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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.124, #1-3)

Biomass pretreatment and hydrolysis by Bruce S. Dien; Quang A. Nguyen (pp. 885-886).

Application of fentonś reaction to steam explosion prehydrolysates from poplar biomass by J. M. Oliva; P. Manzanares; I. Ballesteros; M. J. Negro; A. González; M. Ballesteros (pp. 887-899).
The application of Fenton’s reaction to enhance the fermentability of prehydrolysates obtained from steam explosion pretreatment of poplar biomass was studied. Reaction conditions of temperature and H2O2 and Fe(II) concentrations were studied. The fermentability of prehydrolysate treated by Fenton’s reaction was tested by using different inoculum sizes of thermotolerant strain Kluyveromyces marxianus CECT 10875. The highest percentages of toxic compound degradation (ranging from 71 to 93% removal) were obtained at the highest H2O2 concentration tested (50 mM). However, a negative effect on fermentability was observed at this H2O2 concentration at the lower inoculum loading. An increase in inoculum size to 0.6 g/L resulted in an enhanced ethanol fermentation yield of 95% relative to control.

Keywords: Fenton’s reaction; detoxification; poplar prehydrolysate; ethanol production; Kluyveromyces marxianus


Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion-treated corn stover by Bernie Steele; Srini Raj; John Nghiem; Mark Stowers (pp. 901-910).
Both cellulase and cellobiase can be effectively recovered from hydrolyzed biomass using an ultrafiltration recovery method. Recovery of cellulase ranged from 60 to 66.6% and for cellobiase from 76.4 to 88%. Economic analysis shows that cost savings gained by enzyme recycling are sensitive to enzyme pricing and loading. At the demonstrated recovery of 60% and current loading of 15 Filter paper units of cellulase/g of glucan, enzyme recycling is expected to generate a cost savings of approx 15%. If recovery efficiency can be improved to 70%, the savings will increase to >25%, and at 90% recovery the savings will be 50%.

Keywords: Enzyme recycle; biomass; ammonia fiber explosion; ethanol; corn stover


Ammonium hydroxide detoxification of spruce acid hydrolysates by Björn Alriksson; Ilona Sárvári Horváth; Anders Sjöde; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 911-922).
When dilute-acid hydrolysates from spruce are fermented to produce ethanol, detoxification is required to make the hydrolysates fermentable at reasonable rates. Treatment with alkali, usually by overliming, is one of the most efficient approaches. Several nutrients, such as ammonium and phosphate, are added to the hydrolysates prior to fermentation. We investigated the use of NH4OH for simultaneous detoxification and addition of nitrogen source. Treatment with NH4OH compared favorably with Ca(OH)2, Mg(OH)2, Ba(OH)2, and NaOH to improve fermentability using Saccharomyces cerevisiae. Analysis of monosaccharides, furan aldehydes, phenols, and aliphatic acids was performed after the different treatments. The NH4OH treatments, performed at pH 10.0, resulted in a substantial decrease in the concentrations of furfural and hydroxymethylfurfural. Under the conditions studied, NH4OH treatments gave better results than Ca(OH)2 treatments. The addition of an extra nitrogen source in the form of NH4Cl at pH 5.5 did not result in any improvement in fermentability that was comparable to NH4OH treatments at alkaline conditions. The addition of CaCl2 or NH4Cl at pH 5.5 after treatment with NH4OH or Ca(OH)2 resulted in poorer fermentability, and the negative effects were attributed to salt stress. The results strongly suggest that the highly positive effects of NH4OH treatments are owing to chemical conversions rather than stimulation of the yeast cells by ammonium ions during the fermentation.

Keywords: Ethanol; lignocellulose; detoxification; NH4OH; nitrogen


Detoxification of actual pretreated corn stover hydrolysate using activated carbon powder by R. Eric Berson; John S. Young; Sarah N. Kamer; Thomas R. Hanley (pp. 923-934).
A technique for the removal of acetic acid from an actual pretreated corn stover hydrolysate was investigated. A powdered form of activated carbon previously shown to be effective in the removal of acetic acid from a synthetic hydrolysate was utilized. The method proved to be effective at lowering acetic acid levels while exhibiting minimal adsorption of the desired sugars from the hydrolysate, although at a lower efficiency in the actual hydrolysate than in the synthetic hydrolysate. Results are obtained for temperatures between 25 and 35°C and agitation rates between 150 and 350 rpm in shake flasks. Adsorption isotherm and kinetic rate date are presented. Temperature differences over this range did not have an effect on adsorption characteristics. Five stages of detoxification were necessary to lower acetic acid concentration to the maximum 2 g/L desired for fermentation.

