Insights into cell robustness against lignocellulosic inhibitors and insoluble solids in bioethanol production processes

Effect of WIS and/or inhibitors on yeast fermentation

This study assessed how the presence of inhibitors and WIS may influence yeast fermentation under the conditions stated in Table 2. As shown in Fig. 1A, no differences were observed in terms of glucose consumption rates or residual glucose in fermentation experiments with 50% (v/v) inhibitor mix or 5–10% (w/v) of WIS when compared to control assays without insoluble solids and inhibitors. In these cases, no lag phase was detected and glucose was exhausted within the first 5 h of fermentation. This result agrees with Koppram and co-workers that showed no differences in the consumption of 20 g/L glucose when control fermentation (with no WIS in the medium) was compared to fermentations in the presence of 2, 5, 10, and 12% WIS (w/w)²⁶. The presence of 100% (v/v) of inhibitor mix reduced, however, the glucose consumption rates, reaching glucose exhaustion at 24 h (Fig. 1A) and corroborating the well-known effect that high concentration of inhibitors exerts on yeast cells, which in turns hampers glucose utilization^27,28.

In contrast to glucose conversion, the presence of lignocellulose-derived inhibitors exhibited a strong inhibition effect during the xylose conversion phase (Fig. 1B). In this case, the addition of 50% and 100% (v/v) of inhibitor mix resulted in restricted xylose assimilation by cells, which only consumed 18% and 12% of the initial xylose concentration, respectively (Table 3). The higher susceptibility of xylose fermentation to lignocellulose-derived inhibitors compared to that of glucose fermentation has already been shown in several studies^29,30. Since xylose utilization has been proven to provide less energy in the form of ATP compared to glucose³¹, and response to inhibitors requires high energy levels, the presence of inhibitors may have a stronger effect on yeast when xylose is the utilized carbon source. Furthermore, it is likely that the genetic modifications needed to construct xylose-fermenting yeasts alter their cell metabolic homeostasis affecting the inhibitor tolerance².

By contrast, the presence of 5% (w/w) or 10% (w/w) WIS slightly increased xylose consumption when compared to control assays (Fig. 1B). Tricarboxylic acids (TCA) cycle was identified as one of the targets of transcriptional regulation to optimize xylose utilization. Thus, intensive TCA cycle was assigned to be important for xylose metabolism in xylose-recombinant S. cerevisiae strains³². In the same context, regulation of the stress response and amino acid metabolism have been shown as two important strategies for an effective xylose utilization in a recombinant xylose-fermenting S. cerevisiae strain^32,33. Strikingly, Moreno and co-workers identified amino acids biosynthesis and carboxylic acid metabolic processes among the major overexpressed biological processes in S. cerevisiae F12 grown in glucose media with insoluble solids¹². Thus, WIS may affect yeast cells by promoting xylose utilization when no other lignocellulose-derived inhibitor is present.

Despite the increase in xylose consumption, ethanol yields in presence of WIS were 0.20–0.21 g/g. This value was 25–30% lower than the obtained in control assays (0.28 g/g) (Table 3). Lower ethanol yields are commonly linked to an increase in xylitol production³⁴. Nevertheless, similar xylitol concentrations (< 0.1 g/L) were found in control and fermentation assays with only WIS. Thus, slight differences in cell growth in presence of WIS or redistribution of metabolic fluxes to cope with the challenging conditions imposed by WIS may result in lower ethanol yields.

As mentioned before, Koppram and co-workers²⁶ did not observed differences in ethanol yields when adding up to 12% (w/w) of WIS to fermentation media with 20 g/L glucose, reaching ethanol yields of 0.32 g/g. However, when adding 40% (w/w) and 60% (w/w) insoluble solids, Moreno and colleagues¹² showed a decrease in ethanol yield in glucose media from 0.37 g/g without solids to 0.35 g/g and 0.22 g/g, respectively. It is worth mentioning that previous studies only utilized glucose as carbon source. In spite of promoting xylose consumption in presence of 5% (w/w) and 10% (w/w) of WIS, the reduced ethanol yields obtained in this study indicated that xylose fermentation was more prone to be affected by stressful conditions.

Lower ethanol yields than those obtained for control assays were also found when lignocellulosic inhibitors were present, reaching 0.22 g/g and 0.19 g/g with 50% (v/v) and 100% (v/v) of the inhibitor mix, respectively (Table 3). As previously commented, less than 20% of the initial xylose concentration was consumed by non-evolved yeast cells (Fig. 1B). In addition, when increasing the inhibitor content from 50% (v/v) to 100% (v/v), the glucose consumption rates decreased by threefold (from 1.8 g/L h to 0.6 g/L h) at the initial stages of the fermentation process (5 h) (Fig. 1A). This result is indicative of the high inhibitory potential of lignocellulose-derived inhibitors, especially during the xylose assimilation phase.

