Improve Lipid Production Research Articles

Use of plant growth regulators and their combined application to enhance lipid productivity in the filamentous microalga Tribonema sp.

Oleaginous Tribonema sp. has shown great potential in biodiesel production. However, effective strategies are still needed to increase overall lipid productivity for cost-effectiveness. Although the effects of plant growth regulators (PGRs) on enhancing microalgal growth and lipid content have been widely demonstrated, the synergistic effects of their combinations on improving lipid productivity in Tribonema sp. remain unclear. In this study, we investigated the comparative effects of five PGRs (e.g., fulvic acid, melatonin, gibberellin, γ-aminobutyric acid [GABA], and indoleacetic-3-acid [IAA]) at different concentrations (0.1–10 mg/L) on the growth, nitrogen uptake, and lipid content of Tribonema sp. The results showed that 10 mg/L GABA and 0.1 mg/L IAA were optimal conditions, contributing to an approximately 33.00 % increase in lipid productivity compared with the control (without PGRs). Based on these findings, the combination of optimized concentrations of GABA and IAA, especially in equal proportions, resulted in a more desirable fatty acid profile and produced the highest biomass, lipid content, and lipid productivity, with increases of 23.55 %, 25.62 %, and 54.95 %, respectively, over the control. This combination also significantly increased nitrogen removal efficiency in the medium, which was 46.24 % higher that of the control. These results were attributed to the significant upregulation of genes associated with nitrogen metabolism mediated by the synergy of IAA and GABA, promoting nitrate uptake by algal cells in the medium. Genes involved in photosynthesis and lipid synthesis were also upregulated, facilitating simultaneous biomass and lipid accumulation to maximize lipid productivity. Overall, this study is the first to report the combined application of GABA and IAA as an effective strategy to enhance lipid productivity in Tribonema sp., providing insights into the underlying mechanisms. These findings offer a promising solution for economically improving microalgal lipid production.

Improving antistress capacity and lipid productivity in the green alga Chlorella pyrenoidosa by adding abscisic acid under salt stress conditions

In this study, the regulation of abscisic acid (ABA) on cell growth and lipid biosynthesis was investigated under saltinduced stress in Chlorella pyrenoidosa. It is found that as suffering from only salt stress, although the lipid content of single cell was improved, the inhibitory effects of stress on cell proliferation was visible. When the algal cells were exposed to salt stress and ABA conditions, lipid productivity was increased (45.35 mg L-1 d-1) by 1.17-fold compared to that of control cells (20.91 mg L-1 d-1), and the inhibition to cell growth was relieved. Transcriptomic analysis revealed that after adding ABA, these genes involved in antioxidant activity, jasmonic acid (JA) biosynthesis, and lipid biosynthesis were upregulated. Subsequently, we observed that the levels of glutathione, total antioxidant capacity, trehalose, and JA were elevated and the levels of reactive oxygen species were reduced. This study presents an effective approach to improve lipid production in algal cells, a new mechanism on that ABA alleviates intracellular oxidative stress through JA signaling pathway was elaborated.

Effects of Temperature and Nitrogen Limitation on Growth and Lipid Production of Oleaginous Microalgae from Hot Springs

Oleaginous microalgae have gained increasing attention as an alternative feedstock for biodiesel production due to the increasing demand of fuel, climate change, and global warming. This study aimed to isolate and screen robust microalgal strains from hot springs for cultivation in subtropical and tropical areas. The newly isolated oleaginous microalgae were cultivated at 30, 35 and 40 °C. Among mesophilic and thermophilic strains tested, Chlamydomonas sp. HP59 is considered the most robust strain as it showed high cell growth in a broad range of temperatures (30 - 40 °C), with the maximum dried cell weight at 40 °C. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. To increase lipid production, the 2-stage cultivation, in which nitrogen-rich was applied to promote cell growth in the 1st stage and nitrogen-limitation was applied to stimulate lipid accumulation in the 2nd stage, was performed. The high temperature combined with nitrogen-limitation did improve lipid production by all microalgae. With this strategy, Chlamydomonas sp. HP59 showed the highest dried cell weight of 4.33 g/L and lipid production of 1.72 g/L. This study has shown the potential use of the newly isolated oleaginous microalgae from hot springs to be cultivated at high temperatures and under nitrogen-limited conditions for the production of biodiesel feedstocks. HIGHLIGHTS Mesophilic and thermophilic oleaginous microalgae were isolated from hot springs. Un-optimal temperatures for cell growth did improve lipid content by 2 - 4 folds. Combined effect of high temperature and nitrogen limitation effectively increased lipid content. Two-stage cultivation gave high dried cell weight and hence overall high lipid production. GRAPHICAL ABSTRACT

Regulation of carbon metabolic fluxes to enhance lipid and succinate production in oleaginous fungus Mortierella alpina.

