Search

Agricultural residues (e.g. corn stover (CS)) representing a huge lignocellulosic biomass waste are regarded as a promising renewable resource that can be converted to fuels and chemicals via biochemical route. Nevertheless, the unfavorable properties, such as low bulk density, contamination by microorganisms, fluffy and thereby difficult to handle, cause huge problems for biomass logistics and biomass conversion. Furthermore, traditional biomass pretreatment methods often use severe conditions, consume much energy, difficult to scale up and generate a feedstock with many toxic degradation products that inhibit fermentation. In this study, we developed a novel, low-cost and easy-to-implement pretreatment method: “Densifying Lignocellulosic biomass with acidic Chemicals (DLC)” on CS. The DLC-CS owning a uniform shape showed a bulk density 4 times higher compared to loose CS and was highly resistant to microbial contamination, which greatly facilitates biomass handling, transportation and storage. DLC-CS after regular steam autoclave treatment at 121 ○C exhibited high enzymatic digestibility and much higher fermentability compared to traditional dilute acid pretreatment. An ethanol titer as high as 68.1 g/L was achieved without washing or detoxification of the pretreated biomass. The superior performances of DLC for biomass logistics and biomass conversion render it very promising for industrial use.

Heparinases (Hepases) are critical tools for the studies of highly heterogeneous heparin (HP)/heparan sulfate (HS). However, exolytic heparinases urgently needed for the sequencing of HP/HS chains remain undiscovered. Herein, a type of exolytic heparinases (exoHepases) is identified from the genomes of different bacteria. These exoHepases share almost no homology with known Hepases and prefer to digest HP rather than HS chains by sequentially releasing unsaturated disaccharides from their reducing ends. The structural study of an exoHepase (BIexoHep) shows that an N-terminal conserved DUF4962 superfamily domain is essential to the enzyme activities of these exoHepases, which is involved in the formation of a unique L-shaped catalytic cavity controlling the sequential digestion of substrates through electrostatic interactions. Further, several HP octasaccharides have been preliminarily sequenced by using BIexoHep. Overall, this study fills the research gap of exoHepases and provides urgently needed tools for the structural and functional studies of HP/HS chains., Exolytic heparinases are needed for sequencing of heparin and heparan sulfate (HP), but have not yet been reported. Here, the authors identify exolytic heparinases from different bacteria and show that the heparinases preferentially digest HP, determine the crystal structure of the exoheparinase BlexoHep and perform sequencing of HP octasaccharides using the enzyme.

Cellulosic ethanol has attracted much attention as it is of great benefit to social and environmental sustainability. However, the adverse properties of lignocellulosic biomass (i.e. low bulk density, fluffy/difficult to handle, and being easily contaminated by microbes), and issues related to biomass pretreatment (i.e. high energy consumption, difficulty in scale-up and low fermentability of pretreated biomass), largely impede the commercialization of cellulosic ethanol. To address these issues, we invented a novel pretreatment method, Densifying Lignocellulosic biomass with alkaline Chemicals (DLC), and studied DLC on corn stover (CS). DLC-CS has high density and high durability, with contamination prevented and sugar fully preserved, which greatly facilitates biomass logistics. DLC-CS showed high enzymatic digestibility and high fermentability after storage. DLC-CS containing calcium hydroxide yielded 21.4 g of ethanol per 100 g of CS, which was further enhanced to 25.3 g of ethanol per 100 g of CS using a regular steam autoclave. An ethanol titer as high as 70.6 g L−1 was achieved, for the first time, without washing or detoxification of the pretreated biomass. These promising results demonstrated the great potential of DLC pretreatment for lignocellulosic biofuel production.

A novel pretreatment, Densifying Lignocellulosic biomass with acidic/alkali Chemicals (DLC), was recently invented and owns unique advantages for biomass logistics and fermentation. The pretreatment was largely completed during biomass storage, which renders the storage conditions critical. In this study, the effects of storage temperature (-80 °C to 60 °C) and storage time (up to half a year) on the enzymatic digestibility and fermentability of DLC corn stover (CS) were investigated. DLC-CS containing calcium hydroxide(ch) showed increased enzymatic digestibility with increased storage temperature and time. High glucan conversions (>90%) and ethanol titers (e.g. 73.1 g/L) were achieved after regular steam autoclave of DLC(ch)-CS, without washing or detoxification. DLC-CS containing sulfuric acid(sa) was sensitive to storage conditions, and autoclaved DLC(sa)-CS reached the highest ethanol titer (66.6 g/L) when DLC(sa)-CS was stored at room temperature for 14 days. Results indicated that different ambient temperatures in different regions and seasons have a far-reaching impact on DLC-CS for bioconversion.

