Front-end recovery of protein from lignocellulosic biomass and its effects on chemical pretreatment and enzymatic saccharification

Front-end protein recovery from biomass at different maturities, and its effects on chemical pretreatment and enzyme hydrolysis of partially deproteinized fiber were investigated. The protein recovery from alfalfa and switchgrass biomass using sodium dodecyl sulfate and potassium hydroxide treatments was ~50–65 % of initial biomass protein. When hot water was used as extraction media, the protein recovery was 52.9 and 43.7 % of total protein in switchgrass and alfalfa, respectively. For any treatment, relative protein recovery was higher from switchgrass than from alfalfa. Only approximately half the total protein was recovered from relatively mature (early fall) biomass compared with midsummer harvested biomass. When protein was recovered partially using sodium dodecyl sulfate or potassium hydroxide, and leftover fiber pretreated, aqueous ammonia pretreatment removed 58.5–60.1 % of lignin and retained more cellulose in the fiber compared with acid pretreatment (nearly no lignin removal). Protein removal was helpful in the enzyme digestibility of fibers. Delignification of ammonia pretreated partially deproteinized alfalfa fiber was in the range of 34.4–45 %, while dilute sulfuric acid did not remove lignin effectively. Overall, the higher delignification and enzyme digestibilities were observed in aqueous ammonia pretreated partially deproteinized alfalfa fibers regardless of biomass type.     

Microwave‐based alkali pretreatment of switchgrass and coastal bermudagrass for bioethanol production

Switchgrass and coastal bermudagrass are promising lignocellulosic feedstocks for bioethanol production. However, pretreatment of lignocelluloses is required to improve production of fermentable sugars from enzymatic hydrolysis. Microwave‐based alkali pretreatment of switchgrass and coastal bermudagrass was investigated in this study. Pretreatments were carried out by immersing the biomass in dilute alkali reagents and exposing the slurry to microwave radiation at 250 W for residence times ranging from 5 to 20 min. Simons' stain method was used to quantify changes in biomass porosity as a result of the pretreatment. Pretreatments were evaluated based on yields of total reducing sugars, glucose, and xylose. An evaluation of different alkalis identified sodium hydroxide as the most effective alkali reagent for microwave‐based pretreatment of switchgrass and coastal bermudagrass. 82% glucose and 63% xylose yields were achieved for switchgrass and 87% glucose and 59% xylose yields were achieved for coastal bermudagrass following enzymatic hydrolysis of biomass pretreated under optimal conditions. Dielectric properties for dilute sodium hydroxide solutions were measured and compared with solid losses, lignin reduction, and reducing sugar levels in hydrolyzates. Results indicate that dielectric loss tangent of alkali solutions is a potential indicator of the severity of microwave‐based pretreatments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Simultaneous saccharification and fermentation of lime-treated biomass

Simultaneous saccharification and fermentation (SSF) was performed on lime-treated switchgrass and corn stover, and oxidatively lime-treated poplar wood to determine their compatibility with Saccharomyces cerevisiae. Cellulose-derived glucose was extensively utilized by the yeast during SSF. The ethanol yields from pretreated switchgrass, pretreated corn stover, and pretreated-and-washed poplar wood were 72%, 62% and 73% of theoretical, respectively, whereas those from -cellulose were 67 to 91% of theoretical. The lower ethanol yields from treated biomass resulted from lower cellulose digestibilities rather than inhibitors produced by the pretreatment. Oxidative lime pretreatment of poplar wood increased the ethanol yield by a factor of 5.6, from 13% (untreated) to 73% (pretreated-and-washed).     

