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Second generation biorefineries play an important role in the production of renewable energy and fuels, utilizing forest and agro-industrial residues and by-products as raw materials. The integration of novel bioproducts, such as: xylitol, ß-carotene, xylooligosaccharides, and biopigments into the biorefinery's portfolio can offer economic benefits in the valorization of lignocellulosic materials, particularly cellulosic and hemicellulosic fractions. Fungal biopigments, known for their additional antioxidant and antimicrobial properties, are appealing to consumers and can have applications in various industrial sectors, including food and pharmaceuticals. The use of lignocellulosic materials as carbon and nutrient sources for the growth medium helps to reduce production costs, increasing the competitiveness of fungal biopigments in the market. In addition, the implementation of biopigment production in biorefineries allows the utilization of underutilized fractions, such as hemicellulose, for value-added bioproducts. This study deals with the potential of fungal biopigments production in second generation biorefineries in order to diversify the produced biomolecules together with energy generation. A comprehensive and critical review of the recent literature on this topic has been conducted, covering the major possible raw materials, general aspects of second generation biorefineries, the fungal biopigments and their potential for incorporation into biorefineries.

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Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83.

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Β-Carotene is a red-orange pigment that serves as a precursor to important pharmaceutical molecules like vitamin A and retinol, making it highly significant in the industrial sector. Consequently, there is an ongoing quest for more sustainable production methods. In this study, glucose and xylose, two primary sugars derived from sugarcane bagasse (SCB), were utilized as substrates for ß-carotene production by Rhodotorula glutinis CCT-2186. To achieve this, SCB underwent pretreatment using NaOH, involved different concentrations of total solids (TS) (10%, 15%, and 20%) to remove lignin. Each sample was enzymatically hydrolyzed using two substrate loadings (5% and 10%). The pretreated SCB with 10%, 15%, and 20% TS exhibited glucose hydrolysis yields (%wt) of 93.10%, 91.88%, and 90.77%, respectively. The resulting hydrolysate was employed for ß-carotene production under batch fermentation. After 72 h of fermentation, the SCB hydrolysate yielded a ß-carotene concentration of 118.56 ± 3.01 mg/L. These findings showcase the robustness of R. glutinis as a biocatalyst for converting SCB into ß-carotene.

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This study investigated the diversity of yeast species associated with rotting wood in Brazilian Amazonian rainforests. A total of 569 yeast strains were isolated from rotting wood samples collected in three Amazonian areas (Universidade Federal do Amazonas-Universidade Federal do Amazonas [UFAM], Piquiá, and Carú) in the municipality of Itacoatiara, Amazon state. The samples were cultured in yeast nitrogen base (YNB)-d-xylose, YNB-xylan, and sugarcane bagasse and corncob hemicellulosic hydrolysates (undiluted and diluted 1:2 and 1:5). Sugiyamaella was the most prevalent genus identified in this work, followed by Kazachstania. The most frequently isolated yeast species were Schwanniomyces polymorphus, Scheffersomyces amazonensis, and Wickerhamomyces sp., respectively. The alpha diversity analyses showed that the dryland forest of UFAM was the most diverse area, while the floodplain forest of Carú was the least. Additionally, the difference in diversity between UFAM and Carú was the highest among the comparisons. Thirty candidates for new yeast species were obtained, representing 36% of the species identified and totaling 101 isolates. Among them were species belonging to the clades Spathaspora, Scheffersomyces, and Sugiyamaella, which are recognized as genera with natural xylose-fermenting yeasts that are often studied for biotechnological and ecological purposes. The results of this work showed that rotting wood collected from the Amazonian rainforest is a tremendous source of diverse yeasts, including candidates for new species.

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Lignocellulosic biomass (LCB) has remained a latent alternative resource to be the main substitute for oil and its derivatives in a biorefinery concept. However, its complex structure and the underdeveloped technologies for its large-scale processing keep it in a state of constant study trying to establish a consolidated process. In intensive processes, enzymes have been shown to be important molecules for the fractionation and conversion of LCB into biofuels and high-value-added molecules. However, operational challenges must be overcome before enzyme technology can be the main resource for obtaining second-generation sugars. The use of additives is shown to be a suitable strategy to improve the saccharification process. This review describes the mechanisms, roles, and effects of using additives, such as surfactants, biosurfactants, and non-catalytic proteins, separately and integrated into the enzymatic hydrolysis process of lignocellulosic biomass. In doing so, it provides a technical background in which operational biomass processing hurdles such as solids and enzymatic loadings, pretreatment burdens, and the unproductive adsorption phenomenon can be addressed.

