3.1 Effect of short-term adaptation on bioconversion of xylose to xylitol
Adapted and non-adapted Candida guilliermondii FTI 20037 cells were grown in the hydrolysates to evaluate the effect of short-term adaptation on the bioconversion of xylose to xylitol. Fermentations carried out with non-adapted cells had the purpose to evaluate the behavior of the yeast against different hydrolysate concentration factors. According to the results in Fig. 1, xylose assimilation occurred from the first hours and a similar consumption profile was observed in all adaptation conditions evaluated. However, it is interesting to observe the favoring of xylose consumption in H2N (two-fold concentered and non-treated) and H5 (five-fold concentered and treated) hydrolysates (Figs. 1a and 1e, respectively) which had the highest content of total phenolic compounds, that are known inhibitors of the fermentative activity of the studied yeast [11]. The favoring of xylose consumption after cell adaptation is supported by an 22.5% increase in QXS that varied from 0.40 (non-adapted cells) to 0.49 gL− 1h− 1 (adapted cells) in the cultivations with H2N hydrolysate (medium with higher inhibitors concentrations, Table 1). Similarly, a 10.4% increase in this parameter also was found (Fig. 1e) in the five-fold concentrated hydrolysate and treated (H5). In the fermentations with more concentrated hydrolysate (H5) a complete consumption of xylose of the medium was observed after 96 h of cultivation. In contrast, when non-adapted cells were used as inoculum there was a negligent consumption of xylose (70.1%) after 96 h (Fig. 1e). In regard to the H2N hydrolysate, the adapted cells promoted a 96.9% xylose consumption in 60 h of fermentation, corresponding to an increase of 15.6% in relation to the consumption by the non-adapted cells (Fig. 1a). These results indicate that short-term adaptation may be a technique capable of helping microorganisms to overcome toxicity caused by high concentrations of toxic compounds found in hemicellulosic hydrolysates (Table 1).
Corroborating with the results found in the present study, Tomás-Pejó and Olsson (2015) [31] described an increase in xylose consumption by a recombinant strain of Saccharomyces cerevisiae when short-term adaptation in wheat straw hydrolysate was performed. The authors observed after 120h fermentation of wheat straw hydrolysate containing 4.25 gL− 1 acetic acid, 0.65 gL− 1 5-HMF, 3.85 gL− 1 furfural and 0.025 gL− 1 vanillin, a xylose consumption between 40 and 98%, respectively, while the consumption of this pentose by the non-adapted yeast did not occur. van Dijk et al. (2019) [25] also reported the increase of xylose consumption by a recombinant Saccharomyces cerevisiae strain in fermentations using wheat straw hydrolysate containing about 3.76 gL− 1 acetic acid, 0.48 gL− 1 5-HMF, and 2.4 gL− 1 furfural. After 48 h fermentation, the authors observed a xylose consumption of 46% by the adapted cells, while the consumption by non-adapted cells was only 22%. Zhang et al. (2019) [32] observed the highest consumption of xylose by cells adapted to sweet sorghum hydrolysate. After 24 h cultivation more than 80% of the xylose had been consumed by adapted recombinant S. cerevisiae, while non-adapted cells consumed only 45%. In another study, an increase in the total xylose consumption (from 26.3 to 62.7 %), was also observed for Escherichia coli adapted in increasing concentrations of kenaf hydrolysate [33].
Positive effect of short-term adaptation at the beginning of fermentation was also found in this work. In the first 12 h fermentation, an increase in the assimilation of xylose was observed, especially in the hydrolysates with higher levels of toxic compounds (H2N and H5). The greater tolerance to inhibitors promoted by the cell adaptation to the hydrolysate results in a faster and more complete xylose consumption [26]. In response to previous exposure to inhibitors, microorganisms reprogram their metabolic pathways and regulatory machineries, developing tolerant phenotypes to harmful environments [34]. In this sense, adapted strains might induce stress response faster than non-adapted, allowing a faster development [19].
The importance of the detoxification stage was also evidenced in this study, since the consumption of xylose in H2N hydrolysate (two-fold concentrated and not treated) by non-adapted cells was 16.1% lower than the consumption in the H2 hydrolysate (two-fold concentrated and treated) at 60 h fermentation. For adapted cells, the decrease in xylose assimilation was only 2.17%, showing the benefit of adaptation strategy against the toxic effect of hydrolysates.
