Optimization of pumpkin peel waste pretreatments and quantification of components

Pumpkin cultivar can influence physical properties of the waste such as density, rheological aspects and can also greatly affect chemical composition [26]. Components of different pumpkin cultivar include organic acids and soluble sugars, mainly fructose, glucose, and sucrose, giving typical tasty traits [27], but presence of starch is also reported [28, 29]. Pumpkin peel waste utilized in our study was composed by a tiny peel, probably containing cellulose/hemicellulose, but some residual pulp was present. For these reasons, enzymatic hydrolysis was considered as pretreatment to increase the amount of sugars available for fermentation. Commercial preparation containing a blend of cellulases, ß-glucosidases, and hemicellulases (Cellic CTec2) was tested to optimize the amount of enzyme and time of hydrolysis suited for pumpkin peels treatment. Different amounts of enzyme blend were added to the feedstock (details reported in “Methods” section). We observed that during the enzymatic treatment, the biomass became more broken up, and a minimum of 24 h was required to obtain an almost liquefied mixture. The reactions of hydrolysis were monitored by quantifying the released glucose (Table 1). The addition of 2.25 μL/mL of enzyme blend allowed the release of 23.6 g/L of glucose in 24 h (Table 1), and higher concentration of enzyme did not release more. Since glucose concentration did not increase after further 24 h (48 h from the start), this time was considered optimal for hydrolysis. On the other hand, sucrose and fructose concentrations remained constant over the time at all the tested enzyme concentrations (sucrose 29 g/L, fructose 5 g/L,). Therefore, these conditions (enzyme blend 2.25 μL/mL, 24 h) were selected to obtain pumpkin peel waste hydrolysate suitable for fermentative processes. Due to the known presence of starch among the pumpkin components, we tested also if addition of commercial amylase (A8220 from Aspergillus oryzae), together with the cellulose blend, could increase the amount of glucose in the hydrolysate, but we did not obtain this result. This was due to the presence of amylase activity in the commercial cellulolytic blend (Cellic CTec2), as we tested using starch as substrate (data not shown). The enzymatic hydrolysate obtained after 24 h was characterized for its composition (Table 2). Analysis by HPLC showed that sucrose was the principal sugar found in the soluble fraction (fraction 1), (Table 2, Additional file 1: Table S1), together with glucose and fructose. Sucrose is mostly present in the pulp, thus it can be found at different levels in the wastes, depending on the type of peeling operation adopted in industrial processing. Oligosaccharides at different molecular weights (DP4 and DP3) were also found in fraction 1 (Additional file 1: Table S1). The presence of unbound galacturonic (Table 2, Additional file 1: Table S1), suggested that pectines are also among the components of pumpkin peel waste. Glucuronic acid was also found. The presence of only 25 mg/g of glucose in fraction 2 (Additional file 1: Table S1), indicated that most of the starch had been hydrolyzed by Cellic CTec2 treatment. The absence of sugars in fraction 4 (Additional file 1: Table S1) confirmed that cellulose/hemicellulose in the pumpkin peel waste had been completely hydrolyzed by Cellic CTec2 treatment. Quantification of acetic acid (Table 2) was also done, because it is known to be an inhibitor of cell growth.

Table 1 Enzymatic pre-treatments performed on pumpkin peel waste (small scale)
Table 2 Concentration of the main components of pumpkin peel hydrolysate (after 24 h of enzymatic digestion, large scale)

Nitrogen (N) is an essential element for biomass production; nevertheless, lipid production is triggered by high C/N ratio [10]. When the C/N ratio is low the cells invest nitrogen and carbon to produce biomass, whereas when the ratio is high the carbon is mainly directed toward lipid production. In the pumpkin peel hydrolysate inorganic nitrogen (NH4+) was found only in traces, but quantification of total nitrogen accounted for 2.4 g/L, then revealing that it contains mainly organic N (Table 2).

Analysis of growth and sugar utilization on mineral defined media

The availability of a pumpkin peel hydrolysate containing sucrose, glucose and fructose as principal sugars lead us to evaluate first the capacity of R. azoricus and C. oleaginosum to grow in their presence. Cultures were performed on mineral defined medium (YNB), to avoid the utilization of other carbon sources that could mask the ability to consume provided sugars. In this way, we could also obtain quantitative data, in terms of sugar consumption rate and biomass yield. These parameters are known as key factors for selecting suitable species and for developing efficient fermentation processes [30].

The cultivation of R. azoricus on YNB containing a mixture of sucrose, glucose and fructose, at concentration similar to pumpkin hydrolysate, showed that this yeast starts to hydrolyze sucrose even in presence of other available sugars (Fig. 1A). The increase of fructose concentration after 24 h suggested extracellular localization of invertase activity necessary for sucrose hydrolysis, similarly to what reported in C. curvatus [31]. However, a limited capacity to utilize the derived glucose and fructose was observed. After 72 h both sugars were partially assimilated (Fig. 1A), and yeast biomass reached a concentration of 4 g/L of dry weight, with a low yield (Table 3). We did not found production of other compounds coming from sugar metabolism, such as ethanol, lactic acid or acetic acid. Cellular viability was then tested, and we found that 30% of cells were not viable after 72 h. In the light of these results, we analyzed the growth in presence of single sugar as sole carbon source. When the cells were cultivated on glucose (Fig. 1B), R. azoricus started to grow exponentially, but we observed an early arrest in consumption of this sugar; after 72 h only 20 g/L were consumed, producing again a low concentration of yeast biomass (4 g/L) with a low yield (Table 3). Cellular viability was tested and, surprisingly, also in this case we found that 40% of cells were not viable after 72 h. A partial utilization of glucose was observed also by decreasing the initial glucose concentration from 50 to 25 g/L (data not shown). By contrast, the cultivation on sole fructose revealed that this sugar did not cause any early arrest of growth (Fig. 1C). By comparing growth parameters on glucose and on fructose containing media (Table 3), it was evident that, despite glucose specific consumption rate was higher than fructose, the growth rates were rather similar (Table 3). The main differences were found in terms of final amount of produced biomass (4 g/L on glucose and 9 g/L on fructose) and on biomass yield (0.2 and 0.3, respectively), indicating that fructose was metabolized in a more efficient way than glucose. However, cultivation on YNB-sucrose (Fig. 1D) showed that the cells were able to hydrolyze sucrose, but the released monomers glucose and fructose were partially metabolized, leading to production of low concentration of biomass (Table 3). These results suggest that the presence of glucose in the medium, even when released by sucrose hydrolysis, can cause a negative effect on sugar utilization and early arrest of growth with low production of biomass. This limited ability by R. azoricus to metabolize glucose observed on mineral defined media (YNB) was unexpected, because it had been previously documented that R. azoricus grows efficiently on glucose-based media used for lipid production [17, 32, 33]. However, those media contained yeast extract or corn steep, and were not exclusively mineral like the one used in the present study. On the other hand, the commercial YNB contains all the compounds necessary for the growth, in the form of salts, vitamins and trace elements. This prompted us to investigate if addition of yeast extract can improve sugar metabolism. By supplementing yeast extract to YNB-glucose cultures (Fig. 2A) as well as to YNB-sucrose cultures (not shown), a positive effect on growth and on sugar utilization was observed. The same positive effect was exerted also in cultures with sugar mixture (Fig. 2B): after 72 h, sucrose was completely hydrolyzed, and glucose and fructose depleted, leading to production of 14 g/L of biomass. These results demonstrate that enrichment of the mineral defined medium by other nutrients, contained in yeast extract but not in YNB, is very important for sugar metabolism in this yeast species. This beneficial effect was, therefore, at the basis of the efficient growth on glucose containing media previously utilized for lipid production processes [17, 32, 33]. Recently it has been reported such a positive role of supplementing amino acids on metabolism of all the carbon sources present in sugar beet pulp hydrolysates by Rhodotorula strains [34].

Fig. 1
figure 1

Cultivation of R. azoricus on YNB medium containing: A mixture of 30 g/L sucrose, 15 g/L glucose and 5 g/L fructose; B 50 g/L of glucose; C 50 g/L of fructose; D 50 g/L of sucrose

Table 3 Growth parameters of R. azoricus and C. oleaginosum cultivated in shaken flasks on YNB media containing different carbon sources
Fig. 2
figure 2

Cultivation of R. azoricus on YNB medium supplemented with 1 g/L of yeast extract: A YNB-glucose medium; B YNB-mixture medium, 30 g/L of sucrose, 15 g/L of glucose, 5 g/L of fructose

When C. oleaginosum was cultivated on YNB containing mixture of sugars (glucose, fructose and sucrose) we observed a different behavior. The cells initially grew using already available monosaccharides, glucose and fructose (Fig. 3A). After their depletion, sucrose was hydrolyzed, indicating that the presence of free sugar monomers delayed sucrose hydrolysis. After 120 h, sucrose (10 g/L) was still present in the medium. However, in this culture a higher amount of biomass was obtained (12 g/L) in comparison to R. azoricus culture, and with a higher yield (Table 3), due to the complete utilization of monosaccharides. On the other hand, cultivation on YNB containing glucose as sole carbon source (Fig. 3B) showed an increase of biomass production consequent to glucose utilization, indicating that in this yeast any early arrest of growth caused by this sugar occurs, in contrast to what observed in R. azoricus.

Fig. 3
figure 3

Cultivation of C. oleaginosum on YNB medium containing: A mixture of 30 g/L sucrose, 15 g/L glucose and 5 g/L fructose; B 50 g/L of glucose

By comparing the results obtained in terms of final biomass concentration and yield (Table 3), we can conclude that C. oleaginosum is able to use glucose on mineral defined media in a more efficient way than R. azoricus. However, it is noteworthy that the metabolic problem relative to glucose utilization by R. azoricus can be eliminated by the addition of yeast extract or peptone. These results reinforce the necessity of studies that carefully analyze growth parameters to identify the basis of metabolic diversity among oleaginous yeasts, as recently reported [35, 36]. This kind of information is often lacking, because mineral defined media are not suitable for industrial applications. The limited ability to metabolize glucose by R. azoricus and its early cellular death could lead to think to a phenomenon of glucose toxicity. In mammalian cells under hyperglycemic conditions, activation of the aldose reductase pathway causes redox unbalance and induction of oxidative stress, upregulating glucose toxicity pathways, as non-enzymatic glycation and disruption of mitochondrial respiratory chain [37]. Aldose reductases are present in yeasts and have been studied mainly for the production of sugar alcohols [38, 39], but their role in regulation of sugar metabolism has been scarcely investigated.

Biomass and lipids production from pumpkin peel waste utilization

With the aim of developing fermentative processes for the production of biomass and lipids from pumpkin peel wastes, shaken flask cultures of R. azoricus and C. oleaginosum were set up. For this purpose, we tested the possibility to utilize for yeast cultivation a medium exclusively derived from pumpkin peel hydrolysate, without any additional nutrient. This medium contains sugars and nitrogen source, and, in addition, also acetic acid (Table 2, Additional file 1: Table S1). R. azoricus and C. oleaginosus are reported not able to grow on glucuronic acid [40, 41], and R. azoricus can metabolize acetic acid [32].

After 42 h of cultivation, we observed that R. azoricus consumed all the main sugars (sucrose, glucose and fructose, Fig. 4A) as well as acetic acid, as expected. However, we observed that also galacturonic acid was used, analogously to what reported by Rhodotorula toruloides [34]. On the other hand, oligosaccharides (DP6–DP3) were partially consumed (5 g/L consumed). We can conclude that the presence of amino acids in pumpkin peel hydrolysate played a positive role for sugar metabolism, analogously to what observed by addition of yeast extract (YE) to YNB media (see paragraph above). As a consequence, and due to the higher carbon and nitrogen content of this medium in comparison to the YNB-based one, yeast biomass reached a dry weight of 28.1 g/L after 64 h. This final biomass contained 39% of lipids, corresponding to a concentration of 11 g/L.

Fig. 4
figure 4

Cultivation of R. azoricus and C. oleaginosum in shaken flasks on pumpkin peel hydrolysate-based medium

When C. oleaginosum was cultivated on the same medium, sucrose was only partially hydrolyzed after 65 h (Fig. 4B), but glucose and fructose were exhausted. Acetic acid was metabolized also. These results confirmed that in C. oleaginosum, as observed in mineral defined medium (see paragraph above), sucrose utilization is delayed by the presence of monosaccharides, and this negative effect is not relieved by the presence of amino acids, in contrast to what observed in R. azoricus. As a consequence, lower levels of biomass (25 g/L) and lipids were obtained; lipids represented 29% of yeast dry weight, that corresponded to a concentration of 7 g/L.

In conclusion, these results indicated that pumpkin peel hydrolysate represents a complete source of nutrients for cultivation and lipid production by R. azoricus and C. oleaginosum. In particular, R. azoricus shows a natural ability to co-metabolize the contained carbon sources, that is an important trait for complex wastes utilization.

Two-stage process in bioreactor for increased lipid production

Based on the observation that R. azoricus exhibited the best performance on pumpkin peel hydrolysate-based medium, we developed a fermentative process to increase lipid production. Usually, the process for lipid production is performed in two stages, the first carried out on media at low C/N ratio to produce high amount of cell biomass, and the second at high C/N ratio to trigger lipid accumulation [10].

The cultivation in bioreactor using pumpkin peel waste hydrolysate as medium (Table 2), under controlled conditions of oxygen and pH, resulted in the production of 30 g/L of biomass, that contained 37% of lipids, after 46 h of cultivation (Fig. 5). At this point, sucrose and glucose were exhausted, whereas some fructose was still present. As observed in flask cultures, acetic acid as well as galacturonic acid were also consumed (data not shown).

Fig. 5
figure 5

Two-stage process of R. azoricus on pumpkin peel hydrolysate-based medium (first stage) and by feeding with candied fruits syrup (second stage)

With the aim to increase lipid content of the biomass, another industrial food waste coming from the manufacture of candied fruits (a syrup from mango processing) was used to feed the culture. This waste contains high concentration of available glucose and fructose (199 and 296 g/L, respectively), without the need of any pretreatment. In addition, it does not contain nitrogen, making it a perfect source of sugars to increase C/N ratio of the medium and trigger lipid accumulation. In the following 44 h of process, all glucose and part of fructose were metabolized (Fig. 5). Sugars were converted into lipids, allowing the yeast biomass to reach a dry weight of 45 g/L with a lipid content of 55%. A lipid concentration of 24 g/L was obtained after 90 h of process (Fig. 5), that corresponded to a lipid productivity of 0.26 g/L/h. Based on the consumed carbon sources the lipid yield was 0.24.

In conclusion, these results represent a very promising starting point for developing a lipid production process. By the exclusive use of food wastes (pumpkin peels and candied fruits syrup), R. azoricus was able to efficiently grow and produce lipids with high productivity and yield. Similar results have been reported using other oleaginous yeast cultivated on agro-food wastes. Orange peel extract supplemented with ammonium sulphate has been shown to allow production of 7.6 g/L of Cryptococcus laurentii biomass containing 59% of lipids and 6.9 g/L of Rhodosporidium toruloides containing 79% of lipids in batch cultures [24]. R. toruloides cultivated on Jerusalem artichoke extract has been reported to produce 40 g/L of biomass with 43% of lipids [42]. In [43] a process under pulsed-feeding cultivation on sugar beet pulp (SBP) and molasses by the oleaginous yeast Lipomyces starkeyi was reported. They obtained 20.5 g/L of final biomass with a lipid content of 49.2%. Other examples of lipid production from organic wastes are reported in the recent review by [38, 44].

Lipid analysis

Lipids synthesized by R. azoricus are known to be mainly composed of long-chain fatty acids with 16 and 18 carbon atoms, with a profile similar to vegetable fats [45]. At the end of the process on pumpkin peel hydrolysate-based medium, the percentages of saturated, monounsaturated and polyunsaturated fatty acids resulted 30.16%, 50.12% and 19.73%, respectively. The main fatty acids produced under this condition were: 49.53% oleic acid (C18:1), 20.65% palmitic acid (C16:0), 16.69% linoleic acid (C18:2), and a small percentage of stearic acid (C18:0, 7.15%), and linolenic (C18:3, 3.03%). In addition, we found a significant amount of omega-3 (3.03%) and omega-6 (16.69%). The complete analysis is reported in Additional file 1: Table S2. On the whole, lipid profile obtained by cultivation on pumpkin peel hydrolysate-based medium appears quite similar to those obtained on a lignocellulosic hydrolysate [32], except for a small increase in oleic acid content (Fig. 6). These results indicate that the composition of pumpkin peel hydrolysate did not significantly affect the fatty acid profile of R. azoricus.

Fig. 6
figure 6

Comparision between major fatty acids produced by R. azoricus on pumpkin peel hydrolysate-based medium (black) and on corn stover hydrolysate [32] (grey)

Conclusions

This proof-of-concept study has shown the feasibility to produce yeast biomass and lipids in an economical way, demonstrating a possible process of upgrading low value industrial food wastes to high value products. The use of two inexpensive residues, i.e. pumpkin peels and syrup from candied fruits processing, for sustaining microbial growth and oil production has been never reported before. These wastes represent a renewable and low-cost feedstock to fulfil nutrient demand by yeasts. In particular, R. azoricus valorized the pumpkin peel residues by reaching higher production of biomass and lipids, due to its better ability to hydrolyze sucrose and metabolize derived monomers contained in this waste.

Lipids can be extracted and used to produce biofuels and chemicals. Yeast biomass can in turn be used as a high-quality animal feed ingredient, because it is considered a well-balanced source of protein, and can provide also vitamins (mainly the B group) [46]. In conclusion, one of the main goals of circular economy, which is the reduction of wastes by recycle, could be addressed.

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