应用固定化里氏木霉糖化玉米秆纤维素的研究

采用多孔聚酯材料固定里氏木霉（TrichodermareeseiRutC30）菌丝细胞,将固定化细胞在生长限制条件下重复分批培养,使纤维酶的合成与玉米秆纤维原料的酶解糖化耦合在一个反应器中同时进行。在30℃、初始pH4.8、摇瓶转速150r／min的条件下,连续重复进行12次分批培养试验。每批玉米秆用量为60g／L,培养周期4．5d,共54d。培养液中含滤纸酶活力平均为0.70IU／ml,还原糖26．41g／L,糖化率达到理论值的89.11％。固定化菌丝形态正常,菌量保持在10g／L左右。在间歇添料条件下,玉米秆原料的总量可提高到120g／L,7d后还原糖浓度达52.81g／L,糖化率为89．20％。利用固定化里氏木霉同时产酶和糖化植物纤维原料,工艺简便、成本低廉、易于连续自动化操作,是一条有效利用可再生纤维素资源的新途径。     

Pretreatment of corn stover by combining ionic liquid dissolution with alkali extraction

Pretreatment plays an important role in the efficient enzymatic hydrolysis of biomass into fermentable sugars for biofuels. A highly effective pretreatment method is reported for corn stover which combines mild alkali-extraction followed by ionic liquid (IL) dissolution of the polysaccharides and regeneration (recovery of the polysaccharides as solids). Air-dried, knife-milled corn stover was soaked in 1% NaOH at a moderate condition (90°C, 1 h) and then thoroughly washed with hot deionized (DI) water. The alkali extraction solublized 75% of the lignin and 37% of the hemicellulose. The corn stover fibers became softer and smoother after the alkali extraction. Unextracted and extracted corn stover samples were separately dissolved in an IL, 1-butyl-3-methylimidazolium chloride (C(4) mimCl), at 130°C for 2 h and then regenerated with DI water. The IL dissolution process did not significantly change the chemical composition of the materials, but did alter their structural features. Untreated and treated corn stover samples were hydrolyzed with commercial enzyme preparations including cellulases and hemicellulases at 50°C. The glucose yield from the corn stover sample that was both alkali-extracted and IL-dissolved was 96% in 5 h of hydrolysis. This is a highly effective methodology for minimizing the enzymatic loading for biomass hydrolysis and/or maximizing the conversion of biomass polysaccharides into sugars.     

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

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

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.     

EFFICIENT PRODUCTION OF BIOETHANOL FROM CORN STOVER BY PRETREATMENT WITH A COMBINATION OF SULFURIC ACID AND SODIUM HYDROXIDE

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid–alkali combination (1% H₂SO₄, 150°C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35°C for 36 hr after presaccharification at 50°C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.     

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.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Using FTIR spectroscopy to model alkaline pretreatment and enzymatic saccharification of six lignocellulosic biomasses

Fourier transform infrared, attenuated total reflectance (FTIR-ATR) spectroscopy, combined with partial least squares (PLS) regression, accurately predicted solubilization of plant cell wall constituents and NaOH consumption through pretreatment, and overall sugar productions from combined pretreatment and enzymatic hydrolysis. PLS regression models were constructed by correlating FTIR spectra of six raw biomasses (two switchgrass cultivars, big bluestem grass, a low-impact, high-diversity mixture of prairie biomasses, mixed hardwood, and corn stover), plus alkali loading in pretreatment, to nine dependent variables: glucose, xylose, lignin, and total solids solubilized in pretreatment; NaOH consumed in pretreatment; and overall glucose and xylose conversions and yields from combined pretreatment and enzymatic hydrolysis. PLS models predicted the dependent variables with the following values of coefficient of determination for cross-validation (Q2): 0.86 for glucose, 0.90 for xylose, 0.79 for lignin, and 0.85 for total solids solubilized in pretreatment; 0.83 for alkali consumption; 0.93 for glucose conversion, 0.94 for xylose conversion, and 0.88 for glucose and xylose yields. The sugar yield models are noteworthy for their ability to predict overall saccharification through combined pretreatment and enzymatic hydrolysis per mass dry untreated solids without a priori knowledge of the composition of solids. All wavenumbers with significant variable-important-for-projection (VIP) scores have been attributed to chemical features of lignocellulose, demonstrating the models were based on real chemical information. These models suggest that PLS regression can be applied to FTIR-ATR spectra of raw biomasses to rapidly predict effects of pretreatment on solids and on subsequent enzymatic hydrolysis.     

