Dry biorefining maximizes the potentials of simultaneous saccharification and co-fermentation for cellulosic ethanol production.

Despite the well-recognized merits of simultaneous saccharification and co-fermentation (SSCF) on relieving sugar product inhibition on cellulase activity, a practical concomitance difficulty of xylose with inhibitors in the pretreated lignocellulose feedstock prohibits the essential application of SSCF for cellulosic ethanol fermentation. To maximize the SSCF potentials for cellulosic ethanol production, a dry biorefining approach was proposed starting from dry acid pretreatment, disk milling, and biodetoxification of lignocellulose feedstock. The successful SSCF of the inhibitor free and xylose conserved lignocellulose feedstock after dry biorefining reached a record high ethanol titer at moderate cellulase usage and minimum wastewater generation. For wheat straw, 101.4 g/L of ethanol (equivalent to 12.8% in volumetric percentage) was produced with the overall yield of 74.8% from cellulose and xylose, in which the xylose conversion was 73.9%, at the moderate cellulase usage of 15 mg protein per gram cellulose. For corn stover, 85.1 g/L of ethanol (equivalent to 10.8% in volumetric percentage) is produced with the overall conversion of 84.7% from cellulose and xylose, in which the xylose conversion was 87.7%, at the minimum cellulase usage of 10 mg protein per gram cellulose. Most significantly, the SSCF operation achieved the high conversion efficiency by generating the minimum amount of wastewater. Both the fermentation efficiency and the wastewater generation in the current dry biorefining for cellulosic ethanol production are very close to that of corn ethanol production, indicating that the technical gap between cellulosic ethanol and corn ethanol has been gradually filled by the advancing biorefining technology.

Engineering of Saccharomyces cerevisiae for the efficient co-utilization of glucose and xylose.

The rapid co-fermentation of both glucose and xylose is important for the efficient conversion of lignocellulose biomass into fuels and chemicals. Saccharomyces cerevisiae is considered to be a potential cell factory and has been used to produce various fuels and chemicals, but it cannot metabolize xylose, which has greatly limited the utilization of lignocellulose materials. Therefore, numerous studies have attempted to develop xylose fermenting strains in past decades. The simple introduction of the xylose metabolic pathway does not enable yeast to rapidly utilize xylose, and several limitations still need to be addressed, including glucose repression and slow xylose transport, cofactor imbalance in the xylose reductase/xylitol dehydrogenase pathway, functional expression of a heterologous xylose isomerase, the low efficiency of downstream pathways and low ethanol production. In this review, we will discuss strategies to overcome these limitations and the recent progress in engineering xylose fermenting S. cerevisiae strains.

High vanillin tolerance of an evolved Saccharomyces cerevisiae strain owing to its enhanced vanillin reduction and antioxidative capacity.

The phenolic compounds present in hydrolysates pose significant challenges for the sustainable lignocellulosic materials refining industry. Three Saccharomyces cerevisiae strains with high tolerance to lignocellulose hydrolysate were obtained through ethyl methanesulfonate mutation and adaptive evolution. Among them, strain EMV-8 exhibits specific tolerance to vanillin, a phenolic compound common in lignocellulose hydrolysate. The EMV-8 maintains a specific growth rate of 0.104 h(-1) in 2 g L(-1) vanillin, whereas the reference strain cannot grow. Physiological studies revealed that the vanillin reduction rate of EMV-8 is 1.92-fold higher than its parent strain, and the Trolox equivalent antioxidant capacity of EMV-8 is 15 % higher than its parent strain. Transcriptional analysis results confirmed an up-regulated oxidoreductase activity and antioxidant activity in this strain. Our results suggest that enhancing the antioxidant capacity and oxidoreductase activity could be a strategy to engineer S. cerevisiae for improved vanillin tolerance.

[Progress in research of pentose transporters and C6/C5 co-metabolic strains in Saccharomyces cerevisiae].

