Pretreatment of corn stover and hybrid poplar by sodium hydroxide and hydrogen peroxide

Sodium hydroxide and its derivatives are used as pulping reagents, wherein the spent NaOH is recovered in salt form and reused. In this study, use of low concentration NaOH (1–5%) in pretreatment of corn stover and hybrid poplar was investigated. It was done with the understanding that NaOH can be recovered. One of the main objectives in this study is to explore the potential of H₂O₂ with NaOH for pretreatment of high lignin substrate such as hybrid poplar. Pretreatment time has not been optimized in this study but held constant at 24 h. Corn stover, after treatment with NaOH under moderate conditions, attains near quantitative glucan digestibility. On the other hand, hybrid poplar requires treatment at higher temperature and NaOH concentration to attain acceptable level of digestibility. Supplementation of hydrogen peroxide in the pretreatment significantly raises delignification and digestibility of hybrid poplar. It was also helpful in retaining the carbohydrates in the treated solids. Retention of hemicellulose after pretreatment provides a significant economic benefit as it eliminates the need for detoxifying hemicellulose sugars. As the residual xylan remaining after pretreatment is an impediment to enzymatic digestion of glucan, supplementation of xylanase has significantly increased the digestibility of glucan as well as xylan of the treated hybrid poplar. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment

Barley hull, a lignocellulosic biomass, was pretreated using aqueous ammonia, to be converted into ethanol. Barley hull was soaked in 15 and 30 wt.% aqueous ammonia at 30, 60, and 75 °C for between 12 h and 11 weeks. This pretreatment method has been known as “soaking in aqueous ammonia” (SAA). Among the tested conditions, the best pretreatment conditions observed were 75 °C, 48 h, 15 wt.% aqueous ammonia and 1:12 of solid:liquid ratio resulting in saccharification yields of 83% for glucan and 63% for xylan with 15 FPU/g-glucan enzyme loading. Pretreatment using 15 wt.% ammonia for 24–72 h at 75 °C removed 50–66% of the original lignin from the solids while it retained 65–76% of the xylan without any glucan loss.

Addition of xylanase along with cellulase resulted in synergetic effect on ethanol production in SSCF (simultaneous saccharification and co-fermentation) using SAA-treated barley hull and recombinant E. coli (KO11). With 3% w/v glucan loading and 4 mL of xylanase enzyme loadings, the SSCF of the SAA treated barley hull resulted 24.1 g/L ethanol concentration at 15 FPU cellulase/g-glucan loading, which corresponds to 89.4% of the maximum theoretical yield based on glucan and xylan.

SEM results indicated that SAA treatment increased surface area and the pore size. It is postulated that these physical changes enhance the enzymatic digestibility in the SAA treated barley hull.     

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     

Factors Involved in the Regulation of a Ligninase Activity in Phanerochaete chrysosporium

The regulation of an H₂O₂-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O₂. Elevated levels of ligninase and, in some cases, of extracellular H₂O₂ production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO₂) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.     

Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus — lignin and polysaccharide alteration and production of related enzymatic activities

Summary The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of side-chains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Lactase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H₂O₂-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. Offprint requests to: A. T. Martínez     

Characterisation of Authentic Lignin Biorefinery Samples by Fourier Transform Infrared Spectroscopy and Determination of the Chemical Formula for Lignin

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C₉H_10.2O_3.4 with slight variations depending on the biomass feedstock and processing conditions (between C₉H_9.5O_2.8 and C₉H_11.1O_3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm^?1 relative to the strong band at ~1120 cm^?1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.     

Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium

Cultural conditions affecting lignin degradation by Phanerochaete chrysosporium in various lignocellulosic materials were studied in comparison to an isolated lignin preparation. With shallow mycelial cultures, the degradation of lignin in wood proceeded more slowly in a 100% O₂-atmosphere than in an air atmosphere, indicating that pure oxygen was toxic to the fungus. The organism was able to degrade lignin efficiently even under 30% CO₂ and 10% O₂ concentrations. Evolution of ¹⁴CO₂ from labelled lignocellulosic materials was shown not to be representative of total lignin degradation. Addition of glucose to the culture did not affect lignin degradation measured by ¹⁴CO₂ evolution, whereas lignin degradation measured by Klason lignin method stopped completely (poplar) or slowed considerably (straw). Due to partial depolymerization of lignin to soluble products, measuring only the evolution of ¹⁴CO₂ results in an underestimation of the total amount of lignin bioaltered. The soluble products from all of the tested lignocellulosic materials and from the isolated lignin had an average molecular weight of about 1,000 and the products could be further fractionated by ion exchange chromatography. The relative amount of these products could be varied from 15 to 45% from the original lignin.     

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).     

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     

Relationship Between Lignin Degradation and Production of Reduced Oxygen Species by Phanerochaete chrysosporium

The relationship between the production of reduced oxygen species, hydrogen peroxide (H₂O₂), superoxide (O₂), and hydroxyl radical (·OH), and the oxidation of synthetic lignin to CO₂ was studied in whole cultures of the white-rot fungus Phanerochaete chrysosporium Burds. The kinetics of the synthesis of H₂O₂ coincided with the appearance of the ligninolytic system; also, H₂O₂ production was markedly enhanced by growth under 100% O₂, mimicking the increase in ligninolytic activity characteristic of cultures grown under elevated oxygen tension. Lignin degradation by whole cultures was inhibited by a specific H₂O₂ scavenger, catalase, implying a role for H₂O₂ in the degradative process. Superoxide dismutase also inhibited lignin degradation, suggesting that O₂ is also involved in the breakdown of lignin. The production of ·OH was assayed in whole cultures by a benzoate decarboxylation assay. Neither the kinetics of ·OH synthesis nor the final activity of its producing system obtained under 100% O₂ correlated with that of the lignin-degrading system. However, lignin degradation was inhibited by compounds which react with ·OH. It is concluded that H₂O₂, and perhaps O₂, are involved in lignin degradation; because these species are relatively unreactive per se, their role must be indirect. Conclusions about a role for ·OH in ligninolysis could not be reached.     

Generation of active oxygen species (AOS) and induction of β-Glucanase activity by fungal elicitor xylanase in the suspension cultured cells of Tobacco

It was investigated that active oxygen species (AOS) involved in the plant defense responses induced by fungal elicitor xylanase. When xylanase from the fungusTrichoderma viridae was treated to tobacco suspension cultured cells as an elicitor, β-glucanase activity was increased markedly. Lignin biosynthesis was also increased and peaked at 72 h after the treatment with xylanase. The treatment of H₂O₂ also dramatically increased β-glucanase activity at 24 h, which was much earlier than that of xylanase did. Using lucigenin-and luminol-dependent chemiluminescence, the effects of xylanase on oxidative burst were examined. Superoxide anion (O₂) production was peaked at 40 h and 52 h after xylanase treatment and hydrogen peroxide (H₂O₂) release was peaked at 44 h and 56 h, suggesting H₂O₂ burst was followed by O₂ generation. The scavengers of AOS, n-propyl gallate (PG) and mannitol, inhibited xylanase-induced β-glucanase activity by 85% and 50%, respectively. The activity of superoxide dismutase (SOD), which catalyzes the dismutation of O₂ to H₂O₂, began to increase from 24 h and reached to maximum at 48 h after xylanase treatment. Pretreatment of N,N,-diethyldithiocarbamate (DDC), known as a SOD inhibitor, caused the inhibition of H₂O₂ generation by 80% and reduced the β-glucanase activity by 60%. Treatment of 2,5-norbonadiene (NBD), a specific ethylene-action inhibitor, did not have any significant effect on xylanase-induced β-glucanase activity. This result suggested that ethylene did not involve in xylanase-induced response. Our results strongly suggest that the AOS generation is an essential component in plant defense response, in which cell wall degrading enzyme, glucanase, contributes to remove the necrotic tissue induced by pathogens.     