Acetone–butanol–ethanol production from corn stover pretreated by alkaline twin-screw extrusion pretreatment

In this study, the alkaline twin-screw extrusion pretreated corn stover was subjected to enzymatic hydrolysis after washing. The impact of solid loading and enzyme dose on enzymatic hydrolysis was investigated. It was found that 68.2 g/L of total fermentable sugar could be obtained after enzymatic hydrolysis with the solid loading of 10 %, while the highest sugar recovery of 91.07 % was achieved when the solid loading was 2 % with the cellulase dose of 24 FPU/g substrate. Subsequently, the hydrolyzate was fermented by Clostridium acetobutylicum ATCC 824. The acetone–butanol–ethanol (ABE) production of the hydrolyzate was compared with the glucose, xylose and simulated hydrolyzate medium which have the same reducing sugar concentration. It was shown that 7.1 g/L butanol and 11.2 g/L ABE could be produced after 72 h fermentation for the hydrolyzate obtained from enzymatic hydrolysis with 6 % solid loading. This is comparable to the glucose and simulated hydrozate medium, and the overall ABE yield could reach 0.112 g/g raw corn stover.     

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.     

Fast and efficient alkaline peroxide treatment to enhance the enzymatic digestibility of steam-exploded softwood substrates.

The enzymatic digestibility of steam-exploded Douglas-fir wood chips (steam exploded at 195 degrees C, 4.5 min, and 4.5% (w/w) SO(2)) was significantly improved using an optimized alkaline peroxide treatment. Best hydrolysis yields were attained when the steam-exploded material was post-treated with 1% hydrogen peroxide at pH 11.5 and 80 degrees C for 45 min. This alkaline peroxide treatment was applied directly to the water-washed, steam-exploded material eliminating the need for independent alkali treatment with 0.4% NaOH, which has been traditionally used to post-treat wood samples to try to remove residual lignin. Approximately 90% of the lignin in the original wood was solubilized by this novel procedure, leaving a cellulose-rich residue that was completely hydrolyzed within 48 h, using an enzyme loading of 10 FPU/g cellulose. About 82% of the originally available polysaccharide components of the wood could be recovered. The 18% of the carbohydrate that was not recovered was lost primarily to sugar degradation during steam explosion.     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Calculating sugar yields in high solids hydrolysis of biomass

Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in yield calculations can lead to significant errors. In this paper, a mathematical method was developed for the estimation of liquid volume change and thereafter the sugar yield. The information needed in the calculations include the compositions of the substrate, initial solids loading, initial liquid density, and sugar concentrations before and after hydrolysis. All of these variables are measurable with conventional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in the literature, this method is relatively easy to use and provides good accuracy.     

Concentrated HCl Catalyzed 5-(Chloromethyl)furfural Production from Corn Stover of Varying Particle Sizes

5-(Chloromethyl) Furfural (CMF) is a potential chemical building block for replacing petroleum-derived chemicals derived from lignocellulosic feedstocks. In this study, hand harvested corn stover and mechanically forage chopped corn stover was processed in a 1 L hydrolysis reactor to produce CMF in a biphasic, two solvent system. Both 1,2 dichloroethane (DCE) and dichloromethane (DCM) were tested as organic solvents. The results showed that DCE performed better than DCM due to temperature and pressure limitations of the reactor system. Using DCE as the extracting solvent, the effects of solids loading, particle size, and moisture content of the corn stover on the hydrolysis efficiency were determined. One liter acid hydrolysis reactor provides consistent and reproducible yields of 63% CMF from hand harvested corn stover as feedstock at solid loading of 10% wt/v, 100C for 1 h. For the forage chopped corn stover, increasing particle size brings an increase in the feedstock sugar content. Foraged chopped corn stover (FCCS) particle sizes larger than 19 mm (0.75 in.) results in significant reduction in CMF yield from 43 to 35%.     

Optimizing harvest of corn stover fractions based on overall sugar yields following ammonia fiber expansion pretreatment and enzymatic hydrolysis

Background  

Corn stover composition changes considerably throughout the growing season and also varies between the various fractions of the plant. These differences can impact optimal pretreatment conditions, enzymatic digestibility and maximum achievable sugar yields in the process of converting lignocellulosics to ethanol. The goal of this project was to determine which combination of corn stover fractions provides the most benefit to the biorefinery in terms of sugar yields and to determine the preferential order in which fractions should be harvested. Ammonia fiber expansion (AFEX) pretreatment, followed by enzymatic hydrolysis, was performed on early and late harvest corn stover fractions (stem, leaf, husk and cob). Sugar yields were used to optimize scenarios for the selective harvest of corn stover assuming 70% or 30% collection of the total available stover.     

