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Future biorefineries will integrate biomass conversion processes to produce fuels, power, heat and value-added chemicals. Due to its low price and wide distribution, lignocellulosic biomass is expected to play an important role toward this goal. Regarding renewable biofuel production, bioethanol from lignocellulosic feedstocks is considered the most feasible option for fossil fuels replacement since these raw materials do not compete with food or feed crops. In the overall process, lignin, the natural barrier of the lignocellulosic biomass, represents an important limiting factor in biomass digestibility. In order to reduce the recalcitrant structure of lignocellulose, biological pretreatments have been promoted as sustainable and environmentally friendly alternatives to traditional physico-chemical technologies, which are expensive and pollute the environment. These approaches include the use of diverse white-rot fungi and/or ligninolytic enzymes, which disrupt lignin polymers and facilitate the bioconversion of the sugar fraction into ethanol. As there is still no suitable biological pretreatment technology ready to scale up in an industrial context, white-rot fungi and/or ligninolytic enzymes have also been proposed to overcome, in a separated or in situ biodetoxification step, the effect of the inhibitors produced by non-biological pretreatments. The present work reviews the latest studies regarding the application of different microorganisms or enzymes as useful and environmentally friendly delignification and detoxification technologies for lignocellulosic biofuel production. This review also points out the main challenges and possible ways to make these technologies a reality for the bioethanol industry.

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Operating the saccharification and fermentation processes at high-substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high-substrate loadings. Water-insoluble solids fraction from steam-exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity.

Development of biofuels such as lignocellulosic ethanol represents a sustainable alternative in the transport sector. Wheat straw is a promising feedstock for bioethanol production in Europe due to its large production and high carbohydrates content. In a process to produce cellulosic ethanol, previous to the enzymatic hydrolysis to obtain fermentable sugars and the subsequent fermentation, a pretreatment step to break down the recalcitrance of lignocellulose fiber is essential. In this work, a range of steam explosion pretreatment conditions were evaluated according to different parameters: sugars recovery, degradation products generation, and enzymatic hydrolysis yields. Moreover, the enzymatic hydrolysis process was also studied at high substrate loadings, since operating at high solids loading is crucial for large scale development of ethanol production. Pretreatment at 200°C - 10 min resulted in higher enzymatic hydrolysis yield (91.7%) and overall glucose yields (35.4 g glucose/100 g wheat straw) but also higher production of toxic compound. In turn, the characteristics of the pretreated wheat straw at lower severity (Log R0=3.65) correspond to 190°C and 10 min, with minimal sugars degradation and toxics formation indicated a great potential for maximizing total sugars production by using optimal enzyme combinations including accessory enzymes in the enzymatic hydrolysis step.

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BackgroundConceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality.ResultsTHSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4–7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (kcat/KM = 1050 s−1 mM−1) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg−1, respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg−1). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality.ConclusionThe discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0550-x) contains supplementary material, which is available to authorized users.