Xylotriose

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Barley straw was subjected to hydrothermal pretreatment (steam explosion) processing to evaluate its potential as a raw material to produce xylooligosaccharides (XOS) suitable for use as a prebiotic. The steam explosion pretreatment generated a liquid fraction containing solubilised hemicellulose. This fraction was purified using gel permeation chromatography to obtain a fraction rich in XOS DP2-DP6. The sample was characterised through analytical techniques such as HPAEC-PAD, FTIR and MALDI-TOF-MS. The prebiotic activity was evaluated using in vitro fermentation in human faecal cultures through the quantification of short-chain fatty acid (SCFA) and lactate production, the evolution of the pH and the consumption of carbon sources. The total SCFA production at the end of fermentation (30 h) was 90.1 mM. Positive significant differences between the amount of XOS from 

Background: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. Results: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of “arabinoxylan” degradation in R. cellulolyticum, which challenged this paradigm. This “selfish” system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. Conculsion: The primary degrader model Ruminiclostridium cellulolyticum has developed a “selfish” strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.

The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-D-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50°C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP₂) to six (DP₆), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig1⁸⁸, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig1⁸⁸ overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig1⁸⁸, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.

Xylans, a family of xylose-based polysaccharides, are dietary fibers resistant to digestion. They therefore reach the large intestine intact; there, they are utilized by members of the gut microbiota. They are initially broken down by primary degraders that utilize extracellular xylanases to cleave xylan into smaller oligomers. The resulting xylooligosaccharides (XOS) can either be further metabolized directly by primary degraders or cross-feed secondary consumers, including Bifidobacterium. While several Bifidobacterium species have metabolic systems for XOS, most grow poorly on longer-chain XOS and xylan substrates. In this study, we isolated strains of Bifidobacterium pseudocatenulatum and observed that some, including B. pseudocatenulatum ED02, displayed growth on XOS with a high degree of polymerization (DP) and straight-chain xylan, suggesting a primary degrader phenotype that is rare in Bifidobacterium. In silico analyses revealed that only the genomes of these xylan-fermenting (xylan⁺) strains contained an extracellular GH10 endo-β-1.4 xylanase, a key enzyme for primary degradation of xylan. The presence of an extracellular xylanase was confirmed by the appearance of xylan hydrolysis products in cell-free supernatants. Extracellular xylanolytic activity was only detected in xylan⁺ strains, as indicated by the production of XOS fragments with a DP of 2 to 6, identified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Additionally, in vitro fecal fermentations revealed that strains with a xylan⁺ phenotype can persist with xylan supplementation. These results indicate that xylan⁺ B. pseudocatenulatum strains may have a competitive advantage in the complex environment of the gastrointestinal tract, due to their ability to act as primary degraders of xylan through extracellular enzymatic degradation.

This study explores the production of prebiotic xylooligosaccharide (XOS) from cassava pulp waste and its effectiveness for the growth of Lactobacillus acidophilus (L. acidophilus). We successfully produced and characterized XOS from cassava pulp xylan using a Bacillus sp. endo-β-1,4-D-xylanase. The XOS was added to modify the MRS medium (MRSm) in various concentrations (0, 1, 3 and 5%) in which the L. acidophilus was inoculated. The growth of L. acidophilus was observed every 12 h for 2 days, and the fermentation products were analyzed for pH, sugar content, and short-chain fatty acids (SCFA) in terms of types and amount. The study showed that L. acidophilus grew well in MRSm. The optimum XOS concentration in MRSm was 5%, indicated by the highest growth of L. acidophilus (8.61 log CFU mL⁻¹). The profile of SCFA products is 14.42 mM acetic acid, 0.25 mM propionic acid, 0.13 mM isobutyric acid, 0.41 mM n-butyric acid, 0.02 mM n-valeric acid, 0.25 mM isovaleric acid, and 25.08 mM lactic acid.