Lime pretreatment and enzymatic hydrolysis of corn stover

Corn stover was pretreated with an excess of 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 optimal condition is 55 degrees C for 4 weeks with aeration. Glucan (91.3%) and xylan (51.8%) were converted to glucose and xylose respectively, when the treated corn stover was enzymatically hydrolyzed with 15 FPU/g cellulose. Only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Of the initial lignin, 87.5% was maximally removed. Almost all acetyl groups were removed. After 4 weeks at 55 degrees C with aeration, some cellulose and hemicellulose were solubilized as monomers and oligomers in the pretreatment liquor. When considering the dissolved fragments of glucan and xylan in the pretreatment liquor, the overall yields of glucose and xylose were 93.2% and 79.5% at 15 FPU/g cellulose. The pretreatment liquor has no inhibitory effect on ethanol fermentation.     

Changes in plant cell-wall structure of corn stover due to hot compressed water pretreatment and enhanced enzymatic hydrolysis

Corn stover is a potential feedstock for biofuel production. This work investigated physical and chemical changes in plant cell-wall structure of corn stover due to hot compressed water (HCW) pretreatment at 170–190 °C in a tube reactor. Chemical composition analysis showed the soluble hemicellulose content increased with pretreatment temperature, whereas the hemicellulose content decreased from 29 to 7 % in pretreated solids. Scanning electron microscopy revealed the parenchyma-type second cell-wall structure of the plant was almost completely removed at 185 °C, and the sclerenchyma-type second cell wall was greatly damaged upon addition of 5 mmol/L ammonium sulfate during HCW pretreatment. These changes favored accessibility for enzymatic action. Enzyme saccharification of solids by optimized pretreatment with HCW at 185 °C resulted in an enzymatic hydrolysis yield of 87 %, an enhancement of 77 % compared to the yield from untreated corn stover.     

Partial flow of compressed-hot water through corn stover to enhance hemicellulose sugar recovery and enzymatic digestibility of cellulose

Flowthrough pretreatment with compressed-hot water can significantly increase the removal of xylan and lignin and enhance xylose sugar yields and cellulose digestibility, especially at high flow rates. However, continuous flowthrough operations that realize these benefits suffer from a large amount of water consumption that leads to high energy requirements for pretreatment and downstream processing. Because high flow rates are particularly effective early in hemicellulose hydrolysis and less effective later, flow with compressed-hot water was applied at selected intervals, and performance was compared with that of batch and flowthrough operations for corn stover pretreated with compressed-hot water at 200 degrees C. Partial flow reduced water consumption by 60% compared with continuous flowthrough operation but still achieved higher xylose sugar yields (84-89%) compared to batch pretreatment (46.6%). In addition, corn stover cellulose pretreated by partial flow had higher enzymatic digestibility (88-90%) than batch operations (approximately 85%) at otherwise identical conditions, apparently due to much higher lignin removal for the former (40-45% vs 10-12%). Partial flow also reduced degradation, with recovery of xylose and glucose in the solids and hydrolyzate increased to 90-92% vs only about 76% for batch operation. The partial flow approach could be further improved by optimizing the operating strategy and reaction conditions, suggesting that this novel pretreatment could lead to advanced biomass pretreatment technology.     

Improvement of radio frequency (RF) heating-assisted alkaline pretreatment on four categories of lignocellulosic biomass

Pretreatment plays an important role in making the cellulose accessible for enzyme hydrolysis and subsequent conversion because it destroys more or less resistance and recalcitrance of biomass. Radio frequency (RF)-assisted dielectric heating was utilized in the alkaline pretreatment on agricultural residues (corn stover), herbaceous crops (switchgrass), hardwood (sweetgum) and softwood (loblolly pine). Pretreatment was performed at 90 °C with either RF or traditional water bath (WB) heating for 1 h after overnight soaking in NaOH solution (0.2 g NaOH/g Biomass). Pretreated materials were characterized by chemical compositional analysis, enzyme hydrolysis, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The glucan yields of RF-heated four categories of hydrolysates were 89.6, 72.6, 21.7, and 9.9 %. Interestingly, RF heating raised glucan yield on switchgrass and sweetgum but not on corn stover or loblolly pine. The SEM images and FTIR spectra agreed with results of composition analysis and hydrolysis. GC–MS detected some compounds only from RF-heated switchgrass. These compounds were found by other researchers only in high-temperature (150–600 °C) and high-pressure pyrolysis processes.