3.1. Characterization of woody biomass
The complex nature of residual woody biomass is a well-known initial hurdle in defining its application in a microbial-based biorefinery 1,36,37. Additionally, the intrinsic biodiversity, in terms of plant species and parts, is key to highlight differences in carotenoids production possible arising from the procedure steps as hydrolysis and fermentation (SHF). For these reasons, the composition of the biomass selected for this study, i.e., sessile oak wood (SOW) and bark (SOB), as well as mulberry tree wood (MTW) and bark (MTB) was initially characterized. Table 2 summarises the main components constituting these lignocellulosic biomasses, in terms of the sugar-fraction vs. the lignin content, and considering the extractable compounds from the three solvent consecutive extractions.
Table 2
Composition of biomasses used in this study. Cellulose & hemicellulose [% (w/w)] are calculated as the difference of the sum of lignin (acid soluble), lignin (acid insoluble) and total extractives. Total extractives [% (w/w)] is given by the sum of DCM, EtOH/tol and acetone consecutive extractions.
Biomass component | SOW | SOB | MTW | MTB |
|---|
Cellulose & hemicellulose [% (w/w)] | 61 ± 11 | 55 ± 5 | 59 ± 9 | 53 ± 5 |
Lignin (acid soluble) [% (w/w)] | 4 ± 1 | 3.7 ± 0.8 | 1.5 ± 0.1 | 2.8 ± 0.6 |
Lignin (acid insoluble) [% (w/w)] | 28 ± 3 | 31 ± 2 | 32 ± 3 | 35 ± 2 |
Total extractives [% (w/w)] | 6.4 ± 0.2 | 10.5 ± 0.3 | 7.4 ± 0.4 | 8.9 ± 0.8 |
Overall, the delineated compositions are in line with what is expected from hardwoods, with less cellulose contents in the barks 38 Bulk sugar contents are rather similar, both between the wood components and the bark ones.
The analysis of the lignin content of the various biomasses (Table 2) shows values that seem high for hardwood species. It can be assumed, based on literature data (Kögel-Knabner 2021), that a low percentage of the determined insoluble Klason lignin content is actually comprised of humins, known to form upon sugar degradation during Klason analysis and co-precipitating with the insoluble lignin. Interesting is the fact that in the barks is found more lignin than in the bulk. This must be explained by the residual wood in the bulk bark material and the presence of suberins that behave eventually similar to the lignin in the applied NREL procedure 40
Detailed analysis of the various extractives obtained was performed using gas-chromatography coupled to mass spectrometry to provide an in-depth characterization of their composition. Indeed, rather scant information is reported in the literature on the composition of the trees, wood and bark under study. The results, detailed in Table S1, show the expectable range of bioactive small molecules, with a slightly richer composition found for two bark samples, i.e., SOB and MTB. Several molecular classes of compounds have been identified in the extracts, mostly fatty acids and sterols, but also marker species belonging to other chemical classes. Mulberry tree resulted to be richer in extractives than sessile oak, showing high amount and variety of fatty acids, phenolic compounds, vitamins and coumarins especially in the bark. The three isomers of tocopherol (δ-tocopherol, γ-tocopherol, α-tocopherol), an antioxidant compound known as vitamin E, has been already observed in mulberry fruit and bark 41, as well as coumarins especially in leaves 42 but not those detected in our study as umbelliferone, methyl ostruthin, ostruthin and esculetin typical of umbelliferae family of plants. As regards sessile oak, SOW profile is particularly poor while SOB interestingly presents the wider variety of terpenoids of all the four samples. The identified terpenes belong to the family of diterpenes (copalol), tritepenes (β-amyrone, 24-norursa-3,12-diene, lupeol, β-amyrin, glutinol, copalol and simiarenol) and sesquiterpenes (Humulenol-II). SOB extractives show also the unique presence of alcohols, alkenes and other compounds such as benzaldehyde and benzaldehyde diethylacetal, compared to the other biomasses.
3.2. Enzymatic hydrolysis of woody biomasses
To unlock the potential of lignocellulosic biomasses in microbial-based biorefineries, an enzymatic cocktail able to release both hexose and pentose into the liquid phase sugars is commonly used. As described in Section 2.6, powdered biomasses, i.e., SOW, SOB, MTW, and MTB, were subjected to enzymatic hydrolysis. Data reported in Fig. 1A clearly show the effect of the action of such an enzymatic cocktail in terms of total reducing sugars, with an increase in the carbohydrates released from the sole pre-treatment.