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS^·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

BACKGROUND: Corn arabinoxylan (AX) is a complicated and multi-branched antinutritional factor, thereby proving the use of endo-xylanase (EX) to be marginally valid. This study focused on specific types of AX-degrading enzymes (ADE) to exert the synergy of debranching enzymes and track prebiotic potential of enzymatic hydrolysates. It aimed to investigate the effects of ADE on growth performance, intestinal histomorphology, absorption functions, changes of polysaccharide components, fermentation, and gut microbiota of broilers. To this purpose, 576 five-day-old Arbor Acres male broilers were randomly allocated into eight treatments with six replicates each. Corn basal diets supplemented with or without enzymes were fed for a 21-day period, specifically including EX, its compatible use with arabinofuranosidase (EXA) or ferulic acid esterase (EXF), and compound groups with the above three enzymes (XAF). RESULTS: Specific ADE stimulated the jejunal villus height and goblet cell number, and evidently decreased the crypt depth (P < 0.05), while the ratio of ileal villus height to crypt depth was significantly increased in EXF (P < 0.05). Maltase activities of ileal mucosa in XAF groups were extremely enhanced (P < 0.01), and EX boosted the activity of Na⁺ -K⁺ ATPase in the small intestine (P < 0.01). The insoluble AX concentrations comparatively lessened, thereby notably raising the sundry xylooligosaccharide (XOS) yield in the ileal chyme (P < 0.05), which was dominant in xylobiose and xylotriose. Improvements in the abundance and diversity of ileal microbial communities within EXA, EXF, XAF treatments were observed (P < 0.05). Positive correlations between microbiota and XOS were revealed, with xylobiose and xylotriose being critical for ten beneficial bacteria (P < 0.05). EXF escalated the BWG and FCR of broilers in this phase (P < 0.05), attributing to the thriving networks modified by Lactobacillus. The intracecal contents of acetic acid, butyric acid, and propionic acid were greatly enhanced in most ADE groups such as EXF (P < 0.05). CONCLUSIONS: Debranching enzymes appreciably targeted corn AX to release prebiotic XOS in the posterior ileum and facilitated intracaecal fermentation. It was beneficial for improving gut development, digestion and absorption, and modulating the microflora to promote early performance of broilers.

In the human gut microbiota, Bacteroidetes break down dietary and endogenous glycosides through highly specific polysaccharide utilization loci (PULs). PULs encode a variety of sensor regulators, binding proteins, transporters, and carbohydrate-active enzymes (CAZymes). Surface glycan-binding proteins (SGBPs) are essential for the efficient capture of the glycosides present on the cell surface, providing Bacteroidetes with a competitive advantage in colonizing their habitats. Here, we present the functional and structural characterization of a SusD-like protein encoded by a xylooligosaccharide (XOS) PUL from an uncultured human gut Bacteroides strain. This locus is also conserved in Bacteroides vulgatus, thereby providing new mechanistic insights into the role of SGBPs in the metabolism of dietary fiber of importance for gut health. Various in vitro analyses, including saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, revealed that the SusD-like protein cannot bind to the cognate substrate of the XOS PUL, although its presence is essential for the PUL to function. Analysis of the crystal structure of the SusD-like protein reveals an unfolded binding surface and the absence or inappropriate orientation of several key residues compared with other known SusD-like structures. These results highlight the critical role of the SusD-like protein in the transport of oligosaccharides and provide fundamental knowledge about the structure-function of SusC/D-like transporters, revealing that the binding specificity of SusD-like SGBPs does not necessarily reflect the uptake specificity of the transporter.

