Elia Tomás-Pejó

Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review

Biofuel produced from lignocellulosic materials, so-called second generation bioethanol shows energetic, economic and environmental advantages in comparison to bioethanol from starch or sugar. However, physical and chemical barriers caused by the close association of the main components of lignocellulosic biomass, hinder the hydrolysis of cellulose and hemicellulose to fermentable sugars. The main goal of pretreatment is to increase the enzyme accessibility improving digestibility of cellulose. Each pretreatment has a specific effect on the cellulose, hemicellulose and lignin fraction thus, different pretreatment methods and conditions should be chosen according to the process configuration selected for the subsequent hydrolysis and fermentation steps. This paper reviews the most interesting technologies for ethanol production from lignocellulose and it points out several key properties that should be targeted for low-cost and advanced pretreatment processes.

Comparison of SHF and SSF processes from steam‐exploded wheat straw for ethanol production by xylose‐fermenting and robust glucose‐fermenting Saccharomyces cerevisiae strains

In this study, bioethanol production from steam‐exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose‐fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced. Biotechnol. Bioeng. 2008;100: 1122–1131.

Lignocellulosic ethanol production at high-gravity: challenges and perspectives

In brewing and ethanol-based biofuel industries, high-gravity fermentation produces 10-15% (v/v) ethanol, resulting in improved overall productivity, reduced capital cost, and reduced energy input compared to processing at normal gravity. High-gravity technology ensures a successful implementation of cellulose to ethanol conversion as a cost-competitive process. Implementation of such technologies is possible if all process steps can be performed at high biomass concentrations. This review focuses on challenges and technological efforts in processing at high-gravity conditions and how these conditions influence the physiology and metabolism of fermenting microorganisms, the action of enzymes, and other process-related factors. Lignocellulosic materials add challenges compared to implemented processes due to high inhibitors content and the physical properties of these materials at high gravity.

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.

Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes.

This study was aimed to study the effect of commercial cellulases (Celluclast 1.5 LFG) on Kluyveromyces marxianus CECT 10875 growth and ethanol production in SSF processes. Preliminary tests carried out in glucose (50 g/L) fermentation medium showed that high enzyme amounts (2.5-3.5 FPU/mL) could cause a negative effect on K. marxianus growth rate and viable cells number. However, the maximum ethanol production was not affected and about 86% of the theoretical (22 g/L) was reached in all cases independently of the enzyme dosage. In SSF experiments, cell viability was always affected by enzyme loading. Nevertheless, slight differences observed on cell viability during glucose fermentation processes with the detected concentrations of the additives did not justify the negative effect observed in SSF experiments.

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.

Strategies of xylanase supplementation for an efficient saccharification and cofermentation process from pretreated wheat straw

Ethanol production from lignocellulosic raw materials includes a pretreatment step before enzymatic hydrolysis (EH). Pretreated substrates contain complex hemicelluloses in the solid fraction that can protect the cellulose from enzymatic attack. In addition, soluble xylooligomers are contained in the pretreated materials and may have an inhibitory effect on cellulase activity. In this context, several approaches for xylanase supplementation have been studied to increase EH yields. In this study, the whole slurry obtained after steam explosion pretreatment of wheat straw has been used as substrate. EH experiments were performed using commercial cellulase preparations supplemented with an endoxylanase (XlnC) from Aspergillus nidulans. Among different strategies of XlnC supplementation, the 24‐h xylanase treatment before cellulase addition yielded an increase of 40.1 and 10.1% in glucose and xylose production, respectively. Different XlnC addition strategies were integrated in a simultaneous saccharification and cofermentation process (SSCF) using the xylose fermenting strain Saccharomyces cerevisiae F12. Ethanol production in SSCF was 28.4% higher when comparing to a simultaneous saccharification and fermentation process.

Influence of the propagation strategy for obtaining robust Saccharomyces cerevisiae cells that efficiently co-ferment xylose and glucose in lignocellulosic hydrolysates

Development of xylose‐fermenting yeast strains that are tolerant to the inhibitors present in lignocellulosic hydrolysates is crucial to achieve efficient bioethanol production processes. In this study, the importance of the propagation strategy for obtaining robust cells was studied. Addition of hydrolysate during propagation of the cells adapted them to the inhibitors, resulting in more tolerant cells with shorter lag phases and higher specific growth rates in minimal medium containing acetic acid and vanillin than unadapted cells. Addition of hydrolysate during propagation also resulted in cells with better fermentation capabilities. Cells propagated without hydrolysate were unable to consume xylose in wheat straw hydrolysate fermentations, whereas 40.3% and 97.7% of the xylose was consumed when 12% and 23% (v/v) hydrolysate, respectively, was added during propagation. Quantitative polymerase chain reaction revealed changes in gene expression, depending on the concentration of hydrolysate added during propagation. This study highlights the importance of using an appropriate propagation strategy for the optimum performance of yeast in fermentation of lignocellulosic hydrolysates.

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.