Impact of Dilute Sulfuric Acid,Ammonium Hydroxide,and Ionic Liquid Pretreatments on the Fractionation and Characterization of Engineered Switchgrass

Improving plant characteristics for better environmental resilience and more cost-effective transformation to fuels and chemicals is one of the focus areas in biomass feedstock development. In order to bridge lignin engineering and conversion technologies, this study aimed to fractionate and characterize lignin streams from wild-type and engineered switchgrass using three different pretreatment methods, i.e., dilute sulfuric acid (DA), ammonium hydroxide (AH), and aqueous ionic liquid (IL). Results demonstrate the low lignin content and high S/G ratio switchgrass mutant (4CL) was more susceptible to pretreatment and subsequently more digestible by enzymes as compared to wild-type switchgrass and AtLOV1 mutant. In addition, when compared to DA and AH pretreatment, aqueous IL (cholinium lysinate) was demostrated to be an efficient lignin solvent, as indicated by the high (> 80%) lignin solubility and reduced lignin molecular weight. FTIR and differential scanning calorimetry measurements suggest that pretreatment chemistry greatly influenced the structural and compositional changes and thermal properties of the pretreated switchgrass and recovered lignin-rich streams. The comparative data obtained from this work deepen our understanding of how lignin modification impacts the fractionation and properties of biomass feedstocks.     

Greenhouse and field assessment of phytoremediation for petroleum contaminants in a riparian zone

Greenhouse and field studies were conducted to evaluate the feasibility of phytoremediation for clean-up of highly contaminated sediments from Indiana Harbor. In the greenhouse study, plant species evaluated were willow (Salix exigua), poplar (Populus spp.), eastern gamagrass (Tripsacum dactyloides), arrowhead (Sagitaria latifolia), switchgrass (Panicum virgatum), and sedge (Carex stricta). Sediments with sedge, switchgrass, and gamagrass had significantly less residual total petroleum hydrocarbons (TPH) after one year of growth (approximately 70% reduction) than sediments containing willow, poplar, or no plants (approximately 20% reduction). Although not all polycyclic aromatic hydrocarbons (PAH) had concentration differences due to the presence of plants, residual pyrene concentrations in the unvegetated pots were significantly higher than in pots containing sedge, switchgrass, arrowhead, and gamagrass. As evaluated by TPH dissipation in the upper section of the pots, the sedge, switchgrass, and gamagrass treatments had higher TPH degradation than the unvegetated, willow and poplar treatments. These trends were similar for soil at the bottom of the pots, with the exception that in the switchgrass treatment, degradation was not significantly different than in the unvegetated soil. Two target contaminants, pyrene and benzo[b]fluoranthene, showed differences in degradation between planted and unvegetated treatments. In the field study, phytoremediation plant species were eastern gamagrass (T. dactyloides), switchgrass (P. virgatum), and sedge (C. stricta). In addition, rhizosphere characteristics of arrowhead (S. latifolia) and sedge were assessed. Arrowhead- and sedge-impacted soils were found to contain significantly more PAH-degrading bacteria than unvegetated soils. However, over the 12-month field study, no significant differences in contamination were found between the planted and unplanted soils for TPH and PAH concentrations. TPH concentrations near the canal were greater than concentrations further from the canal, indicating that the canal may have served as a continuous source of contamination during the study.     

Soil phosphorus drawdown by perennial bioenergy cropping systems in the Midwestern US

Without fertilization, harvest of perennial bioenergy cropping systems diminishes soil nutrient stocks, yet the time course of nutrient drawdown has not often been investigated. We analyzed phosphorus (P) inputs (fertilization and atmospheric deposition) and outputs (harvest and leaching losses) over 7 years in three representative biomass crops—switchgrass (Panicum virgatum L.), miscanthus (Miscanthus × giganteus) and hybrid poplar trees (Populus nigra × P. maximowiczii)—as well as in no-till corn (maize; Zea mays L.) for comparison, all planted on former cropland in SW Michigan, USA. Only corn received P fertilizer. Corn (grain and stover), switchgrass, and miscanthus were harvested annually, while poplar was harvested after 6 years. Soil test P (STP; Bray-1 method) was measured in the upper 25 cm of soil annually. Harvest P removal was calculated from tissue P concentration and harvest yield (or annual woody biomass accrual in poplar). Leaching was estimated as total dissolved P concentration in soil solutions sampled beneath the rooting depth (1.25 m), combined with hydrological modeling. Fertilization and harvest were by far the dominant P budget terms for corn, and harvest P removal dominated the P budgets in switchgrass, miscanthus, and poplar, while atmospheric deposition and leaching losses were comparatively insignificant. Because of significant P removal by harvest, the P balances of switchgrass, miscanthus, and poplar were negative and corresponded with decreasing STP, whereas P fertilization compensated for the harvest P removal in corn, resulting in a positive P balance. Results indicate that perennial crop harvest without P fertilization removed legacy P from soils, and continued harvest will soon draw P down to limiting levels, even in soils once heavily P-fertilized. Widespread cultivation of bioenergy crops may, therefore, alter P balances in agricultural landscapes, eventually requiring P fertilization, which could be supplied by P recovery from harvested biomass.     

