Evaluation of total sugar content in Camelina meal and optimization of the enzymatic hydrolysis
The content of water, insoluble components, acetic acid and sugars was quantified in Camelina meal. As shown in Table 1, total acid hydrolysis (see Materials and Methods) revealed that almost one third (31%) of Camelina meal is composed by sugars: by HPLC analysis it was determined that the main fraction of carbohydrates is represented by hexoses, namely glucose and fructose, accounting for more than 2/3 of the total. Despite acid hydrolysis can be used for saccharification, it poses limitations related to the conditions of the reaction itself: the low final pH, which needs to be neutralized prior to provide sugars to the cells, and the release of inhibitory compounds such as furfurals [2,25]. Taken together, these issues can limit the sustainability of the overall biorefinery process, for example considering the use and disposal of acid solutions . Therefore, to release monomeric sugars from their polymeric form an enzymatic instead of an acid hydrolysis was performed, testing different conditions (see below) and comparing the fraction of the released sugars with the value of 31%, considered as 100%. The other main components of Camelina meal, as reviewed by , are crude proteins, which account for 35.2-46.9% of the total biomass, and crude fats, spanning from 4.9 to11.9%. In addition, micronutrients such as vitamins are present in Camelina meal . Overall, Camelina meal is a potential good substrate for the growth of R. toruloides and the consequent production of carotenoids, never reported in literature before.
The optimization of the enzymatic hydrolysis, whose mild operative parameters are generally more compatible to the subsequent growth of microbial cell factories and that can take advantage from the broad range of commercial available enzymatic cocktails [26,27], was performed with the cocktail NS22119 (Novozymes A/S), to release both hexose and pentose sugars. Initial different concentrations of Camelina meal were tested in order to determine the effect of solids loading on sugar release. After autoclaving, the measured pH was 5.5, which is compatible with the enzymatic catalysis, since NS22119 would display up to 90% of its maximum activity, according to the indication of the producers. Remarkably, the pH remained constant during time until the end of the hydrolysis. Both the economic and environmental impact of the procedure can be therefore reduced, since neutralization or addition buffer are both avoidable. As shown in Figure 1A, pre-treatment of the biomass by autoclaving resulted in an initial concentration of released sugars spanning from 1.8 ± 0.03 g/L to 9.27 ± 0.32 g/L, where the increase in sugar concentration is essentially due to the concentration of the initial loaded biomass. After enzymes treatment (11.9% w/wCamelina meal), the concentration of free sugars doubled (at least), compared to the initial amount, independently from the quantity of the loaded biomass (Figure 1A). No additional release of sugar was detectable over time from negative control samples, were 3% or 15% of the initial biomass were incubated in shaking water bath at 50°C, but no enzymes were added (Additional File 1, Figure S1). Sugar titer increased during time until 24 hours, with a linear proportionality (R2=0.98, p < 0,001, calculated with R) in respect to the initial quantity of biomass; therefore, the yield of sugars released by enzymatic hydrolysis was constant regardless of the concentration of the provided Camelina meal (Figure 1B). The maximum yield of sugars over total biomass was 20% after 24 hours from the start; considering the original amount of carbohydrates, a sugars recovery of 65% was calculated, which is in accordance with common reported values for lignocellulose enzymatic hydrolysis [28,29].
With this setting, about 25 g/L of monomeric sugars were released, with a recovery of 53.3%. Notwithstanding this, the fraction of residual carbohydrates not hydrolyzed could be considered as an added value to the final product: in fact, a feed with Camelina meal enriched in carotenoids by fermentation of R. toruloides would still contain fibers of nutritional value.
At the best of our knowledge, these are the first data reporting an enzymatic hydrolysis protocol for Camelina meal to make sugars accessible for a subsequent microbial biotransformation.
Inhibitory compounds in Camelina meal hydrolysate
Biomass hydrolysis is efficacious in releasing sugars from lignocellulose, minimizing the accumulation of high content of inhibitory compounds. Nevertheless, there are some drawbacks related to other compounds detached from these complex matrixes (Jönsson and Martín, 2016; Sitepu et al., 2014). Acetic acid is the most common inhibitor released by hydrolysis of the hemicellulose fraction composing lignocellulosic biomasses: it can easily impair microbial growth and metabolism due to its generic and specific toxicity , therefore reducing the Key Performance Indicators (KPI) of the production process [2,31,32]. Nevertheless, it is important to underline that the toxicity of acetic acid is sharpened at low pH: extracellular pH values higher than its pKa (4.76) reduce its diffusion across the membrane and, therefore, the cellular damages that it can trigger [33,34]. As aforementioned, the operative pH (5.5) is higher than the pKa of acetic acid, lowering the detrimental effect of this molecule towards cells. Moreover, it has been previously described that R. toruloides can withstand acetic acid as additive to defined media or even as sole carbon source up to 20 g/L at pH 6 [35–37]. Figure 3 shows that acetic acid titer increases during time, due to the action of the enzymatic cocktail, reaching 1.80 ± 0.01 g/L after 24 hours from start. This amount has been described as bearable by diverse yeasts [32,35,36], including R. toruloides.
Given these data, the hydrolysate of Camelina meal has all the trumps to be a suitable feedstock for establishing yeast cell factory-based biorefineries, exploiting yeasts biodiversity and possible engineering strategies for obtaining different products of interest.
