Effective application of immobilized second generation industrial Saccharomyces cerevisiae strain on consolidated bioprocessing.

Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts.

Xylooligosaccharides: A Bibliometric Analysis and Current Advances of This Bioactive Food Chemical as a Potential Product in Biorefineries' Portfolios.

Xylooligosaccharides (XOS) are nondigestible compounds of great interest for food and pharmaceutical industries due to their beneficial prebiotic, antibacterial, antioxidant, and antitumor properties. The market size of XOS is increasing significantly, which makes its production from lignocellulosic biomass an interesting approach to the valorization of the hemicellulose fraction of biomass, which is currently underused. This review comprehensively discusses XOS production from lignocellulosic biomass, aiming at its application in integrated biorefineries. A bibliometric analysis is carried out highlighting the main players in the field. XOS production yields after different biomass pretreatment methods are critically discussed using Microsoft PowerBI® (2.92.706.0) software, which involves screening important trends for decision-making. Enzymatic hydrolysis and the major XOS purification strategies are also explored. Finally, the integration of XOS production into biorefineries, with special attention to economic and environmental aspects, is assessed, providing important information for the implementation of biorefineries containing XOS in their portfolio.

Improvement of functional properties of cow's milk peptides through partial proteins hydrolysis.

Allergy by cow's milk proteins is among the major food allergies and could be reduced by the partial hydrolysis of these proteins by proteases, without significantly affecting its physicochemical properties. In addition, the peptides generated through enzymatic hydrolysis of the cow's milk can present prebiotic and bioactive properties. In this work, the cow's milk proteins were submitted to a controlled hydrolysis by Novo-Pro D® and the influence of the degree of hydrolysis (DH) on peptide size distribution was evaluated, as well as the prebiotic and antimicrobial properties of milk hydrolysates. It was shown that for DH-10%, all the peptides have sizes lower than 12 kDa which is the size of the most allergenic proteins, without apparent changes in the milk, as long as heating of the hydrolysate is avoided. The protein hydrolysis promoted a great improvement in the milk functional properties. In addition, the obtained milk peptides presented great prebiotic activities, as indicated by the significant improvement of the growth of prebiotic L. acidophilus and L. reuteri and by the production of bacteriocins indicated by the inhibition halos in the growth of a pathogenic microorganism. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05533-x.

Cell Immobilization Using Alginate-Based Beads as a Protective Technique against Stressful Conditions of Hydrolysates for 2G Ethanol Production.

The development of biorefineries brings the necessity of an efficient consumption of all sugars released from biomasses, including xylose. In addition, the presence of inhibitors in biomass hydrolysates is one of the main challenges in bioprocess feasibility. In this study, the application of Ca-alginate hybrid gels in the immobilization of xylose-consuming recombinant yeast was explored with the aim of improving the tolerance of inhibitors. The recombinant yeast Saccharomyces cerevisiae GSE16-T18SI.1 (T18) was immobilized in Ca-alginate and Ca-alginate-chitosan hybrid beads, and its performance on xylose fermentation was evaluated in terms of tolerance to different acetic acid concentrations (0-12 g/L) and repeated batches of crude sugarcane bagasse hemicellulose hydrolysate. The use of the hybrid gel improved yeast performance in the presence of 12 g/L of acetic acid, achieving 1.13 g/L/h of productivity and reaching 75% of the theoretical ethanol yield, with an improvement of 32% in the xylose consumption rate (1:1 Vbeads/Vmedium, 35 °C, 150 rpm and pH 5.2). The use of hybrid alginate-chitosan gel also led to better yeast performance at crude hydrolysate, yielding one more batch than the pure-alginate beads. These results demonstrate the potential of a hybrid gel as an approach that could increase 2G ethanol productivity and allow cell recycling for a longer period.

High stabilization and hyperactivation of a Recombinant ß-Xylosidase through Immobilization Strategies.

Attainment of a stable and highly active ß-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant ß-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. ß-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L-1 h-1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized ß-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor.

BACKGROUND: The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. RESULTS: This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 gethanol/gsugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. CONCLUSIONS: The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.

Eucalyptus xylan: An in-house-produced substrate for xylanase evaluation to substitute birchwood xylan.

Over the past decades, most studies with xylanases have used Birchwood xylan as the standard substrate for activity assays. However, recently, Birchwood xylan production was discontinued by major suppliers, creating an important demand for a substitute. Ongoing and future studies require a substrate with characteristics equivalent to the discontinued xylan, in order to enable the comparison of results. In this context, a protocol for the production of a substrate similar to the discontinued commercial Birchwood xylan is reported. Obtained from bleached Eucalyptus cellulose pulp, xylan was extracted using 4% w/v NaOH solution at 25 °C, precipitated with glacial acetic acid (HOAc), and freeze-dried. A thermal pretreatment in an autoclave for 15 min increased its solubility. The resulting xylan was characterized by infrared spectroscopy, thermogravimetry, and NMR. When assessing the activity of xylanases, the results were the same as those for commercial Birchwood xylan.

Hemicellulosic ethanol production by immobilized cells of Scheffersomyces stipitis: effect of cell concentration and stirring.

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).

Rice bran extract: an inexpensive nitrogen source for the production of 2G ethanol from sugarcane bagasse hydrolysate.

Selection of the raw material and its efficient utilization are the critical factors in economization of second generation (2G) ethanol production. Fermentation of the released sugars into ethanol by a suitable ethanol producing microorganism using cheap media ingredients is the cornerstone of the overall process. This study evaluated the potential of rice bran extract (RBE) as a cheap nitrogen source for the production of 2G ethanol by Scheffersomyces (Pichia) stipitis NRRL Y-7124 using sugarcane bagasse (SB) hemicellulosic hydrolysate. Dilute acid hydrolysis of SB showed 12.45 g/l of xylose and 0.67 g/l of glucose along with inhibitors. It was concentrated by vacuum evaporation and submitted to sequential detoxification (neutralization by calcium hydroxide and charcoal adsorption). The detoxified hydrolysate revealed the removal of furfural (81 %) and 5-hydroxymethylfurfural (61 %) leading to the final concentration of glucose (1.69 g/l) and xylose (33.03 g/l). S. stipitis was grown in three different fermentation media composed of detoxified hydrolysate as carbon source supplemented with varying nitrogen sources i.e. medium #1 (RBE + ammonium sulfate + calcium chloride), medium #2 (yeast extract + peptone) and medium #3 (yeast extract + peptone + malt extract). Medium #1 showed maximum ethanol production (8.6 g/l, yield 0.22 g/g) followed by medium #2 (8.1 g/l, yield 0.19 g/g) and medium #3 (7.4 g/l, yield 0.18 g/g).