Densifying lignocellulosic biomass with chemicals (calcium hydroxide) (DLC(ch)) followed by regular steam autoclave treatment (DLCA(ch)) is a novel pretreatment method, which unlocks highly fermentable sugars and showed great potential. This study investigated the economics of DLCA(ch) based biorefinery for cellulosic ethanol production and studied the effects of solid loading, enzyme dosage, enzyme price and biorefinery scale on the economics. Compared with dilute acid pretreatment based biorefinery, DLCA(ch) based biorefinery showed much lower feedstock logistics cost, and the savings in feedstock logistics almost offset the DLC(ch) pretreatment cost. DLCA(ch) based biorefinery exhibited higher ethanol yield and lower pretreatment costs. When the biorefinery scale was 2000 Mg rice straw per day, the total capital investment and minimum ethanol selling price of DLCA(ch) based biorefinery were 17.9% and 30.0% lower, respectively, compared to dilute acid pretreatment based biorefinery. This work demonstrated the great potential of DLCA(ch) pretreatment for the commercial production of cellulosic ethanol.

Background For bioethanol production from lignocellulosic biomass, phenolics derived from pretreatment have been generally considered as highly inhibitory towards enzymatic hydrolysis and fermentation. As phenolics are produced from lignin degradation during pretreatment, it is likely that the pretreatment will exert a strong impact on the structure of phenolics, resulting in varied levels of inhibition of the bioconversion process. Despite the extensive studies on pretreatment, it remains unclear how pretreatment process affects the properties of generated phenolics and how the inhibitory effect of phenolics from different pretreatment varies on enzymatic hydrolysis and fermentation. Results In this study, the structural properties of phenolic compounds derived from four typical pretreatment [dilute acid (DA), liquid hot water pretreatment (LHW), ammonia fiber expansion (AFEX) and alkaline pretreatment (AL)] were characterized, and their effect on both enzymatic hydrolysis and fermentation were evaluated. The inhibitory effect of phenolics on enzymatic hydrolysis followed the order: AFEX > LHW > DA > AL, while the inhibitory effect of phenolics on Zymomonas mobilis 8b strain fermentation followed the order: AL > LHW > DA > AFEX. Interestingly, this study revealed that phenolics derived from AFEX showed more severe inhibitory effect on enzymatic hydrolysis than those from the other pretreatments at the same phenolics concentrations (note: AFEX produced much less amount of phenolics compared to AL and DA), while they exhibited the lowest inhibitory effect on fermentation. The composition of phenolics from different pretreatments was analyzed and model phenolics were applied to explore the reason for this difference. The results suggested that the amide group in phenolics might account for this difference. Conclusions Pretreatment process greatly affects the properties of generated phenolics and the inhibitory effects of phenolics on enzymatic hydrolysis and fermentation. This study provides new insight for further pretreatment modification and hydrolysate detoxification to minimize phenolics-caused inhibition and enhance the efficiency of enzymatic hydrolysis and fermentation.

Additional file 1. Additional figures and tables.

Microbial lipids derived from lignocellulosic biomass are attractive feedstocks for biodiesel production. However, toxic degradation products generated by pretreatment greatly inhibited lipid fermentation. Herein, two pretreatment methods, i.e. densifying lignocellulosic biomass with sulfuric acid (DLC(sa)) and densifying lignocellulosic biomass with sulfuric acid followed by autoclave (DLCA(sa)), were developed on rice straw (RS) for lipid production. The conditions of DLCA(sa) pretreatment, including sulfuric acid dosage, dry biomass loading and autoclave time, were optimized by response surface methodology. High fermentable sugar yields with low inhibitor concentrations were obtained on DLC(sa) pretreated RS (DLC(sa)-RS) and DLCA(sa) pretreated RS (DLCA(sa)-RS) via enzymatic hydrolysis at a high solid loading of 30 wt%. Lipid titers of as high as 28.9 g/L and 31.0 g/L, with lipid yields of 0.22 g/g and 0.24 g/g consumed sugar, were achieved when RS was pretreated by DLC(sa) and DLCA(sa), respectively. This study demonstrated efficient microbial lipid production from lignocellulosic biomass without washing or detoxification steps.