Mortierella alpina is popular for lipid production, but the low carbon conversion rate and lipid yield are major obstacles for its economic performance. Here, external addition of organic acids involved in tricarboxylic acid cycle was used to tune carbon flux and improve lipid production. Citrate was determined to be the best organic acid that can be used for enhancing lipid production. By the addition of citrate, the lipid titer and content were approximately 1.24 and 1.34 times higher, respectively. Meanwhile, citrate supplement also promoted the accumulation of succinate, an important value-added platform chemical. Owing to the improved lipid and succinate production through adding citrate, the carbon conversion rate of M. alpina reached up to 52.17%, much higher than that of the control group (14.11%). The addition of citrate could redistribute carbon flux by regulating the expression level of genes related to tricarboxylic acid cycle metabolism. More carbon fluxes flow to lipid and succinate synthesis, which greatly improved the carbon conversion efficiency of M. alpina. This study provides an effective and straightforward strategy with potential economic benefits to improve carbon conversion efficiency in M. alpina.

Engineering Fatty Acid Biosynthesis in Microalgae: Recent Progress and Perspectives.

Microalgal lipids hold significant potential for the production of biodiesel and dietary supplements. To enhance their cost-effectiveness and commercial competitiveness, it is imperative to improve microalgal lipid productivity. Metabolic engineering that targets the key enzymes of the fatty acid synthesis pathway, along with transcription factor engineering, are effective strategies for improving lipid productivity in microalgae. This review provides a summary of the advancements made in the past 5 years in engineering the fatty acid biosynthetic pathway in eukaryotic microalgae. Furthermore, this review offers insights into transcriptional regulatory mechanisms and transcription factor engineering aimed at enhancing lipid production in eukaryotic microalgae. Finally, the review discusses the challenges and future perspectives associated with utilizing microalgae for the efficient production of lipids.

Dissecting Holistic Metabolic Acclimatization of Mucor circinelloides WJ11 Defective in Carotenoid Biosynthesis.

Mucor circinelloides WJ11 is a lipid-producing strain with industrial potential. A holistic approach using gene manipulation and bioprocessing development has improved lipid production and the strain's economic viability. However, the systematic regulation of lipid accumulation and carotenoid biosynthesis in M. circinelloides remains unknown. To dissect the metabolic mechanism underlying lipid and carotenoid biosynthesis, transcriptome analysis and reporter metabolites identification were implemented between the wild-type (WJ11) and ΔcarRP WJ11 strains of M. circinelloides. As a result, transcriptome analysis revealed 10,287 expressed genes, with 657 differentially expressed genes (DEGs) primarily involved in amino acid, carbohydrate, and energy metabolism. Integration with a genome-scale metabolic model (GSMM) identified reporter metabolites in the ΔcarRP WJ11 strain, highlighting metabolic pathways crucial for amino acid, energy, and nitrogen metabolism. Notably, the downregulation of genes associated with carotenoid biosynthesis and acetyl-CoA generation suggests a coordinated relationship between the carotenoid and fatty acid biosynthesis pathways. Despite disruptions in the carotenoid pathway, lipid production remains stagnant due to reduced acetyl-CoA availability, emphasizing the intricate metabolic interplay. These findings provide insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides that are valuable in applied research to design optimized strains for producing desired bioproducts through emerging technology.

Improving lipid production by Rhodotorula glutinis for renewable fuel production based on machine learning

Improving lipid production by Rhodotorula glutinis for renewable fuel production based on machine learning

Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity

BackgroundPreviously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The first gene, named CDMT1, encodes a protein with a calcium-dependent membrane association domain; the second gene, named DMAN1, encodes endo-1,4-β-mannanase, while the third gene, named AATPL1, encodes a plastidic ATP/ADP antiporter-like protein.ResultsTo determine which of these mutant genes are directly responsible for the phenotype of strain PK4, we delivered Cas9-gRNA ribonucleoproteins targeting each of the three genes into the wild-type cells by electroporation and successfully disrupted these three genes separately. The lipid productivity in the disruptants of CDMT1 and DMAN1 was similar to and lower than that in the wild-type strain, while the disruptants of AATPL1 exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions.ConclusionsWe succeeded in improving the lipid productivity of P. kessleri by CRISPR/Cas9-mediated gene disruption of AATPL1. The effective gene-editing method established in this study will be useful to improve Parachlorella strains for industrial applications.

Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)2SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain.

Impact of Ascorbic Acid on Zero-Valent Iron Nanoparticle and UV-B Mediated Stress in the Cyanobacterium, Fremyella diplosiphon.

Fremyella diplosiphon is an ideal third-generation biofuel source due to its ability to produce transesterified lipids. While nanofer 25s zero-valent iron nanoparticles (nZVIs) improve lipid production, an imbalance between reactive oxygen species (ROS) and cellular defense can be catastrophic to the organism. In the present study, the effect of ascorbic acid on nZVI and UV-induced stress in F. diplosiphon strain B481-SD was investigated, and lipid profiles in the combination regimen of nZVIs and ascorbic acid compared. Comparison of F. diplosiphon growth in BG11 media amended with 2, 4, 6, 8, and 10 mM ascorbic acid indicated 6 mM to be optimal for the growth of B481-SD. Further, growth in 6 mM ascorbic acid combined with 3.2 mg/L nZVIs was significantly higher when compared to the combination regimen of 12.8 and 51.2 mg/L of nZVIs and 6 mM ascorbic acid. The reversal effect of UV-B radiation for 30 min and 1 h indicated that ascorbic acid restored B481-SD growth. Transesterified lipids characterized by gas chromatography-mass spectrometry indicated C16 hexadecanoate to be the most abundant fatty acid methyl ester in the combination regimen of 6 mM ascorbic acid and 12.8 mg/L nZVI-treated F. diplosiphon. These findings were supported by microscopic observations in which cellular degradation was observed in B481-SD cells treated with 6 mM ascorbic acid and 12.8 mg/L nZVIs. Our results indicate that ascorbic acid counteracts the damaging effect of oxidative stress produced by nZVIs.

Stramenopile-type Lipid Droplet Protein Functions as a Lipid Droplet Scaffold Protein in the Marine Diatom Phaeodactylum tricornutum.

Oleaginous microalgae gain great attention as feedstock for biofuels because of their substantial accumulation capacity of neutral lipids in the cytosolic compartment called lipid droplet (LD). Understanding the regulation mechanism of neutral lipid accumulation and degradation, which is mediated by LD-associated proteins, is an important issue in improving lipid productivity. However, LD-associated proteins vary among species and wait to be characterized in many microalgae. Stramenopile-type lipid droplet protein (StLDP) was previously identified as a primary LD protein in the marine diatom Phaeodactylum tricornutum. We produced a knockout mutant of StLDP by CRISPR/Cas9 genome editing. Also, we tried to complement this mutant by expressing recognition site-modified StLDP (RSM-StLDP) which is designed to avoid an attack by Cas9 nuclease expressing in the mutant. The RSM-StLDP:EGFP was localized to both LDs and the outer chloroplast-endoplasmic reticulum. Decrease of LD number per cell, increase of LD size, and no alteration of neutral lipid content in the mutant under nitrogen deficiency clearly indicate that StLDP acts as an LD scaffold protein. The number of LD per cell increased in the complemented strain compared to wild-type cells. The LD morphology in the mutant is probably over-rescued in the complemented strain by the strong function of the nitrate reductase promoter, which is also supported by high neutral lipid content in the complemented strain. The growth of stldp mutant showed a long lag phase relative to wild-type cells, suggesting that the low surface-to-volume ratio of fused LD decreased the efficiency of LD hydrolysis during the initial growth phase.

Bioinformatics-Based Screening Approach for the Identification and Characterization of Lipolytic Enzymes from the Marine Diatom Phaeodactylum tricornutum.

Oleaginous diatoms accumulate lipids of biotechnological interest when exposed to nutrient stress conditions such as nitrogen starvation. While accumulation mechanisms are well-known and have been engineered to improve lipid production, degradation mechanisms remain poorly investigated in diatoms. Identifying lipid-degrading enzymes is the initial step to understanding the catabolic processes. In this study, an in silico screening of the genome of Phaeodactylum tricornutum led to the identification of 57 putative triacylglycerol lipases (EC 3.1.1.3) grouped in 4 families. Further analysis revealed the presence of conserved domains and catalytic residues of lipases. Physico-chemical characteristics and subcellular localization predictions highlighted that a majority of these putative proteins are hydrophilic and cytosolic, suggesting they could be recruited to lipid droplets directly from the cytosol. Among the 57 identified putative proteins, three lipases were identified as possibly involved in lipophagy due to a potential vacuolar localization. The expression of the mRNA corresponding to the 57 proteins was then searched in 3 transcriptomic datasets obtained under nitrogen starvation. Nine genes were highly regulated and were considered as encoding enzymes with a probable important function in lipid catabolism. A tertiary structure prediction of these nine candidates yielded eight functional 3D models. Among those, two downregulated enzymes, Phatr3_J54974 and Phatr3_EG00720, were highlighted as good targets for future functional genomics and purification studies to investigate their role in lipid degradation.