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Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sugarcane straw), synthetic (urea, ammonium sulfate, and peptone), and non-synthetic (soybean meal, rice, and corn meal) nitrogen sources and inoculum size were evaluated by one parameter at-a-time strategy. Aureobasidium pullulans LB83 showed maximum cellulase activity (FPase, 2.27 U/mL; CMCase, 7.42 U/mL) on sugarcane bagasse. Among the nitrogen sources, soybean meal as a non-synthetic nitrogen sources showed a maximum cellulase activity (FPase 2.45 U/mL; CMCase, 6.86 U/mL) after 60 hr. The inoculum size of 1.6 × 106 CFU/mL had the maximum FPase and CMCase activities of 3.14 and 8.74 U/mL, respectively. For the enzymatic hydrolysis, both the commercial cellulase (10 FPU/g of Cellic CTec 2 (#A) and 10 FPU/g of crude enzyme extract (CEE) (#B), and varying ratio of CTec 2 and CEE in combination #C (5 FPU/g of CTec 2 + 5 FPU/g CEE), combination #D (2.5 FPU/g of CTec 2 + 7.5 FPU/g CEE), and combination #E (7.5 FPU/g of CTec 2 + 2.5 FPU/g CEE) were assessed for enzymatic hydrolysis of delignified sugarcane bagasse. Enzyme combination #C showed maximum hydrolysis yield of 92.40%. The study shows the hydrolytic potential of cellulolytic enzymes from A. pullulans LB83 for lignocellulosic sugars production from delignified sugarcane bagasse.

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Bioemulsifiers are surface active compounds which could be potentially used in food processing, cosmetic sector and oil recovery. Sugarcane straw (SS), was used as the raw substrate for the production of bio-emulsifiers (BE) by Cutaneotrichosporon mucoides. Three different delignification strategies using dilute sodium hydroxide, sodium sulfite and ammonium hydroxide followed by enzymatic hydrolysis (Cellic CTec 2, 7.5% total solids, 15 FPU/g, 72 h) were studied. Enzyme hydrolysis of ammonium hydroxide pretreated SS showed a maximum of 62.19 ± 0.74 g/l total reducing sugars with 88.35% hydrolytic efficiency (HE) followed by sodium hydroxide (60.06 ± 0.33 g/l; 85.40% HE) and sodium sulfite pretreated SS (57.22 ± 0.52 g/l; 84.71% HE), respectively. The ultrastructure of SS (native and delignified) by fourier transform-infrared and near infrared spectroscopy, revealed notable structural differences. The fermentation of hydrolysates by C. mucoides into bioemulsifiers showing emulsification index (EI) of 54.33%, 48.66% and 32.66% from sodium sulfite, sodium hydroxide, and ammonium hydroxide pretreated SS, respectively.

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This study evaluated D-xylose-assimilating yeasts that are associated with rotting wood from the Galápagos Archipelago, Ecuador, for xylitol production from hemicellulose hydrolysates. A total of 140 yeast strains were isolated. Yeasts related to the clades Yamadazyma, Kazachstania, Kurtzmaniella, Lodderomyces, Metschnikowia and Saturnispora were predominant. In culture assays using sugarcane bagasse hemicellulose hydrolysate, Candida tropicalis CLQCA-24SC-125 showed the highest xylitol production, yield and productivity (27.1 g L(-1) xylitol, Y p/s (xyl) = 0.67 g g(-1), Qp = 0.38 g L(-1). A new species of Cyberlindnera, strain CLQCA-24SC-025, was responsible for the second highest xylitol production (24 g L(-1), Y p/s (xyl) = 0.64 g g(-1), Qp = 0.33 g L(-1) h(-1)) on sugarcane hydrolysate. The new xylitol-producing species Cyberlindnera galapagoensis f.a., sp. nov., is proposed to accommodate the strain CLQCA-24SC-025(T) (=UFMG-CM-Y517(T); CBS 13997(T)). The MycoBank number is MB 812171.

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Bioconversion of hemicellulosic hydrolysate into ethanol plays a pivotal role in the overall success of biorefineries. For the efficient fermentative conversion of hemicellulosic hydrolysates into ethanol, the use of immobilized cells system could provide the enhanced ethanol productivities with significant time savings. Here, we investigated the effect of 2 important factors (e.g., cell concentration and stirring) on ethanol production from sugarcane bagasse hydrolysate using the yeast Scheffersomyces stipitis immobilized in calcium alginate matrix. A 2(2) full factorial design of experiment was performed considering the process variables- immobilized cell concentration (3.0, 6.5 and 10.0 g/L) and stirring (100, 200 and 300 rpm). Statistical analysis showed that stirring has the major influence on ethanol production. Maximum ethanol production (8.90 g/l) with ethanol yield (Yp/s) of 0.33 g/g and ethanol productivity (Qp) of 0.185 g/l/h was obtained under the optimized process conditions (10.0 g/L of cells and 100 rpm).

