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.     

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.     

不同方法提取杜仲中桃叶珊瑚苷等4种高活性成分的比较研究

为研究生物化学方法对杜仲中高活性成分的提取率,本文以高效液相色谱法检验样品中提取出活性成分的含量差异,比较了在相同条件下酶解法、水提法、醇提法对杜仲树皮中桃叶珊瑚苷、京尼平苷、京尼平苷酸与绿原酸这4种活性成分的提取量,并对4种活性成分的不同提取效果进行了比较研究。结果发现三种方法的提取量存在明显差异,其中桃叶珊瑚苷提取量为:酶解法22. 42μg/g,水提法8. 27μg/g,醇提法9. 13μg/g;京尼平苷提取量为:酶解法77. 89μg/g,水提法33. 19μg/g,醇提法7. 76μg/g;京尼平苷酸提取量为:酶解法110. 05μg/g,水提法36. 63μg/g,醇提法47. 40μg/g;绿原酸提取量为:酶解法345. 35μg/g,水提法118. 85μg/g,醇提法172. 04μg/g,即酶解法提取量均比水提法、醇提法高出数倍。实验表明酶解法是3种方法中提取效果最好的,且无化学污染。     

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

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

Process modeling of the lipase-catalyzed dynamic kinetic resolution of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-suprofen 2,2,2-trifluoroethyl thioester in a hollow-fiber membrane

A Candida rugosa lipase immobilized on polypropylene powder was employed as the biocatalyst for the enantioselective hydrolysis of (R, S)-suprofen 2,2,2-trifluorothioester in cyclohexane, in which trioctylamine was added as the catalyst to perform in situ racemization of the remaining (R)-thioester. A hollow-fiber membrane was also integrated with the dynamic kinetic resolution process in order to continuously extract the desired (S)-suprofen into an aqueous solution containing NaOH. A kinetic model for the whole process (operating in batch and feed-batch modes) was developed, in which enzymatic hydrolysis and deactivation, lipase activation, racemization and non-enantioselective hydrolysis of the substrate by trioctylamine, and reactive extraction of (R)- and (S)-suprofen into the aqueous phase in the membrane were considered. Theoretical predictions from the model for the time-course variations of substrate and product concentrations in each phase were compared with experimental data.     

中和剂对稀酸蒸爆玉米秸秆酶解效果的影响

以稀酸蒸爆的玉米秸秆为研究对象,考察直接水洗、Ca(OH)2、NaOH、氨水中和物料至pH 5,在固液比1∶10、酶添加量为每克纤维素14 U(滤纸酶活)的酶解条件下对纤维素转化率的影响。结果表明:水洗、Ca(OH)2、NaOH、氨水中和物料酶解72 h后,纤维素转化率分别为91.7%、80.7%、83.1%及81.7%。同时对影响纤维素酶解效率的各种因素进行了探讨。从综合成本及后续发酵过程考虑,用氨水中和稀酸蒸爆物料更适合于工业化生产。     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. I. Chemical composition,viscosity, and molecular-weight distribution of chitosan products

Ten chitosan products were manufactured from dry shrimp hulls under differing process conditions and compared to a commercially available product. Manufacturing variables tested were: alkali versus enzymatic deproteination; acid demineralization versus no treatment; air versus nitrogen atmosphere; 5 min vs. 15 min deacetylation period: and varying the particle size of the dry starting material. Deproteination by alkali of enzymatic extraction did not substantially affect the nitrogen and ash compositions of dry chitosan samples. However, the viscosity was reduced in samples deproteinated by enzymatic hydrolysis. Elimination of the demineralization step resulted in products having 31–36% ash, as expected. Some differences in viscosity were observed between deminiralized and undemineralized samples, but on important differences in the molecular-weight distribution of these samples were evident. Purging the reaction vessel with nitrogen resulted in chitosan preparations having higher viscosities and molecular-weight distributions than those prepared in an air atmosphere. The degradative effect of air became more proshrimp hulls to 1 mm prior to any treatment resulted in a chitosan product of both higher viscosity and molecular weight than when ground to either 2 or 6.4 mm. Viscosity was not always a direct indicator of molecular weight, for although the presence of colloidal particles increased the viscosity of some samples, the molecular-weight distribution after filtration was essentially the same as in other less viscous samples.     

Enhanced enzymatic hydrolysis of spruce by alkaline pretreatment at low temperature

Alkaline pretreatment of spruce at low temperature in both presence and absence of urea was studied. It was found that the enzymatic hydrolysis rate and efficiency can be significantly improved by the pretreatment. At low temperature, the pretreatment chemicals, either NaOH alone or NaOH-urea mixture solution, can slightly remove lignin, hemicelluloses, and cellulose in the lignocellulosic materials, disrupt the connections between hemicelluloses, cellulose, and lignin, and alter the structure of treated biomass to make cellulose more accessible to hydrolysis enzymes. Moreover, the wood fiber bundles could be broken down to small and loose lignocellulosic particles by the chemical treatment. Therefore, the enzymatic hydrolysis efficiency of untreated mechanical fibers can also be remarkably enhanced by NaOH or NaOH/urea solution treatment. The results indicated that, for spruce, up to 70% glucose yield could be obtained for the cold temperature pretreatment (-15 degrees C) using 7% NaOH/12% urea solution, but only 20% and 24% glucose yields were obtained at temperatures of 23 degrees C and 60 degrees C, respectively, when other conditions remained the same. The best condition for the chemical pretreatment regarding this study was 3% NaOH/12% urea, and -15 degrees C. Over 60% glucose conversion was achieved upon this condition.     

