Optimization of the enzyme loading for Camelina meal hydrolysis
Glycosidic enzymes are cost items when assessing the economics of a bioprocess, therefore reducing the loading of enzymatic cocktails can be crucial to reduce the overall costs. Nevertheless, the yield of the sugars released must be considered to evaluate the performances of the enzymatic hydrolysis itself and the subsequent cellular production of interest. In a previous work we hydrolyzed 15% (w/v) of Camelina meal with 11.9% w/wCamelina meal of enzymatic cocktail for carotenoid production by R. toruloides [11]. In this work, enzyme loadings were gradually decreased from 11.9–0.56% w/wCamelina meal, pairing with the lower dose the specific usage indications by the manufacturer.
As shown in Fig. 1 (first set of bars, from the left), a significant reduction in sugar release could be measured when comparing 11.90% and 8.93% w/wCamelina meal to the lower enzymatic loadings. This can be related to the inhibitory effect of the biomass on enzymatic hydrolysis, both in terms of compounds released by the pre-treatment and the sequestration by LCBs on the enzymes themselves. Nevertheless, starting from 5.95% down to 0.56% w/wCamelina meal of enzyme loading, no significant difference could be observed, as also the lowest dose resulted in the release of about 15 g/L of total sugar.
Starting from the same samples, we considered only the sugars preferentially metabolized by R. toruloides (Figure S1, S2) [16, 27] as carbon and energy source (i.e. sucrose, glucose and fructose; Fig. 1, central set of bars). Despite we can see a similar trend, it is clear that higher amounts of enzymes mainly promoted the release of sugars such as arabinose and galacturonic acid, as starting from 5.95% of enzyme loading the sum of glucose fructose and sucrose does not significantly differ from the quantity released from the use of higher amounts of enzymes. Arabinose and galacturonic acid are not promptly consumed by R. toruloides when the other sugars are present (Figure S1) [11, 28]. Furthermore, when only glucose is considered, that is the first sugar to be consumed by R. toruloides and imposes catabolite repression on the other sugars (Figure S1, S2), its total release from the initial biomass is similar with all the different loadings (Fig. 1, right set of bars). Therefore, a reduction in the enzymatic loading by 95% (from 11.90–0.56% w/wCamelina meal) provided a comparable amount of glucose (and fructose plus sucrose too) in the Camelina meal hydrolysate, thus suggesting a way for reducing the cost of the process, which should be further tested and implemented in terms of carotenoid production.
Effect of Camelina meal solid loadings on the production of carotenoids by R. toruloides
We previously demonstrated that R. toruloides was able to grow and accumulate carotenoids when provided with 15% w/v Camelina meal hydrolyzed with 11.9% w/wbiomass of enzymatic cocktail [11]. Maintaining this enzyme loading, we here explored to increase the amount of solid loading, to understand the impact on sugar release, and how this would affect R. toruloides in a separated hydrolysis and fermentation (SHF) process.
We firstly increased the amount of Camelina meal up to 20% w/v, obtaining 35.1 ± 0.07 g/L of total sugars by hydrolysis, therefore maintaining a yield (17.5 ± 0.04%) very similar to the one obtained when starting from 15% w/v of biomass (16.4 ± 1.69% respectively).
Therefore, increasing the solid loading does not impair the enzymatic hydrolysis. Nonetheless, as shown in Fig. 2A, the growth of R. toruloides was reduced when provided with 20% w/v Camelina hydrolysate compared to 15% w/v in terms of OD. Arguably, this can be related to the presence of higher titers of growth inhibitors in the medium. In respect to the production of carotenoids, with 20% w/v Camelina meal it resulted significantly lower compared to the use of 15% w/v of initial biomass (Fig. 2B), suggesting that providing higher quantities of initial biomass is not beneficial for the obtainment of the product of interest.