Keywords: Acetic acid; activated carbon; adsorption isotherms; detoxification; pretreated corn stover hydrolysate


Use of computational fluid dynamics simulations for design of a pretreatment screw conveyor reactor by R. Eric Berson; Thomas R. Hanley (pp. 935-945).
Computational fluid dynamics simulations were employed to compare performance of various designs of a pretreatment screw conveyor reactor. The reactor consisted of a vertical screw used to create cross flow between the upward conveying solids and the downward flow of acid. Simulations were performed with the original screw design and a modified design in which the upper flights of the screw were removed. Results of the simulations show visually that the modified design provided favorable plug flow behavior within the reactor. Pressure drop across the length of the reactor without the upper screws in place was predicted by the simulations to be 5 vs 40 kPa for the original design.

Keywords: Biomass; pretreatment reactor; computational fluid dynamics simulations; screw conveyor reactor; plug flow; porous media model


Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics by Yun-Chin Chung; Alan Bakalinsky; Michael H. Penner (pp. 947-961).
The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180°C, and 0.5 min for switchgrass and 1% acid, 180°C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.

Keywords: Lignocellulosic; poplar; switchgrass; corn stover; simultaneous saccharification and fermentation; cellulase; cellulose; xylose


Fermentability of water-soluble portion to ethanol obtained by supercritical water treatment of lignocellulosics by Hisashi Miyafuji; Toshiki Nakata; Katsunobu Ehara; Shiro Saka (pp. 963-971).
The water-soluble (WS) portion obtained by supercritical water treatment of lignocellulosics was studied for its fermentability to ethanol. A fermentation test of the WS portion showed it was not fermented to ethanol. Therefore, a wood characoal treatment was applied to the WS portion to remove furan and phenolic compounds, which are thought to be the inhibitors to sugar fermentability. It was found that treatment with wood charcoal can be effective at removing these inhibitors and improving the fermentability of the WS portion without reducing the levels of fermentable sugars.

Keywords: Lignocellulosics; supercritical water; inhibitor; wood charcoal; ethanol fermentation


Application of sequential aqueous steam treatments to the fractionation of softwood by Abolghasem Shahbazi; Yebo Li; Michele R. Mims (pp. 973-987).
The FIRST (Feedstock Impregnation and Rapid Steam Treatment) approach was used in this study to isolate extractives, hemicellulose, lignin, fibers, and cellulosic fines of softwood. With hydrolysis and fermentation of the hemicellulose and cellulosic fines fractions, this approach produces four co-products: extractives, cellulose, lignin, and ethanol. The first unit operation uses aqueous/alcohol to remove and recover the extractive rich fraction. The second unit operation uses steam treatment to destructure the matrix and solubilize a large fraction of the hemicelluloses. The third unit operation uses alkaline delignification to dissolve a lignin fraction. The fourth unit operation uses the refining process to separate fibers from cellulosic fines. The fibers are bleached. The yields of lignin and bleached cellulose were about 20.0 kg and 38.3 kg out of 100 kg initial dry pine, respectively. The recovered hemicelluloses were 23.3 kg (containing 16.1 kg hexoses and 5.0 kg pentoses) and the cellulose fines derived hexoses amounted to 3.4 kg out of 100 kg initial dry pine. When the two liquors containing the hemicellulose sugars and the cellulose fines-derived hexoses were fermented for ethanol production, an ethanol yield of 6.8 kg was obtained.

Keywords: Pretreatment; softwood; hydrolysis; steam explosion; fractionation; ethanol


Acidic sugar degradation pathways by Xianghong Qian; Mark R. Nimlos; David K. Johnson; Michael E. Himmel (pp. 989-997).
Ab initio molecular dynamics (MD) simulations were employed to elucidate xylose and glucose degradation pathways. In the case of xylose, a 2,5-anhydride intermediate was observed leading to the formation of furfural through elimination of water. This pathway agrees with one of the mechanisms proposed in the literature in that no open chain intermediates were found. In the case of glucose, a series of intermediates were observed before forming the 2,5-anhydride intermediate that eventually leads to hydroxymethylfurfural (HMF). One of these intermediates was a very short-lived open-chain form. Furthermore, two novel side-reaction pathways were identified, which lead to degradation products other than HMF.