Alkali and acid pretreatment based processes for lignocellulosic biofuel production typically consume substantial amount of alkali and acid. In addition, alkali pretreatment generates compounds with high toxicity rendering washing/detoxification inevitable. In this work, alkali (AL) and dilute acid (DA) pretreatment based processes were compared side by side for ethanol production from corn stover (CS) using commercial enzymes and Zymomonas mobilis 8b strain. Next, AL and DA pretreated CS slurries (without removing liquid stream) were mixed to neutralize for enzymatic hydrolysis and ethanol fermentation. The digestibility and fermentability of the mixed pretreated biomass were systematically studied and compared to unmixed pretreated biomass. Results showed that mixed biomass process saved 40–50% alkali/acid use and enhanced fermentability compared to AL-CS. This work suggests that it could be beneficial to have both alkali and acid pretreatments operating in a biorefinery, which could accommodate various feedstocks.

Microbial lipid production from lignocellulosic biomass is largely impeded by unsatisfactory fermentation performance due to the presence of toxic degradation products generated by pretreatment. DLC (Densifying Lignocellulosic biomass with Chemicals) pretreatment completed at room temperature, generating fewer degradation products, showed great potentials for lignocellulosic biorefineries. This work developed DLC pretreatment with sodium hydroxide and calcium hydroxide on rice straw (RS) for microbial lipid production. Conditions for steam autoclave on DLC-RS (i.e. DLCA treatment) were optimized using response surface methodology. High solid loading enzymatic hydrolysis and lipid fermentations by Trichosporon dermatis 32,903 together with mass balances were performed. Lipid fermentations were successfully conducted, for the first time, on pretreated biomass at a solid loading as high as 35 wt.% without washing or detoxification. Lipid titers as high as 28.3 g/L and 29.5 g/L were achieved on DLC-RS and DLCA-RS, respectively, with lipid yields of 0.24 and 0.23 g/g consumed sugar.

The presence of toxic degradation products in lignocellulosic hydrolysate typically reduced fermentation rates and xylose consumption rate, resulting in a decreased ethanol productivity. In the present study, Zymomonas mobilis 8b was investigated for high cell density fermentation with cell recycling to improve the ethanol productivity in lignocellulosic hydrolysate. The fermentation performances of Z. mobilis 8b at various conditions were first studied in yeast extract-tryptone medium. It was found that nutrient level was essential for glucose and xylose co-fermentation by Z. mobilis 8b and high cell density fermentation with cell recycling worked well in yeast extract-tryptone medium for 6 rounds fermentation. Z. mobilis 8b was then studied in enzymatic hydrolysates derived from dilute acid (DA) pretreated corn stover (CS) and ammonia pretreated CS for high cell density fermentation with cell recycling. Ethanol productivity obtained was around three times higher compared to traditional fermentation. Ethanol titer and metabolic yield were also enhanced with high cell density fermentation. Z. mobilis 8b cells showed high recyclability in ammonia pretreated CS hydrolysate.

Glycosaminoglycan (GAG) sulfatases, which catalyze the hydrolysis of sulfate esters from GAGs, belong to a large and conserved sulfatase family. Bacterial GAG sulfatases are essential in the process of sulfur cycling and are useful for the structural analysis of GAGs. Only a few GAG-specific sulfatases have been studied in detail and reported to date. Herein, the GAG-degrading Photobacterium sp. FC615 was isolated from marine sediment, and a novel Δ4,5hexuronate-2-O-sulfatase (PB2SF) was identified from this bacterium. PB2SF specifically removed 2-O-sulfate from the unsaturated hexuronate residue located at the non-reducing end of GAG oligosaccharides produced by GAG lyases. A structural model of PB2SF was constructed through a homology-modeling method. Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis. N113A, K141A, K141H, H143A, H143K, H205A, and H205K mutants exhibited only feeble activity, while the H310A, H310K, and D52A mutants were totally inactive, indicating that these conserved residues, particularly Asp52 and His310, were essential in the catalytic mechanism. Furthermore, bioinformatic analysis revealed that GAG sulfatases with specific degradative properties clustered together in the neighbor-joining phylogenetic tree. Based on this finding, 60 Δ4,5hexuronate-2-O-sulfatases were predicted in the NCBI protein database, and one with relatively low identity to PB2SF was characterized to confirm our prediction. Moreover, the signature sequences of bacterial Δ4,5hexuronate-2-O-sulfatases were identified. With the reported signature motifs, the sulfatase sequence of the Δ4,5hexuronate-2-O-sulfatase family could be simply identified before cloning. Taken together, the results of this study should aid in the identification and further application of novel GAG sulfatases.

