Homo- and heterofermentative lactobacilli are distinctly affected by furanic compounds.

PURPOSE: Second generation (2G) ethanol is produced using lignocellulosic biomass. However, the pre-treatment processes generate a variety of molecules (furanic compounds, phenolic compounds, and organic acids) that act as inhibitors of microbial metabolism, and thus, reduce the efficiency of the fermentation step in this process. In this context, the present study aimed to investigate the effect of furanic compounds on the physiology of lactic acid bacteria (LAB) strains that are potential contaminants in ethanol production. METHODOLOGY: Homofermentative and heterofermentative strains of laboratory LAB, and isolated from first generation ethanol fermentation, were used. LAB strains were challenged to grow in the presence of furfural and 5-hydroxymethyl furfural (HMF). RESULTS: We determined that the effect of HMF and furfural on the growth rate of LAB is dependent on the metabolic type, and the growth kinetics in the presence of these compounds is enhanced for heterofermentative LAB, whereas they are inhibitory to homofermentative LAB. Sugar consumption and product formation were also enhanced in the presence of furanic compounds for heterofermentative LAB, who displayed effective depletion kinetics when compared to the homofermentative LAB. CONCLUSION: Homo- and heterofermentative LAB are affected differently by furanic compounds, in a way that the latter type is more resistant to the toxic effects of these inhibitors. This knowledge is important to understand the potential effects of bacterial contamination in 2G bioprocesses.

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.

Analysis of carbohydrate-active enzymes and sugar transporters in Penicillium echinulatum: A genome-wide comparative study of the fungal lignocellulolytic system.

Penicillium echinulatum 2HH is an ascomycete well known for its production of cellulolytic enzymes. Understanding lignocellulolytic and sugar uptake systems is essential to obtain efficient fungi strains for the production of bioethanol. In this study we performed a genome-wide functional annotation of carbohydrate-active enzymes and sugar transporters involved in the lignocellulolytic system of P. echinulatum 2HH and S1M29 strains (wildtype and mutant, respectively) and eleven related fungi. Additionally, signal peptide and orthology prediction were carried out. We encountered a diverse assortment of cellulolytic enzymes in P. echinulatum, especially in terms of ß-glucosidases and endoglucanases. Other enzymes required for the breakdown of cellulosic biomass were also found, including cellobiohydrolases, lytic cellulose monooxygenases and cellobiose dehydrogenases. The S1M29 mutant, which is known to produce an increased cellulase activity, and the 2HH wild type strain of P. echinulatum did not show significant differences between their enzymatic repertoire. Nevertheless, we unveiled an amino acid substitution for a predicted intracellular ß-glucosidase of the mutant, which might contribute to hyperexpression of cellulases through a cellodextrin induction pathway. Most of the P. echinulatum enzymes presented orthologs in P. oxalicum 114-2, supporting the presence of highly similar cellulolytic mechanisms and a close phylogenetic relationship between these fungi. A phylogenetic analysis of intracellular ß-glucosidases and sugar transporters allowed us to identify several proteins potentially involved in the accumulation of intracellular cellodextrins. These may prove valuable targets in the genetic engineering of P. echinulatum focused on industrial cellulases production. Our study marks an important step in characterizing and understanding the molecular mechanisms employed by P. echinulatum in the enzymatic hydrolysis of lignocellulosic biomass.

Sequential process of solid-state cultivation with fungal consortium and ethanol fermentation by Saccharomyces cerevisiae from sugarcane bagasse.

Solid-state cultivation (SSC) is the microbial growth on solid supports, producing a nutrient-rich solution by cell enzymes that may be further used as a generic microbial medium. "Second-generation" ethanol is obtained by fermentation from mainly the acid hydrolysates of lignocellulosic wastes, generating several microbial growth inhibitors. Thus, this research aimed at evaluating the feasibility of ethanol fermentation from sugarcane bagasse hydrolysate after SSC with vinasse as the impregnating solution by a consortium of A. niger and T. reesei as opposed to the conventional method of acid hydrolysis. Fermentation of the hydrolysate from SSC leading to the yield of 0.40 g g-1, i.e., about 78% of maximum stoichiometric indicating that the nonconventional process allowed the use of two by-products from sugarcane processing in addition to ethanol production from glucose release.

