Effect of structural features on enzyme digestibility of corn stover

Corn stover was pretreated with excess calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The enzymatic digestibility of lime-treated corn stover was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. Extensive delignification required oxidative treatment and additional consumption of lime (up to 0.17 g Ca(OH)2/g biomass). Deacetylation reached a plateau within 1 week and there were no significant differences between non-oxidative and oxidative conditions at 55 degrees C; both conditions removed approximately 90% of the acetyl groups in 1 week at all temperatures studied. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed (or solubilized), but cellulose was not affected by lime pretreatment in mild temperatures (25-55 degrees C), even though corn stover was contacted with alkali for a long time, 16 weeks. The degree of crystallinity slightly increased from 43% to 60% with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzymatic hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The non-linear models for 3-d hydrolysis yields of glucan (Y(g)), xylan (Y(x)), and holocellulose (Y(gx)) were empirically established as a function of the residual lignin (L) for the corn stover pretreated with lime and air.     

Effect of stover fraction and storage method on glucose production during enzymatic hydrolysis

One avenue for overcoming the economic challenges associated with the production of ethanol from renewable resources is to reduce the cost of the biomass feedstock. The balance between storage costs and benefits depend on the storage method and composition changes of individual stover fractions. Corn stover from bales stored inside and outside of a barn was separated into an interior and exterior layer after approximately 10 months of storage. The cobs, stalks, and leaves and husks were separated, dried, and ground through a 2 mm screen. Stover, sodium acetate (buffer), cellulase, and deionized water were added to 125 ml flasks. The mixture was held at 50 degrees C in an incubator and samples taken for glucose determination. The average glucose concentration after 60 h of hydrolysis from cobs, leaves and husks, and stalks was 10.5, 9.6, and 3.1 g/l, respectively. Cobs, leaves, and husks produced over 300% more glucose than stalks. Storage outside of the barn decreased the glucose production from individual stover components between 4% and 8%. The effect of stover fraction type on glucose production was significant, while the storage treatment effect was not significant. Fractionation of corn stover may be a method to increase the value of corn stover as a feedstock for glucose production.     

Aerobic and Anaerobic Storage of Single-pass,Chopped Corn Stover

Corn stover has great potential as a biomass feedstock due its widespread availability. However, storage characteristics of moist corn stover harvested from single-pass harvesters have not been well quantified. In 2007, whole-plant corn stover at 19.1–40.3% (w.b.) moisture content was stored for 237 days in aerobic piles, one covered and one uncovered, as well an anaerobic silo bag. In 2008, two stover materials—whole plant and cob/husk from 31.7% to 58.1% (w.b.) moisture—were stored for 183 or 204 days in covered and uncovered anaerobic piles, ventilated bags, or anaerobic silo bags. Stover stored in uncovered piles was rehydrated by precipitation, which increased biological activity resulting in dry matter (DM) losses from 8.2% to 39.1% with an average of 21.5%. Stover in covered piles was successfully conserved when the average moisture was less than 25% (w.b.) with DM losses of 3.3%. Stover above 36% (w.b.) moisture and piled under a plastic cover had DM losses from 6.4% to 20.2% with an average of 11.9%. Localized heating occurred in the aerobic piles when moisture was above 45% (w.b.) which lead to temperatures where spontaneous combustion might be a concern (i.e., >70°C). Ambient air blown through a center tube in the ventilated storage bag dried stover near the tube to an average of 24.2% (w.b.), but the remainder of the bag averaged 46.8% (w.b.) at removal. Loss of DM ranged from 7.4% to 22.0% with an average of 11.8% with this storage method. Stover was most successfully conserved in the bags where anaerobic conditions were maintained. Under anaerobic conditions, DM losses ranged from 0.2% to 0.9%. When anaerobic conditions were not maintained in the silo bag, DM losses averaged 6.1% of DM. Anaerobic storage is the best solution for conserving the value of moist corn stover.     

