Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

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     

Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass

Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free‐fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (A_w) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low‐field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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     

Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids

Sugarcane bagasses from three experimental sugarcane hybrids and a mill‐reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β‐glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70–100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944–951, 2018     

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.     

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

Dilute acid pretreatment of rye straw and bermudagrass for ethanol production

Ethanol production from lignocellulosic materials provides an alternative energy production system. Rye and bermudagrass that are used in hog farms for nutrient uptake from swine wastewater have the potential for fuel ethanol production because they have a relative high cellulose and hemicellulose content. Dilute sulfuric acid pretreatment of rye straw and bermudagrass before enzymatic hydrolysis of cellulose was investigated in this study. The biomass at a solid loading rate of 10% was pretreated at 121 degrees C with different sulfuric acid concentrations (0.6, 0.9, 1.2 and 1.5%, w/w) and residence times (30, 60, and 90 min). Total reducing sugars, arabinose, galactose, glucose, and xylose in the prehydrolyzate were analyzed. In addition, the solid residues were hydrolyzed by cellulases to investigate the enzymatic digestibility. With the increasing acid concentration and residence time, the amount of arabinose and galactose in the filtrates increased. The glucose concentration in the prehydrolyzate of rye straw was not significantly influenced by the sulfuric acid concentration and residence time, but it increased in the prehydrolyzate of bermudagrass with the increase of pretreatment severity. The xylose concentration in the filtrates increased with the increase of sulfuric acid concentration and residence time. Most of the arabinan, galactan and xylan in the biomass were hydrolyzed during the acid pretreatment. Cellulose remaining in the pretreated feedstock was highly digestible by cellulases from Trichoderma reesei.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

The effect of biomass moisture content on bioethanol yields from steam pretreated switchgrass and sugarcane bagasse

This study aimed to determine the effect of moisture content of three different feedstocks on overall ethanol yield. Switchgrass and sugarcane bagasse from two sources were either soaked in water (∼80% moisture) or left dry (∼12% moisture), and half each of these were impregnated with 3% w/w SO₂ and all were steam pretreated. The twelve resulting substrates were compared based on overall sugar recovery after pretreatment, cellulose conversion following enzymatic hydrolysis, and ethanol yield following simultaneous saccharification and fermentation. The overall ethanol yield after simultaneous saccharification and fermentation of hexoses was 18-28% higher in samples that were soaked prior to SO₂ addition than in SO₂-catalyzed samples that were not soaked. In samples that were uncatalyzed, soaking made little difference, indicating that the positive effect of increased moisture content may be related to increased permeability of the biomass to SO₂.     

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     

Butanol production from sweet sorghum bagasse with high solids content: Part I—comparison of liquid hot water pretreatment with dilute sulfuric acid

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H₂SO₄ (2 g L^?1) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250–300 g L^?1. This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L^?1 sugars, 7.60 g L^?1 acetic acid, 0.33 g L^?1 furfural, and 0.07 g L^?1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L^?1 additional sugars, 2.40 g L^?1 acetic acid, zero g L^?1 furfural, and zero g L^?1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone‐butanol‐ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L^?1 ABE of which butanol was the main product. Use of 2 g L^?1 H₂SO₄ as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6–93.8% (w/w) sugars from 250 to 300 g L^?1 SSB, respectively. LHW or dilute H₂SO₄ were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960–966, 2018     

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.     

Pretreatment Of Hardwood (Eucalyptus Regnans) Sawdust By Autohydrolysis Explosion And Its Saccharification By Trichodermal Cellulases

Autohydrolysis explosion pretreatment of hardwood (Eucalyptus regnans) sawdust at 200°C and 6.9 MPa gas pressure (steam + nitrogen) for 5 min solubilized 85% of the total hemicellulose components and produced a pulp that was highly accessible to attack by cellulases from Trichoderma reesei C-30 and by a commercial preparation, Meicelase. The autohydrolysis liquor, representing 15% of the original weight of the sawdust on a solids basis, consisted mainly of xylose, xylose oligomers and minor amounts of galactose, mannose, arabinose, glucose and uronic acids. Enzymic hydrolysis of pretreated E. regnans pulps using Trichodermal cellulases resulted in saccharification yields of <50% within 24 h from 10% (w/v) substrate slurries and 20 cellulase (FPU) units per g of pretreated pulp. The cellulose-to-glucose conversions were lower and this was attributable to the production of a compound(s) during enzymic hydrolysis that was inhibitory to the β-glucosidase component, but not the cellulases, in the Trichodermal cellulase preparations. Enzymic digests supplemented with Novozym 188 β-glucosidase showed >70% cellulose-to-glucose conversion within 24 h under similar conditions of hydrolysis. The inhibitor compound was not inhibitory to the Novozym 188 β-glucosidases. Alkali-extracted autohydrolysis-exploded pulps were less susceptible to hydrolysis than unextracted pulps. Factors that influenced the extent of cellulose conversion into glucose such as enzyme-substrate and cellulase-to-β-glucosidase ratios are also discussed.     

Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse

Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na₂SO₃ and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na₂SO₃, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l^?1 Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na₂SO₃), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %.     

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.     

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).     

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.