Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Does change in accessibility with conversion depend on both the substrate and pretreatment technology?

The accessibility of cellulase and xylanase enzymes to glucan and xylan, respectively, and its change with conversion were measured for pure Avicel glucan and poplar solids that had been pretreated by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute acid, and lime. Avicel and pretreated solids were digested to various degrees by cellulase together with β-glucosidase enzymes and then cleaned of residual protein via a biological method using Protease. Glucan accessibility was determined by purified CBHI (Cel7A) adsorption at 4 °C, and 4 and 24 h hydrolysis yields were determined for solids loading containing equal amounts of glucan (1.0% w/v) and lignin (1.0% w/v), in two separate sets of experiments. Consistent with our previous study and in contrast to some in the literature, little change in glucan accessibility was observed with conversion for Avicel, but glucan and xylan accessibility for real biomass varied with the type of pretreatment. For example, AFEX pretreated solids showed a negligible change in glucan accessibility for conversion up to 90%, although xylan accessibility seemed to decline first and then remained constant. On the other hand, a substantial decline in glucan and xylan accessibility with conversion was observed for lime pretreated poplar solids, as shown by initial hydrolysis rates. Yet, an increase in CBHI adsorption with conversion for lime pretreated poplar solids suggested the opposite trend, possibly due to increased lignin exposure and/or reduced effectiveness of adsorbed enzyme.     

Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies

In order to investigate changes in substrate chemical and physical features after pretreatment, several characterizations were performed on untreated (UT) corn stover and poplar and their solids resulting pretreatments by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough, lime, and SO₂ technologies. In addition to measuring the chemical compositions including acetyl content, physical attributes determined were biomass crystallinity, cellulose degree of polymerization, cellulase adsorption capacity of pretreated solids and enzymatically extracted lignin, copper number, FT-IR responses, scanning electron microscopy (SEM) visualizations, and surface atomic composition by electron spectroscopy of chemical analysis (ESCA). Lime pretreatment removed the most acetyl groups from both corn stover and poplar, while AFEX removed the least. Low pH pretreatments depolymerized cellulose and enhanced biomass crystallinity much more than higher pH approaches. Lime pretreated corn stover solids and flowthrough pretreated poplar solids had the highest cellulase adsorption capacity, while dilute acid pretreated corn stover solids and controlled pH pretreated poplar solids had the least. Furthermore, enzymatically extracted AFEX lignin preparations for both corn stover and poplar had the lowest cellulase adsorption capacity. ESCA results showed that SO₂ pretreated solids had the highest surface O/C ratio for poplar, but for corn stover, the highest value was observed for dilute acid pretreatment with a Parr reactor. Although dependent on pretreatment and substrate, FT-IR data showed that along with changes in cross linking and chemical changes, pretreatments may also decrystallize cellulose and change the ratio of crystalline cellulose polymorphs (Iα/Iβ).     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Application of cellulase and hemicellulase to pure xylan, pure cellulose, and switchgrass solids from leading pretreatments

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.     

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.     

Effect of additives on the digestibility of corn stover solids following pretreatment by leading technologies

Bovine serum albumin (BSA), Tween‐20, and polyethylene glycol (PEG6000) were added to washed corn stover solids produced by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute sulfuric acid (DA), lime, controlled pH, and sulfur dioxide (SO₂) pretreatments and to untreated corn stover (UT) and pure Avicel glucan prior to adding cellulase supplemented with β‐glucosidase at an activity ratio of 1:2/g and a moderate enzyme loading of 16.1 mg/g glucan in the raw corn stover. The additives were applied individually at 150, 300, and 600 mg/g glucan in the pretreated solids and in combinations of equal amounts of each that totaled 600 mg/g. The greatest increase in total sugar release was by Tween‐20 with SO₂ pretreated solids followed by PEG6000 with ARP solids and Tween‐20 with lime solids. The effectiveness of the additives was observed to depend on the type of sugars left in the solids, suggesting that it may be more beneficial to use the mixture of these additives to realize a high total sugar yield. In addition, little enhancement in sugar release was possible beyond a loading of 150 mg additives/g glucan for most pretreatments, and combinations did not improve sugar release much over use of additives alone for all except SO₂. Additives were also found to significantly increase concentrations of cellobiose and cellooligomers after 72 h of Avicel hydrolysis. Biotechnol. Bioeng. 2009;102: 1544–1557. © 2008 Wiley Periodicals, Inc.     