Gelatinization and freeze-concentration effects on recrystallization in corn and potato starch gels

Freeze-concentration of starch gels was controlled by temperature and gelatinization with glucose and lactose. The aim of the study was to evaluate the effects of freezing temperature and gel composition on starch recrystallization behaviour of corn and potato starch gels (water content 70%, w/w) in water or glucose or lactose (10%, w/w) solutions. Starch gels were obtained by heating in differential scanning calorimetry (DSC). Samples of starch gels were frozen at -10 degrees C, -20 degrees C and -30 degrees C for 24h and, after thawing, stored at +2 degrees C for 0, 1, 2, 4 and 8 days. The extent of starch recrystallization was taken from the enthalpy of melting of the recrystallized starch by DSC. Freezing temperatures, glucose, lactose and the origin of the starch affected the recrystallization behaviour greatly. The recrystallization of amorphous starch during storage was enhanced by freeze-concentration of gels at temperatures above T'(m). Molecular mobility was enhanced by unfrozen water and consequently molecular rearrangements for nucleation could take place. Further storage at a higher temperature enhanced the growth and the maturation of crystals. In particular, glucose decreased the T'(m) of the gels and consequently lower freezing temperatures were needed to reduce enhanced recrystallization during storage. Freeze-concentration temperatures also showed a significant effect on the size and the perfection of crystals formed in starch recrystallization.     

Factors affecting the sporulation capacity during long-term storage of the aphid-pathogenic fungus Pandora neoaphidis grown on broomcorn millet

Aphid-pathogenic fungus, Pandora neoaphidis, grown on broomcorn millet possesses greater sporulation capacity (C(s)) than aphid cadavers. The most sporulating cultures (32.0x10(4) spores millet(-1) grain) with water content (C(w)) of 48.7% were prepared by incubation at 20 degrees C for 15 days and used to study the effect of temperature and humidity on C(s) during long-term storage. Cultures were sealed with paper to retain ambient humidity, with parafilm for saturated humidity, or kept in 85% and 98% RH chambers. The C(w) and C(s) were monitored during 200-day storage at 5-20 degrees C. The paper-sealed cultures at 5 degrees C, associated with 21-25% of C(w), were best preserved and their 120-day C(s) was similar to that of the fresh cadavers. Consistently or variably high RH at 5 degrees C resulted in significantly higher C(w) and lower C(s) despite longer viability. The regimes at 10 degrees C preserved the cultures for 40 days. The observations fit well to the logistic model C(s)=35.28/{1+exp[-2.36+(-0.003C(w)+0.001C(w)T)t]} (r(2)=0.95) for all regimes of temperature (T) or C(s)=35.55/[1+exp(-2.33+0.001C(w)t)] (r(2)=0.93) at 5 degrees C only. The rate of decline of C(s) of -0.003C(w)+0.001C(w)T or 0.001 C(w) over days (t) highlights the primary effect of C(w). The daily C(s)-decline rates obtained for the best-stored cultures and air-dried cadavers stored at 5 degrees C were surprisingly identical. The results suggest a possible cheap method for preparing and storing large quantities of P. neoaphiodis inocula.     

Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.     

Alkali treatment of corn stover to improve sugar production by enzymatic hydrolysis

Alkali treatment of corn stover improves the avaliability of cellulose and hemicellulose for enzymatic attack. Treatments were carried out for 1 to 60 min at temperatures and NaOH concentrations ranging from 100 to 150 degrees C and 0 to 2%, respectively. Solubilization of the stover and sugar production by enzymatic hydrolysis (Trichoderma viride cellulase) of the solid residue and the dissolved solids were used to measure the effect of caustic treatment. At 150 degrees C and 2% NaOH concentration, 65% of the original stover was dissolved after 5 min and 52% saccharificatin (g sugar/g stover) of the residue and dissolved solids by enzymatic hydrolysis was achieved compared to 20% for untreated corn stover.     