Synergism of cellulase,pectinase and xylanase on hydrolyzing differently pretreated sweet potato residues

Abstract

The synergism of cellulase (C), pectinase (P), and xylanase (X) for the saccharification of sweet potato residues (SPR) was investigated. The removal of starch from SPR was easily achieved by using amylase, but the cellulose conversion of de-starched SPR was relatively low, thus dilute H₂SO₄, NaOH, and H₂O₂ pretreatment was conducted to improve the enzymatic digestibility. The lignin content of NaOH pretreated SPR was the lowest, whereas H₂SO₄ pretreatment resulted in the lowest contents of hemicellulose and pectin. The combination of C, P, and X exhibited different sugar production patterns, C–P displayed synergistic action on glucose and galactose production from each type of SPR, C–X also exhibited synergistic effect on glucose production except when H₂SO₄ pretreated SPR was used, whereas no synergism between P–X on monosaccharide production was observed. The presence of synergism between cellulase and mixed accessory enzymes [C–(PX)] on glucose formation was determined by C–X, and the degree of synergism between C–P and C–(PX) on glucose production had a positive relationship with pectin content. The highest cellulose conversion of 96.2% was obtained from NaOH pretreated SPR using mixed enzymes comprising C, P, and X with the ratio of 8:1:1.     

Lignin solubilisation by Thermomonospora mesophila

Summary Thermomonospora mesophila degraded [¹⁴C]lignin-labelled wheat lignocellulose to yield high molecular weight water-soluble products and a small amount of ¹⁴CO₂. Solubilisation of [¹⁴C]lignin was found to be extracellular and inducible by growth on lignocellulose (straw) and hemicellulose (xylan), but was not correlated with xylanase or cellulase production.The acid-precipitable product of straw degradation by T. mesophila was found to be a complex of lignin, pentose-rich carbohydrate and protein with some similarity to humic acids. Solid-state ¹³C-NMR spectra of the dried product were generally similar to those of chemically extracted milled straw lignin but showed an increased content of carbonyl groups.The relationship between degradation and solubilisation of lignin is discussed and a role suggested for actinomycetes in humification and the exploitation of lignocellulose bioconversion.     

Biochemistry of the oxidation of lignin by Phangerochate chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus . We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H₂O₂) and possibly superoxide (O₂ ·^-) in lignin degradation. Involvement of hydroxyl radical (·OH) or singlet oxygen (¹O₂) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H₂O₂ for activity.     

The effects of xylanase type enzymes on the different layers of birch wood ORGANOSOLV pulp fibre walls

Some effects of the xylanase treatment on the separate birch ORGANOSOLV pulp fibre wall morphological layers were examined. These investigations were focused on the outer layers, i.e. the primary wall (P) and the outer layer of the secondary wall (S₁), as well as the central layers, i.e. the central layer of the secondary wall (S₂) and the tertiary wall (T). Step by step, the fractionation of the pulp components in the polar solvents N,N-dimethylformamide (DMFA), dimethylsulphoxide (DMSO) and DMSO/H₃PO₄ was used as a mild technique for the isolation of the lignin-carbohydrate complexes. The different residual amounts of lignin and hemicelluloses in the outer and central pulp fibre wall layers as well as the different lignin-hemicellulose ratios were determined. The size-exclusion chromatographical (SEC) analysis showed a higher initial lignin content in the region of the high molecular mass (MM) fibre wall fraction extracted with “DMSO/H₃PO₄” than the outer cell wall layers. In the central layers, the amounts of soluble lignin (calculated on the mass of total dissolved substance) were approximately the same for all the three solvents. The xylanase treatment brought the most considerable changes in the high MM part of the residual lignin (the lignin carbohydrate complex). This was true for both the P-S₁ and S₂-T layers. The careful brightness comparison of the outer and central fractions after the X-E-P-P bleaching sequence showed a surprisingly low bleachability of the outer layer fraction. The xylanase action depended on the composition of the lignin-carbohydrate complex (LCC) and the extent of the maintenance of the outer layers during the pulping process.     

