Engineering Corynebacterium glutamicum for synthesis of poly(3-hydroxybutyrate) from lignocellulose biomass.

Lignocellulose is the only feasible carbohydrates feedstock for commercial scale and carbon neutral production of poly(3-hydroxybutyrate) (PHB) biopolymer by its great abundance and availability. Microbial cell factories for fermentative PHB synthesis are highly restricted by the growth suppression of inhibitors from lignocellulose pretreatment. This study targeted a potential PHB-producing cell factory Corynebacterium glutamicum owing to its strong inhibitors tolerance. A systematic metabolic engineering was conducted starting with the stable PHB synthesis pathway construction from glucose and xylose, followed by the enhancement of PHB synthesis on PHA synthase activity and stability, cell morphology modification, and growth factors regulation. The relocation of the PHA synthase on the cell membrane guided by secrete signal peptides and cell membrane display motifs increased the PHB content by 2.4 folds. Excessive nitrogen preferentially promoted the PHB synthesis capacity and resulted in the PHB content increased by 13.3 folds. Modification of the genes responsible for cell division changed the cell morphology but the cell size was not enlarged to a PHB accumulation favorable environment. The metabolic engineering of C. glutamicum resulted in a high fermentative production of PHB using wheat straw as feedstock. This study provided an important microbial cell factory choice for PHB production using lignocellulose feedstock.

Improving cellulosic ethanol fermentation efficiency by converting endogenous water-soluble carbohydrates into citric acid before pretreatment.

Water-soluble carbohydrates in raw lignocellulosic biomass are converted into hydroxymethylfurfural (HMF) in the acid-based pretreatment, thus increasing the detoxification intensity and reducing the fermentation efficiency of cellulosic ethanol. Therefore, reducing water-soluble carbohydrates in raw corn stover is crucially important to reduce the inhibitors' generation and improve the ethanol fermentation efficiency. In this study, aerobic solid-state fermentation of corn stover by inoculating Aspergillus niger spores converted 83% of the endogenous water-soluble carbohydrates into citric acid, leading to the decrease of 41% of HMF generation and 8% of sulfuric acid usage during the dry acid pretreatment. The reduced inhibitor generation improved the ethanol fermentability by 11% more ethanol than that of the corn stover without water-soluble carbohydrates' removal. This suggests that the removal of the water-soluble carbohydrates before pretreatment significantly reduced the inhibitors' generation in pretreatment and improved the fermentation efficiency of cellulosic ethanol.

Upgrading dry acid pretreatment by post-hydrolysis for carbon efficient conversion of lignocellulose.

Dry acid pretreatment (DAP) as a promising process for industrial biorefinery provide an efficient bioconversion of cellulose without free wastewater, although the partial xylan and lignin degrade to inhibitors or recondense. A biorefinery strategy for carbon efficient conversion of lignocellulose into bioethanol, xylose, and reactive lignin was developed by upgrading DAP with post-hydrolysis. The results showed that lignocellulose after mild DAP (175 °C, acid dosage of 15 mg/g dry material) obtained higher xylan recovery and lower inhibitors than that of general DAP. Subsequently, post-hydrolysis, simultaneous saccharification and ethanol fermentation were performed at solids loading of 20 wt% without detoxification and sterilization, resulting in xylose and ethanol yield of 71.8 % and 67.6 %. The fractionated lignin presented more reactive ß-aryl ether linkages and less condensation than that from DAP. 66 % of lignocellulose carbon was recovered as ethanol, xylose and reactive lignin. This upgrading biorefinery strategy provided an easy-to-operate process for integrated utilization of lignocellulose.

Enhanced cellulosic d-lactic acid production from sugarcane bagasse by pre-fermentation of water-soluble carbohydrates before acid pretreatment.

After several rounds of milling process for sugars extraction from sugarcane, certain amounts of water-soluble carbohydrates (WSC) still remain in sugarcane bagasse. It is a bottleneck to utilize WSC in sugarcane bagasse biorefinery, since these sugars are easily degraded into inhibitors during pretreatment. Herein, a simple pre-fermentation step before pretreatment was conducted, and 98 % of WSC in bagasse was fermented into d-lactic acid. The obtained d-lactic acid was stably preserved in bagasse and 5-hydroxymethylfurfural (HMF) generation was sharply reduced from 46.0 mg/g to 6.2 mg/g of dry bagasse, after dilute acid pretreatment. Consequently, a higher d-lactic acid titer (57.0 g/L vs 33.2 g/L) was achieved from the whole slurry of the undetoxified and pretreated sugarcane bagasse by one-pot simultaneous saccharification and co-fermentation (SSCF), with the overall yield of 0.58 g/g dry bagasse. This study gave an efficient strategy for enhancing lactic acid production using the lignocellulosic waste from sugar industry.

Formulating a fully converged biorefining chain with zero wastewater generation by recycling stillage liquid to dry acid pretreatment operation.