Specifically, given the same initial amount of biomass processed per volume (15% w/v), data show that MTB is the raw material providing the highest amount of released sugars (16.4 ± 0.9 g/L), whereas on MTW basis the lowest content is obtained, reaching approx. only 50% of that seen for the MTB sample (8.3 ± 0.5 g/L). Interestingly, the bark moiety provided also for the SO-biomass consistently more sugars compared to the corresponding wood, i.e., SOW. This fact can be related to the different structural characteristics of bark with respect to wood. Scanning electron microscopy (SEM) imagines collected on the various biomasses after grounding show that the bark samples seem slightly more porous, exposing thus a larger surface for the enzymes to work on (Fig. 2). Nevertheless, these data are in contrast with previous research suggesting a negative impact of bark presence towards enzymatic saccharification, using as biomass source residues of spruce, birch, and douglas-fir 43,44
In order to investigate the composition of the carbohydrate mixture in the hydrolysates of the various biomasses, 1H-13C HSQC analyses were performed alongside quantification by wet-chemical assessment by the use of enzymatic assays of most of the sugars identified. Tables 4 and 5 report the results obtained in the assays, whereas Fig. 3 shows the HSQC spectra obtained for the various hydrolysates.
Table 4
Carbohydrates and inhibitors identified in hydrolysates after 6 hours of the biomasses used in this study by means of 1H-13C HSQC analyses (HSQC) and. A full list of identified signals and their respective shifts is given in the Supporting Information.
Carbohydrate | colour code | SOW | SOB | MTW | MTB |
|---|
Arabinose | | --- | --- | traces | traces |
Fructose | | X | X | X | X |
Galactose | | X | X | X | X |
Glucose | | X | X | X | X |
Mannose | | X | X | X | X |
Methyl gluconoric acid | | traces | traces | traces | traces |
Xylose | | traces | traces | X | X |
Other compounds | | | | | |
Alkanoyl residues | | X | X | X | X |
Alkyl residues | | traces | traces | traces | traces |
Gallate residues | | X | X | traces | traces |
Pyrogalloyl residues | | X | X | X | X |
Table 5
Carbohydrates identified in hydrolysates after 6 hours of the biomasses used in this study by means of tested enzymatic assays. Values are the means ± SD of three independent experiments.
Titer (g/L) | SOW | SOB | MTW | MTB |
|---|
Glucose | 2.4 ± 0.1 | 1.5 ± 0.1 | 3.4 ± 0.4 | 5.3 ± 1.7 |
Fructose | 2.2 ± 0.1 | 2.8 ± 0.1 | 2.5 ± 1.3 | 4.0 ± 1.0 |
Xylose | 1.3 ± 0.2 | 1.0 ± 0.1 | 0.6 ± 0.1 | 0.8 ± 0.1 |
Galactose | 1.0 ± 0.1 | 0.7 ± 0.2 | 0.7 ± 0.2 | 0.6 ± 0.2 |
The wet chemical analysis (Table 5) reveals the presence of glucose, fructose, xylose, and galactose indicating the successful hydrolysis especially of the hemicellulose component of the biomasses studied. The carbohydrates identified are in line with previous findings regarding oak-based hemicelluloses.45–47 These results are confirmed by the HSQC analyses, with the interesting exception of the xylose component in SO samples. While the wet-chemical test shows higher quantities for xylose in SO samples than MT samples, HSQC signal intensity suggests the contrary. The reason for this cannot be delineated precisely on the basis of the data available. More importantly, however, HSQC analyses were able to unveil the presence of additional carbohydrates and derivatives that the wet-chemical test could not target. Via a series of characteristic cross-peaks assigned on the basis of various literature sources 48–57, essentially the same species were identified in all the samples, with variations in relative abundances. SO-derived samples show in HSQC analyses only traces of xylose and methyl glucuronic acid. MT-samples show in HSQC analyses traces in arabinose, whereas this carbohydrate is not clearly detectable in SO samples. Acetylated carbohydrates have been identified in form of acetylated xylose in all samples. This is indicative of the expectable presence of acetylated xylan moieties, especially in MT samples, and thus in line with one of the most famous representatives of hardwood hemicellulose features.
