Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical,Thermal, and Enzymatic (CTE) Treatment

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.     

Use of Spectroscopic and Imaging Techniques to Evaluate Pretreated Sugarcane Bagasse as a Substrate for Cellulase Production Under Solid-State Fermentation

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7 %). However, the high crystallinity (74 %) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g^?1, respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.     

Evaluation and Characterization of Forage Sorghum as Feedstock for Fermentable Sugar Production

Sorghum is a tropical grass grown primarily in semiarid and drier parts of the world, especially areas too dry for corn. Sorghum production also leaves about 58 million tons of by-products composed mainly of cellulose, hemicellulose, and lignin. The low lignin content of some forage sorghums such as brown midrib makes them more digestible for ethanol production. Successful use of biomass for biofuel production depends on not only pretreatment methods and efficient processing conditions but also physical and chemical properties of the biomass. In this study, four varieties of forage sorghum (stems and leaves) were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy and X-ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and the enzymatic hydrolysis process. Forage sorghums with a low syringyl/guaiacyl ratio in their lignin structure were easy to hydrolyze after pretreatment despite the initial lignin content. Enzymatic hydrolysis was also more effective for forage sorghums with a low crystallinity index and easily transformed crystalline cellulose to amorphous cellulose, despite initial cellulose content. Up to 72% hexose yield and 94% pentose yield were obtained using modified steam explosion with 2% sulfuric acid at 140 °C for 30 min and enzymatic hydrolysis with cellulase (15 filter per unit (FPU)/g cellulose) and β-glucosidase (50 cellobiose units (CBU)/g cellulose).     

Surfactant-Assisted Acid Pretreatment of Sugarcane Tops for Bioethanol Production

Sugarcane tops is one of the largest biomass resources in India and in tropical countries such as Brazil in terms of surplus availability. Conversion of this feedstock to ethanol requires pretreatment to make it more accessible for the enzymes used in saccharification. Though several pretreatment regimens have been developed for addressing biomass recalcitrance, very few seem to be promising as an industrial process. A novel hybrid method involving use of mild acid and surfactant was developed which could effectively remove lignin and improve the sugar yield from sugar cane tops. Operational parameters that affect the pretreatment efficiency (measured as yield of sugars) were studied and optimized. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method could yield 0.798?g of reducing sugars per gram of pretreated biomass upon enzymatic hydrolysis.     

Sulfolane pretreatment of shrub willow to improve enzymatic saccharification

Pretreatment has been regarded as the most efficient strategy for conversion of lignocellulosic biomass to fermentable sugars. In this work, sulfolane pretreatment was performed to break the intricate structure of shrub willow for inhabitation of the enzymatic accessibility to holocellulose. The effects of varying pretreatment parameters on enzymatic hydrolysis of shrub willow were investigated. It was found that sulfolane was more compatible with lignin instead of carbohydrate, and the loss of carbohydrate could be attributed to water and acid generated from sulfolane. The optimum conditions leading to maximal sugar recovery from enzymatic saccharification were confirmed. After pretreatment of shrub willow powder in sulfolane at 170 °C for 1.5 h with mass ratio of sulfolane to substrate of 5, the sugar release could reach 555 mg/g raw materials (352 mg glucose, 203 mg xylose) when combining 20 FPU cellulase, 20 CBU β-glucosidase, and 1.5 FXU xylanase, representing 78.2 % of glucose and 56.6 % of xylose in shrub willow. This enhanced enzymatic saccharification was due to delignification and removal of a proportion of hemicelluloses, as confirmed by X-ray diffraction analysis, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, gas chromatography, and ionic chromatography. Thus, these studies prove sulfolane pretreatment to be an effective and promising approach for biomass to biofuel processing.     

Inhibition Effects of Dilute-Acid Prehydrolysate of Corn Stover on Enzymatic Hydrolysis of Solka Floc

Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.     