Huge wastewater generation is the major challenge of biorefinery technology for production of cellulosic ethanol. This study designed and verified a method for completely recycling of wastewater stream (the stillage liquid from the beer column) in cellulosic ethanol production by dry biorefining processing. When the stillage liquid was directly recycled to dry acid pretreatment operation, ethanol production gradually reduced after two recycles primarily because the inorganic compounds accumulated by around 139%. To ultimately solve this technical barrier, the stillage liquid was evaporated and condensed into distillated water, then recycled to the pretreatment for complete dry biorefining process. This strategy supported a stable cellulosic ethanol production, and the overall mass and heat balance confirmed that only 65% of the lignin residue consumption was used for wastewater evaporation with 35% surplus for electricity generation. This study provided a fully converged biorefining process with a closed-loop wastewater recycling.

Acidic deep eutectic solvent assisted isolation of lignin containing nanocellulose from thermomechanical pulp.

Nanocellulose is a promising material but its isolation generally requires unrecyclable hazardous chemicals and high energy consumption and its overall yield is low due to the use of high purity cellulose as precursor. In order to overcome these shortcomings, in this study, thermomechanical pulp (TMP) was investigated as a precursor for isolating lignin containing nanocellulose (LNC) using an environmentally friendly acidic deep eutectic solvent (DES) pre-treatment. Flat "ribbon" like LNCs (around 7.1 nm wide, 3.7 nm thick) with uniformly distributed lignin nanoparticles of 20-50 nm in diameter were successfully obtained at 57 % yield under optimum pre-treatment conditions (90 °C, 6 h, 1:1 oxalic acid dihydrate to choline chloride ratio). The LNCs exhibit cellulose Iß structure, high lignin content (32.6 %), and high thermal stability (Tmax of 358 °C). In general, green acidic DES pre-treatment has shown high efficiency in converting high lignin content biomass into value-added LNC, which benefits both lignocellulose utilization and environmental protection.

Microbial extraction of biotin from lignocellulose biomass and its application on glutamic acid production.

Biotin (vitamin B7) is an important nutrient for various fermentations. It is abundant in agricultural lignocellulose biomass and maintains stable in biorefinery processing chain including acid pretreatment, biodetoxification and saccharification. Here we show a microbial extraction of biotin from biotin-rich corn leaves hydrolysate. Corynebacterium glutamicum was found to have the highest biotin uptake capacity among different biotin auxotrophic microorganisms, and it was further significantly increased by overexpressing the bioYMN gene cluster encoding biotin transporter. Finally 250 folds greater biotin was extracted by recombinant C. glutamicum (303.8 mg/kg dry cell) from virgin corn leaves (1.2 mg/kg), which was far higher than that in commonly used fermentation additives including yeast extract (∼2 mg/kg), molasses (∼1 mg/kg) and corn steep liquor (∼0.75 mg/kg). The biotin extracted from corn leaves was successfully applied to glutamic acid fermentation. This is the first report on microbial extraction of biotin from lignocellulose biomass and fermentation promotion application.

Facilitation of l-Lactic Acid Fermentation by Lignocellulose Biomass Rich in Vitamin B Compounds.

Vitamins are important nutrients for many fermentations, but they are generally costly. Agricultural lignocellulose biomass contains considerable amounts of vitamin B compounds, but these water-soluble vitamins are easily lost into wastewater discharge during pretreatment or detoxification of lignocellulose in biorefinery processes. Here, we showed that the dry acid pretreatment and biodetoxification process allowed the preservation of significant amounts of vitamin B, which promoted l-lactic acid fermentation efficiency significantly. Supplementation with specific vitamin B compounds, VB3 and VB5, into corn stover hydrolysate led to further increases of cellulosic l-lactic acid yield and fermentation rates. This study provided a new solution for the enhancement of biorefinery fermentation efficiency by using vitamin B compounds in lignocellulose biomass.

An Approach of Utilizing Water-Soluble Carbohydrates in Lignocellulose Feedstock for Promotion of Cellulosic l-Lactic Acid Production.

Agricultural lignocellulose biomass generally contains certain amounts of water-soluble carbohydrates (WSC) such as glucose, fructose, or sucrose. These sugars are generally degraded in pretreatment at high temperature or discharged with wastewater in a detoxification process. This study proposed an approach of utilizing frequently ignored water-soluble carbohydrates for promotion of cellulosic l-lactic acid production. A simple solid state fermentation was performed during a corn stover storage period to convert the sugars into l-lactic acid and then a dry biorefining technology was applied to convert cellulose and hemicellulose fractions into the same l-lactic acid product. The 5-hydroxymethylfurfural (HMF) formation in pretreatment was significantly reduced and the consequent biodetoxification time was shortened. l-Lactic acid production was increased from 130.2 g/L to 139.0 g/L, and the minimum l-lactic acid selling price was reduced by 5.9%. This study provided an important option of biorefinery processing technology for production of value added biochemicals.

Oxidative production of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth by Gluconobacter oxydans.

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 59.80g/L of ethanol (no xylose utilization). 65.39g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42g/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.