Keywords: Degradation; pathway; acidic, intermediate, sugar


Studies into using manure in a biorefinery concept by S. Chen; Z. Wen; W. Liao; C. Liu; R. L. Kincaid; J. H. Harrison; D. C. Elliott; M. D. Brown; D. J. Stevens (pp. 999-1015).
Animal manure is an underutilized biomass resource containing a large amount of organic carbon that is often wasted with the existing manure disposal practices. A research project funded by the US Department of Energy explored the feasibility of using manure via the sugar platform in a biorefinery, converting the carbon from fiber to biochemicals. The results showed that (1) fiber was the major component of manure dry material making up approx 50%, 40%, and 36% of the dry dairy, swine, and poultry manure material, respectively; within dairy manure, more than 56% of the dry matter was in particles larger than 1.680 mm; (2) in addition to being a carbon source, manure could provide a variety of nutrient for fungi T. reesei and A. phoenicis to produce cellulase; (3) the hemicellulose component in the manure fiber could be readily converted to sugar through acid hydrolysis; while concentrated acid decrystallization treatment was most effective in manure cellulose hydrolysis; (4) purification and separation was necessary for further chemical conversion of the manure hydrolysate to polyols through hydrogenation; and (5) the manure utilization strategy studied in this work is currently not profitable.

Keywords: Biorefinery; manure


Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure by Wei Liao; Zhiyou Wen; Sharon Hurley; Yan Liu; Chuanbin Liu; Shulin Chen (pp. 1017-1030).
This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.

Keywords: Cellulose; fed-batch enzymatic hydrolysis; glucose; hemicellulose; yield


Critical conditions for improved fermentability during overliming of acid hydrolysates from spruce by Ilona Sárvári Horváth; Anders Sjöde; Björn Alriksson; Leif J. Jönsson; Nils-Olof Nilvebrant (pp. 1031-1044).
Bioethanol can be produced from wood via acid hydrolysis, but detoxification is needed to achieve good fermentability. Overliming was investigated in a factorial designed experiment, in which pH and temperature were varied. Degradation of inhibitory furan aldehydes was more extensive compared to monosaccharides. Too harsh conditions led to massive degradation of sugars and formation of inhibiting acids and phenols. The ethanol productivity and yield after optimal overliming reached levels exceeding reference fermentations of pure glucose. A novel metric, the balanced ethanol yield, which takes both ethanol production and losses of fermentable sugars into account, was introduced and showed the optimal conditions within the investigated range. The findings allow process technical and economical considerations to govern the choice of conditions for overliming.

Keywords: Detoxification; ethanol; inhibitors; lignocellulose; optimization; Saccharomyces cerevisiae


Optimization of dilute-acid pretreatment of corn stover using a high-solids percolation reactor by Yongming Zhu; Y. Y. Lee; Richard T. Elander (pp. 1045-1054).
We have previously demonstrated that pretreatment of corn stover with dilute sulfuric acid can achieve high digestibility and efficient recovery of hemicellulose sugars with high yield and concentration. Further improvement of this process was sought in this work. A modification was made in the operation of the percolation reactor that the reactor is preheated under atmospheric pressure to remove moisture that causes autohydrolysis. This eliminated sugar decomposition during the preheating stage and led to a considerable improvement in overall sugar yield. In addition, liquid throughput was minimized to the extent that only one reactor void volume of liquid was collected. This was done to attain a high xylose concentration in the hydrolyzate. The optimum reaction and operating conditions were identified wherein near quantitative enzymatic digestibilities are obtained with enzyme loading of 15 FPU/g glucan. With a reduced enzyme loading of 5 FPU/g glucan, the enzymatic digestibility was decreased, but still reached a level of 92%. Decomposition of carbohydrates was extremely low as indicated by the measured glucan and xylan mass closures (recovered sugar plus unreacted) which were 98% and 94%, respectively. The data obtained in this work indicate that the digestibility is related to the extent of xylan removal.