The profile of xylitol production followed a similar behavior to xylose consumption for all adaptation conditions evaluated. In addition, the production of this polyol by cells adapted to hydrolysates with higher toxic levels (H2N and H5) was clearly increased (Fig. 1a and 1e). The results showed an increase in xylitol production of about 58.7% (from 23.5 to 37.3 gL− 1) by cells adapted to H5 hydrolysate in 96 h fermentation, corresponding to a yield (YP/S) increase of 15.7% (0.51 to 0.59 gg− 1) (Fig. 2e) and a xylitol volumetric productivity (QP) increase of 62.5% (0.24 to 0.39 gL− 1h− 1) (Fig. 3e). With regard to H2N hydrolysate, the increase in xylitol production by the cell adaptation was of approximately 60.9% (from 6.4 to 10.3 gL− 1) in 60 h of fermentation, which led to an increase in both yield (YP/S: 29.6%) and productivity (QP: 54.5%) (Fig. 3a). No relevant difference was observed in these fermentation parameters in the cultivations with the hydrolysates H2, H3, and H4, making it clear that cell adaptation is not necessary when these hydrolysates are used, since the increase in YP/S and QP was much lower (up to 13.5 %). This is probably due to the lower levels of toxic compounds in these hydrolysates in comparison to H2N and H5 hydrolysates (Table 1). According to Sene et al. (1998) [35], the greater the degree of adaptation of cells, the greater their capacity to metabolize toxic compounds. The production of this polyol by non-adapted cells in H2N hydrolysate fermentation in 60 h was 123.4% lower than that observed in H2 hydrolysate (Fig. 1a and 1b). For adapted cells, the reduction in xylitol production was only 17.5%. There was also a delay in the start of xylitol production when increasing the concentration factor of the hydrolysate (Fig. 1), possibly due to the increased toxic content (Table 1). However, the performance of adapted C. guilliermondii improved in H5 hydrolysate fermentation (Fig. 1e), coinciding with the highest final xylitol concentration, highest volumetric productivity, and maximum xylose consumption in 96 h of fermentation. Regarding the cell growth, there were no differences among the adaptation conditions evaluated (data not shown). Similar behavior was reported by Sene et al. (2001) [36] when adapting C. guilliermondii to increasing concentration factors of sugarcane bagasse hemicellulosic hydrolysate.
An improvement in xylitol production by cell adaptation was also observed by Shah et al. (2020) [33]. These authors carried out the adaptation of a recombinant strain of E. coli in increasing concentrations of kenaf hemicellulosic hydrolysate, and consequently of toxic compounds, observing at the end of the cultivation a two-fold increase in xylitol production and an improvement of 10.7 and 100% in the xylitol yield and volumetric productivity, respectively. Sene et al. (2001) [36] observed a 34% increase in xylitol production by C. guilliermondii adapted to sugarcane bagasse hemicellulosic hydrolysate four-fold concentrated in comparison to non-concentrated hydrolysate. Wang et al. (2011) [37] observed that the number of propagation steps during cell adaptation in increasing concentrations of corn cob hemicellulosic hydrolysate is important to gradually improve the tolerance of Candida tropicalis, increase xylitol yield and decrease residual xylose concentration. Similar behavior was reported by Kim (2019) [34] when adapting C. tropicalis to empty palm fruit bunch fiber hydrolysate. Tomás-Pejó and Olsson (2015) [31] reported a 33.3% increase in ethanol yield when a recombinant strain of S. cerevisiae was adapted in wheat straw hydrolysate (23% vv− 1) in comparison to the adaptation in less concentrated hydrolysate (12% vv− 1). Silva et al. (2014) [38] observed an increase of 22 and 49% in ethanol yield and volumetric productivity, respectively, by Scheffersomyces stipitis adapted to sugarcane bagasse hemicellulosic hydrolysate. An 50% increase in lactic acid volumetric productivity was also observed when Bacillus coagulans was adapted to wheat straw hemicellulosic hydrolysate [39].
Based on the results of xylose consumption and xylitol production, it is possible to observe the existing correlation between the levels of toxic compounds in the hydrolysates and the need for cell adaptation. When comparing to non-adapted cells, adapted C. guilliermondii had a better performance in H2N and H5 hydrolysates, which contained higher inhibitors levels than in H2, H3, and H4 (Table 1). So, it is reasonable to assume that there is a need to adapt C. guilliermondii cells to increase xylitol production from hydrolysates, mainly in that ones with higher inhibitors levels. According to Sene et al. (1998) [35], the higher the concentration factor of the hydrolysate, and consequently of inhibitors levels, the greater the need for adaptation of the cells. Nouri et al. (2018) [40] observed in their study with concentrated and non-concentrated sugarcane bagasse hydrolysate, the importance of cell adaptation in face of the concentration factor of the hydrolysate. According to the authors, Barnettozyma californica adapted to the concentrated hydrolysate exhibited a highest increase in ethanol yield and productivity than in the non-concentrated hydrolysate, as well as in growth rate. The need for adaptation in face of increased toxicity was also observed for S. stipitis grown in semi-defined media containing glucose and xylose and increasing concentrations of acetic acid [41].
