d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

Biomass Production and Composition of Perennial Grasses Grown for Bioenergy in a Subtropical Climate Across Florida, USA

Carbohydrate and lignin composition of feedstock materials are major factors in determining their bioenergy potential. This study was conducted to quantify dry biomass yield and the carbohydrate and lignin composition of six potential biofuel grasses (elephantgrass, energycane, sweetcane, giant reed, giant miscanthus, and sugarcane) across three sites in Florida for plant (2009) and first ratoon (2010) crops. Dry biomass yields ranged from about 30 to 50 Mg ha^?1 and were generally greatest for elephantgrass, energycane, sweetcane, and sugarcane. Accordingly, total plant carbohydrate yields (20 to 25 Mg ha^?1) were comparable among sugarcane, energycane, sweetcane, and elephantgrass, but were generally less for giant reed and even less for giant miscanthus. However, the contribution of total extractable carbohydrates and total fiber carbohydrates to total plant carbohydrate yields differed among species. Sugarcane had the highest concentrations of extractable carbohydrates (219 to 356 mg g^?1), followed by energycane, then sweetcane, elephantgrass, and giant reed, with giant miscanthus having the lowest. Energycane and elephantgrass tended to have significantly more fiber glucose, and elephantgrass less xylose, than other species. Variability in total lignin concentrations on a fiber basis was relatively modest (250 to 285 mg g^?1) across species, but was generally highest in sweetcane and giant reed. Overall, elephantgrass and energycane were prime regional candidates for cellulosic conversion using fermentation processes due to high yields and favorable fiber characteristics, although energycane tended to have higher extractable carbohydrates.     

Biomimetic catalysis for hemicellulose hydrolysis in corn stover

Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3-10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach approximately 95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan).     

Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation

Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass‐to‐ethanol pipeline. Here, the feasibility of scaling‐up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H₂O₂/g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose‐ and xylose‐utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922–931. © 2011 Wiley Periodicals, Inc.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Fermentation of lignocellulosic sugars to acetic acid by <Emphasis Type="Italic">Moorella thermoacetica</Emphasis>

A systematic study of bioconversion of lignocellulosic sugars to acetic acid by Moorella thermoacetica (strain ATCC 39073) was conducted. Four different water-soluble fractions (hydrolysates) obtained after steam pretreatment of lignocellulosic biomass were selected and fermented to acetic acid in batch fermentations. M. thermoacetica can effectively ferment xylose and glucose in hydrolysates from wheat straw, forest residues, switchgrass, and sugarcane straw to acetic acid. Xylose and glucose were completely utilized, with xylose being consumed first. M. thermoacetica consumed up to 62 % of arabinose, 49 % galactose and 66 % of mannose within 72 h of fermentation in the mixture of lignocellulosic sugars. The highest acetic acid yield was obtained from sugarcane straw hydrolysate, with 71 % of theoretical yield based on total sugars (17 g/L acetic acid from 24 g/L total sugars). The lowest acetic acid yield was observed in forest residues hydrolysate, with 39 % of theoretical yield based on total sugars (18 g/L acetic acid from 49 g/L total sugars). Process derived compounds from steam explosion pretreatment, including 5-hydroxymethylfurfural (0.4 g/L), furfural (0.1 g/L) and total phenolics (3 g/L), did not inhibit microbial growth and acetic acid production yield. This research identified two major factors that adversely affected acetic acid yield in all hydrolysates, especially in forest residues: (i) glucose to xylose ratio and (ii) incomplete consumption of arabinose, galactose and mannose. For efficient bioconversion of lignocellulosic sugars to acetic acid, it is imperative to have an appropriate balance of sugars in a hydrolysate. Hence, the choice of lignocellulosic biomass and steam pretreatment design are fundamental steps for the industrial application of this process.     

Tissue chemistry and morphology affect root decomposition of perennial bioenergy grasses on sandy soil in a sub‐tropical environment

Second‐generation biofuels and bio‐based products derived from lignocellulosic biomass are likely to replace current fuels derived from simple sugars and starch because of greater yield potential and less competition with food production. Besides the high aboveground biomass production, these bioenergy grasses also exhibit extensive root systems. The decomposition of root biomass greatly influences nutrient cycling and microbial activity and subsequent accumulation of carbon (C) in the soil. The objective of this research was thus to characterize root morphological and chemical differences in six perennial grass species in order to better understand root decomposition and belowground C cycling of these bioenergy cropping systems. Giant reed (Arundo donax), elephantgrass (Pennisetum purpureum), energycane (Saccharum spp.), sugarcane (Saccharum spp.), sweetcane (Saccharum arundinaceum), and giant miscanthus (Miscanthus × giganteus) were established in Fall 2008 in research plots near Gainesville, Florida. Root decomposition rates were measured in situ from root decomposition bags over 12 months along with initial and final root tissue composition. Root potential decomposition rate constant (K) was higher in elephantgrass (3.64 g kg^?1 day^?1) and sweetcane (2.77 g kg^?1 day^?1) than in sugarcane (1.62 g kg^?1 day^?1) and energycane (1.48 g kg^?1 day^?1). Notably, K was positively related to initial root tissue total C (Total C), total fiber glucose (TFG), total fiber xylose (TFX), and total fiber carbohydrate (TFC) concentrations, but negatively related to total fiber arabinose (TFA) and lignin (TL) concentrations and specific root volume (SRV). Among the six species, elephantgrass exhibited root traits most favorable for fast decomposition: high TFG, high TFX, high TFC, high specific root length (SRL), and a low SRV, whereas giant reed, sugarcane, and energycane exhibited slow decomposition rates and the corresponding root traits. Thus, despite similar aboveground biomass yields in many cases, these species are likely to differentially affect soil C accumulation.     