稀酸和稀碱预处理对四种不同生物质资源制备还原糖的影响

本论文探讨了不同浓度的稀H_2SO_4和稀NaOH预处理对大豆秸秆、水稻秸秆、象草和狼尾草四种不同生物质酶解制备还原糖的影响。结果表明,大豆秸秆、水稻秸秆、象草和狼尾草具有较高的纤维素和半纤维素含量,是制备还原糖的理想原料。与稀H_2SO_4预处理相比,经稀NaOH预处理后的样品表现出较好的酶解性能。通过使用4%的NaOH对大豆秸秆和狼尾草进行预处理,还原糖产量分别为145.8 mg/mL和319.2 mg/mL。此外,以1%NaOH预处理后的水稻秸秆和象草为原料,可以分别获得385.2 mg/mL和231.6 mg/mL还原糖产量。     

Ethanol production from cotton-based waste textiles

Ethanol production from cotton linter and waste of blue jeans textiles was investigated. In the best case, alkali pretreatment followed by enzymatic hydrolysis resulted in almost complete conversion of the cotton and jeans to glucose, which was then fermented by Saccharomyces cerevisiae to ethanol. If no pretreatment applied, hydrolyses of the textiles by cellulase and beta-glucosidase for 24 h followed by simultaneous saccharification and fermentation (SSF) in 4 days, resulted in 0.140-0.145 g ethanol/g textiles, which was 25-26% of the corresponding theoretical yield. A pretreatment with concentrated phosphoric acid prior to the hydrolysis improved ethanol production from the textiles up to 66% of the theoretical yield. However, the best results obtained from alkali pretreatment of the materials by NaOH. The alkaline pretreatment of cotton fibers were carried out with 0-20% NaOH at 0 degrees C, 23 degrees C and 100 degrees C, followed by enzymatic hydrolysis up to 4 days. In general, higher concentration of NaOH resulted in a better yield of the hydrolysis, whereas temperature had a reverse effect and better results were obtained at lower temperature. The best conditions for the alkali pretreatment of the cotton were obtained in this study at 12% NaOH and 0 degrees C and 3 h. In this condition, the materials with 3% solid content were enzymatically hydrolyzed at 85.1% of the theoretical yield in 24 h and 99.1% in 4 days. The alkali pretreatment of the waste textiles at these conditions and subsequent SSF resulted in 0.48 g ethanol/g pretreated textiles used.     

Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse

Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na₂SO₃ and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na₂SO₃, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l^?1 Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na₂SO₃), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %.     

Comparison between solid-state and powder-state alkali pretreatment on saccharification and fermentation for bioethanol production from rice straw

The efficacy of different concentrations of NaOH (0.25%, 0.50%, 0.75%, and 1.00%) for the pretreatment of rice straw in solid and powder state in enzymatic saccharification and fermentation for the production of bioethanol was evaluated. A greater amount of biomass was recovered through solid-state pretreatment (3.74 g) from 5 g of rice straw. The highest increase in the volume of rice straw powder as a result of swelling was observed with 1.00% NaOH pretreatment (48.07%), which was statistically identical to 0.75% NaOH pretreatment (32.31%). The surface of rice straw was disrupted by the 0.75% NaOH and 1.00% NaOH pretreated samples as observed using field-emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). In Fourier-transform infrared (FT-IR) spectra, absorbance of hydroxyl groups at 1,050 cm^?1 due to the OH group of lignin was gradually decreased with the increase of NaOH concentration. The greatest amounts of glucose and ethanol were obtained in 1.00% NaOH solid-state pretreated and powder-state hydrolyzed samples (0.804 g g^?1 and 0.379 g g^?1, respectively), which was statistically similar to the use of 0.75% NaOH (0.763 g g^?1 and 0.358 g g^?1, respectively). Thus, solid-state pretreatment with 0.75% NaOH and powder-state hydrolysis appear to be suitable for fermentation and bioethanol production from rice straw.     

Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods

Simple and cost-effective methods are needed to extract DNA in order to use it in large-scale studies. Blood is an excellent DNA source; however, it is costly and invasive thus an alternative is needed. Several kits and chemical protocols using buccal cells have been proposed for DNA extraction. The objective of the study is to evaluate buccal NaOH chemical protocol and Nucleospin Tissue Kit (BD Biosciences, Macery-Nagel, Germany) for DNA extraction. DNA swab samples were collected from 300 voluntary participants. DNA yields and purity were measured by NaOH and Nucleospin Tissue Kit techniques; the cost and time consumption for DNA extraction per sample were assessed as well. Results have shown that DNA amount and purity extracted by NaOH procedure was compared to that of the kit (p = 0.164; p = 0.249, respectively). NaOH method was considered cheaper and less time consuming (0.06 versus 3.80 USD, and 1.33 versus 3.59 minutes per sample, p < 0.001). Buccal cell derived DNA extracted by NaOH protocol can be considered a feasible substitute for more expensive and time-consuming kits.     