Glycosaminoglycan (GAG) sulfatases, which catalyze the hydrolysis of sulfate esters from GAGs, belong to a large and conserved sulfatase family. Bacterial GAG sulfatases are essential in the process of sulfur cycling and are useful for the structural analysis of GAGs. Only a few GAG-specific sulfatases have been studied in detail and reported to date. Herein, the GAG-degrading Photobacterium sp. FC615 was isolated from marine sediment, and a novel Δ4,5hexuronate-2-O-sulfatase (PB2SF) was identified from this bacterium. PB2SF specifically removed 2-O-sulfate from the unsaturated hexuronate residue located at the non-reducing end of GAG oligosaccharides produced by GAG lyases. A structural model of PB2SF was constructed through a homology-modeling method. Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis. N113A, K141A, K141H, H143A, H143K, H205A, and H205K mutants exhibited only feeble activity, while the H310A, H310K, and D52A mutants were totally inactive, indicating that these conserved residues, particularly Asp52 and His310, were essential in the catalytic mechanism. Furthermore, bioinformatic analysis revealed that GAG sulfatases with specific degradative properties clustered together in the neighbor-joining phylogenetic tree. Based on this finding, 60 Δ4,5hexuronate-2-O-sulfatases were predicted in the NCBI protein database, and one with relatively low identity to PB2SF was characterized to confirm our prediction. Moreover, the signature sequences of bacterial Δ4,5hexuronate-2-O-sulfatases were identified. With the reported signature motifs, the sulfatase sequence of the Δ4,5hexuronate-2-O-sulfatase family could be simply identified before cloning. Taken together, the results of this study should aid in the identification and further application of novel GAG sulfatases.

In this study, a facile method to prepare MnO2 nanodots modified lignin nanocomposite (MnO2@LNP) was developed for efficient dye removal. The MnO2@LNP displayed hierarchical spherical nanostructures, where the MnO2 nanodots were evenly dispersed within the lignin nanosphere. Compared with lignin nanoparticles, the as-prepared MnO2@LNP exhibits higher surface area and can be separated after adsorption. It showed excellent adsorption capacity (806 mg/g) towards a typical cationic dye, methylene blue (MB), at a fast removal rate, where more than 80% of adsorption capacity was reached within 5 min at room temperature. The high adsorption capacity was contributed by the high surface area and negative charge on the adsorbent. The adsorption process is pH-responsive and exothermic, and the spent adsorbent can be reused for at least five cycles. This study displayed an efficient method to prepare MnO2@LNP for the high-value utilization of lignin-derived from lignocellulosic biorefinery.

Background Although various pre-treatment methods have been developed to disrupt the structure of lignocellulosic biomass, high dosage of cellulases is still required to hydrolyze lignocellulose to fermentable sugars. Enzyme recycling via recycling unhydrolyzed solids after enzymatic hydrolysis is a promising strategy to reduce enzyme loading for production of cellulosic ethanol. Results To develop effective enzyme recycling method via recycling unhydrolyzed solids, this work investigated both enzymatic hydrolysis kinetics and enzyme adsorption kinetics on dilute acid and dilute alkali pre-treated corn stover (CS). It was found that most of the hydrolysable biomass was hydrolyzed in the first 24 h and about 40% and 55% of the enzymes were adsorbed on unhydrolyzed solids for dilute alkali-CS and dilute acid-CS, respectively, at 24 h of enzymatic hydrolysis. Lignin played a significant role in such adsorption and lignin materials derived from dilute acid-CS and dilute alkali-CS possessed different enzyme adsorption properties. Enzyme recycling was performed by recycling unhydrolyzed solids after 24 h enzymatic hydrolysis for five successive rounds, and successfully reduced 40% and 50% of the enzyme loadings for hydrolysis of dilute alkali-CS and for hydrolysis of dilute acid-CS, respectively. Conclusions This study presents that the enzymes adsorbed on the unhydrolyzed solids after short-time hydrolysis could be recycled effectively for efficient enzymatic hydrolysis. Lignin derived from dilute acid-CS has higher enzyme adsorption capacity than the lignin derived from dilute alkali-CS, which led to more enzymes recycled. By applying the enzyme recycling strategy developed in this study, the enzyme dosage needed for effective cellulose hydrolysis can be significantly reduced.