Antonio D. Moreno

Different laccase detoxification strategies for ethanol production from lignocellulosic biomass by the thermotolerant yeast Kluyveromyces marxianus CECT 10875.

In this work, laccase enzymes were evaluated to detoxify the whole slurry from steam-exploded wheat straw. For it, two different strategies, laccase treatment before or after enzymatic hydrolysis, were employed. The detoxification efficiency was analyzed on enzymatic hydrolysis and fermentation levels by the thermotolerant yeast Kluyveromyces marxianus. Laccases reduced phenolic compounds without affecting weak acids and furan derivates. A lower glucose recovery was observed when laccase treatments were carried out before enzymatic hydrolysis, phenomenon that was not showed after enzymatic hydrolysis. In contrast, both laccase treatment strategies enhanced ethanol concentrations, reducing significantly the lag phase of the yeast and allowing substrate loading increments of saccharification and fermentation broths.

A review of biological delignification and detoxification methods for lignocellulosic bioethanol production

Abstract 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.

Improving the fermentation performance of saccharomyces cerevisiae by laccase during ethanol production from steam-exploded wheat straw at high-substrate loadings

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.

Comparing cell viability and ethanol fermentation of the thermotolerant yeast Kluyveromyces marxianus and Saccharomyces cerevisiae on steam-exploded biomass treated with laccase

In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to the industrial strain Saccharomyces cerevisiae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was inhibited, and neither growth nor ethanol production occurred during the processes. However, laccase treatment of the whole slurry removed specifically lignin phenols from the overall inhibitory compounds present in the slurry and triggered the fermentation by K. marxianus, attaining final ethanol concentrations and yields comparable to those obtained by S. cerevisiae.

In situ laccase treatment enhances the fermentability of steam-exploded wheat straw in SSCF processes at high dry matter consistencies

This work evaluates the in situ detoxification of inhibitory lignocellulosic broths by laccases to facilitate their fermentation by the xylose-consuming Saccharomyces cerevisiae F12. Treatment of wheat straw slurries with laccases prior to SSCF processes decreased the total phenolic content by 50-80%, reducing the lag phase and increasing the cell viability. After laccase treatment, a negative impact on enzymatic hydrolysis was observed. This effect, together with the low enzymatic hydrolysis yields when increasing consistency, resulted in a decrease in final ethanol yields. Furthermore, when using high substrate loading (20% DM (w/v)), high concentration of inhibitors prevailed in broths and the absence of an extra nitrogen source led to a total cell growth inhibition within the first 24h in non-treated samples. This inhibition of growth at 20% DM (w/v) was overcome by laccase treatment with no addition of nitrogen, allowing S. cerevisiae F12 to produce more than 22 g/L of ethanol.

Fed-batch SSCF using steam-exploded wheat straw at high dry matter consistencies and a xylose-fermenting Saccharomyces cerevisiae strain: effect of laccase supplementation

BackgroundLignocellulosic bioethanol is expected to play an important role in fossil fuel replacement in the short term. Process integration, improvements in water economy, and increased ethanol titers are key considerations for cost-effective large-scale production. The use of whole steam-pretreated slurries under high dry matter (DM) conditions and conversion of all fermentable sugars offer promising alternatives to achieve these goals.ResultsWheat straw slurry obtained from steam explosion showed high concentrations of degradation compounds, hindering the fermentation performance of the evolved xylose-recombinant Saccharomyces cerevisiae KE6-12 strain. Fermentability tests using the liquid fraction showed a higher number of colony-forming units (CFU) and higher xylose consumption rates when treating the medium with laccase. During batch simultaneous saccharification and co-fermentation (SSCF) processes, cell growth was totally inhibited at 12% DM (w/v) in untreated slurries. However, under these conditions laccase treatment prior to addition of yeast reduced the total phenolic content of the slurry and enabled the fermentation. During this process, an ethanol concentration of 19 g/L was obtained, corresponding to an ethanol yield of 39% of the theoretical yield. By changing the operation from batch mode to fed-batch mode, the concentration of inhibitors at the start of the process was reduced and 8 g/L of ethanol were obtained in untreated slurries with a final consistency of 16% DM (w/v). When fed-batch SSCF medium was supplemented with laccase 33 hours after yeast inoculation, no effect on ethanol yield or cell viability was found compared to untreated fermentations. However, if the laccase supplementation (21 hours after yeast inoculation) took place before the first addition of substrate (at 25 hours), improved cell viability and an increased ethanol titer of up to 32 g/L (51% of the theoretical) were found.ConclusionsLaccase treatment in SSCF processes reduces the inhibitory effect that degradation compounds have on the fermenting microorganism. Furthermore, in combination with fed-batch operational mode, laccase supplementation allows the fermentation of wheat straw slurry at high DM consistencies, improving final ethanol concentrations and yields.

