Mika Henrikki Sipponen

Weighing the factors behind enzymatic hydrolyzability of pretreated lignocellulose

The major factors determining enzymatic hydrolyzability of pretreated wheat straw were analyzed and their relative importance quantified. The effects of NaOH-delignification, autohydrolysis and their combination at different severities were analyzed by determining the pore size distribution (DSC-thermoporometry), the cellulose surface area and the accessible phenolic hydroxyls on the lignin surface (adsorption of Congo Red and Azure B; ATR-FTIR) and crystallinity (WAXD). The correlation of these factors with initial and overall enzymatic hydrolyzability was studied and further arranged in order through principal component analysis. The major positive factors affecting hydrolyzability were the cellulose surface area and the accessibility of the pore system, while the lignin content was the major negative factor accompanied by cellulose crystallinity. Autohydrolysis effectively increased the cellulose surface area by hemicellulose dissolution, but the high lignin content associated with small pores led to a lower hydrolyzability compared to delignified straw. Besides the removal of lignin, delignification led to a more accessible pore structure, which was supported by the remaining hemicellulose. Additionally, delignification increased the hydrophilicity of the remaining lignin, which also increased hydrolyzability. All pretreatments decreased cellulose crystallinity, which particularly increased the initial hydrolysis, and also improved the final carbohydrate conversion. The established weighed order of the factors behind enzymatic carbohydrate conversion is an important milestone in the path towards more efficient lignocellulosic sugar utilization in biorefineries.

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.

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.

Autohydrolysis and aqueous ammonia extraction of wheat straw: effect of treatment severity on yield and structure of hemicellulose and lignin

The objective of this study was to elucidate the impact of autohydrolysis severity on the yield and structure of wheat straw hemicellulose and lignin. The autohydrolysis treatments were carried out at maximum temperatures between 170 °C and 200 °C. The autohydrolysis liquors were separated and the solids were successively extracted with aqueous ammonia either in moderate or high intensity extraction conditions to dissolve lignin for analysis. Increasing autohydrolysis severity decreased the molar mass of the aqueous ammonia extracts from 5450 g mol−1 to 1810 g mol−1, and carbohydrate content from 6% to 0.1%. The optimum autohydrolysis severity (log R0 = 3.81) for xylan recovery released mainly oligomeric arabinoxylans at 66% xylan recovery yield. Drastic degradation of pentoses occurred beyond the optimum severity. As an indication of accumulation of “pseudo-lignin” during autohydrolysis, decreasing relative aromaticity in the aqueous ammonia extracts as a function of autohydrolysis severity was shown. The finding was confirmed by quantitative analysis of the cupric oxide oxidation products of lignin suggesting up to 55% decrease in the relative amount of native lignin at the highest severity. These results show the importance of distinguishing between lignin and “pseudo-lignin” in fractions obtained from lignocellulosic materials subjected to acidic pretreatment.

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

AbstractBackgroundExplaining 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.ResultsThe 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.ConclusionsAlthough 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.

Yield optimization and rational function modelling of enzymatic hydrolysis of wheat straw pretreated by NaOH-delignification, autohydrolysis and their combination

A thorough efficacy assessment was performed on three wheat straw saccharification processes including NaOH-delignification, autohydrolysis and their combination, with subsequent enzymatic hydrolysis. Instead of optimizing the process for maximal sugar yield from straw, a novel perspective is provided, allowing optimization of the overall yield against enzyme consumption and reaction volume. At total sugar yields above 60%, NaOH-delignification was the most efficient in terms of enzymatic and volumetric productivity, whereas at lower yields, autohydrolysis showed a comparable enzymatic and a higher volumetric productivity. The double treatment led to improved hydrolysability compared to autohydrolysis, but was the least productive due to reduced solid yields. A threshold in the delignification efficiency between 3% and 6% NaOH-loadings per straw DM resulted from the depletion of alkalinity by the released organic acids. A novel rational function model was developed for the total sugar yield, which is generally superior for describing asymptotic behaviour compared to conventional polynomial models in response surface modelling.