Assessing solid digestate from anaerobic digestion as feedstock for ethanol production

Ethanol production using solid digestate (AD fiber) from a completely stirred tank reactor (CSTR) anaerobic digester was assessed comparing to an energy crop of switchgrass, and an agricultural residue of corn stover. A complete random design was fulfilled to optimize the reaction conditions of dilute alkali pretreatment. The most effective dilute alkali pretreatment conditions for raw CSTR AD fiber were 2% sodium hydroxide, 130 °C, and 3 h. Under these pretreatment conditions, the cellulose concentration of the AD fiber was increased from 34% to 48%. Enzymatic hydrolysis of 10% (dry basis) pretreated AD fiber produced 49.8 g/L glucose, while utilizing 62.6% of the raw cellulose in the AD fiber. The ethanol fermentation on the hydrolysate had an 80.3% ethanol yield. The cellulose utilization efficiencies determined that the CSTR AD fiber was a suitable biorefining feedstock compared to switchgrass and corn stover.     

The effect of calcium and sodium hydroxides and of sodium acrylate on the fermentation and digestibility in vitro of ensiled whole-crop wheat and barley harvested at different stages of maturity

Whole-crop wheat and barley were each harvested at the soft-, medium- and hard-dough stages of grain development. Material from each harvest was ensiled in polythene bag silos without additive or after the addition of calcium hydroxide, sodium hydroxide or sodium acrylate at 50, 50 and 12.5 g kg_?1 of the crop dry matter (DM), respectively. All silages were opened after 60 days.With advancing maturity there was an increase in the content of DM, starch and insoluble-nitrogen, but a reduction in water-soluble carbohydrates (WSC) and ash.When crops were ensiled without additives, only medium-dough barley fermented to give butyric acid. However, the addition of calcium hydroxide to crops of low DM (soft-dough) and medium DM (medium-dough) promoted the activity of clostridial bacteria giving rise to the production of butyric acid, but this did not occur with crops of high DM (hard-dough). Sodium hydroxide gave rise to butyric acid only at low DM, and to restricted fermentation at high DM content. Sodium acrylate restricted fermentation and prevented butyric acid production in all silages.Ensiling led to an average reduction of 5 percentage units in the digestible organic matter (DOM) of the control silages compared to that of the crops. Addition of calcium hydroxide and sodium acrylate gave values similar to the control silages. Only sodium hydroxide consistently increased DOM, the effect becoming more marked as the crops matured. The increases over the control silages were 10, 18 and 26 units for wheat and 15, 21 and 20 units for barley at low, medium and high DM, respectively.     

Cellulosic biofuel crops alter evapotranspiration and drainage fluxes: Direct quantification using automated equilibrium tension lysimeters

An increasing number of crops are being considered as potential sources of biomass for both conventional (e.g., maize/corn) and cellulosic (e.g., switchgrass, miscanthus, and hybrid poplar) biofuels. Studies investigating the hydrologic characteristics of these crops are often conducted at either the field scale with a focus on evapotranspiration (ET) or at the plot scale where experiments generally rely on soil water storage dynamics and residual water balances. While this has led to many important insights into crop–soil water interactions under these crops, there does not appear to be any multiyear direct comparisons of the drainage fluxes under this range of biofuel crops. Furthermore, important advancements in drainage flux measurement technologies have yet to be applied to quantify hydrologic fluxes below a range of biofuel crops. Here, we use soil water content (SWC) probes and automated equilibrium tension lysimeters (AETL) to characterize detailed differences in soil water storage and drainage fluxes under conventional and cellulosic biofuel crops. The results of this study suggest that there are significant differences between subsurface water fluxes under some conventional and cellulosic biofuel crops, such as 75% greater average annual drainage and more rapid drainage accumulation under switchgrass relative to maize.     