Formic Acid as a Potential Pretreatment Agent for the Conversion of Sugarcane Bagasse to Bioethanol

In recent years, growing attention has been focused on the use of lignocellulosic biomass as a feedstock for the production of ethanol, a possible renewable alternative to fossil fuels. Several pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. In this study, effect of various organic solvents and organic acids on the pretreatment of sugarcane bagasse was studied. Among the different organic acids and organic solvents tested, formic acid was found to be effective. Optimization of process parameters for formic acid pretreatment was carried out. The structural changes before and after pretreatment was investigated by scanning electron microscopy, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The X-ray diffraction profile showed that the degree of crystallinity was more for pretreated biomass than that of untreated. The FTIR spectra shown at the stretching of hydrogen bonds of pretreated sugarcane bagasse arose at higher number. It also revealed that the cellulose content in the solid residue increased because the hemicelluloses fraction in raw materials was released by acid hydrolytic reaction.     

Ag+生物吸附的谱学研究

我们曾经研究了地衣芽孢杆菌R08和啤酒酵母废菌体吸附Pd2+以及巨大芽孢杆菌D01吸附Au3+过程的作用机理。有关乳酸杆菌A09吸附Ag+1的作用特点已有报道。本文在此基础上，进一步用谱学技术研究A09菌体吸附还原Ag+的作用机理。     

Simultaneous saccharification and cofermentation of dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fermenting Zymomonas mobilis

Simultaneous saccharification and cofermentation (SSCF) was carried out at approximately 15% total solids using conditioned dilute-acid pretreated yellow poplar feedstock, an adapted variant of National Renewable Energy Laboratory (NREL) xylose-fermenting Zymomonas mobilis and either commercial or NREL-produced cellulase enzyme preparations. In 7 d, at a cellulase loading of 12 filter paper units pergram cellulose (FPU/g), the integrated system produced more than 3% w/v ethanol and achieved 54% conversion of all potentially available biomass sugars (total sugars) entering SSCF. A control SSCF employing Sigmacell cellulose and a commercial cellulase at an enzyme loading of 14 FPU/gachieved 65% conversion of total sugars to ethanol.     

Oxidative lime pretreatment of high-lignin biomass

Lime (Ca[OH]₂) and oxygen (O₂) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150°C, 6 h, 0.1 g of Ca(OH)₂/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of −10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140°C, 3 h, 0.3 g of Ca(OH)₂/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO₂ carbonation.     

提高纤维素酶水解效率和降低水解成本

在我国可大量转化乙醇的是纤维质材料。纤维质材料转化乙醇的关键问题是纤维质转化为糖的过程,提高纤维素酶转化效率的方法有:(1)对纤维质材料进行预处理;(2)研究纤维素酶的最适作用条件;(3)纤维素酶的重复利用;(4)合理的发酵工艺等。本文分析了纤维素的结构以及纤维素酶的作用方式,总结了目前研究较多的几种纤维质材料预处理方法,及其对纤维素酶水解率的影响,并对研究纤维素酶的最适作用条件、纤维素酶的重复利用以及合理的发酵工艺进行了综述和分析。     

On-line cut-off technique and organic modifier addition aided signal enhancement for trace analysis of carbohydrates in cellulase hydrolysate by ion exclusion chromatography-electrospray ionization mass spectrometry

Paper cellulose has been hydrolyzed with calcium alginate immobilized cellulase to produce carbohydrate products and the three trace sugars, galactose, arabinose, and mannose in the cellulase hydrolysate have been analyzed by HPIEC/ESI-MS. Applying the on-line cut-off technique to the HPIEC/ESI-MS can cut the high concentration glucose off to eliminate its interference on the peaks of minor sugars and enhance their signals from 1.1- to 1.6-fold. However, the on-line post column addition of 15% ethanol to the eluate can increase the signal of the three trace sugars, galactose, arabinose, and mannose up to 17-, 23-, and 11-fold, respectively, and make the corresponding detection limits as 0.04, 0.04, and 0.03 ppm. The accuracies of the quantitative analysis for the three trace sugars with the signal enhanced HPIEC/ESI-MS by the two enhancement methods were larger than 95%. The precisions of the analytical results were also greatly improved by the assistance of the two techniques and were less than 6.5%. The quantitative analysis of the three trace sugars was performed with the internal standard method and the internal standard (IS) was sorbitol. Overall, the signal enhancement of HPIEC/ESI-MS and quantification of the three trace sugars by the on-line cut-off technique and organic modifier addition was successful.     

