Cell wall composition and biomass saccharification potential of Sida hermaphrodita differ between genetically distant accessions.

Due to its ample production of lignocellulosic biomass, Sida hermaphrodita (Sida), a perennial forb, is considered a valuable raw material for biorefinery processes. The recalcitrant nature of Sida lignocellulosic biomass towards pretreatment and fractionation processes has previously been studied. However, Sida is a non-domesticated species and here we aimed at expanding the potential of such plants in terms of their processability for downstream processes by making use of the natural variety of Sida. To achieve this goal, we established a collection comprising 16 different Sida accessions obtained from North America and Europe. First, we asked whether their cell wall characteristics are reflected in genetic distance or geographical distribution, respectively. A genotyping-by-sequencing (GBS) analysis resulting in a phylogenic tree based on 751 Single Nucleotide Polymorphisms (SNPs), revealed a high genetic diversity and a clear separation between accessions collected in North America and Europe. Further, all three North American accessions were separated from each other. Of the eleven European accessions, five form individual groups and six others belong to a single group. Clonal plants of seven selected accessions of American and European origin were produced and cultivated under greenhouse conditions and the resulting plant material was used for in-depth wet-chemical and spectroscopic cell wall characterization. Two accessions with contrasting cell wall characteristics were then selected and processed using the OrganoCat technology. Results of the different product yields and chemical compositions are reported. Overall, cell wall analyses revealed contrasting clusters regarding these main components between the accessions that can be related to genetic and, partly, geographical distance. Phenotypically, the accessions clustered into two groups that are not entirely overlapping with geographical origin. These results can be the basis for a targeted selection or cultivation of Sida accessions for biorefinery approaches.

Insights into cell wall disintegration of Chlorella vulgaris.

With their ability of CO2 fixation using sunlight as an energy source, algae and especially microalgae are moving into the focus for the production of proteins and other valuable compounds. However, the valorization of algal biomass depends on the effective disruption of the recalcitrant microalgal cell wall. Especially cell walls of Chlorella species proved to be very robust. The wall structures that are responsible for this robustness have been studied less so far. Here, we evaluate different common methods to break up the algal cell wall effectively and measure the success by protein and carbohydrate release. Subsequently, we investigate algal cell wall features playing a role in the wall's recalcitrance towards disruption. Using different mechanical and chemical technologies, alkali catalyzed hydrolysis of the Chlorella vulgaris cells proved to be especially effective in solubilizing up to 56 wt% protein and 14 wt% carbohydrates of the total biomass. The stepwise degradation of C. vulgaris cell walls using a series of chemicals with increasingly strong conditions revealed that each fraction released different ratios of proteins and carbohydrates. A detailed analysis of the monosaccharide composition of the cell wall extracted in each step identified possible factors for the robustness of the cell wall. In particular, the presence of chitin or chitin-like polymers was indicated by glucosamine found in strong alkali extracts. The presence of highly ordered starch or cellulose was indicated by glucose detected in strong acidic extracts. Our results might help to tailor more specific efforts to disrupt Chlorella cell walls and help to valorize microalgae biomass.

Fractionation of Lignocellulosic Biomass using the OrganoCat Process.

The shift from a petroleum-based to a more sustainable and bio-based economy requires the development of new refinery concepts to maintain the supply of raw materials and energy. For these novel and sustainable biorefinery concepts, it is important to use catalysts and solvents that are aligned with the principles of Green Chemistry. Therefore, the implementation of biogenic alternatives can be a promising solution. The lignocellulose pretreatment and fractionation process presented herein-OrganoCat-is an integrated fractionation of lignocellulose into its main components using biogenic acids such as 2,5-furandicarboxylic acid as catalyst. Hemicelluloses and other non-cellulosic polysaccharides are selectively depolymerized by the diluted acid and dissolved, while the crystalline cellulose remains in the solid pulp. In the presence of a second organic phase consisting of biogenic 2-methyltetrahydrofuran, disentangled lignin is extracted in situ. The process allows for the efficient fractionation of the three main components-lignin, cellulose, and non-cellulosic sugars. This helps to focus on the quality of the lignin, the improvement of enzymatic hydrolysis of the cellulose-enriched pulp, and the mild non-cellulosic sugar extraction with low degradation.