Besides the detrimental effect that the presence of WIS exhibited on ethanol yields in fermentation experiments with 10 g/L glucose and 10 g/L xylose, the influence that the presence of WIS has on the inhibitory tolerance of S. cerevisiae F12 was also studied. For such a goal, 50% (v/v) or 100% (v/v) inhibitor mix were combined with 5% (w/v) or 10% (w/v) of WIS in different fermentation tests. As it is shown in Fig. 2A, when using 50% (v/v) of inhibitor mix, glucose was exhausted within the first 24 h, and 22% of the xylose was consumed after 48 h of fermentation. In this case, the ethanol yield was 0.22 g/g and 0.19 g/g with 5% (w/v) and 10% (w/v) of WIS, respectively (Table 3). These ethanol yields were similar than those obtained when only 50% (v/v) of inhibitor mix was added (Table 3), indicating that yeast tolerance was not significantly affected by the presence of WIS at low inhibitor concentration. On the other hand, when 100% (v/v) of the inhibitor mix was combined with either 5% or 10% (w/v) of WIS neither glucose nor xylose were exhausted in 48-h long fermentation (Fig. 2B). Furthermore, marked differences were observed in ethanol yield in comparison with only 100% (v/v) of the inhibitor mix (Table 3). When 5% WIS (w/v) were added together with 100% (v/v) of the inhibitor mix, about 80% of the initial glucose and 10% of the initial xylose were consumed after 48 h of fermentation, reaching an ethanol yield of 0.16 g/g. However, 10% (w/v) of WIS together with 100% (v/v) inhibitor mix resulted in 80% less ethanol when compared to only 100% (v/v) inhibitor mix. The lower ethanol concentrations were directly linked to a completely hampered xylose consumption and to a limited glucose consumption. These results clearly showed a synergistic effect when combining both lignocellulose-derived inhibitors and WIS and pointed out to the presence of WIS as a crucial factor when yeast cells have to deal with high concentrations of inhibitory compounds.

In the present work, an increase in xylose uptake was observed when 50% (v/v) of inhibitor mix was combined with WIS compared with only 50% (v/v) inhibitors (Table 3). This result supported the hypothesis that the presence of insoluble solids may promote xylose consumption in absence of biomass degradations compounds or when inhibitors are present at low concentrations. In this sense, Koppram and co-workers²⁶ studied the effect of steam-pretreated birch WIS on the glucose consumption and yeast tolerance to either HMF (1 g/L), furfural (1 g/L), syringaldehyde (0.8 g/L) or acetic acid (9 g/L). These authors reported higher glucose uptake rates when low concentrations of these compounds were simultaneously present with WIS compared to those obtained in the absence of solids²⁶. In the same study, a proteomic analysis revealed up-regulation of glycolytic enzymes and ATP synthases in the presence of acetic acid and WIS, strongly indicating an increased generation of energy in the presence of both stressors (WIS and inhibitors) which could be the reason for the increased sugar consumption.

The ALE procedure in WIS-rich and inhibitor-rich media (Table 1) resulted in an evolved S. cerevisiae F12 with improved abilities to cope with the combination of both inhibitors and WIS. When compared with the parental strain, a decrease in the xylose consumption was observed when only WIS (10% w/v) was present in the fermentation broth (Table 3). However, in presence of 100% (v/v) inhibitor mix, xylose consumption increased from 12% with parental S. cerevisiae F12 to 64% with evolved cells which was also translated in an increase of ethanol yield from 0.19 g/g to 0.25 g/g. These results suggest that evolution procedure primarily favored changes to increased tolerance to inhibitors that could be detrimental to cope with the sole presence of insoluble solids. The success of ALE was evident when comparing parental and evolved S. cerevisiae F12 performance at the most challenging conditions (i.e. 100% (v/v) of inhibitor mix and 10% (w/v) of WIS). In this case, parental S. cerevisiae F12 did not consume any xylose and ethanol yield was as low as 0.05 g/g. On the other hand, xylose consumption and ethanol yield increased to 21% and 0.24 g/g, respectively, when using the evolved strain proving the effectiveness of ALE as strategy to increase tolerance to a combination of stressors.

Simultaneous saccharification and fermentation at high substrate loading

Parental S. cerevisiae F12 was used in SSF to evaluate its fermentation performance and cell robustness under high substrate loading. When using the whole slurry at a concentration of 20% TS (w/v), no ethanol was produced during SSF processes (data not shown). Although parental cells were able to cope with 100% (v/v) inhibitory mix in absence of WIS (Fig. 1), the presence of solids and inhibitors in SSF of slurry led to complete cell inhibition. This fact pointed to a reduced tolerance to inhibitors in presence of high solids content. In this case, the progressive liquefaction of the solids during the first hours of SSF was not sufficient to overcome the effect that WIS had on yeast tolerance to inhibitors. Nevertheless, when using 20% WIS (w/v) supplemented with xylose (i.e. absence of inhibitors), parental S. cerevisiae F12 was capable of fermenting both glucose and xylose, reaching a maximum ethanol concentration of 39.3 ± 0.4 g/L (Fig. 3).

