Extraction, characterization and potential applications of cellulose in corn kernels and Distillers’ dried grains with solubles (DDGS)

Cellulose with properties suitable for films and absorbents has been extracted from corn kernels and DDGS. Although DDGS is an inexpensive and abundant co-product that contains valuable components, it is currently not being used for industrial applications. DDGS contains about 9–16% cellulose by weight but the properties of cellulose in DDGS or even in corn kernels such as degree of polymerization (DP), morphology and crystallinity of cellulose have not been studied. In this study, cellulose was extracted from corn kernels and DDGS using alkali and enzymes. A minimum crude cellulose yield of 1.7% and 7.2% with cellulose content of 72% and 81% was obtained from corn kernels and DDGS, respectively. The solids obtained after extraction with cellulose contents ranging from 35% to 81% were made into films with tensile strength and elongation up to 42.5 MPa and 3.3%, respectively, using water and without any additional chemicals. The cellulose obtained holds water up to 9 times its weight and could therefore be used as an absorbent. The cellulose could also be used as paper, composites, lubricant and nutritional supplement.     

Composition of corn and distillers dried grains with solubles from dry grind ethanol processing

Increase in the demand for ethanol has resulted in growth in the dry grind (DG) ethanol industry. In DG processing, the whole corn kernel is fermented, resulting in two main coproducts, ethanol and distillers dried grains with solubles (DDGS). Marketing of DDGS is critical to the economic stability of DG plants. The composition of DDGS can vary considerably; this reduces market value. Factors that cause variation in composition need to be evaluated. The objective was to determine the relationship between composition of corn and composition of DDGS. Samples of corn and DDGS were obtained from a DG ethanol plant and analyzed for protein, fat, starch and other nutrients. Concentrations of protein, fiber and starch were similar to published data for corn but were higher for DDGS. Coefficients of variation for protein fat and fiber concentrations were similar for corn and DDGS. There were no significant correlations between concentrations of components in corn and those in DDGS. Variation in the composition of DDGS was not related to variation in corn composition and probably was due to variation in processing streams or processing techniques. This implies that reducing the variation in composition of DDG will require modification of processing strategies.     

Pilot scale fiber separation from distillers dried grains with solubles (DDGS) using sieving and air classification

Distillers dried grains with solubles (DDGS), the coproduct of fuel ethanol production from cereal grains like corn, is mainly used as cattle feed and is used at low inclusion levels in poultry and swine diets because of high fiber content. Elusieve process, the combination of sieving and air classification (elutriation), was developed in laboratory scale to separate fiber from DDGS to result in a low fiber product which would be more suitable for poultry and swine. In this pilot scale study, DDGS was sieved at a rate of 0.25 kg/s (1 ton/h) into four sieve fractions using a sifter and the three largest sieve fractions were air classified using aspirators to separate fiber on a continuous basis. Results were similar to laboratory scale. Nearly 12.4% by weight of DDGS was separated as Fiber product and resulted in two high protein products that had low fiber contents. Payback period for the Elusieve process in an existing dry grind plant processing corn at the rate of 2030 metric tonnes/day (80,000 bushels/day) would be 1.1 yr.     

Molecular dynamics simulations of the two disaccharides of hyaluronan in aqueous solution

Hyaluronan is an unusually stiff polymer when in aqueous solution,which has important consequences for its biological function.Molecular dynamics simulations of hyaluronan disaccharides havebeen performed, with explicit inclusion of water, to determinethe molecular basis of this stiffness, and to investigate thedynamics of the glycosidic linkages. Our simulations revealthat stable sets of hydrogen bonds frequently connect the neighboringresidues of hyaluronan. Water caging around the glycosidic linkagewas observed to increase the connectivity between sugars, andfurther constrain them. This, we propose, explains the unusualstiffness of polymeric hyaluronan. It would allow the polysaccharideto maintain local secondary structure, and occupy large solutiondomains consistent with the visco-elastic nature of hyaluronan.Simulations in water showed no significant changes on inclusionof the exo-anomeric effect. This, we deduced, was due to hyaluronandisaccharides ordering first shell water molecules. In somecases these waters were observed to transiently induce con-formationalchange, by breaking intramolecular hydrogen bonds. conformation hyaluronan hydrogen bonds molecular dynamics water     

Determination of energy and amino acid digestibility in growing pigs fed corn distillers' dried grains with solubles containing different lipid levels