Isolation of structurally distinct lignin-carbohydrate fractions from maize stem by sequential alkaline extractions and endoglucanase treatment.

Sequential fractionation of extractive-free maize stems was carried out using two mild alkaline extractions (0.5 and 2 M NaOH, 20°C, 24h) before and after endoglucanase treatment. This procedure provided two lignin-carbohydrate fractions (LC1 and LC2) recovered after each alkali treatment. LC1 and LC2 contained 39% and 8% of the total lignin amount, respectively. These two fractions contained structurally distinct lignin molecules. While the content of resistant interunit bonds in lignin was 77% in LC1, it was increased up to 98% in LC2. Not unexpectedly, both alkali-soluble fractions contained substantial amount of p-coumaric and ferulic acids ether-linked to lignins. These results outline heterogeneity of maize stem lignins related to fractionation of grass materials.

Increased Water Resistance of CTMP Fibers by Oat (Avena sativa L.) Husk Lignin

The insertion of oat husk lignin onto chemithermomechanical pulp (CTMP) fibers was studied to increase fiber hydrophobicity. The pretreated pulp samples were subsequently used for preparation of handsheets for characterization. Treatment of CTMP with laccase in the presence of oat husk lignin resulted in a significant increase in hydrophobicity of the handsheet surface, as indicated by dynamic contact angle analysis. Water absorption time of 8 s was obtained with initial contact angle of 118°. Although the handsheets brightness was reduced by 33%, tensile index was only subtly decreased. Neither laccase nor oat husk lignin alone gave much improved water absorption times. Therefore, handsheets made of laccase-treated pulp with and without oat husk lignin were further examined by XPS, which suggested that both laccase and oat husk lignin were inserted onto CTMP fibers. The oat husk lignin was distributed as heterogeneous aggregates on the handsheet surface whereas laccase was uniformly distributed. Evidence was obtained that the adsorbed laccase layer formed a noncovalent base for the insertion of oat husk lignin onto fiber surfaces.

Structural diversity in metal–organic nanoparticles based on iron isopropoxide treated lignin

The magnetic nature of iron-containing nanoparticles enables multiple high-end applications. Metal alkoxides are a highly reactive chemical species, which are widely used in ceramics and sol–gel manufacture. However, their use with organic molecules has been mostly limited to catalytic purposes due to their highly reactive nature. Lignin is the second most abundant biopolymer in the world-rich in OH groups and amenable to highly stable colloid nanoparticle formation by a simple solvent exchange process. Here we show that the reaction between iron isopropoxide and lignin in tetrahydrofuran (THF) solution produces metal–organic nanoparticles with tunable morphologies, ranging from hollow and solid nanospheres to open network structures. The immediate condensation reaction between lignin and iron isopropoxide as well as the resulting structure morphology can be controlled by varying the reaction parameters. Despite iron isopropoxide being highly water sensitive, the formed structures are stable as water suspensions. Our results demonstrate that solution processable metal–organic nanoparticles can be easily produced with macromolecular polyols in an inert solvent, such as THF. This presents a facile method of obtaining various metal–organic nanomaterials, with a wide range of metal alkoxides and organic polyols to choose from. We anticipate that metal–bioorganic sol–gel reactions will produce biocompatible materials with enhanced functionality, such as magnetic, antibacterial and catalytic properties depending on the chosen metal and polyol.

All-lignin approach to prepare cationic colloidal lignin particles: stabilization of durable Pickering emulsions

Surface modification of colloidal lignin particles (CLPs), which are obtained from renewable resources, is a plausible route towards novel biomaterials. Here we show that adsorption of cationic lignin onto spherical CLPs produces positively charged particles with tailored properties for the stabilization of Pickering emulsions. The threshold dosing of cationic lignin needed to achieve colloidally stable cationic dispersions was 4% relative to the dry weight of CLPs. Compared to irregular kraft lignin particles or regular CLPs, cationic CLPs stabilized a broader array of durable Pickering emulsions. This all-lignin adsorption process to prepare cationic CLPs is advantageous because it minimizes the consumption of synthetic polymers, and opens new application opportunities for structurally defined nano- and microscale lignin particles.