Serious corrosion of reactor metal walls by aqueous dilute acid solution is one of the essential drawbacks of dilute acid pretreatment (DAP) in the biorefining of lignocellulose biomass. In this study, the corrosion of pretreatment reactor metal walls was significantly reduced by the dry dilute acid pretreatment (DDAP) of corn stover, a modified DAP, in which the aqueous dilute acid solution is completely absorbed by high solids corn stover feedstock. The iron ion release from the stainless-steel walls of the reactor was measured, and the corrosion degree was quantitatively characterized. The reactor corrosion was reduced by approximately 85% when the solid/liquid ratio increased from the conventional value of 1:10 to the high value of 1:0.5 at the same sulfuric acid concentration in the DDAP. The results provided the simple and practical solution to the key technical barrier of DAP and paved the way for large-scale application.

The high cost of the lignocellulose-degrading enzyme is one of the major bottlenecks for economically producing cellulosic ethanol. To overcome this bottleneck, we performed in-situ vacuum distillation (at 50 °C, 77 mmHg for 30 min) to remove ethanol after simultaneous saccharification and fermentation (SSF) of delignified corncob residues (DCCR). The cellulase and yeast cells left in the stillage were reused by adding fresh DCCR to start a new cycle. This process cycle was repeated five times. Both cellulase and yeast cells were reused five times with a significant cellulase reduction from 16.3 mg of enzyme protein (EP)/g cellulose to as low as 3.3 mg EP/g cellulose. Meanwhile the ethanol in the fermentation broth was maintained as high as 40 g/L before each vacuum distillation. The final ethanol yield after five cycles reached 72.7%. The process economic analysis based on the Aspen Plus model indicated that the cost of the saved cellulase was much higher than that of the additional energy needed to carry out the vacuum distillation. Almost 24% of the ethanol production cost could be reduced by using this new approach compared to batch SSF without cellulase recycling.Keywords: Biofuel; Delignified corncob residues (DCCR); Economic analysis; Enzyme recycling; Ethanol; Microbial fermentation; Vacuum distillation; Yeast recycling;

High product concentration in aerobic biorefining fermentation requires high lignocellulose feedstock loading. However, the high solids content and the consequent high viscosity of the lignocellulose hydrolysate severely limit oxygen transfer and thus lead to low aerobic fermentation rate. This study first investigates the experimental measurement and the computational fluid dynamics (CFD)-based simulation of oxygen transfer properties in the high solids loading and highly viscous lignocellulose hydrolysates. The oxygen mass transfer coefficients kLa values vary with various biorefining processing parameters. A minimum threshold kLa value was required for aerobic fermentation of glucose oxidation into gluconic acid. The rheological properties of the slurry were experimentally determined, and a CFD model was established for the design of aerobic fermentation bioreactors. The kLa values between the CFD calculation and the experimental determination were in good agreement for the high solids loading and highly viscous hydrolysate slurry. The study provided important information and useful tools for achieving efficient aerobic fermentations in high solids loading and highly viscous lignocellulose hydrolysates. The measured kLa value could be applied to general aerobic fermentation using lignocellulose feedstock.Keywords: Aerobic fermentation; Computational fluid dynamics (CFD); High solids loading; High viscosity; Lignocellulose; Oxygen transfer rate;

•Xylose-assimilating pathways were constructed in the chromosome of Pediococcus acidilactici.•Xylose assimilation was significantly accelerated by adaptive evolution.•High corn stover solids content SSCF was conducted.•97.3 g/L of d-lactic acid with 92.6% of xylose conversion ratio was obtained using corn stover.Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3 g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation.Download high-res image (156KB)Download full-size image

