Fibrolytic enzyme treatment prior to ensiling increased press-juice and crude protein yield from grass silage.

Grass is a versatile raw material for green biorefineries and preserving it as silage provides a year-round feedstock. The objective of the current study was to evaluate the effect of fibrolytic enzyme application on silage as a feedstock for a biorefinery. Two batches of grass (mixture of timothy and meadow fescue) silages were ensiled in pilot scale after fibrolytic enzyme was applied to them at four levels. Enzyme application increased fibre degradation linearly during ensiling and increased lactic and acetic acid concentrations in the silage. Simultaneously, silage fermentation quality improved as indicated by decreasing pH and ammonia values. Press-juice and crude protein yields increased in response to the fibrolytic enzyme application, which is beneficial in a biorefinery concept for retrieving valuable nutrients from grass matrix. Optimized ensiling methodology can be considered as a pretreatment for a biorefinery process.

The liquid fraction from hydrothermal pretreatment of wheat straw provides lytic polysaccharide monooxygenases with both electrons and H2O2 co-substrate.

BACKGROUND: Enzyme-aided valorization of lignocellulose represents a green and sustainable alternative to the traditional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important components of the state-of-the art enzyme cocktails for cellulose conversion. Yet, these monocopper enzymes are poorly characterized in terms of their kinetics, as exemplified by the growing evidence for that H2O2 may be a more efficient co-substrate for LPMOs than O2. LPMOs need external electron donors and one key question of relevance for bioprocess development is whether the required reducing power may be provided by the lignocellulosic substrate. RESULTS: Here, we show that the liquid fraction (LF) resulting from hydrothermal pretreatment of wheat straw supports LPMO activity on both chitin and cellulose. The initial, transient activity burst of the LPMO reaction was caused by the H2O2 present in the LF before addition of LPMO, while the steady-state rate of LPMO reaction was limited by the LPMO-independent production of H2O2 in the LF. H2O2 is an intermediate of LF oxidation as evidenced by a slow H2O2 accumulation in LF, despite high H2O2 production rates. This H2O2 scavenging ability of LF is important since high concentrations of H2O2 may lead to irreversible inactivation of LPMOs. CONCLUSIONS: Our results support the growing understanding that fine-tuned control over the rates of H2O2 production and consumption in different, enzymatic and non-enzymatic reactions is essential for harnessing the full catalytic potential of LPMOs in lignocellulose valorization.

Cellulases adsorb reversibly on biomass lignin.

Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments on lignin-rich residues (LRRs) isolated from hydrothermally pretreated spruce (L-HPS) and wheat straw (L-HPWS), respectively. On the basis of adsorption isotherms fitted to the Langmuir model, the ranking of binding affinities was TrCel5A > TrCel6A > TrCel7B > TrCel7A on both types of LRRs. The enzymes had a higher affinity to the L-HPS than to the L-HPWS. Adsorption experiments with dilution after 1 and 24 hr and kinetic modeling were performed to quantify any irreversible binding over time. Models with reversible binding parameters fitted well and can explain the results obtained. The adsorption constants obtained from the reversible models agreed with the fitted Langmuir isotherms and suggested that reversible adsorption-desorption existed at equilibrium. Competitive binding experiments showed that individual types of cellulases competed for binding sites on the lignin and the adsorption data fitted the Langmuir adsorption model. Overall, the data strongly indicate that the adsorption of cellulases onto lignin is reversible and the findings have implications for the development of more efficient cellulose degrading enzymes.

The effect of direct and counter-current flow-through delignification on enzymatic hydrolysis of wheat straw, and flow limits due to compressibility.

This article compares the processes for wheat straw lignocellulose fractionation by percolation, counter-current progressing batch percolation and batch reaction at low NaOH-loadings (3-6% of DM). The flow-through processes were found to improve delignification and subsequent enzymatic saccharification, reduce NaOH-consumption and allow reduction of thermal severity, whereas hemicellulose dissolution was unaffected. However, contrary to previous expectations, a counter-current process did not provide additional benefits to regular percolation. The compressibility and flow properties of a straw bed were determined and used for simulation of the packing density profile and dynamic pressure in an industrial scale column. After dissolution of 30% of the straw DM by delignification, a pressure drop above 100 kPa m-1 led to clogging of the flow due to compaction of straw. Accordingly, the maximum applicable feed pressure and volumetric straw throughput was determined as a function of column height, indicating that a 10 m column can be operated at a maximum feed pressure of 530 kPa, corresponding to an operation time of 50 min and a throughput of 163 kg m-3 h-1 . Biotechnol. Bioeng. 2016;113: 2605-2613. © 2016 Wiley Periodicals, Inc.

Rate-constraining changes in surface properties, porosity and hydrolysis kinetics of lignocellulose in the course of enzymatic saccharification.

BACKGROUND: Explaining the reduction of hydrolysis rate during lignocellulose hydrolysis is a challenge for the understanding and modelling of the process. This article reports the changes of cellulose and lignin surface areas, porosity and the residual cellulase activity during the hydrolysis of autohydrolysed wheat straw and delignified wheat straw. The potential rate-constraining mechanisms are assessed with a simplified kinetic model and compared to the observed effects, residual cellulase activity and product inhibition. RESULTS: The reaction rate depended exclusively on the degree of hydrolysis, while enzyme denaturation or time-dependent changes in substrate hydrolysability were absent. Cellulose surface area decreased linearly with hydrolysis, in correlation with total cellulose content. Lignin surface area was initially decreased by the dissolution of phenolics and then remained unchanged. The dissolved phenolics did not contribute to product inhibition. The porosity of delignified straw was decreased during hydrolysis, but no difference in porosity was detected during the hydrolysis of autohydrolysed straw. CONCLUSIONS: Although a hydrolysis-dependent increase of non-productive binding capacity of lignin was not apparent, the dependence of hydrolysis maxima on the enzyme dosage was best explained by partial irreversible product inhibition. Cellulose surface area correlated with the total cellulose content, which is thus an appropriate approximation of the substrate concentration for kinetic modelling. Kinetic models of cellulose hydrolysis should be simplified enough to include reversible and irreversible product inhibition and reduction of hydrolysability, as well as their possible non-linear relations to hydrolysis degree, without overparameterization of particular factors.

Determination of surface-accessible acidic hydroxyls and surface area of lignin by cationic dye adsorption.

A new colorimetric method for determining the surface-accessible acidic lignin hydroxyl groups in lignocellulose solid fractions was developed. The method is based on selective adsorption of Azure B, a basic dye, onto acidic hydroxyl groups of lignin. Selectivity of adsorption of Azure B on lignin was demonstrated using lignin and cellulose materials as adsorbents. Adsorption isotherms of Azure B on wheat straw (WS), sugarcane bagasse (SGB), oat husk, and isolated lignin materials were determined. The maximum adsorption capacities predicted by the Langmuir isotherms were used to calculate the amounts of surface-accessible acidic hydroxyl groups. WS contained 1.7-times more acidic hydroxyls (0.21 mmol/g) and higher surface area of lignin (84 m(2)/g) than SGB or oat husk materials. Equations for determining the amount of surface-accessible acidic hydroxyls in solid fractions of the three plant materials by a single point measurement were developed. A method for high-throughput characterization of lignocellulosic materials is now available.

Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus.

A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K(m) and V(max) values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg⁻¹, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80 °C was to a great extent reversible.

Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88.

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC-MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.