Lignocellulose Fractionation Using Recyclable Phosphoric Acid: Lignin, Cellulose, and Furfural Production.

The conversion of lignocellulose into its building blocks and their further transformation into valuable platform chemicals (e. g., furfural) are key technologies to move towards the use of renewable resources. This paper explored the disentanglement of lignocellulose into hemicellulose-derived sugars, cellulose, and lignin in a biphasic solvent system (water/2-methyltetrahydrofuran) using phosphoric acid as recyclable catalyst. Integrated with the biomass fractionation, in a second step hemicellulose-derived sugars (mainly xylose) were converted to furfural, which was in situ extracted into 2-methyltetrahydrofuran with high selectivity (70 %) and yield (56 wt %). To further increase the economic feasibility of the process, a downstream and recycling strategy enabled recovery of phosphoric acid without loss of process efficiency over four consecutive cycles. This outlines a more efficient and sustainable use of phosphoric acid as catalyst, as its inherent costs can be significantly lowered.

Multiscale analysis of lignocellulose recalcitrance towards OrganoCat pretreatment and fractionation.

BACKGROUND: Biomass recalcitrance towards pretreatment and further processing can be related to the compositional and structural features of the biomass. However, the exact role and relative importance to those structural attributes has still to be further evaluated. Herein, ten different types of biomass currently considered to be important raw materials for biorefineries were chosen to be processed by the recently developed, acid-catalyzed OrganoCat pretreatment to produce cellulose-enriched pulp, sugars, and lignin with different amounts and qualities. Using wet chemistry analysis and NMR spectroscopy, the generic factors of lignocellulose recalcitrance towards OrganoCat were determined. RESULTS: The different materials were processed applying different conditions (e.g., type of acid catalyst and temperature), and fractions with different qualities were obtained. Raw materials and products were characterized in terms of their compositional and structural features. For the first time, generic correlation coefficients were calculated between the measured chemical and structural features and the different OrganoCat product yields and qualities. Especially lignin-related factors displayed a detrimental role for enzymatic pulp hydrolysis, as well as sugar and lignin yield exhibiting inverse correlation coefficients. Hemicellulose appeared to have less impact, not being as detrimental as lignin factors, but xylan-O-acetylation was inversely correlated with product yield and qualities. CONCLUSION: These results illustrate the role of generic features of lignocellulosic recalcitrance towards acidic pretreatments and fractionation, exemplified in the OrganoCat strategy. Discriminating between types of lignocellulosic biomass and highlighting important compositional variables, the improved understanding of how these parameters affect OrganoCat products will ameliorate bioeconomic concepts from agricultural production to chemical products. Herein, a methodological approach is proposed.

Lignin Precipitation and Fractionation from OrganoCat Pulping to Obtain Lignin with Different Sizes and Chemical Composition.

Fractionation of lignocellulose into its three main components, lignin, hemicelluloses, and cellulose, is a common approach in modern biorefinery concepts. Whereas the valorization of hemicelluloses and cellulose sugars has been widely discussed in literature, lignin utilization is still challenging. Due to its high heterogeneity and complexity, as well as impurities from pulping, it is a challenging feedstock. However, being the most abundant source of renewable aromatics, it remains a promising resource. This work describes a fractionation procedure that aims at stepwise precipitating beech wood (Fagus sp.) lignin obtained with OrganoCat technology from a 2-methyltetrahydrofuran solution, using n-hexane and n-pentane as antisolvents. By consecutive antisolvent precipitation and filtration, lignin is fractionated and then characterized to elucidate the structure of the different fractions. This way, more defined and purified lignin fractions can be obtained. Narrowing down the complexity of lignin and separately valorizing the fractions might further increase the economic viability of biorefineries.