Cellulase enzymes contribute to the largest portion of operation cost on production of cellulosic ethanol. The industrial cellulases available on the industrial enzyme market from different makers and sources vary significantly in hydrolysis and ethanol, and finally lead to the changes of enzyme cost. Therefore, the selection of the proper industrial cellulase enzymes for commercial-scale production of cellulosic ethanol is crucially important in terms of high performance and cost reduction.In this study, three major cellulase enzyme products available on the Chinese industrial enzyme market were selected and evaluated as the biocatalysts for the biorefining process of lignocellulose biomass into high-titer ethanol. The cellulase enzymes included Cellic CTec 2.0 from Novozymes (Beijing), and LLC 4 from Vland (Qingdao), as well as # 7 from an industrial enzyme maker. The detailed assays on the filter paper activity, the cellobiase activity, and the total protein contents of the enzymes were conducted according to the standard protocols. When the cellulase enzymes were applied to the practical hydrolysis and ethanol-fermentation operation under the conditions of high solids loading and low range of cellulase dosage, the hydrolysis yield shows the significant difference, and the difference was narrowed in the final ethanol yield.The commercially available cellulase enzymes showed different performances in the activities, the cellulose hydrolysis yield, and the ethanol fermentation yields based on the protein dosage per gram of cellulose of corn stover. In general, the industrial cellulase products give satisfactory performance and can be applied for the practical cellulosic ethanol production on commercial scale.

•DryPB significantly reduces wastewater generation and energy consumption.•DryPB significantly reduces the MESP of cellulosic ethanol close to corn ethanol.•DryPB significantly elevates the commercial potential of cellulosic ethanol.•Technical and economical gap between cellulosic and corn ethanol is almost filled up.Energy consumption and wastewater generation in cellulosic ethanol production are among the determinant factors on overall cost and technology penetration into fuel ethanol industry. This study analyzed the energy consumption and wastewater generation by the new biorefining process technology, dry acid pretreatment and biodetoxification (DryPB), as well as by the current mainstream technologies. DryPB minimizes the steam consumption to 8.63 GJ and wastewater generation to 7.71 tons in the core steps of biorefining process for production of one metric ton of ethanol, close to 7.83 GJ and 8.33 tons in corn ethanol production, respectively. The relatively higher electricity consumption is compensated by large electricity surplus from lignin residue combustion. The minimum ethanol selling price (MESP) by DryPB is below $2/gal and falls into the range of corn ethanol production cost. The work indicates that the technical and economical gap between cellulosic ethanol and corn ethanol has been almost filled up.Download high-res image (113KB)Download full-size image

Furfural and 5-hydroxymethylfurfural (HMF) are the two major furan aldehyde inhibitors generated from lignocellulose dilute acid pretreatment which significantly inhibit subsequent microbial cell growth and ethanol fermentation. Zymomonas mobilis is an important strain for cellulosic ethanol fermentation but can be severely inhibited by furfural and (or) HMF. Previous study showed that Z. mobilis contains its native oxidoreductases to catalyze the conversion of furfural and HMF, but the corresponding genes have not been identified.This study identified a NADPH-dependent alcohol dehydrogenase gene ZMO1771 from Z. mobilis ZM4, which is responsible for the efficient reduction of furfural and HMF. Over-expression of ZMO1771 in Z. mobilis significantly increased the conversion rate to both furfural and HMF and resulted in an accelerated cell growth and improved ethanol productivity in corn stover hydrolysate. Further, the ethanol fermentation performance was enhanced again by co-expression of the transhydrogenase gene udhA with ZMO1771 by elevating the NADPH availability.A genetically modified Z. mobilis by co-expressing alcohol dehydrogenase gene ZMO1771 with transhydrogenase gene udhA showed enhanced conversion rate of furfural and HMF and accelerated ethanol fermentability from lignocellulosic hydrolysate. The results presented in this study provide an important method on constructing robust strains for efficient ethanol fermentation from lignocellulose feedstock.