Shrinking-bed model for percolation process applied to dilute-acid pretreatment/hydrolysis of cellulosic biomass

For many lignocellulosic substrates, hemicellulose is biphasic upon dilute-acid hydrolysis, which led to a modified percolation process employing simulated two-stage reverse-flow. This process has been proven to attain substantially higher sugar yields and concentrations over the conventional single-stage percolation process. The dilute-acid pretreatment of biomass solubilizes the hemicellulose fraction in the solid biomass, leaving less solid biomass in the reactor and reducing the bed. Therefore, a bed-shrinking mathematic kinetic model was developed to describe the two-stage reverse-flow reactor operated for hydrolyzing biphasic substrates, including hemicellulose, in corn cob/stover mixture (CCSM). The simulation indicates that the shrinking-bed operation increases the sugar yield by about 5%, compared to the nonshrinking bed operation in which 1 reactor volume of liquid passes through the reactor (i.e.,t = 1.0). A simulated optimal run further reveals that the fast portion of hemicellulose is almost completely hydrolyzed in the first stage, and the slow portion of hemicellulose is hydrolyzed in the second stage. Under optimal conditions, the bed shrank 27% (a near-maximum value), and a sugar yield over 95% was attained.     

A further insight into the mechanism of Ag+ biosorption by Lactobacillus sp. strain A09

The mechanism of Ag(+) biosorption by resting cell of Lactobacillus sp. strain A09 has been further investigated at the molecular level using spectroscopic techniques. The values of estimated equilibrium constants, rate constants, half-life periods and apparent enthalpies of the binding reaction were calculated via the determination of Ag(+) adsorbed by the biomass using atomic absorption spectrophotometry (AAS). The reductive ratio of the Ag(+) to Ag(0) by the A09 biomass was examined by X-ray photoelectron spectroscopy (XPS). Analysis for sulfur and nitrogen atomic contents in dry powder of the biomass with EA-1110 elemental analysis (EA) showed that amino acid residues retaining the reductive property of Ag(+) to Ag(0) are very small quantity, whereas glucose content in the hydrolysates of the biomass analyzed by ultraviolet-visible spectrophotometry (UV-vis) indicated that the amount of reducing sugars in the biomass is much larger than 2.71%. The fourier transform infrared (FTIR) spectrophotometry on blank and silver-loaded biomass demonstrated that the chemical functional group such as the free aldehyde group of the hemiacetalic hydroxyl group from reducing sugars, i.e. the hydrolysates of the polysaccharides from the cell wall plays a leading role in serving as the electron donor for reducing the Ag(+) to Ag(0). This result was further supported by characterizations on the interaction of the Ag(+) with glucose using X-ray powder diffractometry (XRD) and FTIR spectroscopy.     

巨大芽孢杆菌D01吸附金(AU3+)的谱学表征

对休眠的巨大芽孢杆菌(Bacillus megatherium)D01菌体吸附Au^3 的作用过程进行了谱学表征．运用AAS考察了pH、时间和温度对D01菌体吸附Au^3 过程的化学动力学和热力学相关参数的影响．D01菌粉中硫元素含量的EDX分析说明该菌体中对Au^3 具有还原作用的L-半胱氨酸和蛋氨酸的含量极少;D01菌体水解后葡萄糖含量的UV-vis测定说明该菌体水解产物中含有一定量的还原糖,空白的和吸附Au^3 的D01菌体的FFIR检测表明该菌体细胞壁肽聚糖层糖类化合物的羟基和肽链侧链氨基酸残基离子化羧基为吸附Au^3 的活性基团;肽聚糖层部分多糖的水解产物低聚糖、二糖及单糖等还原糖的半缩醛羟基游离态醛基为电子供体,将Au^3 原位还原成Au^0．葡萄糖和Au^3 相互作用的XRD和FFIR表征证明Au^3 是在还原糖的醛基上直接被还原成Au^0．     

Oxidative Lime Pretreatment of Dacotah Switchgrass

Oxidative lime pretreatment increases the enzymatic digestibility of lignocellulosic biomass primarily by removing lignin. In this study, recommended pretreatment conditions (reaction temperature, oxygen pressure, lime loading, and time) were determined for Dacotah switchgrass. Glucan and xylan overall hydrolysis yields (72 h, 15 FPU/g raw glucan) were measured for 105 different reaction conditions involving three different reactor configurations (very short term, short term, and long term). The short-term reactor was the most productive. At the recommended pretreatment condition (120 °C, 6.89 bar O₂, 240 min), it achieved an overall glucan hydrolysis yield of 85.2 g glucan hydrolyzed/100 g raw glucan and an overall xylan yield of 50.1 g xylan hydrolyzed/100 g raw xylan. At this condition, glucan oligomers (1.80 g glucan recovered/100 g glucan in raw biomass) and xylan oligomers (25.20 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor, which compensate for low pretreatment yields.     