Chemical and enzymic pretreatment of corn stover to produce soluble fermentation substrates

Corn stover was pretreated with various chemical agents, including sodium hydroxide, sulfuric acid, ethylenediamine, n-butylamine (either alone or in solution with methanol), and acetonitrile or ethanol containing hydrochloric acid. Of these chemicals, n-butylamine was the best reagent for pretreatment of corn stover, considering the degree of loss of total carbohydrate, delignification, cumulative weight loss, cumulative yield of reducing sugars per original total carbohydrate, and the potential ease of recovery and reuse of reagent. In comparison to the other reagents tested, n-butylamine (n-BA) selectively delignified corn stover. The best conditions were as follows: a 12-h presoak of about a 155 g dry wt/L slurry (1 mm average particle size) in 100% n-BA at room temperature, followed by 30 min of refluxing (86.5 degrees C) with 40% (w/w) n-BA-distilled water solution. The cumulative yield of reducing sugars after enzymic hydrolysis was 44.5% of the original total carbohydrate and the cumulative total weight loss (dry basis) was 59%. Degradative loss of total carbohydrate during pretreatment was not detected.     

Maximizing the liquid fuel yield in a biorefining process

Biorefining strives to recover the maximum value from each fraction, at minimum energy cost. In order to seek an unbiased and thorough assessment of the alleged opportunity offered by biomass fuels, the direct conversion of various lignocellulosic biomass was studied: aspen pulp wood (Populus tremuloides), aspen wood pretreated with dilute acid, aspen lignin, aspen logging residues, corn stalk, corn spathe, corn cob, corn stover, corn stover pellet, corn stover pretreated with dilute acid, and lignin extracted from corn stover. Besides the heating rate, the yield of liquid products was found to be dependent on the final liquefaction temperature and the length of liquefaction time. The major compounds of the liquid products from various origins were identified by GC-MS. The lignin was found to be a good candidate for the liquefaction process, and biomass fractionation was necessary to maximize the yield of the liquid bio-fuel. The results suggest a biorefinery process accompanying pretreatment, fermentation to ethanol, liquefaction to bio-crude oil, and other thermo-conversion technologies, such as gasification. Other biorefinery options, including supercritical water gasification and the effectual utilization of the bio-crude oil, are also addressed.     

Photo-fermentation of Rhodobacter sphaeroides for hydrogen production using lignocellulose-derived organic acids

In this study, the corn stover was pretreated, enzymatically hydrolyzed, and then fermented in the lipid production fermentation. The corn stover fermentation effluent was utilized for the photo-fermentation of a Rhodobacter sphaeroides ZX-5 for hydrogen production. The hydrogen production was more than twofolds greater than that in the synthetic medium under the similar organic acid concentration range. The synergism among the pure organic acids was found to facilitate cell growth and hydrogen production, although some organic acid was not utilized for hydrogen production directly. The synergism among the components in the corn stover fermentation effluent was also found. The initial pH value was found to be an important parameter for the photo-fermentation of R. sphaeroide ZX-5 using the corn stover fermentation effluent. The results provided a possible way to utilize lignocellulose-derived organic acids for hydrogen production, and to treat fermentation wastewater in biofuel production using lignocellulose.     

Evaluation of organosolv processes for the fractionation and modification of corn stover for bioconversion

Corn stover was pretreated for compositional fractionation and structural modification for maximum conversion of carbohydrate to soluble sugars. The process scheme consisted of three steps: (1) mild prehydrolysis in dilute sulfuric acid, (2) delignification with various organosolv solvents, and (3) enzymatic hydrolysis in an agitated bead reactor. Prehydrolysis of corn stover can be achieved at temperatures ranging from 95 to 120 degrees C, which is a much milder condition than must be applied to wood. Various organosolv solvents, including several alcohols with acid as catalyst, ethylene glycol, and its derivatives, and amines were used for delignification of the prehydrolyzed corn stover. Aromatic alcohols were found to be more effective in solubilizing the prehydrolyzed corn stover than were the aliphatic alcohols. Butanol was the most effective among the aliphatic alcohols; on the other hand, phenol was the best among the aromatic alcohols. Ethylene glycol, methylcellosolv, and ethylcellosolv were effective in solubilizing the prehydrolyzed corn stover but not for enhancing the enzymatic hydrolysis. Various amines achieved delignification at the mild temperature of 95 degrees C, but they tended to solubilize substantial amounts of carbohydrate in addition to lignin. n-Butylamine was effective in enhancing the conversion during enzymatic hydrolysis; it was a good delignifying agent as well as one that achieved a concomitant swelling of the cellulose structure. The low enzymic conversion (20-37%) of prehydrolyzed and solvent-extracted corn stover that was achieved implies that lignin is not the only major barrier for enzymatic hydrolysis. Modification of cellulose structure also should be accomplished to achieve a high degree of conversion. Enzymatic hydrolysis in the agitated bead system increased the rate and extent of conversion of corn stover substantially compared to systems without beads.     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2)

