Ville Pihlajaniemi

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.

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.

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.

Reduction of surface area of lignin improves enzymatic hydrolysis of cellulose from hydrothermally pretreated wheat straw

Enzymatic hydrolysis of cellulose after pretreatment of wheat straw (WS) was investigated for the first time in relation to lignin surface area (SA). Lignin SA in solid residues from WS autohydrolysis (AH) and successive NH3 (aq) extraction was determined using cationic dye adsorption. AH at increasing severity decreased up to 45% and 53% of WS lignin SA and specific surface area (SSA), respectively. Cellulose-to-glucose conversion from AH solid fractions from 24 h reaction with 15 FPU g−1 cellulase activity increased linearly from 31% to 91% with decreasing lignin SA. When AH solid fractions were extracted with NH3 (aq), both lignin SA and SSA increased in the corresponding solid residues, SSA up to 92%. As a consequence, cellulose-to-glucose conversion decreased in spite of the lower proportion of lignin in the solid residues after the NH3 (aq) extraction. Up to 85% sugar yield was obtained from the single-stage AH process but when combined with NH3 (aq) extraction the two-stage process yielded at most 71% of the original straw sugars. These results show that, independent of the lignin content, reduction of surface area of lignin improves the enzymatic hydrolysis process.

Integrating the opposites of biofuel production: Absorption of short-chain alcohols into oleaginous yeast cells for butanol recovery and wet-extraction of microbial oil

Recovery of two biotechnologically produced fuel components, butanol and microbial oil, is assessed by absorption of the six shortest 1-alcohols into oleaginous yeast cells. We show an unexpectedly high extent of absorption of >C3 alcohols from water into Rhodosporidium fluviale cells with a lipid content of 69% of cell dry weight (CDW). Increasing the carbon chain length of the alcohol boosts both the rate and the quantity of absorption of the alcohol from water containing an initial ratio of 9.5 of CDW to alcohol. Under these conditions, 40% of butanol is removed from water, while the methanol concentration remains unchanged in 48 h incubation with the oleaginous yeast cells. Lower absorption of alcohols into non-oleaginous bakers yeast cells as a reference suggests that a majority of the alcohols combines with the lipid droplets inside oleaginous cells. The partition coefficient of the intracellular microbial oil to butanol exceeds those of oleyl alcohol and rapeseed oil by factors of 4 and 16. The capacity of oleaginous yeast cells to absorb butanol reaches 13% of CDW from 48 g per L butanol solution. Leakage of intracellular microbial oil occurs when the initial butanol concentration exceeds approximately 20 g L−1. Butanol can be recovered after absorption from oleaginous yeast biomass, while microbial oil can be separated by subsequent wet-extraction with the alcohols as solvents. These results suggest that synergistic outcomes can be achieved by process integration both for industry and the environment.

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.