Bioethanol production from dedicated energy crops and residues in Arkansas, USA

Globally, one of the major technologic goals is to achieve cost-effective lignocellulosic ethanol production from biomass feedstocks. Lignocellulosic biomass of four dedicated energy crops [giant reed (Arundo donax L.), elephantgrass (Pennisetum purpureum (Schumach), Miscanthus × giganteus (Illinois clone), and (clone Q42641) {hybrid of Miscanthus sinensis Anderss. and Miscanthus sacchariflorus (Maxim)}, Hack. called giant miscanthus, and sugarcane clone US 84-1028 (Saccharum L. spp. hybrid)] and residues from two crops [soybean (Glycine max (L.) Merr.) litter and rice (Oryza sativa L.) husk] were tested for bioethanol production using cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and enzymatic (cellulase) hydrolysis. Giant miscanthus (Illinois), giant reed, giant miscanthus (Q42641), elephantgrass, and sugarcane all yielded higher amount of glucose on a biomass dry weight basis (0.290-0.331 g/g), than did rice husk (0.181 g/g) and soybean litter (0.186 g/g). To reduce the capital investment for energy consumption in fermentation, we used a self-flocculating yeast strain (SPSC01) to ferment the lignocellulosic biomass hydrolysates. Bioethanol production was ～0.1 g/g in dedicated energy crops and less in two crop residues. These methods and data can help to develop a cost-effective downstream process for bioethanol production.     

Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Engineering of Klebsiella oxytoca for production of 2,3‐butanediol using mixed sugars derived from lignocellulosic hydrolysates

2,3‐Butanediol (2,3‐BDO) is a promising bulk chemical owing to its high potential in industrial applications. Here, we engineered Klebsiella oxytoca for the economic production of 2,3‐BDO using mixed sugars from renewable biomass. First, to improve xylose consumption, the xylose transporter gene (xylE) was integrated into the methylglyoxal synthase A (mgsA)‐coding gene loci, and the engineered CHA004 strain showed much faster consumption of xylose than wild‐type (WT) strain with 1.4‐fold increase of overall sugar consumption rate. To further improve sugar utilization, we performed adaptive laboratory evolution for 90 days. The evolved strain (CHA006) was evaluated by cultivating it in the media containing single‐ or mixed‐sugars, and it was clearly observed that CHA006 has improved sugar consumption and 2,3‐BDO production than those of the parental strain. Finally, we demonstrated the superiority of CHA006 by culturing in two lignocellulosic hydrolysates derived from sunflower or pine tree. Particularly, in the pine tree hydrolysate containing xylose, glucose, galactose, and mannose, the CHA006 strain showed much improved consumption rates for all sugars, and 2,3‐BDO productivity (0.73 g L^?1 hr^?1) increased by 3.2‐fold compared to WT strain. We believe that the engineered CHA006 strain can be a potential host in the development of economic bioprocess for 2,3‐BDO through efficient utilization of mixed sugars derived from lignocellulosic biomass.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.     

Consolidated Pretreatment and Hydrolysis of Plant Biomass Expressing Cell Wall Degrading Enzymes

Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.     

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr⁻¹. The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr⁻¹, strain QU 50 produced 42.6 g L⁻¹ of l -LA with a yield of 0.912 g g⁻¹-consumed sugars, LA yield of 0.655 g g⁻¹ based on mixed sugar-loaded, and a productivity of 4.31 g L⁻¹ hr⁻¹ from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L⁻¹ with LA yield of 0.944 g g⁻¹-consumed sugar and LA productivity of 6.49 ± 0.357 g L⁻¹ hr⁻¹. Besides this, a dramatic increase in LA yield of 0.927 g g⁻¹ based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.     

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

Candida shehatae and Saccharomyces cerevisiae work synergistically to improve ethanol fermentation from sugarcane bagasse and rice straw hydrolysate in immobilized cell bioreactor

Sugarcane bagasse (SCB) and rice straw (RS), abundant lignocellulosic agro‐industrial residues in South‐East Asia, are potent feedstocks for bioethanol production as they contain significant amount of glucose and xylose monomers after fractionation and subsequent enzymatic hydrolysis. To simultaneously convert glucose and xylose to ethanol, it requires co‐cultivation of Saccharomyces cerevisiae and Candida shehatae which are hexose and pentose‐fermenting yeasts, respectively. Xylose‐fermenting strain grows slower than glucose‐fermenting one, therefore low efficiency of xylose‐to‐ethanol conversion was found. To enhance the efficiency of ethanol fermentation, the present work proposed to improve xylose assimilation by using co‐immobilization of two strains in a packed bed bioreactor and to increase oxygenation of the medium by applying a recycled batch system when the recycle stream was intervened by a mixing system in a naturally aerated vessel. Initially, conversion of glucose and xylose to ethanol using pure culture was investigated. Subsequently, influence of different immobilization techniques was investigated. Cells entrapment in Ca‐alginate beads provided considerably high ethanol yield over cells immobilized on delignified cellulose, and thus it was selected to use as inoculum in an immobilized cell bioreactor (ICB). The results showed that continuous ethanol production yielded 0.38 and 0.40 g/g corresponding to 74.5% and 78.4% theoretical yields from SCB and RS hydrolysate, respectively. However, recycled batch system produced significantly improved ethanol yield to 0.49 g/g and 0.50 g/g corresponding to 96.1% and 98.0% theoretical yields for SCB and RS hydrolysate, respectively. In this study, higher ethanol concentration and less unfermented sugar concentration was successfully achieved in the ICB with recycled batch system when using SCB and RS hydrolysate as the substrate.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.