Lignocellulose-based Biorefinery Research Articles

Biorefinery design with combined deacetylation and microwave pretreatment for enhanced production of polyhydroxyalkanoates and efficient carbon utilization from lignocellulose

Lignocellulosic biomass has emerged as a sustainable and economically viable feedstock for polyhydroxyalkanoate (PHA) production. However, it has seldom been used as part of an integrated process for effective carbon utilization. An innovative combined deacetylation and microwave pretreatment strategy (DEA-MW) and a wild-type Cupriavidus necator strain with the potential to utilize a lignocellulose-based carbon source and PHA biosynthesis were selected to set up the module needed to enable a new sustainable platform for biomass pretreatment and waste carbon valorization. DEA-MW pretreatment was introduced to mitigate the concentration of inhibitory acetic acid and phenolic compounds in the biomass hydrolysate. This pretreatment enhanced deacetylation and delignification, resulting in 95.50 % deacetylation and 45.30 % delignification. When DEA-MW pretreated rice husk biomass was coupled with microbial fermentation, C. necator promoted the fermentability of the rice husk hydrolysate. With a 3 g/L of levulinic acid from hydrolysis, as the sole carbon source, we achieved a concentration of PHA 440 mg/L with a content of 65 %. We also successfully recovered 72.22 % of lignin using a combined DEA-MW pretreatment. A detailed structural analysis showed that β-O-4, β–β, and β-5 linkages had been preserved in the recovered lignin. This study has shown that DEA-MW pretreatment plays a role in promoting biomass conversion and enhancing PHA production and opens up new possibilities for efficient and cost-effective lignocellulose-based biorefinery processes.

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Co-production of ethanol-hydrogen by genetically engineered Escherichia coli in sustainable biorefineries for lignocellulosic ethanol production

This work shows the impact of the hydrogen and ethanol co-production –via dark fermentation– using a genetically engineered Escherichia coli in the environmental and economic sustainability of a lignocellulose-based biorefinery. Wheat straw (WS) and corn stover (CS) were used as feedstock. These were treated with either a 1.5% dilute sulphuric acid pretreatment (DAP) or an autohydrolysis followed by a 0.25% dilute sulphuric acid pretreatment (AH-VDAP) to compare the effect of the lignocellulosic matrix and its pretreatment as strategies for obtaining rich hemicellulosic hydrolysates, which were used as substrate in the dark fermentation experiments. The engineered E. coli strain used here increased 30% the co-production of hydrogen and ethanol compared to other genetically modified strains. Further, their impact on the profitability was determined on biorefinery conceptual designs incorporating the pretreatment and dark fermentation stages experimental results. The dark fermentation stage contributed with 20% to 30% of the total ethanol production in the lignocellulose-based biorefinery designs proposed here. Techno economic and sustainability analyses established that the biorefinery design using WS as feedstock and employing AH-VDAP presented the lowest negative environmental impact with the lowest Total Production Cost (TPC). This design produced 187,846 L EtOH/day with a TPC of $1.37 USD/L EtOH. The results showed that co-production schemes could be an alternative for lignocellulosic ethanol biorefineries.

Organosolv pretreated beech wood as a substrate for acetone butanol ethanol extractive fermentation

Abstract Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lignocellulose-based biorefinery. Nevertheless, strong end-product inhibition is a severe limitation of this process, which can be partially solved by in situ product recovery techniques, such as extractive fermentation. In this work, the hydrolyzed cellulose from organosolv pre-treated beech wood (Fagus sylvatica L.) was fermented by Clostridium beijerinckii CECT 508 and compared with synthetic P2 medium in conventional and extractive fermentation processes. The conventional control fermentation yielded 9.12 g l−1 of ABE with C. beijerinckii CECT 508, while ABE overproduction of 40% was observed with cellulose hydrolysate as a substrate. After extractive batch fermentation based on 2-butyl-1-octanol as extractant, the final ABE titer was doubled in the P2 control medium over the conventional batch, with respective increases of 50, 190, and 140% for butanol (B), acetone (A) and ethanol (E). In the case of ABE as a hydrolysate medium, the total production increased by 10% with liquid extraction, while mainly B was responsible for this improvement. The total productivity was nevertheless gradually decreased in extractive fermentation, with a significantly slower production rate and the microbial growth compared to conventional fermentation. Moreover, solvent production and sugar consumption stopped, although the aqueous B concentration was below the inhibition threshold. The mid-term toxicity of the extraction solvent 2-butyl-1-octanol seems responsible for these results and this issue should be considered in future research.