ARP2/3 complex‐mediated actin dynamics is required for hydrogen peroxide‐induced stomatal closure in Arabidopsis

Multiple cellular events like dynamic actin reorganization and hydrogen peroxide (H₂O₂) production were demonstrated to be involved in abscisic acid (ABA)‐induced stomatal closure. However, the relationship between them as well as the underlying mechanisms remains poorly understood. Here, we showed that H₂O₂ generation is indispensable for ABA induction of actin reorganization in guard cells of Arabidopsis that requires the presence of ARP2/3 complex. H₂O₂‐induced stomatal closure was delayed in the mutants of arpc4 and arpc5, and the rate of actin reorganization was slowed down in arpc4 and arpc5 in response to H₂O₂, suggesting that ARP2/3‐mediated actin nucleation is required for H₂O₂‐induced actin cytoskeleton remodelling. Furthermore, the expression of H₂O₂ biosynthetic related gene AtrbohD and the accumulation of H₂O₂ was delayed in response to ABA in arpc4 and arpc5, demonstrating that misregulated actin dynamics affects H₂O₂ production upon ABA treatment. These results support a possible causal relation between the production of H₂O₂ and actin dynamics in ABA‐mediated guard cell signalling: ABA triggers H₂O₂ generation that causes the reorganization of the actin cytoskeleton partially mediated by ARP2/3 complex, and ARP2/3 complex‐mediated actin dynamics may feedback regulate H₂O₂ production.     

Pretreatment of corn stover by aqueous ammonia

Corn stover was pretreated with aqueous ammonia in a flow-through column reactor, a process termed ammonia recycled percolation (ARP). This method was highly effective in delignifying of the biomass, reducing the lignin content by 70-85%. Most lignin removal occurred within the first 20 min of the process. Lignin removal by ARP was further confirmed by FTIR analysis and lignin staining. The ARP process solubilized 40-60% of the hemicellulose but left the cellulose intact. The solubilized carbohydrate existed in oligomeric form. Carbohydrate decomposition during the pretreatment was insignificant. Corn stover treated for 90 min exhibited enzymatic digestibility of 99% with 60 FPU/g of glucan enzyme loading, and 92.5% with 10 FPU/g of glucan. The digestibility of ARP treated corn stover was substantially higher than that of alpha-cellulose. The enzymatic digestibility was related with the removal of lignin and hemicellulose, perhaps due to increased surface area and porosity. The SEM pictures indicated that the biomass structure was deformed and its fibers exposed by the pretreatment. The crystallinity index increased with pretreatment reflecting removal of the amorphous portion of biomass. The crystalline structure of the cellulose in the biomass, however, was not changed by the ARP treatment.     

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.     

Investigation of enzyme formulation on pretreated switchgrass

This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime + ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO₂). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO₂). Xylanase addition also improved overall yields with lime + ball-milling and SO₂ achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime + ball-milling obtained an enzymatic yield of 92.3 kg of sugar digested/kg of protein loaded.     

Lignin-carbohydrate complex formed in isolated cell walls of callus

Cell walls of Pinus elliottii tissue cultures were isolated and incubated with coniferyl alcohol and H₂O₂. Lignin having physical and chemical properties similar to that prepared from wood was formed by the peroxidase attached to the walls. Fractions of the callus lignin isolated enzymatically or chemically contained bound carbohydrate. The lignin was also strongly bound to a protein containing hydroxyproline, probably extensin. This system may be analogous to the earliest stage of normal lignin formation in which monomers are transported from the protoplast into the primary wall and middle lamella, where peroxidase polymerizes monomers and catalyzes bonds to carbohydrate and protein.