Carbamoylethylation of guar gum

Carbamoylethylation of guar gum was carried out with acrylamide in presence of sodium hydroxide under different reaction conditions. Variables studied were concentration of sodium hydroxide, acrylamide, guar gum as well as reaction temperature and time. The nitrogen content, carboxyl content and total ether content were determined. Rheological properties of carbamoylethyl guar gum solutions showed non-Newtonian pseudoplastic behavior regardless of the %N. At a constant rate of shear, the apparent viscosity of carbamoylethyl guar gum solutions decreases with the increase in %N of the product.     

Developmental Control of Lignification in Stems of Lowland Switchgrass Variety Alamo and the Effects on Saccharification Efficiency

The switchgrass variety Alamo has been chosen for genome sequencing, genetic breeding, and genetic engineering by the US Department of Energy Joint Genome Institute (JGI) and the US Department of Energy BioEnergy Science Center. Lignin has been considered as a major obstacle for cellulosic biofuel production from switchgrass biomass. The purpose of this study was to provide baseline information on cell wall development in different parts of developing internodes of tillers of switchgrass cultivar Alamo and evaluate the effect of cell wall properties on biomass saccharification. Cell wall structure, soluble and wall-bound phenolics, and lignin content were analyzed from the top, middle, and bottom parts of internodes at different developmental stages using ultraviolet autofluorescence microscopy, histological staining methods, and high-performance liquid chromatography (HPLC). The examination of different parts of the developing internodes revealed differences in the stem structure during development, in the levels of free and well-bound phenolic compounds and lignin content, and in lignin pathway-related gene expression, indicating that the monolignol biosynthetic pathway in switchgrass is under complex spatial and temporal control. Our data clearly show that there was a strong negative correlation between overall lignin content and biomass saccharification efficiency. The ester-linked p-CA/FA ratio showed a positive correlation with lignin content and a negative correlation with sugar release. Our data provide baseline information to facilitate genetic modification of switchgrass recalcitrance traits for biofuel production.     

干旱胁迫下柳枝稷在露天矿区土壤中的种子萌发和生长特性

将能源植物用作矿区生态修复物种,对矿区的经济发展和生态环境具有重要意义。以能源植物柳枝稷为研究对象,通过盆栽试验,分析其在对照、轻度、中度以及重度干旱胁迫下柳枝稷幼苗在矿区土壤基质和非矿区土壤基质下的生长特性,并结合不同干旱胁迫下矿区土壤基质中种子萌发特征,揭示柳枝稷对干旱胁迫的响应机制和对矿区土壤的生态适应性。试验结果表明：(1)柳枝稷种子发芽总数、发芽率、发芽势以及发芽指数在轻度胁迫下达到最大值,活力指数在对照组最高,而在重度胁迫下,各项指标均达到最小值;种苗各生长指标在轻度胁迫下最小,幼苗根长和鲜重在重度胁迫下最高,芽长和芽重在其余三组胁迫下相差不大。(2)干旱胁迫使两种土壤基质下的柳枝稷株高降低,枯叶率增加,在对照、轻度胁迫和重度胁迫下能够保持叶片水分含量和分蘖数稳定;矿区土壤基质中,柳枝稷根体积在轻度胁迫下最大,中度胁迫下根长最长,重度胁迫下根数最多;根冠比随着干旱胁迫的加剧表现出先减后增的趋势,在轻度胁迫下最小,在重度胁迫下最大。(3)与非矿区土壤基质相比,柳枝稷在矿区土壤基质下根冠比更大,其余生长指标均更小;随着干旱胁迫的加剧,柳枝稷在矿区土壤基质下的株高、枯叶率、叶片相...     

Production of free fatty acids from switchgrass using recombinant Escherichia coli

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake‐flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass‐based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91–98, 2018     

Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies

Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods.     

