioproducts in the so-called carboxylate platform. These compounds can be sustainably produced via anaerobic fermentation (AF) of organic substrates, such as microalgae. However, SCFAs bioconversion efficiency is hampered by the hard cell wall of some microalgae. In this study, one thermal and two enzymatic pretreatments (carbohydrases and proteases) were employed to enhance Chlorella vulgaris biomass solubilization prior to AF. Pretreated and non-pretreated microalgae were assessed in continuous stirred tank reactors (CSTRs) for SCFAs production. Aiming to understand microorganisms' roles in AF depending on the employed substrate, not only bioconversion yields into SCFAs were evaluated but microbial communities were thoroughly characterized. Proteins were responsible for the inherent limitation of raw biomass conversion into SCFAs. Indeed, the proteolytic pretreatment resulted in the highest bioconversion (33.4% SCFAs-COD/CODin), displaying a 4-fold enhancement compared with raw biomass. Population dynamics revealed a microbial biodiversity loss along the AF regardless of the applied pretreatment, evidencing that the imposed operational conditions specialized the microbial community. In fact, a reduced abundance in Euryarchaeota phylum explained the low methanogenic activity, implying SCFAs accumulation. The bacterial community developed in the reactors fed with pretreated microalgae exhibited high acidogenic activities, being dominated by Firmicutes and Bacteroidetes. Firmicutes was by far the dominant phylum when using protease (65% relative abundance) while Bacteroidetes was prevailing in the reactor fed with carbohydrase-pretreated microalgae biomass (40% relative abundance). This fact indicated that the applied pretreatment and macromolecule solubilization have a strong effect on microbial distribution and therefore in SCFAs bioconversion yields.

AbstractIncreasing yeast robustness against lignocellulosic-derived inhibitors and insoluble solids in bioethanol production is essential for the transition to a bio-based economy. This work evaluates the effect exerted by insoluble solids on yeast tolerance to inhibitory compounds, which is crucial in high gravity processes. Adaptive laboratory evolution (ALE) was applied on a xylose-fermentingSaccharomyces cerevisiaestrain to simultaneously increase the tolerance to lignocellulosic inhibitors and insoluble solids. The evolved strain gave rise to a fivefold increase in bioethanol yield in fermentation experiments with high concentration of inhibitors and 10% (w/v) of water insoluble solids. This strain also produced 5% (P > 0.01) more ethanol than the parental in simultaneous saccharification and fermentation of steam-exploded wheat straw, mainly due to an increased xylose consumption. In response to the stress conditions (solids and inhibitors) imposed in ALE, cells induced the expression of genes related to cell wall integrity (SRL1,CWP2,WSC2andWSC4) and general stress response (e.g.,CDC5,DUN1,CTT1,GRE1), simultaneously repressing genes related to protein synthesis and iron transport and homeostasis (e.g.,FTR1,ARN1,FRE1), ultimately leading to the improved phenotype. These results contribute towards understanding molecular mechanisms that cells might use to convert lignocellulosic substrates effectively.

AbstractIncreasing yeast robustness against lignocellulosic-derived inhibitors and insoluble solids in bioethanol production is essential for the transition to a bio-based economy. This work evaluates the effect exerted by insoluble solids on yeast tolerance to inhibitory compounds, which is crucial in high gravity processes. Adaptive laboratory evolution (ALE) was applied on a xylose-fermentingSaccharomyces cerevisiaestrain to simultaneously increase the tolerance to lignocellulosic inhibitors and insoluble solids. The evolved strain gave rise to a fivefold increase in bioethanol yield in fermentation experiments with high concentration of inhibitors and 10% (w/v) of water insoluble solids. This strain also produced 5% (P > 0.01) more ethanol than the parental in simultaneous saccharification and fermentation of steam-exploded wheat straw, mainly due to an increased xylose consumption. In response to the stress conditions (solids and inhibitors) imposed in ALE, cells induced the expression of genes related to cell wall integrity (SRL1,CWP2,WSC2andWSC4) and general stress response (e.g.,CDC5,DUN1,CTT1,GRE1), simultaneously repressing genes related to protein synthesis and iron transport and homeostasis (e.g.,FTR1,ARN1,FRE1), ultimately leading to the improved phenotype. These results contribute towards understanding molecular mechanisms that cells might use to convert lignocellulosic substrates effectively.

AbstractLignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40–60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.

AbstractLignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40–60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.