In SSF from WIS, S. cerevisiae F12 assimilated glucose immediately upon enzymatic hydrolysis, thus maintaining a low glucose concentration during the fermentation process (Fig. 3). In contrast, limited xylose consumption was shown within 72 h of SSF. Recombinant S. cerevisiae cells use the same transport systems to incorporate both glucose and xylose inside the yeast cell^35,36. The uptake of xylose through the transport system has been reported to have significantly lower affinities for xylose than for glucose³⁷. In this sense, the xylose uptake is strongly inhibited when glucose is present. This fact is decisive in mixed sugar fermentations with recombinant S. cerevisiae strains because this yeast does not utilize xylose unless glucose is significantly depleted. In this case, glucose concentration was below 0.5 g/L during SSF process, and the limited xylose consumption could be therefore explained due to the stressful fermentation conditions.

The robustness of the evolved strain was evaluated under the same SSF conditions than the parental strain. Similar to the parental S. cerevisiae F12, the evolved strain was totally inhibited during SSF processes of the whole slurry at 20% TS (w/v) (data not shown). However, in the SSF from WIS, the evolved strain produced a maximum ethanol concentration of 41.5 ± 0.5 g/L, which was 5% higher (P < 0.01) than the obtained by the parental strain (Fig. 3) and represented 50% of the theoretical maximum ethanol that could be obtained in SSF (yield estimated considering the total glucose and xylose that can be potentially available during SSF process and a maximum sugar-to-ethanol conversion yield of 0.51 g/g). The evolved cells also exhibited improved xylose uptake rates, which increased the xylose consumption by about 10% (32% of xylose was consumed after 72 h of SSF). The high xylose:glucose ratio utilized during ALE was decisive for the success of the process since the utilization of xylose as carbon source during the evolution procedure is a key factor to increase the yeast affinity for this sugar. This improved xylose fermenting capacity could be due to improved xylose transport kinetics^38,39. As a matter of fact, increased expression of hexose transporters was reported in evolved xylose-utilizing yeasts^39,40,41, as may be the case for the resulting evolved strain in this study as well.

Differential gene expression of the improved phenotype

A total of 196 genes were found upregulated (130 genes) or downregulated (66 genes) in evolved cells in the presence of both solids (20% w/w) and inhibitors (80% v/v of inhibitory mix) (Fig. 4A). These conditions of solids and inhibitors were the most challenging conditions to which cells were evolved in the ALE and thus they were selected for differential gene expression analysis. The differences between parental and evolved cells were also analyzed by hierarchical clustering, which clearly plotted two different groups (Fig. 4B): i) one corresponding to parental cells and ii) another one corresponding to evolved cells. This result supported the differences between S. cerevisiae F12 and the corresponding evolved strain.

Differentially expressed genes (parental vs evolved) were subsequently analyzed by gene ontology (GO) analysis to determine the biological processes induced and repressed. This analysis highlighted cell cycle (e.g., cytokinesis, regulation of cell cycle, reproductive process) and cell wall organization or biogenesis (e.g., fungal-type cell wall organization, sexual sporulation) as major upregulated biological processes, while maltose metabolic process, transport (e.g., ion transport, amino acid transport, water transport) and homeostatic process (e.g., iron ion homeostasis) were the main biological processes downregulated (Table 4). In spite of identifying several biological processes induced and repressed in the improved phenotype, enrichment analysis identified no metabolic pathway statistically upregulated or downregulated. It is also important to remark that a significant number of identified upregulated (53 genes, ca. 40%) and downregulated (19 genes, ca. 30%) genes had an unknown molecular function (Supplementary Table S1). Furthermore, about 90% of these genes have a Log2-fold change above one order. These results might indicate the potential role of these genes during the cell response to insoluble solids, and therefore, they should be further investigated.