Alkaline and peracetic acid pretreatments of biomass for ethanol production

Prehydrolysis with dilute acid and steam explosion constitute the most promising methods for improving enzymatic digestibility of biomass for ethanol production. Despite world wide acceptance, these methods of pretreatment are quite expensive considering costs for the reactor, energy, and fractionation. Using peracetic acid is a lignin-oxidation pretreatment with low-energy input by which biomass can be treated in a silo-type system without need for expensive capitalization. Experimentally, ground hybrid poplar and sugar cane bagasse are placed in plastic bags and a peracetic acid solution is added to the biomass in different concentrations based on ovendried biomass. The ratio of solution to biomass is 6∶1 and a 7-d storage period at ambient temperature (20°C) has been used. As an auxiliary method, a series of pre-pretreatments using stoichiometri camounts of sodium hydroxide and ammonium hydroxide based on 4-methyl-glucuronic acid and acetyl content in the biomass are performed before addition of peracetic acid. The basic solutions are added to the biomass in a ratio of 14∶1 solution to biomass, and mixed for 24 h at the same ambient temperature. Biomass is filtered and washed to a neutral pH before peracetic acid addition. The aforementioned procedures give high xylan content substrates as a function of the selectivity of peracetic acid for lignin oxidation and the mild conditions of the process. Consequently, xylanase/β-glucosidase combinations were more effective than cellulase preparations in hydrolyzing these materials. The pretreatment efficiency was evaluated through enzymatic hydrolysis and simultaneous saccharification and cofermentation (SSCF) tests. Peracetic ac treatment improves enzymatic digestibility of hybrid poplar and sugar cane bagasse with no need of high temperatures. Alkaline treatments are helpful in reducing peracetic acid requirements in the pretreatment.     

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.     

Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity

The overall objective in this European Union-project is to develop cost and energy effective production systems for coproduction of bioethanol and electricity based on integrated biomass utilization. A pilot plan reactor for hydrothermal pretreatment (including weak acid hydrolysis, wet oxidation, and steam pretreatment) with a capacity of 100 kg/h was constructed and tested for pretreatment of wheat straw for ethanol production. Highest hemicellulose (C5 sugar) recovery and extraction of hemicellulose sugars was obtained at 190°C whereas highest C6 sugar yield was obtained at 200°C. Lowest toxicity of hydrolysates was observed at 190°C; however, addition of H₂O₂ improved the fermentability and sugar recoveries at the higher temperatures. The estimated total ethanol production was 223 kg/t straw assuming utilisation of both C6 and C5 during fermentation, and 0.5 g ethanol/g sugar.     

Ethanol production from steam-explosion pretreated wheat straw

Bioconversion of cereal straw to bioethanol is becoming an attractive alternative to conventional fuel ethanol production from grains. In this work, the best operational conditions for steam-explosion pretreatment of wheat straw for ethanol production by a simultaneous saccharification and fermentation process were studied, using diluted acid [H₂SO₄ 0.9% (w/w)] and water as preimpregnation agents. Acid-or water-impregnated biomass was steam-exploded at different temperatures (160–200°C) and residence times (5, 10, and 20 min). Composition of solid and filtrate obtained after pretreatment, enzymatic digestibility and ethanol production of pretreated wheat straw at different experimental conditions was analyzed. The best pretreatment conditions to obtain high conversion yield to ethanol (approx 80% of theoretical) of cellulose-rich residue after steam-explosion were 190°C and 10 min or 200°C and 5 min, in acid-impregnated straw. However, 180°C for 10 min in acid-impregnated biomass provided the highest ethanol yield referred to raw material (140 L/t wheat straw), and sugars recovery yield in the filtrate (300 g/kg wheat straw).