HSQC spectra indicate also the presence of alkanoates, originating eventually from waxes present in the hydrolysate. Other weak cross-peaks could be assigned to alkyl residues, eventually stemming from alkylated sugars. Most importantly, SO-derived hydrolysates SOW and SOB contain very weak cross-peaks in the aromatic region that could potentially indicate the presence of pyrogallyl and gallate units, as such presence in tannins, with the latter especially in the typically oak-derived tannic acid. The cross-peak typical for a gallate unit is more pronounced in the SOB sample, whereas here the pyrogallyl peak is hardly detectable. This is a relevant observation since propyl gallate was demonstrated to increase microbial carotenogenesis in the thraustochytrids.58
The combined NMR analyses do not indicate the presence of lipids, since typical glycerol-derived cross-peaks are absent. Neither amino acids or peptide residues were detected. Samples do contain nitrogen sources, however, as revealed by wet-chemical tests, but contents in amino acids are supposedly too low as that they would exceed the detection threshold of around 1–2% (w/w) typical for NMR analyses. Figure 1B shows the amount of primary nitrogen source found in the hydrolysate. Nitrogen content is not increased by the action of saccharifying enzymes, with MTB as an exception. The measured amount of nitrogen permits to calculate C/N ratio for the different hydrolysate, being 38 for SOW, 80 for SOB, 34 for MTW and 97 for MTB. C/N ratio is one of the factors involved in the triggering to produce lipids and carotenoids in oleaginous yeasts 7 Given the presence of both carbon and nitrogen sources, the ability of R. toruloides to grow in such media without any supplementation of nutrients, or elimination of possible toxic compounds was directly tested, to further improve the sustainability aspects of the present work in respect to published bioprocesses based on this microbial cell factory and residual biomasses (Table 1).
3.3. R. toruloides production of carotenoids from woody hydrolysate
Once the presence of fermentable sugars in the hydrolysates was confirmed, the ability of R. toruloides to grow in such media, withstanding possible inhibitory compounds, and to produce carotenoids was tested. As described in Section 2.7, the yeast was directly inoculated into the woody hydrolysate and sampled over time for growth, sugar consumption, and carotenoids production. Figure 4 summarises the obtained results from the growth kinetics on SOW, SOB, MTW, and MTB hydrolysates.
MTB is the biomass supporting the highest growth in terms of OD (Fig. 4D), which is consistent with having the highest initial sugar content among the hydrolysates (Fig. 1A). While R. toruloides shows a similar growth profile in MTW and SOW (Fig. 4A and C) considering the initial difference in the provided sugars, growth in SOB hydrolysate resulted to be linear until reaching a plateau (Fig. 4D), rather than being exponential. Since the NMR analysis of SOB revealed the presence of substructures typical for tannins, above the NMR detection threshold, which is typically around 5% (w/w); it could be speculated that this tannin presence is interfering with yeast productivity. Similarly, terpenes detected in SOB extractives in higher concentration and variety with respect to the other samples by GC-MS analysis (Table S1), could have an inhibitory effect.
Nevertheless, R. toruloides was able to grow in all cases, consumed the available carbohydrates, despite not completely, and, most importantly, accumulated carotenoids. Specifically, both β-carotene and torulene were detected intracellularly, with the first being the more abundant of the two. The ratio between the two carotenoids in oleaginous yeasts may vary depending not only on strain, but also on the conditions, such as sugar availability, C/N ratio, or light exposure 59–62. The highest production of β-carotene occurred, in light of the above discussed inhibitors, interestingly on SOB hydrolysate after 30 h of growth, reaching 362.7 ± 33.9 mg/L (Fig. 4B), followed by MTB with 165.3 ± 22.6 mg/L of β-carotene (after 30 h of growth) (Fig. 4D). The production of torulene was instead observed to be similar across the provided biomass around 24 and 30 h or growth, ranging from 4.5 to 2.5 mg/L. Calculation and comparison of carotenoid yields in the different conditions are summarized in Table 4, whereas Figure S1 permits to visualize the direct comparison between carotenoids production profiles. These data are in accordance with previous findings disclosing that a C/N ratio of 80, as in SOB hydrolysate, is optimal to maximise β-carotene production over other carotenoids.62 In addition, as already mentioned, the presence of gallates in SOB hydrolysate can be considered an additional triggering element for carotenogenesis 58
Table 4
Quantitative analysis and yields calculations of β-carotene and torulene production by R. toruloides from SOW, SOB, MTW, and MTB hydrolysates, after 30 h of fermentation. Values are the means ± SD of three independent experiments.