酶法破碎裂殖壶菌提取胞内油脂

采用酶法破碎裂殖壶菌提取胞内油脂,进行单因素实验和正交实验优化酶解反应条件,酶解反应的影响因素主次顺序依次为酶用量、温度、时间、pH,最佳酶解工艺参数:55 ℃、pH 9.5、搅拌反应2.5 h、酶用量为菌体生物量的2%.在该条件下,胞内油脂的提取量高达(81.53±0.33) g/L,过氧化值仅为0.15,酸价为0.24.     

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using gas chromatography-mass spectrometry

The aim of the presented study was to identify the metabolites of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using GC-MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4'-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC-MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.     

Isolation of residual lignin from softwood kraft pulp. Advantages of the acetic acid acidolysis method

Lignin in kraft pulp was extracted by enzymatic hydrolysis of the carbohydrates, acidolysis with dioxane-water-HCl (conventional method), and acidolysis with acetic acid-water-ZnCl2. The latter method was shown to extract lignin with a better yield than for conventional acidolysis and with a much lower content in impurities than for enzymatic hydrolysis. It was confirmed by 13C NMR analysis of the lignin samples that conventional hydrolysis modified the lignin polymer, causing the cleavage of some aryl-ether linkages. The cleavage was also observed on a model compound submitted to the same extraction conditions. In that respect, the acetic acid-water-ZnCl2 method was less damaging and consequently more suitable for analytical purposes.     

大贵寺国家森林公园野生青檀檀皮纤维成分分析

分析了湖北大贵寺国家森林公园野生青檀种群64份2～3年生青檀枝条檀皮的纤维成分.结果表明,该野生青檀枝条檀皮纤维的水分、灰分、冷水抽出物、热水抽出物等指标的平均值分别为8.267％、6.273％、24.025％、26.594％;1％NaOH抽出物、苯-醇抽出物、综纤维素、酸不溶木素等指标的平均值分别为60.904％、11.011％、56.759％、9.698％;纤维素吸光值为0.373.酸不溶木素、灰分、纤维素吸光值以及1％ NaOH抽出物4个指标呈现近正态分布,其它指标的分布偏斜度较大;水分与1％ NaOH抽出物、灰分与酸不溶木素、综纤维素与酸不溶木素之间具有显著的相关性(p＜0.05);水分与灰分、水分与热水抽出物、水分与综纤维素、苯-醇抽出物与综纤维素、苯-醇抽出物与酸不溶木素之间具有极显著的相关性(p＜0.01).平均连锁值取5.627时,可以把64个样品分为23类,说明大贵寺国家森林公园野生青檀群体檀皮纤维成分存在着较高的表型多样性,具有广阔的遗传改良前景.     

Multi-step feeding systems for lactic acid production by simultaneous saccharification and fermentation of processed wood

Eucalyptus globulus wood samples were delignified in acetic acid media and swelled with NaOH solutions in a further stage. Solid residues from treatments were used as substrates for lactic acid production by Simultaneous Saccharification and Fermentation (SSF) in media containing Trichoderma reesei cellulases and Lactobacillus delbrueckii cells. The improvements in the overall process derived from adding fresh enzymes and/or substrate during the SSF process were assessed. In order to obtain comparative data on the efficiency of substrate utilization, enzymatic hydrolysis runs (in absence of microorganisms) were also carried out. Lactic acid concentrations in the range 48-62 g/l were obtained in SSF experiments. The solid residues after SSF (made up of microbial biomass and the non-hydrolyzed fraction of substrate) were characterized for measuring their potential as feed additives.     

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO₂ chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO₂ was required in comparison to the enzymatically untreated samples.     

Multi-residue extraction–purification procedure for corticosteroids in biological samples for efficient control of their misuse in livestock production

A fast and efficient multi-residue extraction–purification procedure was developed for 12 corticosteroids in biological matrices (hair, urine and meat), in order to control their illegal use as growth promoters in cattle. Detection and identification of the analytes were achieved using a previously described LC–MS–MS method based on negative electrospray ionisation and a triple quadrupole analyser. The presented procedures included acid (hair) or enzymatic (urine and meat) hydrolysis, C₁₈ reversed-phase SPE, Na₂CO₃ liquid–liquid clean-up and SiOH normal-phase SPE. The detection limits of the developed methods were between 2.9 and 9.3 pg/mg (ppb) for hair samples and in the 40 – 70 pg/g (ppt) range for the urine or meat samples. The acid hydrolysis used for corticosteroid extraction in hair was optimised using an experimental design and response surface methodology. Achieved performances were linked to a physico–chemical approach based on the corticosteroids specific C₁₇ side-chain. This original multi-residue and multi-matrices analytical methodology will be used for further metabolism studies.