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The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.

Background: Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. Results: Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. Conclusions: The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides.

Wheat straw based composting generates a selective substrate for mushroom production. The first phase of this process requires 5 days, and a reduction in time is wished. Here, we aim at understanding the effect of gypsum on the duration of the first phase and the mechanism behind it. Hereto, the regular process with gypsum addition and the same process without gypsum were studied during a 5-day period. The compost quality was evaluated based on compost lignin composition analysed by py-GC/MS and its degradability by a commercial (hemi-)cellulolytic enzyme cocktail. The composting phase lead to the decrease of the pyrolysis products 4-vinylphenol and 4-vinylguaiacol that can be associated with p-coumarates and ferulates linking xylan and lignin. In the regular compost, the enzymatic conversion reached 32 and 39% for cellulose, and 23 and 32% for xylan after 3 and 5 days, respectively. In absence of gypsum similar values were reached after 2 and 4 days, respectively. Thus, our data show that in absence of gypsum the desired compost quality was reached 20% earlier compared to the control process.

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2 > 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin. (Figure Presented).

Background: Lytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity. Results: Here, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds. Conclusions: Several of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity towa

Background: The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-)glucuronic acids (UAme). Hence, α-glucuronidases play an important role in the degradation of UAmexylan structures facilitating the complete utilisation of plant biomass. The characterisation of α-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UAmexylan structures. Results: The mode-of-action of two α-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 °C. ReGH67 was able to release UAme from UAme-substituted xylo-oligosaccharides (UAmeXOS), but only the UAme linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UAmeXOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UAmeXOS (DP 3-4). On the contrary, ReGH115 was able to release UAme from both polymeric UAmexylan and UAmeXOS. ReGH115 cleaved UAme from both internal and non-reducing end xylosyl residues, with the exception of UAme attached to the non-reducing end of a xylotriose oligosaccharide. Conclusion: In this research, and for the first time, we define the mode-of-action of two α-glucuronidases from two different GH families both from

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus degrades compost in the controlled environment in commercial production facilities and utilizes this substrate to produce mushrooms. Using transcriptomics and proteomics linked to analysis of the compositional changes in the compost, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Lignin degradation appears to only occur during the spawning stage. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis, demonstrating the presence of these enzymes during specific stages. The out-of-phase enzyme production in the second flush may explain the reduction in the number of mushrooms during the second flush. Combined with other data generated in our lab we have now generated the first detailed model of the genes and enzymes involved in compost degradation and utilization by A. bisporus, which has provided many leads for a better understanding and improvement of the commercial mushroom cultivation.

The increasing interest in plant biomass based biofuels and chemicals arouses mainly from the increased awareness of a possible finiteness of fuels. The current main challenge to produce such biofuels and biochemicals is economic efficiency, but also knowledge concerning type and effectiveness of both thermally assisted chemical and enzymatic treatments, needed to generate fermentable sugars, is lacking. The subject of this thesis is to gain understanding of the effect of both sulphuric acid and NaOH catalysed pretreatments of sugar cane bagasse and oil palm empty fruit bunches on subsequent enzymatic saccharification, with a focus on the NaOH catalysed pretreatments. The fate of the main polymers present, lignin, cellulose and xylan, was studied and also single-activity xylan degrading enzymes, all from Rasamsonia emersonii, were studied for their mode-of-action.

Thermo-assisted NaOH pretreatment to deconstruct xylan and lignin in sugar cane bagasse (SCB) is poorly understood. Hence, in this research it is was aimed to study the effect of NaOH pretreatment on the insoluble remaining lignin structures. Hereto, SCB milled fibres were pretreated using different dosages of NaOH at different temperatures and residence times. Of untreated SCB about 63% of the lignin compounds were assigned as p-coumarates and ferulates, analysed by pyrolysis-GC/MS as 4-vinyl phenol and 4-vinyl guaiacol, and designated as non-core lignin (NCL) compounds. More severe NaOH pretreatments resulted in lower xylan and lower lignin recoveries in the insoluble residues. Especially, the relative abundance of NCL decreased and this decrease followed a linear trend with the decrease in xylan. Core lignin compounds, analysed as phenol, guaiacol and syringol, accumulated in the residues. The decrease in residual xylan and NCL correlated positively with the enzymatic hydrolysis of the residual glucan.