Keywords: Corn stover; dilute-acid hydrolysis; percolation process; pretreatment; high sugar recovery; low enzyme loading


Optimization of steam pretreatment of SO2-impregnated corn stover for fuel ethanol production by Karin Öhgren; Mats Galbe; Guido Zacchi (pp. 1055-1067).
In this study, corn stover with a dry matter content of 20% was impregnated with SO2 and then steam pretreated for various times at various temperatures. The pretreatment was evaluated by enzymatic hydrolysis of the solid material and analysis of the sugar content in the liquid. The maximum overall yield of glucose, 89% of the theoretical based on the glucan in the raw material, was achieved when the corn stover was pretreated at 200°C for 10 min. The maximum overall yield of xylose, 78%, was obtained with pretreatment at 190°C for 5 min.

Keywords: Steam pretreatment; SO2 ; corn stover; fuel-ethanol production


Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content by Xuejun Pan; Dan Xie; Neil Gilkes; David J. Gregg; Jack N. Saddler (pp. 1069-1079).
Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.

Keywords: Softwood; bioconversion; pretreatment; steam explosion; lignin; cellulose; hydrolysis


Understanding factors that limit enzymatic hydrolysis of biomass by Lizbeth Laureano-Perez; Farzaneh Teymouri; Hasan Alizadeh; Bruce E. Dale (pp. 1081-1099).
Spectroscopic characterization of both untreated and treated material is being performed in order to determine changes in the biomass and the effects of pretreatment on crystallinity, lignin content, selected chemical bonds, and depolymerization of hemicellulose and lignin. The methods used are X-ray diffraction for determination of cellulose crystallinity (CrI); diffusive reflectance infrared (DRIFT) for changes in C-C and C-O bonds; and fluorescence to determine lignin content. Changes in spectral characteristics and crystallinity are statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that govern its enzymatic conversion to monomeric sugars. Models of the hydrolysis initial rate and 72 h extent of conversion were developed and evaluated. Results show that the hydrolysis initial rate is most influenced by the cellulose crystallinity, while lignin content most influences the extent of hydrolysis at 72 h. However, it should be noted that in this study only crystallinity, lignin, and selected chemical bonds were used as inputs to the models. The incorporation of additional parameters that affect the hydrolysis, like pore volume and size and surface area accessibility, would improve the predictive capability of the models.

Keywords: AFEX; corn stover; multilinear regression; statistical model


Steam pretreatment of Salix with and without SO2 impregnation for production of bioethanol by Per Sassner; Mats Galbe; Guido Zacchi (pp. 1101-1117).
In the wood-to-ethanol process, pretreatment of the material is necessary prior to enzymatic hydrolysis to obtain high overall yields of sugar and ethanol. Steam pretreatment of fast-growing Salix either with or without SO2 impregnation has been investigated by varying different parameters. Overall glucose yields of above 90% and overall xylose yields higher than 80% were obtained both with and without impregnation. However, the most favorable pretreatment conditions for the separate yields of glucose and xylose differed to a lower degree using SO2-impregnated wood chips, resulting in higher total sugar yield than that obtained with non-impregnated wood chips.

Keywords: Steam pretreatment; Salix ; willow; ethanol production; enzymatic hydrolysis


Pretreatment of corn stover by soaking in aqueous ammonia by Tae Hyun Kim; Y. Y. Lee (pp. 1119-1131).
Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D5A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.

Keywords: Corn stover; pretreatment; soaking; ammonia; simultaneous saccharification and fermentation (SSF); SSCF


Pretreatment of switchgrass by ammonia fiber explosion (AFEX) by Hasan Alizadeh; Farzaneh Teymouri; Thomas I. Gilbert; Bruce E. Dale (pp. 1133-1141).
The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100°C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.

Keywords: Ammonia fiber explosion (AFEX); switchgrass; enzymatic hydrolysis; simultaneous saccharification and fermentation

Plant biotechnology and feedstock genomics by Daniel Jones; Michael Lassner (pp. 1145-1145).

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots by Gwi-Taek Jeong; Don-Hee Park; Hwa-Won Ryu; Baik Hwang; Je-Chang Woo; Doman Kim; Si-Wonk Kim (pp. 1147-1157).
Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Keywords: Panax ginseng ; transformed hairy roots; elicitation; yeast elicitor; ginseng saponin

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