A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

BackgroundThe filamentous fungus Trichoderma reesei, the most widely used cellulase producer, also has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high value-added products. However, such applications are thwarted by the time-consuming metabolic engineering processes (design–build–test–learn cycle) for T. reesei, resulted from (i) the spore separation-mediated purification as the multinucleate hyphae, (ii) transformant screening for high expression levels since unavailable of episomal expression system, and (iii) cases of inexpressible heterologous proteins.ResultsIn this study, a GFP-fusion coupled fluorescence-activated cell sorting (FACS) platform was established to speed up the build and test process of the DBTL cycle, by enabling rapid selection for expressible heterologous genes and bypassing both laborious spore separation and transformant screening. Here, the feasibility of flow cytometry in analyzing and sorting T. reesei cells harboring GFP-fused expressible protein was proven, as well as the application of the platform for constitutive promoter strength evaluation. As a proof-of-concept, the platform was employed to construct the first T. reesei strain producing fatty alcohol, resulting in up to 2 mg hexadecanol being produced per gram biomass. Pathway construction was enabled through rapid selection of functional fatty acyl-CoA reductase encoding gene Tafar1 from three candidate genes and strains with high expression level from spore pools. As a result of using this method, the total costed time for the build and test cycle using T. reesei, subsequently, reduced by approx. 75% from 2 months to 2 weeks.ConclusionThis study established the GFP-fusion coupling FACS platform and the first filamentous fungal fatty alcohol-producing cell factory, and demonstrated versatile applications of the platform in the metabolic engineering of filamentous fungi, which can be harnessed to potentially advance the application of filamentous fungi in lignocellulose-based biorefinery.

Pure enzyme cocktails tailored for the saccharification of sugarcane bagasse pretreated by using different methods

Abstract The compositions and physical properties of pretreated lignocellulose vary depending on pretreatment methods; therefore, enzyme cocktails specific to pretreatments are desired for efficient saccharification of lignocellulose. Here, enzyme cocktails consisting of three pure lignocellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH) and endoxylanase (XN) with a fixed amount of β-glucosidase were tailored for acid- and alkali-pretreated sugarcane bagasse (ACID and ALKALI, respectively). Based on a mixture design, the optimal mass ratios of EG, CBH, and XN were determined to be 61.25:38.73:0.02 and 53.99:34.60:11.41 for ACID and ALKALI, respectively. The optimized enzyme cocktail yielded a higher or comparable amount of reducing sugars from the hydrolysis of ACID and ALKALI when compared to that obtained using commercial cellulase mixtures. Using the commercial and easily available pure enzymes, this simple method for the in-house preparation of an enzyme cocktail specific to pretreated lignocellulose consisting of only four enzymes with a high level of hydrolysis will be helpful for achieving enzymatic saccharification in the lignocellulose-based biorefinery.

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

Microbial lipids derived from oleaginous yeast could be a promising resource for biodiesel and other oleochemical materials. The objective of this study was to develop an efficient bioconversion process from lignocellulosic biomass to microbial lipids using three types of robust oleaginous yeast: T. oleaginosus, L. starkeyi, and C. albidus. Sorghum stalks and switchgrass were utilized as feed-stocks for lipid production. Among oleaginous yeast strains, T. oleaginous showed better performance for lipid production using sorghum stalk hydrolysates. Lipid titers of 13.1 g·L-1 were achieved by T. oleaginosus, using sorghum stalk hydrolysates with lipid content of 60% (wt·wt-1) and high lipid yield of 0.29 g·g-1, which was substantially higher than the value reported in literature. Assessment of overall lipid yield revealed a total of 14.3 g and 13.3 g lipids were produced by T. oleaginosus from 100 g of raw sorghum stalks and switchgrass, respectively. This study revealed that minimization of sugar loss during pretreatment and selection of appropriate yeast strains would be key factors to develop an efficient bioconversion process and improve the industrial feasibility in a lignocellulose-based biorefinery.