The results obtained by GO analysis regarding induced and repressed biological processes were also supported by the protein–protein interaction networks resulting from STRING analysis. STRING revealed cell cycle process, response to stress and cell wall organization as the main upregulated processes, while homeostasis, ribosome biogenesis and transport were highlighted as major downregulated processes (Table 5, Fig. 5). From these analyses, it is important to highlight the upregulation of genes specifically related with DNA damage and the cell response to stress. These genes included for instance CDC5, CTF18, HTA1, MMS4, PLM2, RNR1, RAD51, DUN1, SSA3, TRR2, CTT1, ALD3, ALD2, PAI3, SIP18, and GRE1. CDC5 is known to prevent the cell-cycle arrest induced by the DNA damage checkpoint, allowing cell division and promoting the adaptation of cells to this cell state⁴². Simultaneously, DUN1, CTF18, RNR1 and RAD51 genes were also induced in the evolved S. cerevisiae F12 strain. These genes are also related with the DNA damage replication checkpoint and DNA repair mechanisms^43,44,45. The overexpression of these genes might prevent cells from having an excess of mutations during cell adaptation, thus encouraging cell survival. The response to stress was also induced through the overexpression of genes involving the protection against oxidative stress (TRR2, CTT1), heat shock (SSA3, SPG4) and osmotic stress (PAI3), as well as genes related to the general response to stress (GRE1, SIP18, ALD2, ALD3). It is worth highlighting that the overexpression of CTT1 improved xylose utilization in recombinant strains³², supporting the overexpression of this gene after ALE that may be responsible of the increased xylose consumption in the evolved S. cerevisiae F12.

Specific genes (8 in total) related with cell wall organization were also induced (Tables 4 and 5). Among them, SRL1, CWP2, WSC2 and WSC4 encode important proteins for the stabilization of the cell wall^46,47,48. The overexpression of these genes might specifically be related with the yeast response against the stress promoted by solids. The presence of insoluble solids during yeast growth promotes the formation of cavities that cause a change in the external morphology of cells from a round-turgid shape to a highly wrinkled morphology¹². Overexpression of the aforementioned cell wall proteins might counteract this effect and maintain cell wall integrity under the stress conditions.

Major downregulated biological processes include ribosome biogenesis and RNA processing, as well as the transport of specific molecules including iron, peptides and water (Table 5). Repression of protein synthesis is one of the first cell responses upon stress exposure (heat shock, osmotic and oxidative stress), as it is a highly energy consuming process^49,50. Nevertheless, although having the general protein synthesis process repressed, cells can simultaneously induce the translation of stress-related genes to face the adverse environmental conditions⁵¹. This was also the case for the evolved S. cerevisiae F12 in this work. The second main downregulated biological process was transport. Most of the transport-related genes are associated to peptide/amino acid transport and to iron ion transport and homeostasis (Tables 4 and 5). In this work, repression of peptide/amino acid transport genes might be linked with the downregulation of protein biosynthesis upon stress exposure. On the other hand, it is highly remarkable the relatively high number of genes (up to 12 genes) that are involved in iron ion transport and homeostasis, including the transporter-encoding genes FIT2, FTR1, SIT1, ARN1 and ARN2, and genes encoding different ferric reductases (FRE1, FRE2, FRE5, FRE6, FRE8). Iron is an essential element required for different biological processes such as respiration, synthesis of nucleic acids, carbon metabolism, as well as photosynthesis and nitrogen fixation⁴⁹. However, iron may be toxic for cells due to its oxidative capacity in the ferrous form, which increases the importance of having a tight control of the iron metabolism. A high intracellular concentration of reactive oxygen species (ROS) under oxidative stress conditions represents a potential threat since the interaction between ROS and iron may end up in the formation of new hydroxyl radicals with increased prooxidant capacity⁵². The simultaneous presence of both insoluble solids and lignocellulose-derived inhibitors during fermentation processes causes a severe oxidative damage in yeast cells, which greatly increases the intracellular ROS levels¹². This high ROS concentration might be responsible for repressing the corresponding iron-related genes as a way to reduce the risks associated to a marked oxidative stress. Yeast cells (and other multicellular organisms) usually promote iron depletion to prevent metal toxicity and the irreversible damage under oxidative stress conditions⁵².

Overall, these results clearly show the complex inhibitory environment that cells have to face during lignocellulosic biomass conversion. In response to a single stressor, specific genes and pathways have been identified as key components to increase yeast robustness. For instance, ZWF1 has been identified as a key element during oxidative stress in S. cerevisiae upon exposure to a wide variety of chemical and environmental stress agents⁵³. During a heat shock, changing ergosterol by fecosterol alters membrane fluidity rendering thermotolerance in yeast⁵⁴. The general response to stress and the cell cycle arrest have been identified as important processes to face a high concentration of insoluble solids¹². By contrast, in lignocellulose-conversion processes cells must simultaneously deal with a bunch of chemical inhibitors and a high concentration of insoluble solids. To cope with such adverse conditions, this study demonstrate that cells should be capable of maintaining cell membrane integrity and preventing oxidative damage. Therefore, upregulation of membrane-related genes (e.g. SRL1, CWP2, WSC2 and WSC4) and induction/repression of genes and pathways involving the oxidative stress and the general response to stress (e.g. CDC5, DUN1, CTT1, GRE1, FTR1, ARN1, FRE1) can be targeted in future studies to evaluate cell robustness in lignocellulose-related bioprocesses.