OrganoCat Fractionation of Empty Fruit Bunches from Palm Trees into Lignin, Sugars, and Cellulose-Enriched Pulp.

The palm oil industry produces large amounts of empty fruit bunches (EFB) as waste. EFB are very recalcitrant toward further processing, although their valorization could create novel incentives and bio-economic opportunities for the industries involved. Herein, EFB have been successfully subjected to the OrganoCat pretreatment-using 2,5-furandicarboxylic acid as the biogenic catalyst-to fractionate and separate this lignocellulosic material into its main components in a single step. The pretreatment of EFB leads to the deacetylation and depolymerization of noncellulosic polysaccharides and to the partial delignification of the cellulosic fiber. The OrganoCat processing of EFB yielded 45 ± 0.5 wt % cellulose-enriched pulp, 20 ± 0.7 wt % extracted lignin, 3.8 ± 0.2 wt % furfural, and 11 ± 0.6 wt % hydrolyzed sugars. The obtained EFB-pulp showed high accessibility to cellulases, resulting in a glucan conversion of 73 ± 2% after 72 h (15 ± 2% after 1 h) with commercial cellulase cocktail (Accellerase 1500). Overall, the results suggest that the treatment of the EFB material using OrganoCat may create promising paths for the full valorization of EFBs.

One-Step Lignocellulose Fractionation by using 2,5-Furandicarboxylic Acid as a Biogenic and Recyclable Catalyst.

To develop novel biorefinery concepts, the use of bio-based catalysts and solvents must be aligned with the principles of green chemistry. In this context, biogenic 2,5-furandicarboxylic acid (FDCA) is a very promising yet underused molecule with high potential for application as an acid catalyst, combining feasibility and sustainability with efficient and straightforward recovery. In this study, FDCA was evaluated as a catalyst in the recently developed OrganoCat pretreatment, a biphasic lignocellulose fractionation system. The catalyst was investigated for the efficient fractionation of the three main components-lignin, cellulose and noncellulosic sugars-with particular focus on the lignin quality, on the effect on enzymatic hydrolysis of the cellulosic residue, and on the noncellulosic sugar extraction. To address recovery of FDCA from the OrganoCat system, a method was developed, leading to the recovery of >97 % of FDCA with a spectroscopic purity of >99 %, maintaining full activity in consecutive runs.

Multi-step biocatalytic depolymerization of lignin.

Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the Cα hydroxyl group in the ß-O-4 structure of lignin. Subsequently, selective ß-O-4 ether cleavage of the oxidized ß-O-4 linkages is achieved with ß-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions.

Chemo-enzymatic conversion of glucose into 5-hydroxymethylfurfural in seawater.

Do you sea water? Water consumption will be a challenge in biorefineries, and the use of non-drinkable sources of water will be preferred. Herein, glucose is converted into 5-hydroxymethylfurfural (HMF) in a chemo-enzymatic one-pot, two-step procedure, involving immobilized glucose isomerase to produce fructose and oxalic acid to dehydrate it to HMF.

Biomass pretreatment affects Ustilago maydis in producing itaconic acid.

BACKGROUND: In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate. RESULTS: U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood. CONCLUSION: The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.

Enzymatic hydrolysis of microcrystalline cellulose in concentrated seawater.

This communication explores the use of seawater (1X) and concentrated seawater (2X and 4X) as reaction media for the enzyme-catalyzed depolymerization of cellulose. The commercially available Accellerase-1500® - a "cocktail" of different glycosidases - is able to depolymerize several amorphous celluloses and microcrystalline cellulose Avicel® in these reaction media, at slightly lower rates (ca. 90%) than those observed when reactions are performed in pure citrate buffer (control reactions). Remarkably, at concentrated seawater effluents enzymes also display significant rates of cellulose hydrolysis. Considering the expected increasing shortages in accessibility to fresh drinkable water, the herein-reported concept may provide novel inspiring leads for a smart use of resources in an environmentally-friendly and efficient manner, and for the genetic development of cellulases highly active and stable in concentrated seawater solutions.