The AlkaPolP process: Fractionation of various lignocelluloses and continuous pulping within an integrated biorefinery concept

The implementation of a lignocellulose-based biorefinery requires an efficient fractionation of its raw material. For this purpose a comprehensive biorefinery concept has been developed based on alkaline polyol pulping (AlkaPolP). It exhibits a high degree of flexibility with respect to the extent of fractionation by adjusting easily controllable process parameters. As shown in this contribution, the AlkaPolP process can be successfully applied for a wide range of lignocellulosic biomass including hardwood, softwood, bark and grasses, all of which yielding high quality product fractions.With essentially complete delignification the obtained pulp fraction is easily degraded by cellulases. The produced hydrolysates are free of inhibitors and can be used as substrates for a variety of fermentation processes. Without further modification, the AlkaPolP lignin fraction can be readily applied as high value substrate for various processes as confirmed by extensive characterisations in industrial and research laboratories (Dynea, Fraunhofer Institute). The remaining product fraction resulting from the degradation of various polysaccharides is being recovered as carboxylic acids.Based on extensive experimental results up to mini-plant scale, an integrated biorefinery concept has been developed including a continuous pulping stage and a full scale recycling of process chemicals thus allowing operations in environmentally sensitive rural areas.

Bioprospecting thermostable cellulosomes for efficient biofuel production from lignocellulosic biomass

The adverse climatic conditions due to continuous use of fossil-derived fuels are the driving factors for the development of renewable sources of energy. Current biofuel research focuses mainly on lignocellulosic biomass (LCB) such as agricultural, industrial and municipal solid wastes due to their abundance and renewability. Although many mesophilic cellulolytic microorganisms have been reported, efficient and economical bioconversion to simple sugars is still a challenge. Thermostable cellulolytic enzymes play an indispensible role in degradation of the complex polymeric structure of LCB into fermentable sugar stream due to their higher flexibility with respect to process configurations and better specific activity than the mesophilic enzymes. In some anaerobic thermophilic/thermotolerant microorganisms, few cellulases are organized as unique multifunctional enzyme complex, called the cellulosome. The use of cellulosomal multienzyme complexes for saccharification seems to be a promising and cost-effective alternative for complete breakdown of cellulosic biomass. This paper aims to explore and review the important findings in cellulosomics and forward the path for new cutting-edge opportunities in the success of biorefineries. Herein, we summarize the protein structure, regulatory mechanisms and their expression in the host cells. Furthermore, we discuss the recent advances in specific strategies used to design new multifunctional cellulosomal enzymes, which can function as lignocellulosic biocatalysts and evaluate the roadblocks in the yield and stability of such designer thermozymes with overall progress in lignocellulose-based biorefinery.