Cost reduction on cellulase enzyme usage has been the central effort in the commercialization of fuel ethanol production from lignocellulose biomass. Therefore, establishing an accurate evaluation method on cellulase enzyme cost is crucially important to support the health development of the future biorefinery industry. Currently, the cellulase cost evaluation methods were complicated and various controversial or even conflict results were presented. To give a reliable evaluation on this important topic, a rigorous analysis based on the Aspen Plus flowsheet simulation in the commercial scale ethanol plant was proposed in this study. The minimum ethanol selling price (MESP) was used as the indicator to show the impacts of varying enzyme supply modes, enzyme prices, process parameters, as well as enzyme loading on the enzyme cost. The results reveal that the enzyme cost drives the cellulosic ethanol price below the minimum profit point when the enzyme is purchased from the current industrial enzyme market. An innovative production of cellulase enzyme such as on-site enzyme production should be explored and tested in the industrial scale to yield an economically sound enzyme supply for the future cellulosic ethanol production.

Particle size of lignocellulose materials is an important factor for enzymatic hydrolysis efficiency. In this study, corn stover was milled and sieved into different size fractions from 1.42, 0.69, 0.34, to 0.21 mm, and the corresponding enzymatic hydrolysis yields were 24.69, 23.96, 25.34, and 26.97 %, respectively. The results indicate that the hydrolysis yield is approximately constant with changing corn stover particle sizes in the experimental range. The overall surface area and the inner pore size measurement show that the overall specific surface area was less than 2 % with the half reduction of particle size due to the greater inner pore surface area. The scanning electron microscope photographs gave direct evidence of the much greater inner pore surface area of corn stover particles. This result provided a reference when a proper size reduction of lignocellulose materials is considered in biorefining operations.

Oleaginous yeast Trichosporon cutaneum CGMCC 2.1374 was found to utilize inulin directly for microbial lipid fermentation without a hydrolysis step. The potential inulinase-like enzyme(s) in T. cutaneum CGMCC 2.1374 were characterized and compared with other inulinase enzymes produced by varied yeast strains. The consolidated bioprocessing (CBP) for lipid accumulated using inulin was optimized with 4.79 g/L of lipid produced from 50 g/L inulin with the lipid content of 33.6 % in dry cells. The molecular weight of the enzyme was measured which was close to invertase in Saccharomyces cerevisiae. The study provided information for inulin hydrolyzing enzyme(s) in oleaginous yeasts, as well as a preliminary CBP process for lipid production from inulin feedstock.

β-1,3-1,4-glucanase is a widely used enzyme in brewing and in animal feed processing. To produce the bacterial enzyme at an industrial scale, the enzyme should be able to be secreted from microbial cells into fermentation broth and be stable in different conditions. In this study, the LicB gene encoding β-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum was secretively expressed in a secretive strain, Bacillus subtilis WB800, with eight extracellular protease deletion which made LicB expressed obviously and reached 1.18 U/g cell mass. The secreted β-1,3-1,4-glucanase was found to be active from 40 °C to 80 °C and achieved the optimal activity at 80 °C. The enzyme also has a wide pH range (pH 4–11). The most common metal ions and chemicals were found to be inert on its activity. The property of LicB-encoded β-1,3-1,4-glucanase and its efficient secretive expression makes it a potential enzyme for industrial production and application.

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.

Biological pretreatment is a promising way to overcome the biorecalcitrance of cleaving the supermolecular structure of lignocellulose by lignin degrading enzymes from microorganisms. Solid state fermentation of corn stover with the white-rot fungus Phanerochaete chrysosporium was carried out and the efficiency of this pretreatment was evaluated. The enzymatic hydrolysis yield reached a maximum when the corn stover was biologically pretreated for nine days, and the hydrolysis yield decreased sharply if the solid state fermentation was carried out for more than nine days. A possible explanation for this sharp decrease is that not only the lignin degrading enzymes (LiP and MnP) were secreted, but also other metabolites, which were toxic or fatal to the hydrolysis enzymes resulting in the lower hydrolysis yield were generated during the prolonged period of biopretreatment. These results are useful to help determine the optimal timing and to understand the lignin structure and degradation mechanism in biological pretreatment processes.