Microwave hydrothermal treatment (MHT) is considered a sustainable technology for the valorization of lignocellulosic materials, enabling the solubilization of hemicellulosic-derived compounds, especially in the form of oligosaccharides that may present potential in the chemical, pharmaceutical or nutraceutical industries. Hence, MHT at 200 and 230°C, at severity (S₀) among 2.92-4.66 were performed. S₀ = 3.98 permitted the recovery of about 80% of the initial xylan as xylooligosaccharides. In order to compare the effectiveness of MHT, conventional hydrothermal treatment (CHT) was performed at conditions leading to the maximum recovery of oligosaccharides (S₀ =3.98, non-isothermal regime at 203°C). Despite the structural features of oligomers in the three liquors were very similar, the spent solids presented different enzymatic digestibility, which implied a different effect of the treatments, reaching up to 80% of glucan to glucose conversion for the solid after MHT at 230°C for 0.5 min. Additionally, CHT consumed 2.1-2.8-fold greater energy than MHT, reflecting that microwave-assisted autohydrolysis is a sustainable and efficient technology to process PW.

The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

For xylooligosaccharides (XOS) production from agricultural residues, highly concentrated xylan liquor (116 g/L) was produced by crushing an oil palm empty fruit bunch (EFB) via continuous hydrothermal treatment and subsequent membrane-based concentration on a pilot scale. The yield of xylan extracted from the raw EFB was 53.7%. To produce XOS with a low degree of polymerization, xylanase-catalyzed hydrolysis was performed and the sum of xylobiose and xylotriose to extracted xylan was 81.6% under lab-scale optimal conditions and 69.3% under pilot-scale conditions. In addition, the crude XOS could be purified through a novel approach involving enzyme-mediated radical polymerization and membrane-based separation, while minimizing XOS loss and waste generation. As a result, 50.2% of the total phenolic compounds in the crude XOS material could be polymerized and precipitated through peroxidase-catalyzed reactions, and an additional 22.6% were removed using the membrane. Furthermore, the amount of activated carbon consumed and the XOS recovery rate after phenolic compound removal via the adsorption method using activated carbon were evaluated and compared with the integrated enzyme-membrane purification results. Consequently, this study provides an ecofriendly biomass refinery process for low-molecular-weight XOS production and purification that produces few toxic substances and little waste.

A thermotolerant Enterococcus faecium s6 strain exhibited homoferementative lactic acid (LA) production at high xylose concentration (≥50 g/L). In batch fermentation at 45°C and controlled pH of 6.5, strain E. faecium s6 produced up to 72.9 g/L of LA with a yield of 0.99 g/g_{-consumed xylose} and productivity of 1.74 g/L.h from 75 g/L xylose. An increased LA concentration and productivities with high yields were obtained in repeated batch fermentation that was conducted for 24 runs. In this mode, the strain could produce LA up 81.1 g/L within 5 h fermentation at a high productivity of 13.5 g/L.h and a yield of 1.02 g/g_{-consumed xylose}. The strain was unable to consume birchwood xylan as sole carbon source. However, it could efficiently utilize xylooligosaccharides of xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose. The intracellular xylosidase activity was induced by xylose. Using xylooligosaccharide (50 g/L)/xylose (5 g/L) mixtures, the strain was able to produce maximum LA at 48.2 g/L within 120 h at a yield of 1.0 g/g_{-consumed sugar} and productivity of 0.331 g/L.h. These results indicate that E. faecium s6 is capable of directly utilizing xylan-hydrolyzate and will enable the development of a feasible and economical approach to the production of LA from hemicellulosic hydrolysate.

Arabinoxylans (AXs) display biological activities that depend on their chemical structures. To structurally characterize and distinguish AXs using a non-enzymatic approach, various TEMPO-oxidized AXs were partially acid-hydrolysed to obtain diagnostic oligosaccharides (OS). Arabinurono-xylo-oligomer alditols (AUXOS-A) with degree of polymerization 2-5, comprising one and two arabinuronic acid (AraA) substituents were identified in the UHPLC-PGC-MS profiles of three TEMPO-oxidized AXs, namely wheat (ox-WAX), partially-debranched WAX (ox-pD-WAX), and rye (ox-RAX). Characterization of these AUXOS-A highlighted that single-substitution of the Xyl unit preferably occurs at position O-3 for these samples, and that ox-WAX has both more single substituted and more double-substituted xylose residues in its backbone than the other AXs. Characteristic UHPLC-PGC-MS OS profiles, differing in OS abundance and composition, were obtained for each AX. Thus, partial acid-hydrolysis of TEMPO-oxidized AXs with analysis of the released OS by UHPLC-PGC-MS is a promising novel non-enzymatic approach to distinguish AXs and obtain insights into their structures.