Influence of xylan on the enzymatic hydrolysis of steam‐pretreated corn stover and hybrid poplar

The focus of this study was to alter the xylan content of corn stover and poplar using SO₂‐catalyzed steam pretreatment to determine the effect on subsequent hydrolysis by commercial cellulase preparations supplemented with or without xylanases. Steam pretreated solids with xylan contents ranging from ～1 to 19% (w/w) were produced. Higher xylan contents and improved hemicellulose recoveries were obtained with solids pretreated at lower severities or without SO₂‐addition prior to pretreatment. The pretreated solids with low xylan content (<4% (w/w)) were characterized by fast and complete cellulose to glucose conversion when utilizing cellulases. Commercial cellulases required xylanase supplementation for effective hydrolysis of pretreated substrates containing higher amounts of xylan. It was apparent that the xylan content influenced both the enzyme requirements for hydrolysis and the recovery of sugars during the pretreatment process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO₂) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with β‐glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8–116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and β‐glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase–xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis,supramolecular structure,and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF‐pretreated corn stover but reached only ～60% for the DA‐pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non‐cellulose accessibility to cellulase (NCAC) based on adsorption of a non‐hydrolytic recombinant protein TGC were measured for the first time. The COSLIF‐pretreated corn stover had a CAC of 11.57 m²/g, nearly twice that of the DA‐pretreated biomass (5.89 m²/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA‐ and COSLIF‐pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility. Biotechnol. Bioeng. 2009;103: 715–724. © 2009 Wiley Periodicals, Inc.     

The pretreatment of corn stover with Gloeophyllum trabeum KU-41 for enzymatic hydrolysis

Background

Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover.

Results

The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover.

Conclusion

G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method.     

Structural changes of corn stover lignin during acid pretreatment

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70?% at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by (31)P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180?°C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.     

Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover

Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms.     

Optimization of enzyme complexes for lignocellulose hydrolysis

The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.     

Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover

Ionic liquid (IL) and ammonia fiber expansion (AFEX) pretreatments were studied to develop the first direct side-by-side comparative assessment on their respective impacts on biomass structure, composition, process mass balance, and enzymatic saccharification efficiency. AFEX pretreatment completely preserves plant carbohydrates, whereas IL pretreatment extracts 76% of hemicellulose. In contrast to AFEX, the native crystal structure of the recovered corn stover from IL pretreatment was significantly disrupted. For both techniques, more than 70% of the theoretical sugar yield was attained after 48 h of hydrolysis using commercial enzyme cocktails. IL pretreatment requires less enzyme loading and a shorter hydrolysis time to reach 90% yields. Hemicellulase addition led to significant improvements in the yields of glucose and xylose for AFEX pretreated corn stover, but not for IL pretreated stover. These results provide new insights into the mechanisms of IL and AFEX pretreatment, as well as the advantages and disadvantages of each.     

Examining the role of particle size on ammonia‐based bioprocessing of maize stover

The role of particle size in carbohydrate fractionation upon pretreatment and glucan yields upon enzymatic hydrolysis was investigated at two different temperatures, to examine the possibility of pretreating under milder conditions smaller particles, in order to satisfy pilot‐scale operational constraints. Maize stover was knife‐milled through 1‐mm and 0.5‐mm screens and pretreated by soaking in aqueous ammonia pretreatment at 60 or 110°C for 6 h. Pretreated solids were analyzed for composition and a material balance calculated for glucan, xylan, and lignin. At 60°C, milling resulted in greater delignification compared to unmilled biomass. Delignification was more uniform at 110°C. Pretreated solids were washed and cellulase hydrolysis carried out at 10% w/w solids loading, with low and high enzyme loadings. Liquid samples were drawn and concentration data developed through HPLC to calculate 48‐h glucan and xylan hydrolytic yields. The differences in hydrolytic yield between milled and unmilled treatments were found to vary with pretreatment temperature and enzyme loading. The results show that while particle size impacts carbohydrate recovery and hydrolytic yield, it is less important in bioprocessing than pretreatment temperature and enzyme loading, possibly owing to the particles’ morphology rather than the size. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:134–140, 2016     

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.