Background: Many agricultural and industrial food by-products are rich in cellulose and xylan. Their enzymatic degradation into monosaccharides is seen as a basis for the production of biofuels and bio-based chemicals. Lytic polysaccharide monooxygenases (LPMOs) constitute a group of recently discovered enzymes, classified as the auxiliary activity subgroups AA9, AA10, AA11 and AA13 in the CAZy database. LPMOs cleave cellulose, chitin, starch and β-(1 → 4)-linked substituted and non-substituted glucosyl units of hemicellulose under formation of oxidized gluco-oligosaccharides. Results: Here, we demonstrate a new LPMO, obtained from Myceliophthora thermophila C1 (MtLPMO9A). This enzyme cleaves β-(1 → 4)-xylosyl bonds in xylan under formation of oxidized xylo-oligosaccharides, while it simultaneously cleaves β-(1 → 4)-glucosyl bonds in cellulose under formation of oxidized gluco-oligosaccharides. In particular, MtLPMO9A benefits from the strong interaction between low substituted linear xylan and cellulose. MtLPMO9A shows a strong synergistic effect with endoglucanase I (EGI) with a 16-fold higher release of detected oligosaccharides, compared to the oligosaccharides release of MtLPMO9A and EGI alone. Conclusion: Now, for the first time, we demonstrate the activity of a lytic polysaccharide monooxygenase (MtLPMO9A) that shows oxidative cleavage of xylan in addition to cellulose. The ability of MtLPMO9A to cleave these rigid regions provides a new paradigm in the understanding of the degradation of xylan-coated cellulose. In addition, MtLPMO9A acts in strong synergism with endoglucanase I. The mode of action of MtLPMO9A is considered to be important for loosening the rigid xylan-cellulose polysaccharide matrix in plant biomass, enabling increased accessibility to the matrix for hydrolytic enzymes. This discovery provides new insights into how to boost plant biomass degradation by enzyme cocktails for biorefinery applications.

In this thesis, the fate of carbohydrates and lignin was studied in industrial wheat straw based compost during composting and growth of Agaricus bisporus. The aim was to understand the availability and degradability of carbohydrates in order to help improve their utilization in the compost. The wheat straw based compost was characterized as being composed mainly of cellulose and lowly substituted xylan. During the first phase of composting, ester-bound substituents were removed from the xylan backbone and during the second phase of composting 50% of carbohydrates present in the original material where metabolized in a uniform manner. Lignin structure, however, remained unaltered during these composting stages. Over the period of A. bisporus mycelium growth, 20% of the original xylan became water soluble while xylan structures remained rather similar and the remaining water insoluble xylan was partially degraded. In addition, 40% of lignin was metabolized during mycelium growth with an increase in the ratio of syringyl to guaiacyl lignin units from 0.5 to 0.7 in mycelium grown compost compared to the basic compost mixture. During the fruiting body formation minor changes in lignin structure occurred, while accumulation of xylan substituents was observed for arabinosyl residues and glucuronic acid substituents. Finally, putative genes encoding carbohydrate degrading enzymes were identified in A. bisporus’ genome. Genes involved in the pentose and hexose catabolic pathway were found to be upregulated in A. bisporus mycelium. A. bisporus was found to produce both xylan and cellulose degrading enzymes and maximum activity was observed during the formation of the 1st flush of mushrooms. But, as observed from the remaining xylan structures analyzed, A. bisporus lacks the enzymatic activity to degrade xylan substituted with two arabinosyl- residues and glucuronic acid substituted xylan. Edita Jurak How Mushrooms feed on compost: Conversion of carbohydrates and lin

