Samarthya Bhagia

Effects of dilute acid and flowthrough pretreatments and BSA supplementation on enzymatic deconstruction of poplar by cellulase and xylanase

To help understand factors controlling the recalcitrance of lignocellulosic biomass to deconstruction to sugars, poplar was pretreated with liquid hot water (LHW) and extremely dilute acid (EDA) at 140°C and 180°C in batch and flowthrough reactors. The resulting solids were then subjected to enzymatic hydrolysis by eight combinations of cellulase, xylanase, and bovine serum albumin (BSA). Co-addition of xylanase to cellulase resulted in up to 11 percentage points higher overall sugar yield than their sequential addition. In general, supplementation of BSA to enzymes had a larger impact on flowthrough solids with reduced lignin content than batch solids with high lignin content. BSA did not affect xylan yields and while it had low impact on LHW solids, it caused large increases in sugar yields from EDA solids. Flowthrough pretreatment produced less recalcitrant solids than did batch operation, but using very dilute acid reduced recalcitrance even more.

Robustness of two-step acid hydrolysis procedure for composition analysis of poplar

The NREL standard procedure for lignocellulosic biomass composition has two steps: primary hydrolysis in 72% wt sulfuric acid at 30°C for 1h followed by secondary hydrolysis of the slurry in 4wt% acid at 121°C for 1h. Although pointed out in the NREL procedure, the impact of particle size on composition has never been shown. In addition, the effects of primary hydrolysis time and separation of solids prior to secondary hydrolysis on composition have never been shown. Using poplar, it was found that particle sizes less than 0.250mm significantly lowered the glucan content and increased the Klason lignin but did not affect xylan, acetate, or acid soluble lignin contents. Composition was unaffected for primary hydrolysis time between 30 and 90min. Moreover, separating solids prior to secondary hydrolysis had negligible effect on composition suggesting that lignin and polysaccharides are completely separated in the primary hydrolysis stage.

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

Cellulose-hemicellulose interactions at elevated temperatures increase cellulose recalcitrance to biological conversion

It has been previously shown that cellulose-lignin droplets’ strong interactions, resulting from lignin coalescence and redisposition on cellulose surface during thermochemical pretreatments, increase cellulose recalcitrance to biological conversion, especially at commercially viable low enzyme loadings. However, information on the impact of cellulose–hemicellulose interactions on cellulose recalcitrance following relevant pretreatment conditions are scarce. Here, to investigate the effects of plausible hemicellulose precipitation and re-association with cellulose on cellulose conversion, different pretreatments were applied to pure Avicel® PH101 cellulose alone and Avicel mixed with model hemicellulose compounds followed by enzymatic hydrolysis of resulting solids at both low and high enzyme loadings. Solids produced by pretreatment of Avicel mixed with hemicelluloses (AMH) were found to contain about 2 to 14.6% of exogenous, precipitated hemicelluloses and showed a remarkably much lower digestibility (up to 60%) than their respective controls. However, the exogenous hemicellulosic residues that associated with Avicel following high temperature pretreatments resulted in greater losses in cellulose conversion than those formed at low temperatures, suggesting that temperature plays a strong role in the strength of cellulose–hemicellulose association. Molecular dynamics simulations of hemicellulosic xylan and cellulose were found to further support this temperature effect as the xylan–cellulose interactions were found to substantially increase at elevated temperatures. Furthermore, exogenous, precipitated hemicelluloses in pretreated AMH solids resulted in a larger drop in cellulose conversion than the delignified lignocellulosic biomass containing comparably much higher natural hemicellulose amounts. Increased cellulase loadings or supplementation of cellulase with xylanases enhanced cellulose conversion for most pretreated AMH solids; however, this approach was less effective for solids containing mannan polysaccharides, suggesting stronger association of cellulose with (hetero) mannans or lack of enzymes in the mixture required to hydrolyze such polysaccharides.

Sugar Yield and Composition of Tubers from Jerusalem Artichoke (Helianthus tuberosus) Irrigated with Saline Waters.

Currently, major biofuel crops are also food crops that demand fertile soils and good‐quality water. Jerusalem artichoke (Helianthus tuberosus, Asteraceae) produces high tonnage of tubers that are rich in sugars, mainly in the form of inulin. In this study, plants of the cultivar “White Fuseau” grown under five salinity levels were evaluated for tuber yield. Results indicated that this cultivar is moderately salt‐tolerant if the goal is tuber production. Hydraulic pressings of the tubers produced juice that contained 15% (wet weight) or 55% (dry weight) free sugars, with 70% of these in the form of inulin and the rest as fructose, sucrose, and glucose. Importantly, salinity did not affect the total free sugar or inulin content of the tubers. Tubers were composed of about 12% dry washed bagasse (wet weight) or 44% (dry matter basis) and bagasse retained such high quantities of free sugars after pressing that washing was required for complete sugar recovery. Chemical composition analysis of tuber bagasse suggested that it had low lignin content (11–13 wt%), and its structural sugar composition was similar to chicory root bagasse. Because of the high hemicellulose and pectin content of the bagasse, adding xylanase and pectinase to cellulase substantially improved sugar yields from enzymatic hydrolysis compared to at the same protein loading as cellulase alone. In addition to the high total sugar yield of tuber, these first findings on the sugar and lignin content and enzymatic hydrolysis of tuber bagasse can lead to low‐cost production of ethanol for transportation fuels.

Ultrastructure and Enzymatic Hydrolysis of Deuterated Switchgrass

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media.

In situ Rheological Method to evaluate Feedstock Physical Properties throughout Enzymatic Deconstruction

Feedstock physical properties determine not only downstream flow behavior, but also downstream process yields. Enzymatic treatment of pretreated feedstocks greatly dependent on upstream feedstock physical properties and choice of pre-processing technologies. Currently available enzyme assays have been developed to study biomass slurries at low concentrations of ≤ 1% w/w. At commercially relevant biomass concentrations of ≥ 15% w/w, pretreated feedstocks have sludge-like properties, where low free water restricts movement of unattached enzymes. This work is an account of the various steps taken to develop a method that helps identify the time needed for solid-like biomass slurries transition into liquid-like states during enzymatic hydrolysis. A pre-processing technology that enables feedstocks in achieving this transition sooner will greatly benefit enzyme kinetics and thereby overall process economics. Through this in situ rheological properties determining method, we compared a model feedstock, Avicel®PH101 cellulose, and acid pretreated corn stover. We determined that 25% (w/w) Avicel when treated with Novozymes Cellic®CTec2 (80 mg protein/g glucan) can reduce from solid-like to liquid-like state in 5.5 h, as the phase angles rise beyond 45° at this time. The same slurry needed 5.3 h to achieve liquid-like state with Megazyme endoglucanase (40 mg protein/g glucan). After 10.8 h, CTec2 slurry reached a phase angle of 89° or complete liquid-like state but Megazyme slurry peaked only to 64.7°, possibly due to inhibition by cello-oligomers. Acid pretreated corn stover at 30% (w/w) with a CTec2 protein loading of 80 mg/g glucan exhibited a solid-like to liquid-like transition at 37.8 h, which reflects the combined inhibition of low water activity and presence of lignin. The acid pretreated slurry also never achieved complete liquid-like state due to the presence of biomass residue. This method is applicable in several scenarios comparing varying combinations of pre-processing technologies, feedstock types, pretreatment chemistries, and enzymes. Using this method, we can generate a process chain with optimal flow behavior at commercially-relevant conditions.