Carotenoids production from Camelina meal hydrolysate in SHF and SSF processes
Having established a protocol for obtaining Camelina meal hydrolysate, the carotenoids production was investigated by mean of the Separate Hydrolysis and Fermentation (SHF). The first experiment considered as medium the sole clarified supernatant, collected after 6 hours of enzymatic hydrolysis: it resulted to sustain the growth of R. toruloides, as indicated by the accumulation of biomass and sugars consumption (Figure 4A, dotted and dashed lines, respectively). The accumulation of carotenoids increased over time, reaching 5.51 ± 0.67 mg/L after 96 hours of fermentation (Figure 4A, white bars). These data are in accordance to the previous observation that R. toruloides, and carotenogenic microorganisms in general, produces carotenoids mainly in response to stressful or sub-optimal conditions, such as the stationary phase of growth [17,18,38,39]. Time lapse of the fermentation was first chosen as 96 hours mainly to compare this work with similar ones from the literature, when R. toruloides was provided with defined media or other/different residual biomasses (Table 2). Indeed, after 96 hours we observed a reduction of extracted carotenoids from the cells (Additional File 1, Figure S2): this could correlate to the export/release of the molecules from the cells o to an imbalance of nutrients that may promote their consumption/corruption. The production achieved in the here described SHF process are competitive compared to shake flasks carotenoid productions that have been reported for R. toruloides provided with other complex matrixes (Table 2).
In order to overcome the need to clarify the media after enzymatic hydrolysis, we provided to the cells the entire Camelina meal hydrolysate, comprising also the water-insoluble solid (WIS) fraction remained after the enzymatic hydrolysis. WIS may be troublesome for microorganisms, both considering the uneven homogenization of the liquid media, due to the presence of solid components, and the toxicity of some of the molecules there comprised . These two aspects combined might impair microbial growth and production. In the conditions that here we named “SHF with WIS”, R. toruloides was not only able to consume sugars and to produce carotenoids (Figure 4B), but remarkably it was observed a higher titer of intracellular carotenoids, reaching 12.64 ± 2.57 mg/L after 96 hours from the inoculum. It is important to underline that the carotenoids measured in this as in the following experiments in the presence of WIS are due to microbial metabolism: indeed, the measured amount of carotenoids extracted from Camelina meal with and without the addition of enzymes, at the beginning and at the end of the reaction, remained constant over time (Additional File 1, Figure S3).
Often proposed as alternative to SHF, the Simultaneous Saccharification and Fermentation (SSF) displays a single and co-current step of hydrolysis and microbial fermentation. The two processes have several pros and cons compared to each other, considering parameters such as efficiency, time, presence/release of inhibitory molecules and downstream of the final product [40,41]. SHF and SSF have been proposed and compared for several 2nd generation biorefineries, based for an example on Arundo donax, grass or wheat straw [28,42,43]. A potential drawback of incubating enzymes and cells in the same environment is the compromise to be reached between the optimum conditions for them both. In the present study, since 50°C was not a viable temperature for R. toruloides, 30°C was selected as operative temperature, while an increased shaking was imposed with the aim to partially compensate for the reduced activity by augmenting the probability of interactions between the matrix and the enzymes. Remarkably, the release of sugars in these conditions was comparable with the previous setting (Additional File 1, Figure S4). Figure 4C shows that after the first 6 hours of hydrolysis the amount of sugars in the media was lower compared to the amount of the SHF, very likely due to the initial growth (and therefore sugar consumption) of R. toruloides. After 24 hours from the start, there is a clear consumption of sugars, which in parallel led to an accumulation of carotenoids. After 96 hours, the concentration reached is 15.97 ± 1.93 mg/L. Therefore, SSF process seems to be even more effective than “SHF with WIS”. Significantly, the higher amount of carotenoids was registered when WIS was left into the medium. Diverse environmental variables can influence carotenoids production, and sub-lethal concentrations of insoluble solids might from the one hand still impair microbial growth but on the other hand can trigger the accumulation of metabolites that natural producers, such as microalgae and yeasts, synthetize for their own defense [39,44]. A more specific example was reported, where β-phenol can trigger carotenoid production in yeast .
The titers reached from SSF process also indicate an efficacious concurrent hydrolysis and fermentation, suggesting that a simplified procedure involving a single vessel can be used. The final productivity remains similar, suggesting that the initial sugar release in the presence of cell do not speed the overall process. Overall, data from SSF and SHF+WIS reveal that with this residual biomass, the often mandatory detoxification step, also indicated for R. toruloides , is avoidable. Moreover, the final product obtained by both SHF with WIS and SSF is a Camelina meal enriched with carotenoids, which has the potential for being directly used in the feed industry.
Therefore, as outcomes to the proposed processes different products can be recovered, such as pure carotenoids and carotenoids-enriched Camelina meal. In particular, the latter would be an innovative product in the market, since carotenoids are commonly added to animal feed for nutritional and organoleptic reasons [3,12]. In addition, the production of carotenoids from a residual biomass of lower value may increase the economic attractiveness of the proposed process. Therefore, the present work, according to the logic of cascading [47,48], paves the way for an alternative use of Camelina meal as feedstock in second generation biorefineries exploiting microbial cell factories to produce fine chemicals.