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.     

Characterization of beta-glucosidase from corn stover and its application in simultaneous saccharification and fermentation

Purification and characterization of beta-glucosidase from corn stover was performed and the enzyme was tried in SSF to evaluate the suitability of plant glycosyl hydrolases in lignocellulose conversion. A beta-glucosidase with M(w) of 62.4 kDa was purified to homogeneity from post-harvest corn stover. The following physicochemical and kinetic parameters of the beta-glucosidase were studied respectively: optimum temperature, thermal stability, optimum pH, pH stability, K(m), V(max), V(i), cellobiose inhibition, tryptic peptide mass spectrometry and effect of metal ions and other reagents on the activity. The beta-glucosidase activity on salicin was optimal at pH 4.8 and 37 degrees C. The unique property of optimum temperature makes the beta-glucosidase potentially useful in SSF. In SSF of steam explosion pretreated corn stover, the supplementation of the purified beta-glucosidase was more effective than Aspergillus niger beta-glucosidase.     

Kinetic modeling analysis of maleic acid-catalyzed hemicellulose hydrolysis in corn stover

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.     

The role of capacitance in the water balance of Andean giant rosette species

Abstract Pith water storage capacity and its role in plant-water relations were studied in seven giant rosette species of the genus Espeletia from the Venezuelan Andes. Readily available water from the pith was calculated to be capable of sustaining mean transpiration for up to 2.5 h. The relative importance of water stored in the pith, however, differed among species. The species that grow in the higher and colder environments tended to have a greater capacitance than the species that grow in the lower and less extreme environments. The pith volume per unit leaf area (PV/LA) was found to be a good indicator of the relative water storage capacity of the adult individuals of each species. Diurnal fluctuations in leaf water potential were not as pronounced in the species with higher PV/LA values. The species-specific PV/LA was highly correlated with the leaf turgor loss point and with the total resistance to water flow from soil to leaves. These results suggested that species-specific capacitance in the genus Espeletia is a response to temperature-limited soil water availability and that cold tropical environments with frequent subfreezing temperatures tend to select for high water storage capacity in giant rosette plants.     

Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process

A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30–70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40–120 °C) for 48–144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1 g-ammonia + 1.0 g-water per g biomass, 80 °C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan + xylan in corn stover).     

Lateral movement of water and sugar across xylem in sugarcane stalks

Laterally connected vascular bundles in the nodes of sugarcane (Saccharum species cv. Pindar) stalks allow a rapid redistribution of water across the stalk should the vascular continuity be partly disrupted. Tritiated water supplied to the roots exchanged rapidly between the xylem and storage tissue so that net movement up the stalk was slow. The half-time for exchange in a labeled stalk was about 4 hours so that the entire water content of a sugarcane stalk can turn over at least once in a single day. No rapid flux of sugar between xylem and phloem or xylem and storage tissue was detected. Functional xylem contained only low sugar concentrations: less than 0.3% w/v in the stalk and less than 0.02% w/v in the leaf. Previous reports of high sugar levels (9% w/v) in sugarcane stalk xylem reflect some degree of xylem blockage followed by a slow equilibration with free space sugars in the storage tissue.     

Structural changes of corn stover lignin during acid pretreatment

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