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BACKGROUND: Heavy usage of gasoline, burgeoning fuel prices, and environmental issues have paved the way for the exploration of cellulosic ethanol. Cellulosic ethanol production technologies are emerging and require continued technological advancements. One of the most challenging issues is the pretreatment of lignocellulosic biomass for the desired sugars yields after enzymatic hydrolysis. We hypothesized that consecutive dilute sulfuric acid-dilute sodium hydroxide pretreatment would overcome the native recalcitrance of sugarcane bagasse (SB) by enhancing cellulase accessibility of the embedded cellulosic microfibrils. RESULTS: SB hemicellulosic hydrolysate after concentration by vacuum evaporation and detoxification showed 30.89 g/l xylose along with other products (0.32 g/l glucose, 2.31 g/l arabinose, and 1.26 g/l acetic acid). The recovered cellulignin was subsequently delignified by sodium hydroxide mediated pretreatment. The acid-base pretreated material released 48.50 g/l total reducing sugars (0.91 g sugars/g cellulose amount in SB) after enzymatic hydrolysis. Ultra-structural mapping of acid-base pretreated and enzyme hydrolyzed SB by microscopic analysis (scanning electron microcopy (SEM), transmitted light microscopy (TLM), and spectroscopic analysis (X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Fourier transform near-infrared (FT-NIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy) elucidated the molecular changes in hemicellulose, cellulose, and lignin components of bagasse. The detoxified hemicellulosic hydrolysate was fermented by Scheffersomyces shehatae (syn. Candida shehatae UFMG HM 52.2) and resulted in 9.11 g/l ethanol production (yield 0.38 g/g) after 48 hours of fermentation. Enzymatic hydrolysate when fermented by Saccharomyces cerevisiae 174 revealed 8.13 g/l ethanol (yield 0.22 g/g) after 72 hours of fermentation. CONCLUSIONS: Multi-scale structural studies of SB after sequential acid-base pretreatment and enzymatic hydrolysis showed marked changes in hemicellulose and lignin removal at molecular level. The cellulosic material showed high saccharification efficiency after enzymatic hydrolysis. Hemicellulosic and cellulosic hydrolysates revealed moderate ethanol production by S. shehatae and S. cerevisiae under batch fermentation conditions.

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BACKGROUND: Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. RESULTS: OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform-near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). CONCLUSIONS: OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases' ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

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Bioconversion of hemicellulosic hydrolysates into ethanol with the desired yields plays a pivotal role for the overall success of biorefineries. This paper aims to evaluate the ethanol production potential of four native strains of Scheffersomyces shehatae (syn. Candida shehatae) viz. S. shehatae BR6-2AI, CG8-8BY, PT1-1BASP and BR6-2AY, isolated from Brazilian forests. These strains were grown in commercial D-xylose-supplemented synthetic medium and sugarcane bagasse hemicellulose hydrolysate. S. shehatae BR6-2AY showed maximum ethanol production [0.48 ± 0.019 g g-1, 95 ± 3.78 % fermentation efficiency (FE)] followed by S. shehatae CG8-8BY (0.47 ± 0.016 g g-1, 93 ± 3.12 % FE), S. shehatae BR6-2AI (0.45 ± 0.01 g g-1, 89 ± 1.71 % FE) and S. shehatae PT1-1BASP (0.44 ± 0.02 g g-1, 86 ± 3.37 % FE) when grown in synthetic medium. During the fermentation of hemicellulose hydrolysates, S. shehatae CG8-8BY and S. shehatae BR6-2AY showed ethanol production (0.30 ± 0.05 g g-1, 58 ± 0.02 % FE) and (0.21 ± 0.01 g g-1, 40 ± 1.93 % FE), respectively.

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Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing-thawing method. Entrapment experiments were planned according to a 2(3) full factorial design, using the PVA concentration (80, 100, and 120 g L(-1)), the freezing temperature (-10, -15, and -20 degrees C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 degrees C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L(-1), a xylitol yield on consumed xylose of 0.49 g g(-1) and a xylitol volumetric productivity of 0.24 g L(-1) h(-1) were obtained.

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Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2(4-1) fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S(F)) and in the fermentor (S0), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S(F) = 45 g/L, S0 = 40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L x h) and a xylose xylitol yield factor of 0.68 g/g.

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