Impact of Harvest Time and Cultivar on Conversion of Switchgrass to Bio-oils Via Fast Pyrolysis

The study of the effects of harvest time on switchgrass (Panicum virgatum L.) biomass and bioenergy production reported herein encompasses a large study evaluating the harvest of six switchgrass cultivars grown at three northern US locations over 3 years, harvested at upland peak crop (anthesis), post-frost, and post-winter. Delaying harvest of switchgrass until after frost and until after winter has resulted in decreased yields of switchgrass and reduced amounts of minerals in the biomass. This report examines how changes in biomass composition as a result of varying harvest time and other factors affect the distribution of products formed via fast pyrolysis. A subset (50) of the population (n = 864) was analyzed for fast pyrolysis and catalytic pyrolysis (zeolite catalyst) product yields using a pyrolysis-GC/MS system. The subset was used to build calibrations that were successful in predicting the pyrolysis product yield using near-infrared reflectance spectroscopy (NIRS), and partial least squares predictive models were applied to the entire sample set. The pyrolysis product yield was significantly affected by the field trial location, year of harvest, cultivar, and harvest time. Delaying harvest time of the switchgrass crop led to greater production of deoxygenated aromatics improving the efficiency of the catalytic fast pyrolysis and bio-oil quality. The changes in the pyrolysis product yield were related to biomass compositional changes, and key relationships between cell wall polymers, potassium concentration in the biomass, and pyrolysis products were identified. The findings show that the loss of minerals in the biomass as harvest time is delayed combined with the greater proportion in cellulose and lignin in the biomass has significant positive influences on conversion through fast pyrolysis.     

酶促红花油水解中的时间效应

目的:研究无溶剂体系中脂肪酶催化红花油水解反应的时间效应。方法:采用单因素实验法,确定不同反应时间时各因素的最佳水平。结果:反应1h时的最适条件为35℃、n(水)/n(油)比35p、H 7.0、缓冲液浓度0.05mol/L、添加0.2?Cl2;反应24h时,最适条件为26℃、n(水)/n(油)比60p、H 7.6,缓冲液浓度及CaCl2的影响不显著。结论:水解最佳条件随反应时间变化,应根据反应时间长短确定各因素的最佳水平。     

Use of Ascomycete Extracts in Enzymatic Cocktail Formulations Increases Sugar Cane Bagasse Hydrolysis

Hemicellulases and accessory enzymes are essential for supplementation of cellulolytic enzyme extracts, and combinations of these enzymes can lead to high performance in plant biomass hydrolysis. In this work, enzyme extracts rich in hemicellulases and β-glucosidase, produced by the unique ascomycete strains Annulohypoxylon stygium DR47 and Aspergillus niger DR02, were tested for use in formulations with Celluclast 1.5 L. Statistical analysis showed that a mixture based on these enzymes was able to increase the hydrolysis of hydrothermally pretreated sugar cane bagasse. The two A. stygium extracts only effectively increased glucose release when they were combined. These extracts had no positive effect when used together with the A. niger extract, and the findings suggested that a blend based on the commercial cellulose preparation and the xylanase-rich extract from A. niger provided the best carbohydrate solubilization. Supplementation at low cellulolytic loading resulted in 120 and 238 % increases in cellulose and hemicellulose hydrolysis yields.     

Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol

During the course of studies on the oxidative metabolism of d-sorbitol by acetic acid bacteria, it was found that d-sorbitol was almost quantitatively converted to 5-keto-d-fructose via l-sorbose by a certain strain of Gluconobacter suboxydans. In addition to 5-keto-d-fructose, three γ-pyrone compounds, kojic acid, 5-oxymaltol, and 3-oxykojic acid, 2-keto-l-gulonate, and several organic acids such as succinic, glycolic, and glyceric acids were confirmed in the culture filtrate of this bacterium.

• The most suitable carbon source for 5-ketofructose fermentation by Gluconobacter suboxydans Strain 1 was confirmed to be d-sorbitol or l-sorbose using growing and resting cells. d-Fructose had little effect on the formation of this dicarbonylhexose.

• The optimal pH for the formation from l-sorbose by intact cells was found to be at 4.2.

• The activity of the pentose phosphate cycle in the resting cells was calculated as 13~17 μatoms/hr/mg of dry cells by the use of the manometric techniques.

• There was no strain tested so far which could accumulate a large amount of 5- keto-d-fructose from d-sorbitol except this bacterium.

• The experimental results shown in this paper makes the prediction that a certain dehydrogenating system of l-sorbose is functional in the organism, and the metabolic pathways of d-sorbitol via l-sorbose and 5-keto-d-fructose is proposed.