Algal biorefinery: an integrated process for industrial effluent treatment and improved lipid production in bioenergy application

Algal biorefinery: an integrated process for industrial effluent treatment and improved lipid production in bioenergy application

A novel strategy to promote microalgal growth and lipid productivity by supplementation of lignin related phenolic elicitors

An environment-friendly and cost-effective approach to improving lipid productivity without sacrificing microalgal growth was established. Syringic acid (SA) and p-coumaric acid (p-CA) are two major lignin hydrolysates, widely distributed in the wastewater from downstream industries of lignin. In this study, their potential in regulating the growth and valuable metabolites biosynthesis of microalga Euglena gracilis was evaluated. The cell growth was increased by 1.63 and 1.93 times at the optimal dosage of 0.5 g·L-1 SA and 0.3 g·L-1p-CA, respectively. Cell morphology also varied under the two treatments, reflecting the changes in cellular physiological status. Moreover, increased chlorophyll a content was only observed under p-CA treatment, indicating the influences of these compounds on photosynthesis were different. Fourier-transform infrared spectroscopy analysis revealed the fluctuations in the macromolecular pools. The variation trend of carbohydrate content was different under the treatment of the p-CA and SA, demonstrating that different phenolic compounds will determine different biosynthetic pathways and the flow of carbon to metabolites in microalgal cells. However lipid biosynthesis was improved by both of the phenolic acids treatments, which would be favorable to the biofuel production since the cell biomass was not sacrificed for lipid accumulation.

Maximizing unsaturated fatty acids production by using sugarcane agroindustry wastes in cultivation of Desmodesmus sp. in a flat plate photobioreactor

Microalgae lipid accumulation can be accomplished by different strategies rather than naturally reaching the stationary phase. Many studies employ nitrogen (N) depletion to improve lipid production; however, this approach might not be a suitable alternative when growth in wastewater is attempted. Agro-industry effluents in particular can have high concentrations of N, so nutrient removal is also required. This study evaluated two possibilities of achieving stress conditions in Desmodesmus sp. cultivation: light intensity and CO2 concentration. The culture medium also included liquid and solid residues from the sugarcane agro-industry: vinasse and a biofertilizer produced from bagasse biochar. Optimization of growth in a flat plate photobioreactor was conducted by combining a two-level factorial design and simplex methodology. Both the highest biomass and polyunsaturated fatty acid productivities (150.2 and 21.4 mg L−1 day−1, respectively) were achieved near the central points (5% CO2 in air and 1000 μmol m−2 s−1 light intensity). These results show the possibility of microalgae growth in a sustainable medium coupled with high-value lipid production, e.g., omegas-3, − 6, and − 9.

An Approach for Incorporating Glycerol as a Co-Substrate into Unconcentrated Sugarcane Bagasse Hydrolysate for Improved Lipid Production in Rhodotorula glutinis

Sugarcane bagasse is a potential raw material for microbial lipid production by oleaginous yeasts. Due to the limited sugar concentrations in bagasse hydrolysate, increasing carbon the concentration is necessary in order to improve lipid production. We aimed to increase carbon concentration by incorporating glycerol as a co-substrate into unconcentrated bagasse hydrolysate in the cultivation of Rhodotorula glutinis TISTR 5159. Cultivation in hydrolysate without nitrogen supplementation (C/N = 42) resulted in 60.31% lipid accumulation with 11.45 ± 0.75 g/L biomass. Nitrogen source supplementation increased biomass to 26.29 ± 2.05 g/L without losing lipid accumulation at a C/N of 25. Yeast extract improved lipid production in the hydrolysate due to high growth without altering the lipid content of the cells. Mixing glycerol up to 10% v/v into the unconcentrated hydrolysate improved biomass and lipid production. A further increase in glycerol concentrations drastically decreased growth and lipid accumulation by the yeast. By maintaining C/N at 27 using yeast extract as the sole nitrogen source, hydrolysate mixed with 10% v/v glycerol resulted in the highest lipid yield, at 19.57 ± 0.53 g/L with 50.55% lipid content, which was a 2.8-fold increase compared to using the hydrolysate alone. In addition, yeast extracts were superior for promoting growth and lipid production compared to inorganic nitrogen sources.

