Effect of pretreatment severity on the conversion of barley straw to fermentable substrates and the release of inhibitory compounds

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from −1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS −0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Σsugars/Σinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.     

Optimization of microwave-assisted FeCl3 pretreatment conditions of rice straw and utilization of Trichoderma viride and Bacillus pumilus for production of reducing sugars

In this study, Box-Behnken design (BBD) and response surface methodology (RSM) were used to optimize microwave-assisted FeCl₃ pretreatment conditions of rice straw with respect to FeCl₃ concentration, microwave intensity, irradiation time and substrate concentration. When rice straw was pretreated at the optimal conditions of FeCl₃ concentration, 0.14 mol/L; microwave intensity, 160 °C; irradiation time, 19 min; substrate concentration, 109 g/L; and inoculated with Trichoderma viride and Bacillus pumilus, the production of reducing sugars was 6.62 g/L. This yield was 2.9 times higher than that obtained with untreated rice straw. The microorganisms degraded 37.8% of pretreated rice straw after 72 h. The structural characteristic analyses suggest that microwave-assisted FeCl₃ pretreatment damaged the silicified waxy surface of rice straw, disrupted almost all the ether linkages between lignin and carbohydrates, and removed lignin.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

Effect of physical pretreatment on dilute acid hydrolysis of water hyacinth (Eichhornia crassipes)

Effects of different physical pretreatments on water hyacinth for dilute acid hydrolysis process (121 ± 3 °C, 5% H₂SO₄, 60 min) were comparatively investigated. Untreated sample had produced 24.69 mg sugar/g dry matter. Steaming (121 ± 3 °C) and boiling (100 ± 3 °C) for 30 min had provided 35.9% and 52.4% higher sugar yield than untreated sample, respectively. The highest sugar yield (132.96 mg sugar/g dry matter) in ultrasonication was obtained at 20 min irradiation using 100% power. The highest sugar production (155.13 mg sugar/g dry matter) was obtained from pulverized samples. Hydrolysis time was reduced when using samples pretreated by drying, mechanical comminution and ultrasonication. In most methods, prolonging the pretreatment period was ineffective and led to sugar degradations. Morphology inspection and thermal analysis had provided evidences of structure disruption that led to higher sugar recovery in hydrolysis process.     

Enhanced hydrogen production by Rhodopseudomonas palustris CQK 01 with ultra-sonication pretreatment in batch culture

In the present study, the photo-hydrogen production performances by Rhodopseudomonas palustris CQK 01 growing from the inoculated cells with ultra-sonication pretreatment (R. palustris CQK 01-USP) were experimentally investigated in batch culture and compared with that without pretreatment (R. palustris CQK 01-NP). It was found that the ultra-sonication pretreatment modified membrane morphology and broke up part of the cells, resulting in improvement of membrane permeability and bacterial activities and hence, helping the improvement of hydrogen production. The hydrogen production rate, hydrogen yield and energy conversion efficiency with R. palustris CQK 01-USP were increased to be nearly 2 times higher than that with R. palustris CQK 01-NP. The parametric study showed that under the conditions of initial glucose concentration 50 mmol/l, inoculum size 12%, illumination wavelength 590 nm, the photobioreactor with R. palustris CQK 01-USP obtained the optimal hydrogen production rate 0.54 mmol/l/h, hydrogen yield 1.2 mol-H₂/mol-glucose and energy conversion efficiency 9.03%.     

固定化纤维素酶对预处理纤维素原料水解效果的影响

以壳聚糖为载体用交联法制备固定化纤维素酶,考察固定化纤维素酶对蒸爆、球磨、超声波、喷淋、高温预处理玉米秸秆纤维素原料的酶解效果.结果表明:物料经蒸爆预处理后酶水解效率最高可以达到95%,球磨预处理水解效率次之,达到60%.用电镜和FT-IR对处理前后秸秆结构进行表征分析,证明预处理对物料的物理结构及化学组成有一定的影响.蒸爆法和球磨法可以使物料致密的天然结构彻底破坏,从而增加物料的比表面积;蒸爆预处理可以使纤维素内部氢键和官能团改变,使物料更易于酶解.     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.     

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ～1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ～85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450. © 2010 Wiley Periodicals, Inc.     

A functionally based model for hydrolysis of cellulose by fungal cellulase

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.     

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV-Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase.