3.1 Proximate and biochemical analysis of BIB
The biochemical composition of BIB as shown in Table 1 indicates that the holocellulosic content i.e., cellulose content (35.56 % ± 5.26, w/w), hemicellulose content (22.41% ± 1.89, w/w) followed by the lignin content (8.78 % ± 1.17, w/w). The high percentage of total carbohydrates was 59.71% ± 0.90, w/w, which is considered to be a major component of BIB. The proximate composition analysis of BIB implies that the presence of biologically accessible components is exposed by the highest proportion of total volatile solid content that can be used for biofuel production. The content of cellulose, hemicellulose and lignin results were 14.4 ± 0.84, 20.6 ± 1.02, and 14.8 ± 1.15 (%, w/w) obtained in the research carried out by Fingolo et al. [10]. The variances in the composition analysis could be caused by ecological or seasonal variations, and they also rely on the variety of bananas. Whereas, the proximate analysis and total carbohydrate of BIB was showed similar results to study reported by Amutha et al. [9] followed the same methodology as our study. The ability to efficiently utilize the BIB substrate by the yeasts, which is high in fermentable sugars was confirmed by the presence of a high level of holocellulosic content (hemicellulose and cellulose) i.e., 57.97 %, w/w, can further employ to produce SCO and bioethanol. The BIB has a low lignin content, allows effective hydrolysis, increases the reducing sugar yield recovery, generates a limited number of lignin-derived compounds, and can consider a highly desirable feedstock for microbial bioconversions. Due to the low lignin content, phenolic derivative formation during hydrolysis and its inhibition of microbial proliferation are minimal. However, the presence of total solid in a significant, high content of holocellulosic components and less lignin content affirms the BIB biomass potential as a source for biofuel production. Furthermore, hemicellulose and cellulose structural components are readily available as raw materials for bio-based conversion and biorefinery processes due to the high volatile solid and moisture content of BIB.
3.2 Effect of microwave pretreatment on delignification through analytical studies
To investigate the morphological changes associated with delignification, scanning electron microscope (SEM) images of the untreated and treated BIB biomass (160 W for 7 min) were examined (Fig. 2). A smooth, firm, and comparatively clear surface was observed in the untreated BIB sample (Fig.2a). The pretreated BIB surface, in contrast, was distorted, heavily damaged, ruptured, and had a structure dissimilarity (Fig.2b). After MAMT, there was a noticeable change in the surface structure. Therefore, it was assumed that the reduction of surface lignin coverage plays a key role in the breakdown of lignin, producing lignin degradatory by-products that could act as an inhibitor [40]. The breakdown of polyaromatic lignin induced the surface to become distorted and deformed after MAMT, increasing the surface area of cellulose for improved enzymatic saccharification. Similar research was conducted and observed following MAMT [41]. In the current study, the BIB biomass crystallinity index (CrI) increased from 21.44 % (raw biomass) to 24.44 % after MAMT, which could be related to the elimination of lignin (amorphous nature) and the breakage of hydrogen bonds (local hydrolyzation) caused by microwave heating (Fig. 2c). According to the literature, lignocellulosic biomass with a high CrI has a high rate of enzymatic hydrolysis, and vice versa, whereas biomass with a low CrI has a slow rate of hydrolysis [42]. Similar changes in CrI value were examined when various feedstocks were exposed to acid pretreatment [43]. The observations made from XRD were found to be correlative to the FTIR analysis, where the deformation of the amorphous lignin and hemicellulose structure during mild acid pretreatment indicates that pretreated BIB biomass might change to a crystalline state as reflected by the increase of CrI value, thereby improving the accessibility of cellulase for the saccharification process.
These FTIR spectra obtained from pretreated and raw biomass indicated that the appearance and/or disappearance of adsorption bands representing different functional groups (Fig. 2d). The presence of the band with the highest intensity at 1371 cm-1 in the untreated BIB sample (which corresponds to C-H deformation and the aromatic ring vibrations of S type lignin) suggests that the BIB biomass is rich in G/S type lignin. CH stretching in side chains methyl and methylene groups, as well as in aromatic methoxyl groups, is attributed to the wavenumbers 2920 cm-1 and 2852 cm-1 [44]. The ester carbonyl vibration in ferloyl, acetyl, and p-coumaryl groups in hemicelluloses is responsible for the wavenumber 1729 cm-1 present in the pretreated BIB sample. A further drop in peak intensity after MAMT reveals the lignin depolymerization. In a study by Guo et al. [45], lignin was found to be abundant when there was the highest peak (phenolic hydroxyl groups) at about 1371 cm-1. According to Corredor et al. [46], polymeric lignin emits a peak at around 1320 cm-1 due to the phenyl and methyl propane groups, and the disappearance of this peak following pretreatment denotes the removal of lignin. Further evidence that cellulose was exposed after lignin was distorted based on the emergence of a peak of about 3338 cm-1 following pretreatment [42].
3.3 Detection of inhibitors through HPLC
During acid hydrolysis, derivatives of lignocellulosic biomass such as furan ((5-hydroxymethylfurfural (HMF) and furfural)) and lignin derivatives were generated. Acetic acid, which is produced by the deacetylation of hemicellulose, was the second most prevalent component in the hydrolysate. During fermentation, these compounds have growth-inhibitory effects on microorganisms, which decrease the efficiency of their bioconversion. In the current investigation, the liquid hydrolysate formed via MAMT was subjected to a detoxification process in order to eliminate or reduce the inhibitory effects of the toxic compounds generated from lignocellulosic biomass. The concentration of polyphenols in liquid hydrolysate before and after detoxification were estimated. The total polyphenolic compounds in non-detoxified and detoxified BIB hydrolysate were found to be 0.64 ± 0.03 mg/ml and 0.38 ± 0.04 mg/ml respectively. The two-fold decrease of phenolic compounds confirmed the effect of detoxification in BIB liquid hydrolysate. Further, the existence of inhibitors including 5-HMF, furfural, and acetic acid was determined using a quantitative HPLC analysis of the detoxified and non-detoxified liquid hydrolysate of BIB. The standard samples were subjected to single-point calibration at 100 μg/mL concentration. The peak area and standard concentration were then used to compute the response factor. The inhibitor concentration in the NDBBH and DBBH samples was calculated by dividing the peak areas by the response factor of the respective samples. Before and after detoxification corresponds to the chromatogram and the concentration of inhibitors were shown in Table S1 and Fig. S1 (Supplementary data). The type of pretreatment, the origin of the lignocellulosic biomass, and its impact on the microbial cell are all directly related to the concentration of the by-products of lignocellulose degradation. It was noticeable from Table S1 that the toxic inhibitors present in the NDBBH and DBBH, including furfural and HMF, are by-products of pentose and hexose breakdown and acetic acid, derived from hemicellulose. These inhibitory compounds might have an impact on the specific rate of microbial cell proliferation and prevent the synthesis of precursor molecules for fatty acid synthesis. It inhibits the enzymes involved in the synthesis of acetyl-CoA from pyruvate in the molecular pathway of R. toruloides [47]. However, the detoxification led to a 20 % and 27.80 % reduction in furfural and acetic acid, respectively, in the BIB hydrolysate, which may reduce the inhibitor’s adverse effects on lipid synthesis whereas, HMF resulted in a negligible amount after detoxification. Although the levels of inhibitors were observed to be higher in NDBBH than in DBBH, NDBBH had a higher maximal cell biomass production and lipid content. This might be a result of R. toruloides’ inherent capacity or the abundance of fermentable sugars [22]. As shown in Fig. 4, NDBBH had a higher lipid content than DBBH. When compared to previous studies using different hydrolysates produced via diluted acid hydrolysis, the number of inhibitors in BIB liquid hydrolysate obtained during MAMT was lower [23,37,48].
3.4 Effect of pretreatment on cell biomass and reducing sugar concentration
For BIB, an effective MAMT has been done to deal with lignin solubilization, decrease the generation of inhibitors, and increase the reducing sugar yield. The liquid hydrolysate from the MAMT of BIB was detoxified to get rid of the undesirable phenolic and furan derivatives. Liquid BIB hydrolysate that has been detoxified and non-detoxified was employed for R. toruloides NCIM 3547 growth as a carbon source and the consequent lipid production. This is done to determine the inherent resistance to inhibitory compounds of the yeast. Here, a very minimal amount of yeast extract (1.5 g/L) was added along with less nitrogen source (minimal media) to one fraction of NDBBH and DBBH while the supplement (yeast extract) was left out of another. The exclusion of yeast extract was done in order to investigate the role of the NDBBH and DBBH as the sole sources for C and N for R. toruloides growth. Over the period of 96 hours, the growth pattern of R. toruloides NCIM 3547 was observed using five different media formulations, including YBM (Control), DBBH (w) YE, NDBBH (w) YE, NDBBH (w/o) YE and DBBH (w/o) YE as shown in Fig. 3 (a-e). When compared to NDBBH (w/o) YE and DBBH (w/o) YE, it was observed that the inclusion of supplements in NDBBH (w) YE and DBBH (w) YE increased microbial growth. According to Fig. 3 (a-e), the cell biomass production estimated using dry cell weight (DCW), all carbon sources exhibited an initial lag phase within 24 hours and was found to significantly increase during the log (exponential) phase from 1.01-7.68 g/L (YBM-C), 1.68-10.01 g/L ((NDBBH (w) YE)), 1.56- 8.32 g/L ((NDBBH (w/o) YE)), 1.72-8.74 g/L ((DBBH (w) YE)), and 2.79-8.64 g/L ((DBBH (w/o) YE)) within 75 h before reaching stationary phase (78 h-96 h). The cell biomass (DCW) yield of NDBBH (w) YE (10.01 g/L) was found to be higher than that of DBBH (w) YE (8.74 g/L) throughout the exponential growth. However, the cell biomass yield between the sets ((NDBBH (w/o) YE and DBBH (w/o) YE)) and ((NDBBH (w) YE and DBBH (w) YE)) were significantly different. Whereas, YBM (C) showed a similar cell biomass yield (9.05 g/L) to liquid hydrolysates (NDBBH & DBBH). Though YBM contains a high amount of yeast extract (10g/L), peptone (6 g/L) and malt extract (6 g/L) along with the nitrogen source (salts) when it is given to yeast for its growth resulting in 28.44 %, w/w (weight of lipid / weight of dry cell weight) of lipid content with a biomass concentration of 9.05 g/L. Compared to complex media (YBM), liquid hydrolysate with supplementation of minimal yeast extract and nitrogen source resulted in maximum cell biomass concentration (10.01 g/L) and lipid content of 41.59 %, g/g. This proved that liquid hydrolysate acts as a C and N source for R. toruloides growth resulting in maximum cell biomass concentration and lipid content making the process more efficient and cost-effective compared to complex media and also utilising BIB, an agro-waste. The highest cell biomass concentrations were found in the DBBH (w) YE (10.11 g/L) and NDBBH (w) YE (10.94 g/L) samples, which were almost similar to those obtained in the detoxified and non-detoxified hydrolysates of wheat straw, respectively [49]. And a nearly similar result (8.74 ± 0.38 g/L) of biomass yield was observed when sugarcane bagasse hydrolysate was used as a substrate by Rhodotorula pacifica INDKK [50]. DBBH (w) YE and NDBBH (w) YE were reported to have total reducing sugar concentrations of 48.16 and 49.30 g/L, respectively. Similar to earlier studies, DBBH had a lower concentration of reducing sugar than NDBBH, which may have been caused by elevated pH during detoxification [51]. R.toruloides efficiently utilize nitrogen and sulphur-containing medium (YBM) where the reducing sugar concentration was found to be 42.94 g/L but resulted in a lower amount of lipid content compared with liquid hydrolysates. Rice straw (35.2 g/L) was reported to have lower levels of reducing sugar after the chemical treatment compared to BIB which had undergone MAMT [52]. As a result, compared to chemical pretreatment, MAMT increases the concentration of reducing sugar [53]. However, in the current study complete utilisation of the available carbon source was done through enzymatic hydrolysis of the pretreated solid cellulosic fraction using crude cellulolytic enzymes for the production of ethanol, making the process more efficient and novel. The maximum cell biomass concentration and lipid content in NDBBH is due to the existence of more reducing sugar in NDBBH than in DBBH, inhibitor tolerance and the inherent capabilities of R. toruloides in NDBBH that are attributed to larger inoculum size. Additionally, the presence of a significant amount of alkali (KOH) during the detoxification of BIB hydrolysate, subsequent neutralisation to pH 5.5 and reacidification using 1 N sulfuric acid would account for the relatively lower biomass yield, lower reducing sugar concentration that led to the lower lipid content in DBBH. Since detoxification (an additional step) is not necessary, the hydrolysate fermentation process is made simple. Although oleaginous yeast strains have been used in numerous studies to convert hemicellulosic hydrolysate into lipids, the strain used in this study was still able to produce lipids effectively despite the presence of inhibitors in the hydrolysate. Due to the presence of a preceding detoxification procedure, the fermentation process will be costly and time-consuming. Therefore, the detoxification step can be eliminated prior to fermentation. Thus, the outcome demonstrated the viability of R. toruloides growth in NDBBH (w) YE for microbial lipid synthesis.
3.5 Determination of lipid content with various substrates and confirmed through GC-MS analysis
The oleaginous yeasts use building blocks, malonyl and acetyl-CoA for de novo lipid accumulation in their pathway for lipid synthesis of sugar-based substrates. The maximal lipid concentration in the current study with NDBBH (w) YE and DBBH (w) YE were found to be 3.83 g/L, and 3.16 g/L and the lipid content was determined to be 41.59 %, w/w, 37.05 %, w/w, respectively, after 96 hours of fermentation. This outcome is consistent with the cell growth of NDBBH which exhibits the highest reducing sugar consumption and highest cell biomass production. The oleaginous yeast DCW and total lipid yield are directly correlated, with more cells (DCW) achieving the maximum overall lipid content. NDBBH was found to have a marginally higher lipid content than DBBH (Fig. 4), which also reveals a minimal difference between NDBBH (w/o) YE-37.37 %, w/w and DBBH (w/o) YE-35.50 % w/w. The amount of lipid in YBM was found to be 28.44 %, w/w shows that R.toruloides efficiently utilized only nitrogen/sulphur source and accumulated lipids. These results stated that the oleaginous strain was capable of replicating and accumulating lipids, therefore the hydrolysates that had not undergone detoxification had no negative effects on the microbial growth. Here, the R.toruloides strain would regulate the reducing sugar from the BIB hydrolysate by depleting available nitrogen into lipid production. The content of lipid was found to be 42.81%, w/w and 43.65%, w/w when cassava starch hydrolysate and distillery wastewater were used as a substrate by Trichosporon cutaneum B3 and Rhodosporidium toruloides respectively [54,55]. Based on the obtained results as indicated in Fig. 4, BIB hydrolysate without detoxification might be a suitable substrate for effective lipid synthesis. Therefore, lipid produces more efficiently in non-detoxified hydrolysate than in complex media (YBM) and detoxified hydrolysate. However, the lipid yield was calculated by the concentration of lipid from the total sugar consumed during fermentation. The obtained results were (a). YBM- 0.06; (b) NDBBH (w) YE- 0.10; (c) NDBBH (w/o) YE- 0.09; (d) DBBH (w) YE-0.10; (e) DBBH (w/o) YE- 0.08. The lipid yield was found to be higher in the medium containing YE. According to Amirsadeghi et al. [29], the fermentation media and the growth conditions of oleaginous yeast have a significant impact on the fatty acid composition. Oleaginous microbes typically acquire lipids in nitrogen-limited conditions. As a result, the type and amount of nitrogen sources play a crucial role in lipid fermentation. Furthermore, employing R. toruloides for lipid production could be improved by optimising lipid fermentation. Table 4 provides a comparative table showing the lipid accumulation of oleaginous microbes employing different substrates.
Following a fermentation period of 96 hours, the microbial lipids were extracted using Bligh and Dyer method, transesterified, and FAME was analyzed as indicated in Fig. S2 and Table S2. According to previous studies [56], all fatty acids (both unsaturated and saturated) could be transesterified into biodiesel-like fuel, with the inclusion of stearic acid and palmitic acid being effective fatty acids that enhance fuel efficiency. Saturated fatty acids are preferred for the production of biodiesel. In comparison to biodiesel, it may be suggested that C16:0 and C18:0 are economically viable products produced from oleaginous yeasts. The main fatty acid methyl esters from transesterified lipids, according to Fig. S2 (Supplementary data), are palmitic acid and stearic acid. Table S2 demonstrated the transesterified lipids contain an excess level (89.07 %) of total saturated fatty acids (C16:0, C18:0), which may enhance the biodiesel quality in terms of octane number, oxidative stability and nitrogen oxide level [57]. These findings correspond with the highest level of lipid content (41.59 %, g/g) discovered in the NDBBH (w) YE when compared to other samples, showing that R. toruloides NCIM 3547 was also capable of efficiently utilising non-detoxified substrate and accumulating lipid comparable to the detoxified substrate. It is proposed as a suitable target for biodiesel synthesis that a microbial lipid generated from R. toruloides with more saturated fatty acids (89.07 %) could be used. Our results demonstrated with earlier reports that similar fatty acid distributions were observed with R. toruloides [58]. These fatty acids are important building blocks for the production of biodiesel. While the fatty acid composition is crucial to the quality and function of biodiesel, the fatty acid profiles of oils derived from R. toruloides in this and other studies are comparable to those of conventional plant oils [59]. For instance, the melting point and heat of combustion will rise as chain length increases, along with other favourable characteristics like cetane number. Additionally, saturated fatty acids contribute to increased shorter ignition delay times, oxidative stability, and bigger cetane numbers. The obtained saturated fatty acids like stearic acid and palmitic acid may soon be adopted by industries that deal with adhesives, solvents, lubricants, and platform chemicals, where palmitic acid and stearic acid is a natural additive in organic products as well as a component of cosmetics, soap, releasing agents, and processed foods [60].
3.6 Kinetic modelling for product formation and substrate utilization
The kinetic model predicts the performance under various operational conditions and is important in understanding the non-linear behaviours of biological reactions. To identify relevant kinetic constants that would be useful for further scaling up the fermentation process, two alternative models have been used to evaluate their application to these selected substrates. The results of the hybrid Logistic-Monod and Luedeking-Piret equations used to analyse the growth kinetics of the R. toruloides strain are shown in Table 2. The experimental results, as shown in Fig. 5 (a–e), were found to be consistent with the kinetic model prediction for the R.toruloides strain maximum cell growth (Xmax) and initial cell growth (Xo). Where, the maximum specific growth rate (μmax) was higher for NDBBH (w) YE (0.125 h-1), in comparison with YBM (C), NDBBH (w/o) YE, DBBH (w) YE, and DBBH (w/o) YE was 0.122 h-1, 0.102 h-1, 0.113 h-1, and 0.103 h-1 respectively. The maximum value obtained was greater than the value obtained from R. glutinis grown on various carbon substrates (0.05 h-1) [29,61]. The maximum cell biomass output was determined to be 10.94 g/L, despite the fact that the specific growth rate (0.125 h-1) of the R.toruloides strain in NDBBH (w) YE was higher than that of the other carbon sources (Table 2). This might be a result of the availability of yeast extract as an essential nutritional supplement and R. toruloides’ high substrate utilisation rate in NDBBH (w) YE. When compared to other substrates with greater μmax value, the production and yield of cell biomass are mostly influenced by extrinsic factors like nutritional limitations and fermentation conditions. On the other hand, the R. toruloides strain substrate utilisation efficiency performed a kinetic modelling investigation, with the results summarised in Table 2. From the table, it has been observed that the growth of R. toruloides in NDBBH (w) YE was able to use the maximum reducing sugar (substrate), and its kinetic parameters, Yx/s (0.39) and Yp/s (0.98) were higher than those of other substrates. However, R. toruloides strain consumed the reducing sugar to maintain its growth and the generation of its product [62]. The non-growth-associated and growth-associated product formation constants are represented by the factors β and α in the kinetic model, respectively. Yeast provides growth (lipid-free biomass, X) and maintenance to requirements prominence when using the supplied carbon sources. After then, in addition to the basal amount of lipid synthesis for cell membranes, a change in the usage of carbon sources for lipid accumulation occurs, causing the yeast to become "Oleaginous". Eq. 7 & 8 have accounted for the α and β factors in order to clarify the function of the maintenance coefficient in product synthesis. For all of the hydrolysate substrates, it was shown that α > β, demonstrating that the lipid accumulation and synthesis of the R. toruloides strain would be a growth-associated process. Additionally, it was confirmed by the prolonged log phase as shown in Fig. 3 (a-e). All of the kinetic parameters for substrate utilisation and microbial growth determined from model prediction were in excellent agreement with the experimental results, with a coefficient of determination (R2) value ranging from 0.97 to 0.99.