Carotenoids are produced by cells as scavenger molecules in response to environmental and cellular stresses, such as the stationary phase. Therefore, we decided to move toward the opposite direction, namely lowering the amount of biomass, and, hence, of sugars initially provided to cells. In this context cells could experience precociously a starving situation, therefore accumulating carotenoids early and possibly in higher amounts. The tested solid loadings were 3%, 6%, 9% and 12% w/v, with a loading of the enzymatic cocktail NS22119 calculated in order to release increasingly amount of sugars [11]. The derived growth media were able to support the growth of R. toruloides, with a consistent anticipated entrance in stationary phase (Fig. 2A). Interestingly, the use of 6% w/v of substrate sustained a higher growth in terms of OD compared to 9% and 12% w/v, probably due to lower amounts of inhibitors in the media. Figure 2B shows the production of carotenoids over time, for each of the biomass amounts tested in SHF process. Carotenoids accumulation is quite low after 24 h, increasing with the respective entrance into stationary phases. In particular, the most interesting conditions are observed with 9% and 12% w/v, where carotenoids production obtained after 48 h of fermentation are comparable to the production obtained with 15% w/v, but only after 72 h of growth. Consequently, the best productivities were reached using 9% and 12% of Camelina meal hydrolysate (p < 0.05) (Fig. 2C). Figure 2D shows that the carotenoids yield on 9% w/v after 48 h resulted significantly higher compared to the yield on 12% w/v (p < 0.05), but not if compared with the yield with the 6% w/v, although the difference is at the limit of significativity (probably due to the uneven nature of the hydrolysates). Moving to specific productivity, after 48 h of growth this was significantly higher with 9% w/v compared to 6% w/v Camelina meal (2.1 ± 0.39 x 10− 5 h− 1 and 0.5 ± 0.11 x 10− 5 h− 1, p < 0.02), therefore supporting the inferior performance of the latter combination.
Therefore, considering Camelina meal solid loading as the variable parameter of the process, optimized conditions for carotenoids synthesis were the use of 9% w/v of biomass and 48 h of fermentation, with the following performances: production = 6.1 ± 0.85 mg/L, productivity = 0.13 ± 0.017 mg/L/h, specific productivity = 2.1 ± 0.39 x 10− 5 h− 1, yield on CWD = 0.1 ± 0.02%, yield on consumed sugars = 0.1 ± 0.01 %, yield on total sugars provided = 0.02 ± 0.003%. This result highlights the importance to test different conditions when developing bioprocesses, changing single parameters and assessing their effect on the final product.
Combinatory effect of optimized enzymes and biomass titers on the production of carotenoids by R. toruloides
After testing the possibility to reduce the enzymatic loading and the solid loading of Camelina meal, we then combined the two strategies in a single process. Therefore, we run a SHF process in which R. toruloides was provided with 9% w/v of Camelina meal hydrolysed by NS22119 enzymatic cocktail 0.56% w/wCamelina meal as growth medium. The results in terms of OD, sugar consumption and carotenoids production are shown in Fig. 3: samples were collected until 48 h in the light of data shown in the previous section. R. toruloides did not consume all the sugars provided, arguably due to the depletion of fundamental micronutrients in the medium [11]. Furthermore, the reduction of enzymes and the consequent reduction in sugar titer contributed to anticipate the maximum carotenoid accumulation (2.2 ± 0.33 mg/L) to 24 h. Although this value was inferior compared to that obtain from the use of 11.90% w/wCamelina meal at 48 h of fermentation, no significant difference could be observed in terms of productivity and specific productivity after 24 h when 0.56% w/wCamelina meal were used (0.1 ± 0.01 mg/L/h and 2 ± 0.3 x 10− 5 h− 1, respectively). Despite a techno-economic analysis (including downstream processing and possible related issues) was not performed yet, it is reasonable to conclude that reduction in both process time and enzymatic loading would in turn reduce the overall cost of the process.