Production of Ethanol from Lignocellulosic Biomass

Ethanol fuel is leading the transition towards a post-petrol era in the transport sector worldwide. Ethanol is produced via sugar fermentation processes by yeasts or bacteria. Although the current industrial production of ethanol mainly involves the use of starch- and sugar-based feedstocks, lignocellulosic biomass is expected to play a key role as renewable, carbohydrate-rich raw material. With the aim of placing lignocellulosic ethanol into the market, the scientific community has made great efforts to develop and implement efficient conversion technologies. Prior to fermentation, lignocellulosic biomass must be pretreated and hydrolysed to obtain the fermentable sugars. Biomass processing is, however, a major limiting step since it is hindered by the native structure of lignocellulose and generates different biomass-derived compounds that are inhibitors of the subsequent microbial conversion. In this context, different pretreatment, delignification and detoxification methods have been investigated to produce less inhibitory pretreated materials. Furthermore, several strategies such as working at high gravity conditions, high temperatures and/or different process configurations, have been shown to maximize ethanol production from lignocellulosic materials. The development of robust microbial strains tolerant to inhibitory compounds and capable of converting sugar mixtures is also needed for cost-effectiveness of the process. This chapter compiles recent advances in lignocellulosic ethanol production processes, from novel raw materials or fermenting microorganisms to new processing technologies addressed to commercialization.

Complete Genome Sequences of the Xylose-Fermenting Candida intermedia Strains CBS 141442 and PYCC 4715

ABSTRACT Sustainable biofuel production from lignocellulosic materials requires efficient and complete use of all abundant sugars in the biomass, including xylose. Here, we report on the de novo genome assemblies of two strains of the xylose-fermenting yeast Candida intermedia: CBS 141442 and PYCC 4715.

Pretreatment of lignocellulosic Feedstocks

With the aim of reducing greenhouse gas (GHG) emissions and with our dependence on non-renewable fossil fuels, lignocellulose has been proposed to be an alternative sugar-rich raw material for renewable biofuel production, especially for the transportation sector. Biorefineries can efficiently convert lignocellulosic biomass into fuels, value-added chemicals, and other energy forms. When lignocellulose is transformed using biochemical routes, the highly recalcitrant structure of lignocellulosic materials hampers the conversion process by limiting the accessibility of the chemical building blocks. It is therefore imperative to include a pretreatment step to reach high overall yields and productivities in subsequent hydrolysis and fermentation steps. The main purpose of pretreatment is to break down the structure of lignin and/or to solubilize hemicellulose in order to improve the accessibility of cellulose towards hydrolytic enzymes. The effects of pretreatment on the different lignocellulosic polymers depends on the type of pretreatment itself and the conditions used. This chapter outlines the most common physical, chemical, physicochemical, and biological pretreatment technologies that have been developed and optimized over last four decades for disruption and/or fractionation of lignocellulosic feedstocks. The mechanisms of action and the potential benefits and drawbacks of each pretreatment are listed and discussed. Furthermore, the pretreatment technologies are also analyzed from an economic and environmental point of view to evaluate their sustainability.

Starch Biomass for Biofuels, Biomaterials, and Chemicals

The success of modern biorefineries, including those using starch-based feedstocks, should be based on versatile biomass supply chains and on the production of a wide spectrum of competitive bio-based products. This chapter summarizes the current knowledge of bio-based products obtained mainly from biochemical platforms from starch- and sugar-based feedstocks. After an initial review of starch production sources and starch properties as well as starch-based end applications, this chapter reviews the state of the art of starch hydrolytic enzymes, focusing on a bio-based platform for the main value-added (bio)chemicals, biofuels, and biomaterials that can be obtained from sugar-based feedstocks.