Two experiments were conducted to estimate the digestibility of energy, nitrogen and amino acids (AA) in growing pigs fed diets containing one of five corn distillers' dried grains with solubles (DDGS), including three normal oil DDGS (NO-DDGS) and two low oil DDGS (LO-DDGS) samples. Exp. 1 was conducted to determine the digestible energy (DE) and metabolisable energy (ME) content. Six growing barrows (initial body weight [BW]: 35.1 +/- 2.2 kg) were allotted to a 6 x 6 Latin square design, with six periods and six diets. One diet was a corn soybean meal basal diet and the other five diets were based on corn, soybean meal and 28.8% DDGS. The average DE and ME values for the three NO-DDGS samples were 16.0 and 14.9 MJ/kg dry matter (DM). These values were 9 and 13% greater than the LO-DDGS values of 14.7 and 13.2 MJ/kg DM respectively. Exp. 2 was conducted to determine and compare apparent (AID) and standardised (SID) ileal digestibility for crude protein and AA in the five DDGS samples. Six growing barrows (initial BW, 32.2 +/- 1.9 kg) fitted with a simple T-cannula were allotted to a 6 x 6 Latin square design with six periods and six diets. Five of the diets were based on the five DDGS samples, and the remaining one diet was nitrogen-free diet based on cornstarch and sucrose. Titanium dioxide (0.1%) was used as inert marker. The results of the experiment showed the largest variation among the different samples in AID and SID for lysine (from 41.8 to 65.8% and 53.8 to 73.9% respectively) and threonine (from 54.3 to 73.8% and 65.2 to 79.5% respectively). Also, among the indispensable AA, the SID values for arginine, histidine, threonine and tryptophan observed in LO-DDGS were not different from the values derived from NO-DDGS. In conclusion, LO-DDGS may have decreased energy compared with NO-DDGS because of its lower fat content. However, oil removal during the production of DDGS may not affect amino acid digestibility.     

Effects of particle size distribution,compositional and color properties of ground corn on quality of distillers dried grains with solubles (DDGS)

Oftentimes, corn processors believe that ground corn (raw material) and distillers dried grains with solubles (DDGS) are interrelated in certain quality parameters. Yet, previous studies, although rather limited, have not established this relationship. In this study, six ground corn samples and their resulting DDGS were analyzed for particle size distribution (PSD), using a series of six selected US standard sieves: Nos. 8, 12, 18, 35, 60, and 100, and a pan. The original sample and sieve sized fractions were measured for contents of moisture, protein, oil, ash and starch, and surface color. Total carbohydrate (CHO) and total non-starch CHO were also calculated. Results show that the geometric mean diameter (d_gw) of particles varied with individual corn and DDGS samples, and that d_gw of DDGS was larger than that of corn (0.696 vs. 0.479 mm, average values), indicating that during conversion of corn to DDGS, certain particles became enlarged. For d_gw and mass frequency of individual particle size classes, the relationship between ground corn and DDGS varied, but PSD of the whole sample was well correlated between them (r = 0.807). Upon conversion from corn to DDGS, on an average, protein was concentrated 3.59 times; oil, 3.40 times; ash, 3.32 times; and total non-starch CHO, 2.89 times. There were some positive correlations in contents of protein and non-starch CHO and in L value between corn and DDGS. Yet, variations in nutrients and color attributes were larger in DDGS than in corn. For either corn or DDGS, these variations were larger in sieved fractions than in the whole fraction. Raw material, processing method and addition of yeasts are among major factors considered for causing larger variations in these attributes among DDGS. The study partially supports the common belief by processors that quality attributes of corn affect those of DDGS.     

The hydrogen bonding in the crystal structure of raffinose pentahydrate

The crystal structure of raffinose pentahydrate, O-alpha-D-galactopyranosyl-(1----6)-O-alpha-D-glucopyranosyl-(1----2)- beta-D- fructofuranose pentahydrate, C18H32O16.5H2O, has been redetermined using low-temperature, 119 K, CuK alpha X-ray data. All hydrogen atoms were unambiguously located on difference syntheses. The final R-factor is 0.036 for 2423 observed structure amplitudes. The hydrogen bonding is composed of infinite chains, which are linked through the water molecules to form a three-dimensional network containing a chain of five linked water molecules. Three of the infinite chains extend in the directions of the crystallographic axis of the space group P2(1)2(1)2(1). Four of the water molecules accept two hydrogen bonds and one accepts one. All the hydroxyls and the ring and glycosidic oxygen atoms are involved in the hydrogen bonding. With one exception, the ring and glycosidic oxygens are hydrogen-bonded by means of the minor components of unsymmetrical three-center bonds.     