Understanding the differences in 2G ethanol fermentative scales through omics data integration.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.

Genome-wide analysis of general phenylpropanoid and monolignol-specific metabolism genes in sugarcane.

Lignin is the main component of secondary cell walls and is essential for plant development and defense. However, lignin is recognized as a major recalcitrant factor for efficiency of industrial biomass processing. Genes involved in general phenylpropanoid and monolignol-specific metabolism in sugarcane have been previously analyzed at the transcriptomic level. Nevertheless, the number of genes identified in this species is still very low. The recently released sugarcane genome sequence has allowed the genome-wide characterization of the 11 gene families involved in the monolignol biosynthesis branch of the phenylpropanoid pathway. After an exhaustive analysis of sugarcane genomes, 438 haplotypes derived from 175 candidate genes from Saccharum spontaneum and 144 from Saccharum hybrid R570 were identified as associated with this biosynthetic route. The phylogenetic analyses, combined with the search for protein conserved residues involved in the catalytic activity of the encoded enzymes, were employed to identify the family members potentially involved in developmental lignification. Accordingly, 15 candidates were identified as bona fide lignin biosynthesis genes: PTAL1, PAL2, C4H4, 4CL1, HCT1, HCT2, C3'H1, C3'H2, CCoAOMT1, COMT1, F5H1, CCR1, CCR2, CAD2, and CAD7. For this core set of lignin biosynthetic genes, we searched for the chromosomal location, the gene expression pattern, the promoter cis-acting elements, and microRNA targets. Altogether, our results present a comprehensive characterization of sugarcane general phenylpropanoid and monolignol-specific genes, providing the basis for further functional studies focusing on lignin biosynthesis manipulation and biotechnological strategies to improve sugarcane biomass utilization.

Sustainability of sugarcane lignocellulosic biomass pretreatment for the production of bioethanol.

The sustainability of a biofuel is severely affected by the technological route of its production. Chemical pretreatment can be considered the traditional method of decomposition of the lignocellulose into its mono and oligomeric units, which can be further bioconverted to ethanol. The evaluation of the recent advances in chemical pretreatments of sugarcane bagasse, especially diluted acids, alkaline, organosolv and ionic liquids, identified the critical points for sustainability. In this context, chemicals recovery and reutilization or their substitution by green solvents, heat and electricity generation through bioenergy, reutilization of water from evaporators, vinasse concentration and the upgrading of lignin were discussed as strategic routes for developing sustainable chemical-based lignocellulose pretreatment. The advances in the technologies that allow greater fractionation of lignocellulosic biomass should be focused on the minimization of the use of natural resources, effluent generation and energy expenditure.

Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30.

The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, ß-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol.

Integral process assessment of sugarcane agricultural crop residues conversion to ethanol.

This work focuses a whole process assessment on post-harvesting sugarcane residues for 2G ethanol production by different saccharification-fermentation conditions at high solids loading, performed after steam explosion, alkaline and acidic pretreatments. Carbohydrate recoveries and enzymatic digestibility results showed that alkali and steam explosion pretreatments were effective for the biomass assayed. Due to a significant improvement (60%) of the glucose released by combining hemicellulases and cellulases only after the NaOH pretreatment, the most favorable process settled comprised an alkali-based pretreatment followed by a pre-saccharification and simultaneous saccharification and fermentation (PSSF). The produced ethanol reached 4.8% (w/w) as a result of an 80% conversion of the glucose from the pretreated biomass. Finally, an ethanol concentration of 3.2% (w/w) was obtained by means of a steam explosion followed by PSSF, representing a suitable start point to further develop a low environmental impact alternative for ethanol production.