The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.

Empty fruit bunch (EFB), the largest amount of biomass from a palm oil mill, is a promising renewable source for many value-added products. This study aimed to utilize EFB for production of xylanase by Streptomyces thermocarboxydus ME742 and applied the enzyme to the production of xylooligosaccharides (XOS) from EFB derived xylan (EFB-xylan). The strain ME742 produced the maximum xylanase (473.21 ± 6.55 U/mL) in a medium containing 50 g/L EFB supplemented with 3.0 g/L peptone at 40°C for 4 days. Xylanase ME742 was purified 23.68 fold with specificity of 1229 U/mg by acetone precipitation and DEAE-Sepharose chromatography. It was stable in a wide range of pH (4.0-9.5) and in elevated temperatures up to 55°C. Xylanase ME742 effectively hydrolyzed EFB-xylan. HPLC analysis revealed that the hydrolysate was composed of xylose and XOS with degree of polymerization between 2 and 5 and xylobiose was the predominant product. The XOS yield ranging in 95.01-151.19 mg/g EFB-xylan was generated from enzymatic processing for 12-72 h. Interestingly, arabinosyl-xylooligosaccharides were also predicted to be produced from EFB-xylan. XOS mixture exhibited high antioxidant activity (71.36 ± 0.57%) against DPPH at 100 μg/mL concentration and moderate activity (45.18 ± 1.56%) against ABTS radical. These findings reveal the potential use of Streptomyces thermocarboxydus ME742 and xylanase produced in a sustainable bioprocess to add value of EFB waste.

At present, xylooligosaccharides (XOS) from corncob using acid-base conjugate system has not been reported. In this study, XOS production from corncob by two-step acetic acid/sodium acetate (AC/SA) conjugate system hydrolysis and xylanase hydrolysis was optimized, and monosaccharides were subsequently produced from corncob residues by cellulase hydrolysis. The XOS of 19.9 g/L was obtained from corncob (10%, w/v) using 0.15 M AC/SA hydrolysis at a molar ratio of 3.0 at 170 °C for 60 min, followed by xylanase hydrolysis. The second-step AC/SA hydrolysis of hydrolyzed corncob (10%, w/v) produced 3.1 g/L of XOS. Finally, the maximum XOS yield of 74.8% (based on xylan in corncob) was achieved, which is the highest yield among yields reported previously. The purity of XOS was high, whereas the contents of by-products were very low. This work presents a novel and promising strategy for co-production of XOS and monosaccharides from corncob without xylan isolation and purification.

Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions-multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

The aim of this work was to investigate the use of isobutanol as organic solvent for the efficient delignification and fractionation of beechwood through the OxiOrganosolv process in the absence of any catalyst. The results demonstrate that cellulose-rich solid pulp produced after pretreatment is a source of fermentable sugars that can be easily hydrolyzed and serve as a carbon source in microbial fermentations for the production of omega-3 fatty acids and D-lactic acid. The C5 sugars are recovered in the aqueous liquid fractions and comprise a fraction rich in xylo-oligosaccharides with prebiotic potential. The maximum production of optically pure D-lactic from Lactobacillus delbrueckii sp. bulgaricus reached 51.6 g/L (0.57 g/g_biomass), following a simultaneous saccharification and fermentation strategy. Crypthecodenium cohnii accumulated up to 52.1 wt% lipids with a DHA content of 54.1 %, while up to 43.3 % hemicellulose recovery in form of oligosaccharides was achieved in the liquid fraction.