Economic analysis of a modified dry grind ethanol process with recycle of pretreated and enzymatically hydrolyzed distillers' grains

A modification of the conventional dry grind process for producing ethanol from yellow dent corn is considered with respect to its economic value. Process modifications include recycling distillers' grains, after being pretreated and hydrolyzed, with the ground corn and water to go through fermentation again and increase ethanol yields from the corn starch. A dry grind financial model, which has been validated against other financial models in the industry, is utilized to determine the financial impact of the process changes. The hypothesis was that the enhanced process would yield higher revenues through additional ethanol sales, and higher valued dried distillers' grains (DDGS), due to its higher protein content, to mitigate the drop in DDGS yields. A 32% increase in net present value (NPV) for the overall operation is expected when applying the process modifications to a 100million gallon ethanol plant, and an enzyme cost of $0.20 for each additional gallon of ethanol produced. However, there may be no value added to the enhanced dried distillers' grains (eDDGS), even in light of its higher protein levels, as current pricing is expected to be more sensitive to the amino acid profile than the total protein level, and the eDDGS has lower lysine levels, a key amino acid. Thus, there is a decrease in revenue from eDDGS due to the combination of no price change and loss of DDGS yield to ethanol. The financial improvements are a result of the increased revenue from higher ethanol yields outpacing the sum of all added costs, which include higher capital costs, larger loan payments, increased operating costs, and decreased revenues from dried distillers' grains.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

The hydrolysis of cotton cellulose by hydrochloric acid in benzene

The partial hydrolysis of cotton cellulose by hydrogen chloride in benzene containing various proportions of water has been studied by measurements of fluidity, loss of weight, and copper number. Both the rate and site of hydrolysis are strongly dependent on the amount of water present. The determining factors are the partition of the hydrogen chloride between the water in the benzene and that adsorbed by the cellulose, and the relative accessibility of glycosidic linkages near the ends of chains and those far removed from chain ends. With little water present, hydrolysis tends to be confined to the ends of the cellulose chains.     

Product binding varies dramatically between processive and nonprocessive cellulase enzymes

Cellulases hydrolyze β-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.     

Additional properties of a soluble polymer of glucose from cultures of Acetobacter xylinum

The results of differential, thermal analysis of a soluble, beta (1 leads to 2)-branched, beta (1 leads to 4)-D-glucan isolated from cultures of Acetobacter xylinum are consistent with previous conclusions about its structure. The O-acetyl content of the polymer is 8.3% which corresponds to a maximum substitution of one acetyl group per three glucose residues. Proton nuclear magnetic resonance spectra confirm that all the glycosidic bonds are beta linkages. Some preparations of the polymer are contaminated by another polymer containing mannose and rhamnose. No evidence was obtained to support a previous suggestion that the branched D-glucan is a precursor of bacterial cellulose and this suggestion is now withdrawn.     

Modeling the effects of pelleting on the logistics of distillers grains shipping

The energy security needs of energy importing nations continue to escalate. It is clear that biofuels can help meet some of the increasing need for energy. Theoretically, these can be produced from a variety of biological materials, including agricultural residues (such as corn stover and wheat straw), perennial grasses, legumes, algae, and other biological materials. Currently, however, the most heavily utilized material is corn starch. Industrial fuel ethanol production in the US primarily uses corn, because it is readily converted into fuel at a relatively low cost compared to other biomass sources. The production of corn-based ethanol in the US is dramatically increasing. As the industry continues to grow, the amount of byproducts and coproducts also increases. At the moment, the nonfermentable residues (which are dried and sold as distillers dried grains with solubles – DDGS) are utilized only as livestock feed. The sale of coproducts provides ethanol processors with a substantial revenue source and significantly increases the profitability of the production process. Even though these materials are used to feed animals in local markets, as the size and scope of the industry continues to grow, the need to ship large quantities of coproducts grows as well. This includes both domestic as well as international transportation. Value-added processing options offer the potential to increase the sustainability of each ethanol plant, and thus the industry overall. However, implementation of new technologies will be dependent upon how their costs interact with current processing costs and the logistics of coproduct deliveries. The objective of this study was to examine some of these issues by developing a computer model to determine potential cost ramifications of using various alternative technologies during ethanol processing. This paper focuses specifically on adding a densification unit operation (i.e., pelleting) to produce value-added DDGS at a fuel ethanol manufacturing plant. We have examined the economic implications of pelleting DDGS for varying DDGS production rates (100–1000 tons/d) and pelleting rates (0–100%), for a series of DDGS sales prices ($50–$200/ton). As the proportion of pelleting increases, the cost of transporting DDGS to distant markets drastically declines, because the rail cars can be filled to capacity. For example, at a DDGS sales price of $50/ton, 100% pelleting will reduce shipping costs (both direct and indirect) by 89% compared to shipping the DDGS in bulk form (i.e., no pelleting), whereas at a DDGS sales price of $200/ton, it will reduce costs by over 96%. It is clear that the sustainability of the ethanol industry can be improved by implementing pelleting technology for the coproducts, especially at those plants that ship their DDGS via rail.     