Therefore, optimized conditions for the synthesis of carotenoids by R. toruloides were the use of 9% (w/v) Camelina meal hydrolysed with 0.56% w/wCamelina meal of NS22119 as medium for 24 h of growth in a SHF process. Furthermore, in these conditions the specific growth rate (µ) of R. toruloides was calculated to be 0.25 h− 1, with a duplication time of 2.74 h (Figure S3). Entrance in exponential phase was evaluated to be reached after about 5 h from start, whereas the entrance in stationary phase after 21 h, coherently with what disclosed in Fig. 3.
We further explored the use of such conditions in SSF and SSF + presaccharification processes, to evaluate the effect of the presence of water insoluble components (WIS) as potential stressing agent triggering carotenoids production. Figure 4 shows that R. toruloides was able to consume sugars and to produce carotenoids in both settings, reaching the maximum after 48 h of fermentation time (4.6 ± 0.21 mg/L for SSF + presaccharification, 4.9 ± 0.39 mg/L for SSF). The delayed production compared to SHF may be related to the harsher conditions that cells had to face in the presence of WIS [29]. Overall, WIS do not significantly interfere with the production of carotenoids, and this fermentation conditions lead to a Camelina meal enriched in carotenoids, which may be directly used as enriched feed supplement.
Carotenoids Production In Batch Bioreactors
To test the reliability of the protocols in larger volume and to acquire data for quantitative analysis, we moved the process to stirred tank bioreactor. As starting point, the volume of the enzymatic hydrolysis was increased to 1 L: due to the uneven nature of lignocellulosic biomasses and their inhibitory effect towards enzymatic activity, scaling up this step may lead to a decrease in hydrolysis yield. Remarkably, after 6 h of hydrolysis of 9% w/v of Camelina meal by NS22119 0.56% w/wCamelina meal the amount of released sugars (7.2 ± 0.84 g/L, see T0 Fig. 3) was comparable with that obtained from lower volumes of hydrolysis, demonstrating the scalability of the first step of the process.
The obtained medium was used to test the production of carotenoids in batch bioreactors, where R. toruloides cells were inoculated at initial OD of 0.4, pH 5.6 ± 0.09 and oxygenation of 25%. Cells reached the stationary phase already after T16, as shown by the profile of the optical density and the cellular dry weight (Fig. 5A). Bioreactor cultivation permitted also to monitor pH and dissolved oxygen (Fig. 5A): the first increased over time to reach the value of 6.7 ± 0.03 at T48, whereas the second increased after T16 (up to 80%), witnessing a strong decrease in cellular growth, as from the reduction of oxygen consumption. The increase in pH could be related to the accumulation of ammonia in the medium, initially consumed by cells (Fig. 5B), but then produced probably as a consequence of amino acid catabolism triggered in response to starvation. In fact, as shown in Fig. 5B, cells already consumed sugars and nitrogen (in the form of primary amines) at T16, although the incomplete consumption of both (that remained till the end of the fermentation) may be related to the depletion in the medium of micronutrients pivotal to cellular sustainment.
The exhaustion of nutrients, the increase in pH and pO2 and the growth curve profiles (in terms of OD and CDW) suggest an early entrance in stationary phase, which is of interest as aforementioned for the production of secondary metabolites such as carotenoids.
Consistently, regarding the production of carotenoids, from T16 on there was no statistically significant increase in their accumulation, reaching 3.6 ± 0.69 mg/L after T24 (Fig. 5B), with a productivity (0.13 ± 0.03 mg/L/h) comparable with the one obtained in the same conditions in shake flasks (Fig. 3). Therefore, shake flask tests showed to be predictable on the behavior of R. toruloides in bioreactor.
The data here disclosed are the first reports of bioreactor scale fermentation of Camelina meal hydrolysate, and therefore they can pave the way for further optimization. To maximize the production of interest several modifications influencing lipid and carotenoid production in yeasts, like C/N ratio, initial CDW, pH and oxygenation [30–33], can be operated.