Evaluation of resource recovery potential of the Pseudoscillatoria coralii BERC01 under variable compositions of wastewater to produce biomass for cyanobacterium biorefinery

This study employed the cyanobacterium Pseudoscillatoria coralii BERC01 for the biological retrieval of the nutrients from sullage wastewater for the resource recovery and recycling of water. The strain was cultured in the synthetic wastewater (mBG11) containing varying concentrations of nitrogen, phosphorous, sulfur, and carbon. Interestingly, real sullage wastewater (SWW) improved the growth and CO2 utilization productivities by 2.35-fold. The carbohydrates, proteins, and lipid productivities were improved 2.33-fold, 2.39-fold, and 2.33-fold, respectively. Interestingly, 28.8 mg.L−1 of phosphorous resulted in a remarkably higher (372.74 ± 3.93 mg.g−1) content of the high-value phycobilin proteins. The light utilization efficiency was improved to 95 % in the presence of 49.35 mg.L−1 sulfur. The biodiesel properties of the SWW-produced biomass’s lipids showed improvement in the monounsaturated fatty acid content by 7.44 %. Besides, P. coralii removed phosphorous, nitrogen, COD, BOD, and TDS from the wastewater by 91 %, 86.76 %, 78.24 %, 80 %, and 52.50 % resulting the suitability of recycled water for irrigation purposes according to WHO standards. Correlation analyses showed that the cultivation media was significantly correlated with improved lipid productivity and negatively correlated with carbohydrate productivity. This study will benefit towards better understanding of the growth and metabolic behavior of cyanobacteria in response to variable pollution load of urban wastewater.

Silencing 1,3-β-glucan synthase gene promotes total lipid production and changes fatty acids composition by affecting carbon flow distribution in Phaeodactylum tricornutum

To clarify the effect of 1,3-β-glucan (known as chrysolaminarin) synthase ( Pt BGS) on carbon flow distribution, expression of 1,3-β-glucan synthase gene ( Ptbgs ) was silenced by RNA interference in Phaeodactylum tricornutum . The results showed that the chrysolaminarin content in all transgenic strains was significantly lower than that in the wild-type (WT), while the total lipid content increased to varying degrees. Importantly, the decrease in chrysolaminarin content was significantly correlated with the increase in total lipid content. There was no significant difference in the protein content between all the transgenic strains and WT. Thus, Pt BGS mainly affected the distribution of carbon flow in total lipids and polysaccharides , mainly chrysolaminarin. Silencing Ptbgs changed the fatty acids composition , and specifically promoted polyunsaturated fatty acids production and reduced monounsaturated fatty acids content, but had only a little effect on saturated fatty acids content. Interestingly, the growth of transgenic strains was not influenced while the lipid content increased, indicating that Ptbgs silencing may be an effective strategy to improve lipid production in diatoms. • Distribution of carbon flow can be affected by 1,3-β-glucan synthase ( Pt BGS). • Inhibiting the expression of Ptbgs promoted lipid accumulation. • Silencing Ptbgs changed the fatty acids composition. • Interfering with Ptbgs exerted little effect on growth in Phaeodactylum tricornutum . • Ptbgs silencing may be an effective strategy to improve lipid production in diatoms.

Using gel microdroplets to develop a simple high-throughput screening platform for oleaginous microorganisms

The oleaginous yeast Lipomyces starkeyi is expected to be a new lipid source since this microorganism is capable of accumulating more than 85% lipid per dry cell weight. For effective utilization of oleaginous yeast, mutants with improved lipid production compared to the wild-type have been screened by methods such as single-cell sorting and Percoll density gradient centrifugation. Because these methods need to reculture all mutated oleaginous yeasts together in a flask, it is difficult to evaluate the growth of each individual mutant. Thus, screening for the slow-growing mutants with high-throughput has never been performed by conventional methods. In this study, we developed a high-throughput method using gel microdroplets (GMD). With this method, the growth and lipid production of L. starkeyi can be evaluated simultaneously. L. starkeyi grew in GMD and the size of these microcolonies was evaluated by scattered light. Finally, a mutant with a 10-fold delay in growth compared to the wild-type was obtained. Analysis of genetic information in this mutant could reveal valuable information about critical genes involved in the growth of these microorganisms, which could then be utilized further.

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification.

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.