Large waste water disposal was the major problem in microbial lipid fermentation because of low yield of lipid. In this study, the repeated batch fermentation was investigated for reducing waste water generated in the lipid fermentation of an oleaginous yeast Trichosporon cutaneum CX1 strain. The waste fermentation broth was recycled in the next batch operation after the cells were separated using two different methods, centrifugation and flocculation. Two different sugar substrates, glucose and inulin, were applied to the proposed operation. The result showed that at least 70% of the waste water was reduced, while lipid production maintained satisfactory in the initial four cycles. Furthermore, it is suggested that T. cutaneum CX1 cells might produce certain naturally occurring inulin hydrolyzing enzyme(s) for obtaining fructose and glucose from inulin directly. Our method provided a practical option for reducing the waste water generated from microbial lipid fermentation.

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30 % solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48 g/L and yield of 65.6 %. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.

Dry distiller’s grain and solubles (DDGS) is a major by-product of corn-based ethanol production and is usually used as animal feed. Here, it was added to the simultaneous saccharification and ethanol fermentation (SSF) carried out at high solids loading of steam explosion pretreated corn stover using a mutant strain Saccharomyces cerevisiae DQ1. The performance of SSF process with DDGS was comparable to those using the expensive yeast extract supplementation. With 30% (w/w) solids plus the addition of cellulase and 1 g DDGS l−1, the final ethanol reached 55 g l−1 (7% v/v). The results indicated that the expensive supplement of yeast extract could be replaced by DDGS.

Degradation of the toxic compounds generated in the harsh pretreatment of lignocellulose is an inevitable step in reducing the toxin level for conducting practical enzymatic hydrolysis and ethanol fermentation processes. Various detoxification methods have been tried and many negative outcomes were found using these methods, such as the massive freshwater usage and wastewater generation, loss of the fine lignocellulose particles and fermentative sugars and incomplete removal of inhibitors. An alternate method, biodetoxification, which degrades the toxins as part of their normal metabolism, was considered a promising option for the removal of toxins without causing the above problems.A kerosene fungus strain, Amorphotheca resinae ZN1, was isolated from the microbial community growing on the pretreated corn stover material. The degradation of the toxins as well as the lignocelluloses-derived sugars was characterized in different ways, and the results show that A. resinae ZN1 utilized each of these toxins and sugars as the sole carbon sources efficiently and grew quickly on the toxins. It was found that the solid-state culture of A. resinae ZN1 on various pretreated lignocellulose feedstocks such as corn stover, wheat straw, rice straw, cotton stalk and rape straw degraded all kinds of toxins quickly and efficiently. The consequent simultaneous saccharification and ethanol fermentation was performed at the 30% (wt/wt) solid loading of the detoxified lignocellulosic feedstocks without a sterilization step, and the ethanol titer in the fermentation broth reached above 40 g/L using food crop residues as feedstocks.The advantages of the present biodetoxification by A. resinae ZN1 over the known detoxification methods include zero energy input, zero wastewater generation, complete toxin degradation, processing on solid pretreated material, no need for sterilization and a wide lignocellulose feedstock spectrum. These advantages make it possible for industrial applications with fast and efficient biodetoxification to remove toxins generated during intensive lignocellulose pretreatment.

High cost of triacylglycerol lipid feedstock is the major barrier for commercial production of biodiesel. The fermentation of oleaginous yeasts for lipid production using lignocellulose biomass provides a practical option with high economic competitiveness. In this paper, the typical oleaginous yeast strains were screened under the pressure of lignocellulose degradation compounds for selection of the optimal strains tolerant to lignocellulose. The inhibitory effect of lignocellulose degradation products on the oleaginous yeast fermentation was carefully investigated. Preliminary screening was carried out in the minimum nutritious medium without adding any expensive complex ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute sulfuric acid. Seven typical lignocellulose degradation products formed in various pretreatment and hydrolysis processing were selected as the model inhibitors, including three organic acids, two furan compounds, and two phenol derivatives. The inhibition of the degradation compounds on the cell growth and lipid productivity of the selected oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors. Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose degradation compounds.