     

Crop Management Impacts Biofuel Quality: Influence of Switchgrass Harvest Time on Yield, Nitrogen and Ash of Fast Pyrolysis Products

Although upgrading bio-oil from fast pyrolysis of biomass is an attractive pathway for biofuel production, nitrogen (N) and mineral matter carried over from the feedstock to the bio-oil represents a serious contaminant in the process. Reducing the N and ash content of biomass feedstocks would improve process reliability and reduce production costs of pyrolytic biofuels. This study investigated: (1) How does switchgrass harvest date influence the yield, N concentration ([N]), and ash concentration of biomass and fast pyrolysis products? and (2) Is there a predictive relationship between [N] of switchgrass biomass and [N] of fast pyrolysis products? Switchgrass from five harvest dates and varying [N] from central Iowa were pyrolyzed using a free-fall reactor. Harvestable biomass peaked in August (8.6 Mg ha^?1), dropping significantly by November (6.7 Mg ha^?1, P?=?0.0027). Production of bio-oil per unit area mirrored that of harvested biomass at each harvest date; however, bio-oil yield per unit dry biomass increased from 46.6 % to 56.7 % during the season (P?=?0.0018). Allowing switchgrass to senesce lowered biomass [N] dramatically, by as much as 68 % from June to November (P?<?0.0001). Concurrently, bio-oil [N] declined from 0.51 % in June to 0.17 % by November (P?<?0.0001). Significant reductions in ash concentration were also observed in biomass and char. Finally, we show for the first time that the [N] of switchgrass biomass is a strong predictor of the [N] of bio-oil, char, and non-condensable gas with R ² values of 0.89, 0.94, and 0.88, respectively.     

Transcriptional Analysis of Flowering Time in Switchgrass

Over the past two decades, switchgrass (Panicum virgatum) has emerged as a priority biofuel feedstock. The bulk of switchgrass biomass is in the vegetative portion of the plant; therefore, increasing the length of vegetative growth will lead to an increase in overall biomass yield. The goal of this study was to gain insight into the control of flowering time in switchgrass that would assist in development of cultivars with longer vegetative phases through delayed flowering. RNA sequencing was used to assess genome-wide expression profiles across a developmental series between switchgrass genotypes belonging to the two main ecotypes: upland, typically early flowering, and lowland, typically late flowering. Leaf blades and tissues enriched for the shoot apical meristem (SAM) were collected in a developmental series from emergence through anthesis for RNA extraction. RNA from samples that flanked the SAM transition stage was sequenced for expression analyses. The analyses revealed differential expression patterns between early- and late-flowering genotypes for known flowering time orthologs. Namely, genes shown to play roles in photoperiod response and the circadian clock in other species were identified as potential candidates for regulating flowering time in the switchgrass genotypes analyzed. Based on their expression patterns, many of the differentially expressed genes could also be classified as putative promoters or repressors of flowering. The candidate genes presented here may be used to guide switchgrass improvement through marker-assisted breeding and/or transgenic or gene editing approaches.     

蛋白质的酶水解过程研究

进行了蛋白质酶水解过程的研究。结果表明木瓜蛋白酶对混合蛋白质的亲和力最强 ,而 1398蛋白酶的亲和力最弱。也表明作用位点和亲和力之间有一定的对应关系 ,Km值和作用位点氨基酸含量比例的相关系数为 0 .90 9。温度影响结果表明温度较低时温度升高加速水解反应过程处主要地位 ;当温度较高时 ,酶失活过程处主导地位。在一定水解时间内的讨论最适温度条件具有更明确的针对性 ,从本研究的采用胰酶 (胰蛋白酶和胰凝乳蛋白酶 )水解 4h的条件下 ,反应温度控制在45～ 5 0℃之间最适     

Biomass Yield and Composition of Switchgrass Bales on Marginal Land as Influenced by Harvest Management Scheme

Switchgrass (Panicum virgatum L.) is well suited to marginal croplands, but is difficult to manage sustainably both for maximum yield and optimal biomass composition. Quality can be improved by overwintering switchgrass in the field, but more information is needed on amount and consistency of yield recovery in spring. Two cultivars of switchgrass were sown on separate fields in Freeville, NY, and mowed and baled in late fall (FALL), mowed in fall and baled in spring (WINTER), or mowed and baled in spring (SPRING), using conventional field harvesting equipment. Samples were collected for analysis of plant morphological components and for determining the influence of harvest stubble height on yield and composition. Recovery of FALL biomass yields the following spring ranged from 52 to 82% and was related to both total winter snowfall and to the spring date when soil was dry enough to allow equipment traffic. Approximately 1% of dry matter yield was left in the field for each centimeter of stubble height following mowing. Bale moisture content was very low in spring, averaging 7.3%, but was much more variable and higher in the fall, averaging 22% for “Cave-in-Rock”. Inflorescence and leaf blade were the primary morphological components lost in standing switchgrass over winter. The SPRING treatment can be mowed and baled on the same day without other field operations and has higher quality than WINTER, with no consistent yield advantage for either spring baling treatment. The large and variable yield loss due to overwintering switchgrass in the field makes the practice questionable.     

Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis

Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of plant cell walls is a powerful tool for characterizing changes in cell wall chemistry during the hydrothermal pretreatment process of wheat straw for second-generation bioethanol production. One-bond ¹³C–¹H NMR correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1?→?2)-linked 4-O-methyl-α-d-glucuronic acid of xylan. In the polysaccharide anomeric region, decreases in the minor β-d-mannopyranosyl, and α-l-arabinofuranosyl units were observed in the NMR spectra from hydrothermally pretreated wheat straw. The aromatic region indicated only minor changes to the aromatic structures during the process (e.g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic hydrolysis incurred further deacetylation of the xylan, leaving approximately 10 % of acetate intact based on the weight of original wheat straw.     

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin,Lignin Monomers,and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.     

Production of Autopolyploid Lowland Switchgrass Lines Through In Vitro Chromosome Doubling

Switchgrass is considered one of the most promising energy crops. However, breeding of elite switchgrass cultivars is required to meet the challenges of large scale and sustainable biomass production. As a native perennial adapted to North America, switchgrass has lowland and upland ecotypes, where most lowland ecotypes are tetraploid (2n?=?4x?=?36), and most upland ecotypes are predominantly octoploid (2n?=?8x?=?72). Hybridization between lowland and upland switchgrass plants could identify new cultivars with heterosis. However, crossing between tetraploid and octoploid switchgrass is rare in nature. Therefore, in order to break down the cross incompatibility barrier between tetraploid lowland and octoploid upland switchgrass lines, we developed autoployploid switchgrass lines from an anueploid lowland cv. Alamo. In this study, colchicine was used in liquid and solid mediums to chemically induce chromosome doubling in embryogenic calli derived from cv. Alamo. Thirteen autopolyploid switchgrass lines were regenerated from seedlings and identified using flow cytometry. The autoplyploid switchgrass plants exhibited increased stomata aperture and stem size in comparison with the cv. Alamo. The most autooplyploid plants were regenerated from switchgrass calli that were treated with 0.04 % colchicine in liquid medium for 13 days. One autopolyploid switchgrass line, VT8-1, was successfully crossed to the octoploid upland cv. Blackwell. The autoployploid and the derived inter-ecotype hybrids were confirmed by in situ hybridization and molecular marker analysis. Therefore, the results of this study show that an autopolyploid, generated by chemically induced chromosome doubling of lowland cv. Alamo, is cross compatible with upland octoploid switchgrass cultivars. The outcome of this study may have significant applications in switchgrass hybrid breeding.     

直接加热膨化蔗渣酶法水解的研究

采用加热时间为２０ｍｉｎ,压力为２．０ＭＰａ,温度为１２０℃的直接加热膨化甘蔗渣为水解底物、日本ｙｓｋｕｌｔ生物化学试剂公司生产的ｏｎｏｚｕｋＲＳ型纤维纯洁酶粉进行蔗渣的酶法水解实验,考察了蔗渣中纤维素的酶解还原糖得率与反应时间、酶浓度、ｐＨ值、缓冲液种类、离子强度以及固液比的关系,结果表明：当固液比为５％（ｗ／ｖ）,酶浓度＞１．２０ｍｇ／ｍｌ时,还原糖得率随酶浓度的增加变化不显著;本实验条件下,缓冲液种类和离子强度对还原糖得率几乎没有影响;水解最适宜ｐＨ值为４．２～４．９;最佳反应温度为５０℃。     

Hormonal Effects on Sugar Release from Vine Canes

Disbudded sections were prepared from 1-year-old vine caneswhich had been collected during the winter and stored at 1 °C.Effects of indole-3yl-acetic acid, gibberellic acid, and 6-benzy-laminopurine on the rate of sugar release into effluent solutionspassed longitudinally through the sections were measured. Of the three hormones tested, indole-3yl-acetic acid was themost effective in promoting sugar release, but in all casesthe largest effects on sugar release were associated with anhormonal stimulation of cambial activity. Treatments which ledto an increased outflow of sugar also resulted in a lower extractablestarch level at the end of treatment. The results are discussed in relation to hormone effects onsugar outflow, starch dissolution, and cambial activation. Itis concluded that starch dissolution is predominantly a consequenceof hormonal influence on cambial activation.     