3.7 Ethanol production using enzymatic hydrolysate of BIB through different fermentation strategies and confirmation by GC-MS analysis
The solid cellulosic fraction of BIB obtained after MAMT was saccharified using the crude cellulolytic enzyme secreted by the Aspergillus sp. using sugarcane bagasse as a substrate to produce reducing sugars. The saccharification of delignified BIB was found to be 69.99 ± 0.30 (%). Nearly similar results were observed with delignified aloe vera leaf rind biomass (63.60 ± 0.16 %) with crude cellulolytic enzyme (CMC- 5.25 U/ mL, FPA- 1.75 U/mL) [41]. The enzymatic hydrolysate obtained from saccharification was subjected to fermentation for ethanol production using Saccharomyces cerevisiae. The fermentation was carried out through two different fermentation strategies i.e., Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). Fig. 6 (a & b) represented the reducing sugar consumption by S. cerevisiae and ethanol yield during fermentation. The maximum reducing sugar after saccharification of a cellulosic fraction of BIB was found to be 27.22 g/L in the enzymatic hydrolysate with 462.74 mg/g of reducing sugar in biomass. However, some amount of reducing sugar was found in the hemicellulosic hydrolysate (liquid hydrolysate) and might be lost during washings of a cellulosic fraction. According to the authors’ knowledge, this is the first report on saccharifying the cellulosic portion of BIB and utilising it for the production of bioethanol. From Fig. 6 (a & b), the maximum level of ethanol produced using the hydrolysate obtained from the enzymatic saccharification of BIB through SHF and SSF were 12.70 g/L and 10.38 g/L, respectively, after 96 hours of fermentation. The ethanol productivity in SHF was 0.132 g/L/h while in SSF it was 0.108 g/L/h (Table 3). In the study, reducing sugar consumption in SHF was found to be 26.81 g/L during fermentation resulting in high ethanol production of about 12.70 g/L, whereas, in SSF, the reducing sugar level started increasing during saccharification, reaching the maximum of about 34.61 g/L and started consuming 25.05 g/L by Saccharomyces cerevisiae, resulting in ethanol production of 10.38 g/L. When utilizing enzymatic hydrolysates of banana leaves and water hyacinth, the maximum ethanol content was 6.18 and 8.1 g/L respectively (Shankar et al., 2020). These results showed lesser content of ethanol compared to our present study. However, in the study, the enzyme cocktail contains the majority of the lignocellulolytic enzymes, which facilitates saccharification. SSF were less effective in producing bioethanol than SHF. Although SHF is a multi-step process and SSF is a one-step procedure, the latter method is more expensive since it necessitates the use of expensive, highly advanced instruments in order to maintain the pH and temperature. This outcome is comparable to the study conducted by Shankar et al. [24], which found that employing lignocellulosic agro-wastes, SHF produced a higher content of ethanol than SSF. Furthermore, the yeast’s ability to ferment can be limited by the greater solid loadings at SSF. Results revealed that SHF produced more ethanol with a fermentation efficiency of 92.74 % as compared to SSF (81.71 %). However, it was found that the average fermentation efficiency was considerably higher than the ethanol production using wheat straw hydrolysate (65.6-78.7%) [63]. The product yield coefficient (Yp/s), which determines the amount of ethanol generated with the supplied sugar solution at a given time, is another crucial factor that plays a key role in scaling up were found to be 0.473 (g/g) and 0.414 (g/g) in SHF and SSF respectively. The amount of ethanol produced with the supplied biomass is determined by the substrate yield co-efficient (Yp/x), which is also essential in fermentation was found to be 0.211 and 0.104 in SHF and SSF respectively. The maximum reducing sugar consumption under SHF and SSF was determined to be 26.81 g/L and 25.05 g/L, respectively, with the rate of saccharified liquid usage being found to be 0.279/h and 0.260/h. It's noteworthy to observe that ethanol productivity has a higher maximum substrate utilization/uptake rate than SSF. Therefore, after 96 hours of fermentation, the fermentation broth has undergone distillation and the distillate was given for GC-MS analysis for the confirmation of ethanol as given in Fig. S3 and Table S3. The obtained results through the GC profile were similar to that of the results observed theoretically. The integrated production of microbial lipid from the liquid hydrolysate makes the process efficient and demonstrates the significance of biomass for sustainability.
3.8 Mass balance
The lignocellulosic biomass, BIB had undergone MAMT where the resultant liquid fraction containing sugars was fermented to microbial lipid production by Rhodosporidium toruloides. The maximum lipid yield was found to be 38.3 mg of lipid for 92.7 mg of biomass with a lipid content of 41.59 %, w/w proves its application in biodiesel with the presence of 89.07% of total saturated fatty acids. Further, the solid fraction containing cellulose was saccharified using crude cellulolytic enzymes produced by Aspergillus niger and an enzymatic hydrolysate was fermented to ethanol production using Saccharomyces cerevisiae. The yield of ethanol was found to be 127 mg of ethanol with 0.132 g/L/h of ethanol productivity. The mass flow of BIB during pretreatment, saccharification and fermentation for the co-production of biodiesel and bioethanol was represented in Figure. S4.