The increased use of fiber-rich feedstuffs in pig and poultry diets requires an optimal utilization of these feed ingredients. Hence, the animal feed industry explores opportunities to improve degradability of these feedstuffs and maximize their inclusion levels in pig and poultry diets. Processing and enzyme technologies can modify the physicochemical characteristics of fiber fractions from feed ingredients, thereby affecting their degradability. In this way, fermentability of non-starch polysaccharides (NSP) and thus their potential energetic utilization might be enhanced. In addition, technologies can be aimed at alleviation of adverse effects on digestion and absorption of other nutrients, which might be particularly of interest for young pigs and poultry. However, to understand modifications that occur during processing detailed information on the composition of fiber structures is required. This thesis aimed at identifying limiting factors in the degradability of fiber fractions in pigs and poultry and at development of technologies to improve their degradation. Focus was on recalcitrant fiber structures as found in in maize dried distillers grain with solubles (DDGS) and rapeseed meal (RSM). Fiber degradation in growing pigs and broilers was studied in detail and limiting structures in the degradation of NSP were identified (Chapter 5 to 7). Effects of processing and enzyme technologies on fiber-rich feedstuffs were evaluated based on literature and in vitro and in vivo studies in growing pigs and broilers (Chapter 2 to 7). In addition, marker methods to study digestibility of fiber-rich diets in broilers were discussed (Chapter 8). Furthermore, interactive effects between specific fermentable fiber sources and the digestive utilization of the diet were investigated (Chapter 9). In the final chapter, results of the thesis were summarized and synthesized. Methods to analyze fiber components and evaluate fiber degradation in vitro and in vivo were discussed, su

Cell wall polysaccharides (CWPs) contribute to the water holding capacity (WHC) of fibre rich feeds, such as pressed potato fibres (PPF). However, the role of CWPs on the WHC of PPF was unidentified so far. PPF was characterized to be abundant in arabinogalactan (AG) linked rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and cellulose, next to which xyloglucan (XG) contributed the most of the hemicellulosic CWPs. The CWP network in potatoes was loosened upon starch extraction of potatoes and solubilized HG-RG-I-AG. Analyses of the WHCs upon enzyme treatments indicated that the WHC of PPF was mainly caused by a network of insoluble, non-cellulosic CWPs such as pectic CWPs (HG-RG-I-AG) and XG. Findings in this thesis showed that AGs were better degraded than xyloglucans (XGs). Since XGs were found to be equally important in contributing to the WHC as AGs, the substantial removal of AGs, as well as XGs, should be advantageous to lower the WHC. Other than lowering the WHC, the use of a pectinase-rich preparation improved the recovery of starch from potatoes by the degradation of mainly pectic CWPs, in particular pectic AG side chains and HG. The degradation of arabinan was observed to be inhibited by components in potato juice (PJ).

In this thesis, the fate of non-starch polysaccharides (NSP) from rapeseed meal (RSM) during fermentation in vitro and in vivo was studied. The aim was to understand and improve the fermentation of NSP from RSM in poultry and pigs, by processing and enzyme addition. First, the NSP-structures in RSM were characterized as being branched arabinan, arabinogalactan type II, homogalacturonan, glucurono-xylan, XXGG- and XXXG-type xyloglucan, and cellulose. Second, RSM was processed using shear, heat, and acid prior to in vitro incubation, in the presence or absence of pectolytic enzymes. Acid-treatment combined with pectolytic enzymes was the best option to improve NSP-solubilization in vitro. Unprocessed and acid-extruded RSM with or without addition of enzymes were fed to broilers. In broilers, 22% of the NSP in unprocessed RSM could be fermented, which only significantly improved to 38% by addition of commercial pectolytic enzymes. In broilers’ excreta, XXXG-type xyloglucan, (glucurono-)xylan, arabinan, and cellulose remained unfermented. Unprocessed and acid-extruded RSM was also fed to growing pigs and NSP-fermentation was followed along the digestive tract. In pigs, at the terminal ileum 22% of the NSP was cumulatively fermented and total tract around 70% was fermented. Acid-extrusion improved total tract NSP-fermentability in pigs numerically by 4% points. Water-soluble carbohydrates were nearly completely fermented. In the feces some rhamnogalacturonan, (branched) arabinan, linear xylan, XXXG-type xyloglucan, galactomannan, and cellulose remained. Surprisingly, during alkaline extraction of the broilers’ excreta and pigs’ feces, around 40% (w/w) of the insoluble carbohydrates was released as glucosyl- and/or uronyl-rich carbohydrates, probably originally present via ester-linkages or hydrogen-bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP-fermentation.