北京地区14份柳枝稷生物量及分配差异研究

采用14份柳枝稷开展盆栽试验,研究了在北京地区条件下其生物量差异及分配规律。结果表明,低地型柳枝稷Kanlow生物量最高,其茎秆、地上部和整株生物量分别达到175.48 g/株、299.18 g/株和447.66 g/株,而高地型柳枝稷Nebraska生物量最低,其茎秆、地上部和整株生物量分别为29.86 g/株、58.08 g/株和140.51 g/株。就柳枝稷整株植株而言,Kanlow地上部生物量分配比例最高,达到63.13%,S2最低,为40.55%,Kanlow地上部营养器官生物量分配比例最高,达到48.67%,Nebraska最低,为31.88%。就柳枝稷地上部而言,Alamo、Kanlow和Trailblazer茎秆生物量分配比例及茎叶比均较高,分别为35.91%和2.75,37.09%和2.56,34.39%和2.48。起源纬度显著影响了柳枝稷的生物量及其分配,就柳枝稷整株植株而言,起源纬度与柳枝稷生物量显著负相关,与地下部生物量分配比例显著正相关,与地上部、种子和茎生物量分配比例显著负相关。就柳枝稷地上部而言,起源纬度与茎生物量分配比例及茎叶比显著负相关,与叶和鞘生物量分配比例显著正相关。生物量的差异及其分配规律反映出柳枝稷对生态环境长期适应的生殖与生长策略。本研究为柳枝稷遗传资源引种和品种选育提供了依据。     

Arabinoxylan fractionation on DEAE-cellulose chromatography influenced by protease pre-treatment

An arabinoxylan mixture was extracted with saturated barium hydroxide from a water unextractable residue of rye bran. The mixture was fractionated on an anion exchange column which was eluted with water, 0.0025 M sodium borate, 0.025 M sodium borate and 0.4 M sodium hydroxide. It was possible to fractionate the arabinoxylan mixture into fractions with different structural features. The fractionation was repeated with prior protease treatment of the arabinoxylan mixture, but most of the arabinoxylan did not bind to the column by any mechanism that the protease treatment affected, As the largest fraction was still eluted with 0.4 M sodium hydroxide. Protease treatment affected the proportion of disubstituted xylose residues (dXyl) in the water, 0.0025 M sodium borate and 0.025 M sodium borate fractions, indicating that protein associated with arabinoxylans with a high dXyl content is more liable to the protease treatment or that protein is mainly associated with these structures. This study gives indications that protein is involved in the separation mechanism of rye arabinoxylan on a DEAE–cellulose column.     

Pretreatment of corn stover and hybrid poplar by sodium hydroxide and hydrogen peroxide

Sodium hydroxide and its derivatives are used as pulping reagents, wherein the spent NaOH is recovered in salt form and reused. In this study, use of low concentration NaOH (1–5%) in pretreatment of corn stover and hybrid poplar was investigated. It was done with the understanding that NaOH can be recovered. One of the main objectives in this study is to explore the potential of H₂O₂ with NaOH for pretreatment of high lignin substrate such as hybrid poplar. Pretreatment time has not been optimized in this study but held constant at 24 h. Corn stover, after treatment with NaOH under moderate conditions, attains near quantitative glucan digestibility. On the other hand, hybrid poplar requires treatment at higher temperature and NaOH concentration to attain acceptable level of digestibility. Supplementation of hydrogen peroxide in the pretreatment significantly raises delignification and digestibility of hybrid poplar. It was also helpful in retaining the carbohydrates in the treated solids. Retention of hemicellulose after pretreatment provides a significant economic benefit as it eliminates the need for detoxifying hemicellulose sugars. As the residual xylan remaining after pretreatment is an impediment to enzymatic digestion of glucan, supplementation of xylanase has significantly increased the digestibility of glucan as well as xylan of the treated hybrid poplar. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

人胎盘组织液氨基氮含量检测方法的建立

目的：建立检测人胎盘组织液氨基氮含量的方法,为产品质量控制和生产工艺稳定性提供判断手段。方法：加入福尔马林溶液后,以麝香草酚蓝为指示剂,用NaOH滴定液滴定。结果：当福尔马林溶液为中性、NaOH滴定液浓度为0.04mol/L时,甲醛滴定氨基氮的方法可靠。结论：本方法回收率高,操作简便,可以作为检测人胎盘组织液氨基氮含量的方法。