Naturally p-Hydroxybenzoylated Lignins in Palms

The industrial production of palm oil concurrently generates a substantial amount of empty fruit bunch (EFB) fibers that could be used as a feedstock in a lignocellulose-based biorefinery. Lignin byproducts generated by this process may offer opportunities for the isolation of value-added products, such as p-hydroxybenzoate (pBz), to help offset operating costs. Analysis of the EFB lignin by nuclear magnetic resonance (NMR) spectroscopy clearly revealed the presence of bound acetate and pBz, with saponification revealing that 1.1 wt% of the EFB was pBz; with a lignin content of 22.7 %, 4.8 % of the lignin is pBz that can be obtained as a pure component for use as a chemical feedstock. Analysis of EFB lignin by NMR and derivatization followed by reductive cleavage (DFRC) showed that pBz selectively acylates the γ-hydroxyl group of S units. This selectivity suggests that pBz, analogously with acetate in kenaf, p-coumarate in grasses, and ferulate in a transgenic poplar augmented with a feruloyl-CoA monolignol transferase (FMT), is incorporated into the growing lignin chain via its γ-p-hydroxybenzoylated monolignol conjugate. Involvement of such conjugates in palm lignification is proven by the observation of novel p-hydroxybenzoylated non-resinol β–β-coupled units in the lignins. Together, the data implicate the existence of p-hydroxybenzoyl-CoA:monolignol transferases that are involved in lignification in the various willows (Salix spp.), poplars and aspen (Populus spp., family Salicaceae), and palms (family Arecaceae) that have p-hydroxybenzoylated lignins. Even without enhancing the levels by breeding or genetic engineering, current palm oil EFB ‘wastes’ should be able to generate a sizeable stream of p-hydroxybenzoic acid that offers opportunities for the development of value-added products derived from the oil palm industry.

Microbial exo-xylanases: a mini review.

Exo-xylanases are a class of glycosyl hydrolases and play an important role in hydrolysis of xylan to xylose. They belong to glycosyl hydrolase (GH) family 8 with a characteristic (α/α)6 barrel fold in their molecular structures. These enzymes are generally produced by bacteria. Exceptionally, the endo-xylanases from Trichoderma reesei Rut C-30 and a few bacterial strains also show considerable exo-xylanase activities. Exo-xylanases are active on natural xylan substances, hydrolyzing long-chain xylo-oligomers from the reducing end to produce short-chain xylo-oligomers and xylose. Exo-xylanases usually show multiple enzyme functions such as β-xylosidase, exo-glucanase, β-glucosidase, and arabinofuranosidase activities, which are helpful for more efficient hydrolysis of xylan. The combined use of exo- and endo-xylanases can increase the xylose yield compared to using either of them alone. Screening new exo-xylanase-producing microbes, mining the enzyme coding sequences, genetically engineering the enzymes, and producing them in a large scale are recommended for their commercial applications in lignocellulose-based biorefinery.

Microbial cellulases: Engineering, production and applications

Depolymerization of cellulose to glucose requires the synergistic action of three key cellulases, endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21). They belong to the glycoside hydrolase (GH) family and catalyze the hydrolysis of glyosidic linkages depolymerizing cellulose to fermentable sugars. Cellulases are naturally produced by a wide spectrum of bacteria and fungi. These enzymes usually exist as cellulosomes attached to the cell wall of bacteria but are secreted into environments in fungi. They exist either as monomers or multimers with each monomer having a simple architecture containing a cellulose binding domain (CBD) and a catalytic domain (CD) interlinked by a linker peptide. Thermophilic, mesophilic and psychrophilic cellulases are quite different in their structures and amino acid compositions. Post-translational modifications such as glycosylation contribute to enzyme function, multiplicity and stability. Recent advances in recombinant DNA technology allow fast identification of novel cellulase genes, large scale production of cellulases and their genetic modifications to make tailor-made enzymes for various applications. Cellulases have been traditionally used in food processing and textile industries. The rapid depletion of fossil fuels and production of cheap fermentable sugars from abundant renewable resources have increased the demand for cellulases in lignocellulose-based biorefinery. However, the lack of a better understanding of the mechanisms of individual cellulases, their synergistic actions and their high prices are the major bottlenecks yet to be overcome for large scale commercial applications of cellulases in lignocellulose-based biorefinery.

One-pot pretreatment, saccharification and ethanol fermentation of lignocellulose based on acid–base mixture pretreatment

One-pot pretreatment, saccharification and ethanol fermentation of lignocellulose, which is based on acid–base mixture pretreament, will greatly reduces the overall processing costs not only for the production of cellulosic ethanol but also for the lignocellulose-based biorefinery.