This study examined the inhibition performance by the major lignocellulose degradation products, including organic acids, furan derivatives, lignin derivatives, and ethanol, on a broadly used commercial cellulase enzyme Spezyme CP (Genencor International, Rochester, NY, USA) to cellulose hydrolysis at both the well-mixing state (shaking flask) and the static state (test tube). The cellulase activity in the cellulase complex of Spezyme CP was assumed to be one single “cellulase”, and the apparent kinetic parameters of this cellulase enzyme were measured as an approximate index of the inhibitory effect to the industrial cellulase enzyme. The inhibition performance of these degradation products was compared and analyzed using the determined apparent kinetic parameters. All the degradation products strongly inhibit the cellulose hydrolysis by cellulase enzyme, and the inhibitions on cellulase were all competitive type. The order of the inhibition strength by the lignocellulose degradation products to cellulase is lignin derivatives > furan derivatives > organic acids > ethanol. This study gave a quantitative view to the enzymatic hydrolysis of lignocellulose under the inhibition performance of the lignocellulose degradation products and will help to understand the lignocellulose recalcitrance to enzyme hydrolysis.

•A novel process for short-chain polyols production from corn stover was developed.•Purification by decolorization and desalting worked well for stover sugars.•Hydrogenolysis of stover sugars to polyols was competitive to corn-based polyols.Currently short-chain polyols such as ethanediol, propanediol, and butanediol are produced either from the petroleum feedstock or from the starch-based food crop feedstock. In this study, a combinational process of enzymatic hydrolysis with catalytic hydrogenolysis for short-chain polyols production using corn stover as feedstock was developed. The enzymatic hydrolysis of the pretreated corn stover was optimized to produce stover sugars at the minimum cost. Then the stover sugars were purified and hydrogenolyzed into polyols products catalyzed by Raney nickel catalyst. The results show that the yield of short-chain polyols from the stover sugars was comparable to that of the corn-based glucose. The present study provided an important prototype for polyols production from lignocellulose to replace the petroleum- or corn-based polyols for future industrial applications. Download full-size image

•High citric acid accumulation was obtained from corn stover feedstock.•100.04 g/L of citric acid with yield of 94.11% were obtained from corn stover.•No extra inorganic salts or inducers are required to corn stover hydrolysate.•Zero waste water was generated from pretreatment to citric acid fermentation.The aim of this work is to study the citric acid fermentation by a robust strain Aspergillus niger SIIM M288 using corn stover feedstock after dry dilute sulfuric acid pretreatment and biodetoxification. Citric acid at 100.04 g/L with the yield of 94.11% was obtained, which are comparable to the starch or sucrose based citric acid fermentation. No free wastewater was generated in the overall process from the pretreatment to citric acid fermentation. Abundant divalent metal ions as well as high titer of potassium, phosphate, and nitrogen were found in corn stover hydrolysate. Further addition of extra nutrients showed no impact on increasing citric acid formation except minimum nitrogen source was required. Various fermentation parameters were tested and only minimum regulation was required during the fermentation. This study provided a biorefining process for citric acid fermentation from lignocellulose feedstock with the maximum citric acid titer and yield.Download high-res image (86KB)Download full-size image