Regarding the consumption of glucose and arabinose, it was found that short-term adaptation favored both of them (data not shown). However, while glucose was completely consumed in the first 12 h cultivation, regardless of hydrolysate concentration factor, arabinose was slowly and partially consumed. Tomás-Pejó and Olsson (2015) [31] also observed the favoring of glucose consumption by an adapted S. cerevisiae recombinant strain. The yeast consumed all the glucose present in the wheat straw hydrolysate in less than 24 h, while the consumption of this hexose by non-adapted cells was partial. Sene et al. (2001, 1998) [35, 36] also observed a slow assimilation of arabinose by C. guilliermondii adapted in different concentration factors of sugarcane bagasse hemicellulosic hydrolysate. This behavior may be justified by the catabolite repression of arabinose assimilation caused by xylose. Pichia guilliermondii, the teleomorph form of Candida guilliermondii, and Candida arabinofermentans have two arabinose transportation systems: proton symport and facilitated diffusion. The first one has a high arabinose affinity, however it is strongly inhibited by xylose in the case of C. arabinofermentans, while for P. guilliermondii the presence of xylose leads to a reduction in arabinose transportation once the proton symport system has similar affinities for both pentoses. In facilitated diffusion transport, although xylose does not causes inhibition, it has a low affinity by arabinose [42]. In another work, arabinose assimilation by Kluyveromyces marxianus and P. guilliermondii induced by arabinose transporters expressed in S. cerevisiae was strongly inhibited by xylose (75 and 100 %, respectively), indicating that this pentose is preferentially transported than arabinose [43]. The consumption of arabinose by some yeasts seems to be attached to the reduction of xylose levels in the medium, as this pentose can inhibit arabinose assimilation. In this sense, xylose consumption over the fermentation could relieve the repression in arabinose assimilation, favoring its use as observed in other studies [44, 45]. In this work, QAS was 250 % and 117 % increased by short-term adaptation in H2N and H5 hydrolysates, respectively. According to the results, the improvement in xylose assimilation may be the reason of the favoring of arabinose assimilation.
3.3 Effect of short-term adaptation on ethanol and glycerol production
The formation of by-products such as ethanol and glycerol were observed during fermentations, regardless the concentration factor of the hydrolysate and yeast adaptation (Fig. 4). Ethanol formation occurred from the first fermentation hours, with its maximum production between 12 and 24 h fermentation. The highest ethanol concentration (2.14 gL− 1) was verified in 12 h in the hydrolysate with the highest toxic level (H5). This higher ethanol production is related to the increase of glucose concentration in the hydrolysates, as demonstrated by C. tropicalis in synthetic medium containing xylose and increasing concentrations of glucose [46, 47]. Sene et al. (2001) [36] also reported an increase in ethanol formation by C. guilliermondii adapted to increasing concentrations of sugarcane bagasse hydrolysate. An increase of 40% in ethanol production was found with cells adapted to the four-fold concentrated hydrolysates in comparison to the not concentrated one. In addition, the production of ethanol coincides with the period of glucose consumption (first 12 h of fermentation - data not shown). Consumption of ethanol over fermentation time was also observed. Likewise, there was production of glycerol in the first hours of fermentation in all cultivation conditions, especially in hydrolysates with higher concentrations of toxics. Maximum glycerol production (2.52 gL− 1) was verified for non-adapted cells in 60 h fermentation in the media based on H2N hydrolysate (Fig. 4a). There is a tendency of increasing glycerol generation by non-adapted cells due to the increase of the concentration factor of the hydrolysates and consequently of inhibitors. The increase in glycerol production due to the increase in the concentration of toxic compounds in hydrolysates has been reported by Zhang et al. (2019) [32]. It is worth to highlight the lower production of this by-product by adapted cells, when compared to non-adapted, notably in the fermentations of hydrolysates with higher concentrations of toxic substances. A decrease of approximately 300% in glycerol production was observed by cells adapted to the hydrolysate H2N after 60 h fermentation (Fig. 4a), while a decrease of 102% was observed for the hydrolysate H5 after 96 h fermentation (Fig. 4e). Even with the increased toxicity of the hydrolysates, the production of glycerol by adapted cells was still lower. Unlike ethanol, glycerol was not consumed by the cells, thus accumulating in the medium throughout the entire fermentation.
Glycerol is a compatible solute typically produced by yeasts under stress conditions. The lower glycerol formation by adapted cells indicates that short-term adaptation made C. guilliermondii more tolerant to the toxic compounds present in the hydrolysates, promoting a decrease in its toxic effects on cell metabolism and favoring fermentative performance. According to Nevoigt and Stahl (1997) [48], in addition to cell osmoregulation, glycerol generation plays an important role in the regeneration of NAD+ and maintenance of the cell redox balance. Zhang et al. (2019) [32] reported a higher glycerol production by the recombinant strain of S. cerevisiae non-adapted to sweet sorghum hydrolysate than by the adapted yeast. This byproduct was produced in highest amounts by the yeast in fermentations of hydrolysates of greater toxicity, corroborating to the relationship between increased cellular stress and glycerol production. Narayanan et al. (2016) [49] and Sànchez I Nogué et al. (2013) [50] also observed a decrease in glycerol production by adapted cells of S. cerevisiae in a semi-defined medium containing inhibitors usually found in hydrolysates.