| | Production (mg/L) | Yield on initial biomass (10− 5g/g) | Yield on hydrolysate sugars (g/g) | Yield on consumed sugars (g/g) |
|---|
β-carotene | Torulene | β-carotene | Torulene | β-carotene | Torulene (10− 2) | β-carotene | Torulene (10− 2) |
|---|
SOW | 123.8 ± 10.6 | 3.6 ± 0.5 | 83 ± 7 | 2.4 ± 0.4 | 1.61 ± 0.20% | 4.7 ± 0.8% | 2.6 ± 0,4% | 7.6 ± 1.6% |
SOB | 362.7 ± 33.8 | 4.0 ± 0.2 | 241 ± 23 | 2.7 ± 0.9 | 2.62 ± 0.25% | 2.9 ± 0.1% | 9,2 ± 1,1% | 10.1 ± 0.9% |
MTW | 89.9 ± 14.8 | 1.5 ± 0.2 | 60 ± 10 | 1.0 ± 0.1 | 0.99 ± 0.16% | 1.6 ± 0.1% | 1.7 ± 0.3% | 2.9 ± 0.3% |
MTB | 163.5 ± 22.6 | 2.6 ± 0.4 | 109 ± 15 | 1.7 ± 0.3 | 1.04 ± 0.16% | 1.7 ± 0.3% | 1.6 ± 0.3% | 2.5 ± 0.5% |
Table 4 shows, however, that, considering the yield on hydrolysate sugars, SOW hydrolysate can be calculated to represent the best option, after 30 h of growth, for torulene production (0.047 ± 0.008% g/g), and the second best option for β-carotene production (1.61 ± 0.20% g/g). SOB hydrolysate remains the best choice considering β-carotene yields on both initial sugars in the medium and on consumed sugars (Table 4). MT-derived samples, in this calculation mode, fall behind the SO-derived ones, for both β-carotene and torulene production.
This is consistent with previous data on R. toruloides growth on residual biomasses hydrolysates, where the maximum production of carotenoids was reached between the end of the exponential phase and the entrance of the stationary one.34,63 In case of SOB the presence of a linear growth rather than an exponential one can be seen as indicative of the fact that the cells face hurdles, potentially due to the gallate presence (vide supra). Indeed, stressful compounds (such as phenolic ones), or more generally stressful growth conditions, are known to act as a trigger to production of carotenoids, while being detrimental to growth.8,64 Therefore, when developing bioprocesses based on residual lignocellulosic biomasses, a trade-off between these actors has to be considered to maximise both growth and carotenogenesis.
In order to investigate whether also components comprised in the extractives could represent inhibitors, dedicated studies in this direction were performed. In fact, such compounds are typically present in woody biomasses (like those valorised in this study), while being limited or absent in other organic / lignocellulosic materials, such as wheat bran, tea or molasses.
3.4. Effect of extractives on the production of carotenoids
To study the importance of extractives and/or impurities, MTB and SOB were chosen as biomasses since the numbers and respective amounts of extractives in these bark samples were higher compared to the ones found for the wood samples (Table 2). Extractives were removed as described in Section 2.13 and extracted MTB and SOB, i.e., eMTB and eSOB, respectively, were subjected to enzymatic hydrolysis and SHF following the established procedures. Data obtained from growth in terms of optical density (OD), sugar concentration over time, and carotenoids produced allowed for determining the impact of the presence or absence of extractives in the biomasses. Data are summarised in form of Fig. 6.
Figure 6 shows that the elimination of extractives did not have an impact on enzymatic hydrolysis since the amount of released sugars present in the starting medium is comparable regardless of their presence. We expected the elimination of extractives might improve microbial performance and, in turn, the production of carotenoids. Indeed, eSOB hydrolysate permits for a higher OD compared to SOB, despite a comparable sugar consumption, suggesting that the extractives are responsible of impairing yeast growth. On the other hand, no significant difference can be observed between growth on MTB and eMTB. Surprisingly and independently from growth recovering, the absence of extractives in eSOB and eMTB resulted in a reduced accumulation of carotenoids (Fig. 6CD), compared to SOB and MTB derived carotenoids, implying that the presence of extractives, while being inhibitors, acts as a trigger for carotenogenesis. In fact, the production of carotenoids on eMTB and eSOB hydrolysates is comparable to previous studies whose starting feedstock for R. toruloides do not typically contain significant amount of such extractives (e.g. wheat bran, tea, molasses) (Table 1). Indeed, some of the molecules identified in either SOB or MTB extractives (Table S1) are associated with antimicrobial activity, like in the case of lupeol 65, β-amyrin 66 and benzaldehyde 67 possibly causing a stressful environment leading to carotenogenesis. These observations provide novelty in terms of understanding that extractive components of lignocellulosic biomasses can act as promoting elements for the production of the desired molecule, especially when it is triggered by stressful conditions (like carotenogenesis). These findings are in line with previous literature, which suggest the role of different types of stress in the production of carotenoids by R. toruloides.8 Furthermore, in terms of biorefinery development, detoxification of the initial biomass is not needed in this case, making the process simpler.
These data also suggest that growth and production of carotenoids are not strictly correlated, depending on the environmental conditions. We can thus correlate microbial nutritional needs and biomass composition to the production of the desired compounds, which is definitely a relevant issue in the field of bioprocesses and biorefineries. Further investigations, beyond the scope of this work, might involve genomic and transcriptomic analysis of carotenogenesis related genes in R. toruloides, to better understand the effect of extractives (as a cohort or as single compounds) on cellular behaviour and regulation.