Enzymic activities of endo-1,4-beta-D-glucanases purified from Trichoderma viride

Endoglucanases II, III and IV (EC 3.2.1.4) from Trichoderma viride are highly active in degrading CM-cellulose or phosphoric acid swollen cellulose, and only slightly active on Avicel. The specific activities of the endoglucanases increase with the length of the cellooligosaccharide substrates. By rate and product analyses using high pressure liquid chromatography the mode of action of Endoglucanase III was differentiated from that of Endoglucanases II and IV. Endoglucanase III has a low affinity for cellobiose, reacts rapidly with cellotriose, and gradually increases in reactivity with cellooligosaccharides as degree of polymerization increases from four to six. In addition to cleaving internal glycosidic bonds of polymeric substrates, it preferentially cleaves cellobiosyl units from the non-reducing end of oligosaccharides. The cellobiosyl units are often, under initial reaction conditions, transferred to the substrate-acceptor. Endoglucanases II and IV show a preference for internal glycosidic bonds of cellooligosaccharides. The soluble products from the initial action of Endoglucanases II and IV on swollen cellulose are glucose, cellobiose, and cellotriose, which are slowly converted to glucose and some cellobiose.     

Particle size distribution of distillers dried grains with solubles (DDGS) and relationships to compositional and color properties

Eleven distillers dried grains with solubles (DDGS), processed from yellow corn, were collected from different ethanol processing plants in the US Midwest area. Particle size distribution (PSD) by mass of each sample was determined using a series of six selected US standard sieves: Nos. 8, 12, 18, 35, 60, and 100, and a pan. The original sample and sieve sized fractions were measured for surface color and contents of moisture, protein, oil, ash, and starch. Total carbohydrate (CHO) and total non-starch CHO were also calculated. Results show that there was a great variation in composition and color among DDGS from different plants. Surprisingly, a few DDGS samples contained unusually high amounts of residual starch (11.1-17.6%, dry matter basis, vs. about 5% of the rest), presumably resulting from modified processing methods. Particle size of DDGS varied greatly within a sample and PSD varied greatly among samples. The 11 samples had a mean value of 0.660mm for the geometric mean diameter (d(gw)) of particles and a mean value of 0.440mm for the geometric standard deviation (S(gw)) of particle diameters by mass. The majority had a unimodal PSD, with a mode in the size class between 0.5 and 1.0mm. Although PSD and color parameters had little correlation with composition of whole DDGS samples, distribution of nutrients as well as color attributes correlated well with PSD. In sieved fractions, protein content, L and a color values negatively while contents of oil and total CHO positively correlated with particle size. It is highly feasible to fractionate DDGS for compositional enrichment based on particle size, while the extent of PSD can serve as an index for potential of DDGS fractionation. The above information should be a vital addition to quality and baseline data of DDGS.     

Evaluation of value-added components of dried distiller's grain with solubles from triticale and wheat

This study focused on the detection of value-added co-products in dried distiller’s grain plus soluble (DDGS), a possibility that could open new avenues for further processing and marketing of DDGS and improving economic sustainability of ethanol industry. Varieties of triticale, wheat and two benchmarks, CPS wheat and Pioneer Hi-Bred corn, were fermented using two very high gravity (VHG) fermentation approaches: jet-cooking and raw starch processing (STARGEN fermentation). DDGS from STARGEN fermentation could be promising sources of value-added co-products. Pronghorn triticale DDGS (STARGEN fermentation) had the highest concentration of sterols (3.7 mg/g), phenolic compounds (13.61 mg GAE/g), and β-glucan (2.07%). CDC Ptarmigan DDGS (STARGEN fermentation) had the highest concentration of tocopherols and tocotrienols (107.0 μg/g), 1.93% of β-glucan, and 53.0 mg/g of fatty acids. AC Reed DDGS (STARGEN method) showed 1.97% of β-glucan. This study shows that proper choice of fermentation approach and feedstock for ethanol production could improve commercial quality of DDGS.     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation

Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross‐correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water‐supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β‐sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond.     

Effect of fiber orientation in dynamic FTIR study on native cellulose

The properties of cellulose materials are highly dependent on the interactions between and within the cellulose chains mainly related to inter- and intramolecular hydrogen bonds. To investigate the deformation behavior of cellulose and its relation to molecular straining, cellulose sheets with different fiber orientations were studied by dynamic FTIR spectroscopy. The sheets were stretched sinusoidally at low strains while being irradiated with polarized infrared light. It is shown that the polarization direction determines the dynamic IR response to a higher extent than the fiber direction in the sample sheets. Different polarization modes give different dynamic signals, allowing conclusions to be drawn on the structural orientation of submolecular groups in the cellulose molecules. The bands in the spectra mainly affected by the deformation of the sheets were derived from skeletal vibrations that include the C-O-C bridge connecting adjacent rings and from the hydrogen bonds. The conclusion that these groups are the ones that are mainly deformed under load has thereby experimentally demonstrated the theoretical calculations from Tashiro and Kobayashi [Tashiro, K.; Kobayashi, M. Polymer 1991, 32, 1516-1526].     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.