One of the requirements for increasing the economic profitability on the large-scale production of second-generation ethanol and other bio-chemicals using lignocellulose biomass as raw materials is efficient hexose and pentose utilization. Saccharomyces cerevisiae, the traditional ethanol producer, is an attractive chassis cell due to its robustness towards harsh environmental conditions and inherent advantages. But S. cerevisiae cannot utilize pentose. The precision construction of suitable strains for second-generation bio-ethanol production has been taken for more than three decades based on the principle of metabolic engineering and synthetic biology. The resulting strains have improved significantly co-fermentation of glucose and xylose. Recently, much attentions have been focused on sugar transport, which is one of the limiting but formerly ignored step for ethanol production from both glucose and xylose, to get the desired state that different sugars could efficiently delivered by their individual specific transporters. In this paper, the progress on sugar transporters of S. cerevisiae was reviewed, and the research status of xylose and/or L-arabinose metabolic engineering in S. cerevisiae were also presented.

High selective delignification using oxidative ionic liquid pretreatment at mild conditions for efficient enzymatic hydrolysis of lignocellulose.

Herein, the oxidative ionic liquid (IL) pretreatment for overcoming recalcitrance of lignocellulose with selective delignification was investigated, and the subsequent enzymatic hydrolysis was evaluated. IL pretreatment incorporating oxygen delignification could enhance lignin extraction with high selectivity at low carbohydrate loss. The dual-action of oxidative decomposition and dissolution by 1-butyl-3-methlimidazolium chloride (BmimCl) on biomass were synergistically acted, accounting for efficient recalcitrance removal. In addition, the mild oxidative IL treatment only slightly converted crystalline cellulose into amorphous structure, and the extensive extraction of the amorphous lignin and carbohydrate resulted to the expose of cellulose with high susceptibility. Correspondingly, the enzymatic hydrolysis of the pretreated lignocellulose was greatly enhanced. The oxidative IL treatment at mild conditions, collaborating BmimCl treatment with oxygen delignification is a promising and effective system for overcoming the robust structure of lignocellulose.

Fractionation of oil palm empty fruit bunch by bisulfite pretreatment for the production of bioethanol and high value products.

In this work, fractionation of empty fruit bunch (EFB) by bisulfite pretreatment was studied for the production of bioethanol and high value products to achieve biorefinery of EFB. EFB was fractionated to solid and liquor components by bisulfite process. The solid components were used for bioethanol production by quasi-simultaneous saccharification and fermentation. The liquor components were then converted to furfural by hydrolysis with sulfuric acid. Preliminary results showed that the concentration of furfural was highest at 18.8g/L with 0.75% sulfuric acid and reaction time of 25min. The conversion of xylose to furfural was 82.5%. Furthermore, we attempted to fractionate the liquor into hemicellulose sugars and lignin by different methods for producing potential chemicals, such as xylose, xylooligosaccharide, and lignosulfonate. Our research showed that the combination of bisulfite pretreatment and resin separation could effectively fractionate EFB components to produce bioethanol and other high value chemicals.

Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production.

To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h(-1) in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g(-1) consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production.

[Inhibitors and their effects on Saccharomyces cerevisiae and relevant countermeasures in bioprocess of ethanol production from lignocellulose--a review].

The pretreatment of raw materials is necessary for ethanol production from lignocellulose, however, a variety of compounds which inhibit the fermenting microorganism such as Saccharomyces cerevisiae are inevitably formed in this bioprocess. Based on their chemical properties, the inhibitors are usually divided into three major groups: weak acids, furaldehydes and phenolic compounds. These compounds negatively affect the growth of S. cerevisiae, ethanol yield and productivity, which is one of the significant hurdles for the development of large-scale ethanol production from lignocellulose. We address here the origins of the three kinds of inhibitors and their mechanisms to S. cerevisiae. We also discuss the strategies of improving the fermentation performance of yeast, including detoxification of the pretreated substrates, enhancement of yeast tolerance and also fermentation control to reduce the effects of the inhibitors. The methods used in enhancing the yeast tolerance are traditional mutagenic breeding integrated with strains evolution under the suitable selective pressure, and metabolic engineering by introducing and/or overexpressing genes encoding enzymes such as furfural reductase, laccase and phenylacrylic acid decarboxylase, that confer the S. cerevisiae strains resistance towards specific inhibitors.