•Dry dilute acid pretreatment and biodetoxification are applied on corn stover biorefining.•75.22 g/L of xylonic acid is produced using xylose in cellulosic ethanol distillation stillage.•59.80 g/L of ethanol is also obtained before xylose fermentation.•Significant reduction of wastewater generation and energy consumption are achieved.•Aspen Plus modeling is conducted on the flowsheet simulation of the proposed process.An oxidative production process of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth was designed, experimentally investigated, and evaluated. Dry dilute acid pretreated and biodetoxified corn stover was simultaneously saccharified and fermented into 59.80 g/L of ethanol (no xylose utilization). 65.39 g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42 g/L of xylonic acid by Gluconobacter oxydans. The rigorous Aspen Plus modeling shows that the wastewater generation and energy consumption was significantly reduced comparing to the previous xylonic acid production process using xylose in pretreatment liquid. This study provided a practical process option for xylonic acid production from lignocellulose feedstock with significant reduction of wastewater and energy consumption.Download high-res image (158KB)Download full-size image

Directed regulation of the promiscuity of pyruvate kinase (PK) on different deoxynucleoside diphosphate (dNDP) substrates is an effective way to improve the process efficiency of biosynthesis of deoxynucleoside triphosphates (dNTP). The rabbit muscle PK was found to be highly specific on adenosine diphosphate (ADP) substrate, and the activity on dNDP substrates were reduced significantly. It was deduced that the bacteria PKs might have better promiscuity on dNDP substrates because of the less demand for fast energy conversion from ADP to ATP by PK. Two bacteria sourced PK genes from Bacillus sp. ATCC 21616 and Zymomonas mobilis ATCC 31821 were cloned and expressed in the E. coli strain BL21 (DE3). The results showed that the promiscuity of B. sp. and Z. mobilis PKs on dNDP substrates was improved significantly, which is in agreement with the deduction of bacteria PKs have better substrate promiscuity. The maximum reaction velocities and the Michaelis constants of Z. mobilis and B. sp. PKs on dNDP substrates were within one order of magnitude difference, respectively, comparing to two order of magnitude difference for rabbit muscle PK. The dNDP conversion experiments showed that the process efficiency was improved when the bacteria PKs were used for the dNTP biosynthesis.

•Oleaginous yeast T. cutaneum CGMCC 2.1374 possesses excellent inhibitor tolerance.•The genome benefits for mechanism of lipid production and inhibitor degradation.•The genome improves microbial lipid fermentation using lignocellulose biomass.Oleaginous yeast Trichosporon cutaneum demonstrated excellent lipid accumulation performance and inhibitor tolerance derived from lignocellulose pretreatment. Here we firstly report a 30.45 Mb assembly genome of T. cutaneum ACCC 20271 for understanding the microbial lipid biosynthesis and mechanism of inhibitor tolerance and degradation.

A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose–fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml−1, and 0.70 U mg−1, respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn2+ was found to be most effective by inhibiting the Entner–Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 g l−1 and the sorbitol yield was increased to almost 100% from 89%. The Ca2+ enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier.

•Two robust P. acidilactici strains for l-/d-lactic acid production using lignocellulose biomass as feedstock were constructed.•Over 76 g L−1 of l-/d-lactic acid was produced from 25% (w/w) corn stover content.•l-/d-Lactic acid yield and productivity using lignocellulose as feedstock highly depend on the lignocellulose derived inhibitor levels.Pediococcus acidilactici TY112 producing l-lactic acid and P. acidilactici ZP26 producing d-lactic acid, were engineered from the wild-type P. acidilactici DQ2 by ldhD or ldh gene disruption, and the robustness of the wild-type strain to the inhibitors derived from lignocellulose pretreatment was maintained well. In simultaneous saccharification and fermentation (SSF), 77.66 g L−1 of l-lactic acid and 76.76 g L−1 of d-lactic acid were obtained at 25% (w/w) solids content of dry dilute acid pretreated and biodetoxified corn stover feedstock. l- and d-Lactic acid yield and productivity were highly dependent on the inhibitor removal extent due to the significant down-regulation on the expressions of ldh and ldhD encoding lactate dehydrogenase by inhibitor, especially syringaldehyde and vanillin at the low concentrations. This study provided a prototype of industrial process for high titer l- and d-lactic acid production from lignocellulose feedstock.Download full-size image