Enhancement of Enzymatic Adipyl-7-ADCA Hydrolysis

We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.     

Enhancement of Enzymatic Adipyl-7-ADCA Hydrolysis

We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was considerably enhanced by addition of phenylglycine amide, the side-chain donor used for cephalexin synthesis; unlike reactions carried out with free enzyme. The rate enhancing effect was not specifically related to phenylglycine amide; we found a linear relationship between the reaction rate and the buffering capacity of the added substance. These observations can be explained by a pH-gradient in the immobilised enzyme, the pH inside the particle being lower (corresponding to low enzyme activity) than outside. It was concluded that the buffer reduced the pH-gradient inside the biocatalyst, and therewith, caused the reaction rate enhancing effects. Further, chloride ions decreased the reaction rate strongly, while sodium, magnesium, sulphate, and potassium did not influence the reaction rate much. For an actual process, it is important to use a buffer that is appropriate for the reaction-pH. In that way the amount of enzyme required in a process can be reduced considerably, in our case a factor of three was found.     

酶解骨胶原多肽的抗氧化特性研究

本文主要研究了酶解法制备的骨胶原多肽的总抗氧化能力、羟自由基清除作用和抑制超氧阴离子自由基的能力.通过与谷胱甘肽(GSH)的比较发现,溶液浓度在1130～150 mg/mL时,该骨胶原多肽的总抗氧化能力为GSH的71.92%.溶液浓度在2.5～20 mg/mL时,该多肽羟自由基清除作用为谷胱甘肽的1.36倍.溶液浓度为10～150 mg/mL时,多肽抑制超氧阴离子自由基的能力低于谷胱甘肽.     

Enzymatic Hydrolysis of Esters of Alkali Labile Carotenols

Esters of alkali labile carotenols were hydrolyzed enzymatically with pig liver esterase in Tris-HCl buffer containing 10-85% methanol or acetone.

The natural acetates peridinin, fucoxanthin, 19'-butanoyloxyfucoxanthin, pyrrhoxanthin and synthetic actinioerythrol diacetate provided the corresponding carotenols in 85%, 45%, (5 + 5 + 30)%, 65% and (30 + 3)% of the recovered carotenoid with total pigment recoveries 60%, 73%, 88%, 52% and 66%, respectively.

(3RS, 3'RS)-Astaxanthin dipalmitate was converted enzymatically with lipase in low yield to the monopalmitate and free astaxanthin (5% + 6%, pigment recovery 93%) with a preferred hydrolysis of the S-ester. The (R,R:R,S:S,S) ratio of substrate dipalmitate and product astaxanthin changed from 1:2:1 to 1:5:3.     

Effect of Potassium and Nitrogen Fertilizer on Switchgrass Productivity and Nutrient Removal Rates under Two Harvest Systems on a Low Potassium Soil

Biomass demand for energy will lead to utilization of marginal, low fertility soil. Application of fertilizer to such soil may increase switchgrass (Panicum virgatum L.) biomass production. In this three-way factorial field experiment, biomass yield response to potassium (K) fertilizer (0 and 68 kg?K?ha^?1) on nitrogen (N)-sufficient and N-deficient switchgrass (0 and 135 kg?N?ha^?1) was evaluated under two harvest systems. Harvest system included harvesting once per year after frost (December) and twice per year in summer (July) at boot stage and subsequent regrowth after frost. Under the one-cut system, there was no response to N or K only (13.4 Mg?ha^?1) compared to no fertilizer (12.4 Mg?ha^?1). Switchgrass receiving both N and K (14.6 Mg?ha^?1) produced 18 % greater dry matter (DM) yield compared to no fertilizer check. Under the two-cut harvest system, N only (16.0 Mg?ha^?1) or K only (14.1 Mg?ha^?1) fertilizer produced similar DM to no fertilizer (15.1 Mg?ha^?1). Switchgrass receiving both N and K in the two-cut system (19.2 Mg?ha^?1) produced the greatest (P?<?0.05) DM yield, which was 32 % greater than switchgrass receiving both N and K in the one-cut system. Nutrient removal (biomass?×?nutrient concentration) was greatest in plots receiving both N and K, and the two-cut system had greater nutrient removal than the one-cut system. Based on these results, harvesting only once during winter months reduces nutrient removal in harvested biomass and requires less inorganic fertilizer for sustained yields from year to year compared to two-cut system.