The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 ºC to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2 3 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil. Novozymes ( FAPESP )Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

<p>Abstract</p> <p>Background</p> <p>The description of new hydrolytic enzymes is an important step in the development of techniques which use lignocellulosic materials as a starting point for fuel production. Sugarcane bagasse, which is subjected to pre-treatment, hydrolysis and fermentation for the production of ethanol in several test refineries, is the most promising source of raw material for the production of second generation renewable fuels in Brazil. One problem when screening hydrolytic activities is that the activity against commercial substrates, such as carboxymethylcellulose, does not always correspond to the activity against the natural lignocellulosic material. Besides that, the macroscopic characteristics of the raw material, such as insolubility and heterogeneity, hinder its use for high throughput screenings.</p> <p>Results</p> <p>In this paper, we present the preparation of a colloidal suspension of particles obtained from sugarcane bagasse, with minimal chemical change in the lignocellulosic material, and demonstrate its use for high throughput assays of hydrolases using Brazilian termites as the screened organisms.</p> <p>Conclusions</p> <p>Important differences between the use of the natural substrate and commercial cellulase substrates, such as carboxymethylcellulose or crystalline cellulose, were observed. This suggests that wood feeding termites, in contrast to litter feeding termites, might not be the best source for enzymes that degrade sugarcane biomass.</p

Conversion of rice straw to bio-based chemicals: an integrated process using Lactobacillus brevis

Commercialization of lignocellulosic biomass as a feedstock for bio-based chemical production is problematic due to the high processing costs of pretreatment and saccharifying enzymes combined with low product yields. Such low product yield can be attributed, in large part, to the incomplete utilization of the various carbohydrate sugars found in the lignocellulosic biomass. In this study, we demonstrate that Lactobacillus brevis is able to simultaneously metabolize all fermentable carbohydrates in acid pre-processed rice straw hydrolysate, thereby allowing complete utilization of all released sugars. Inhibitors present in rice straw hydrolysate did not affect lactic acid production. Moreover, the activity of exogenously added cellulases was not reduced in the presence of growing cultures of L. brevis. These factors enabled the use of L. brevis in a process termed simultaneous saccharification and mixed sugar fermentation (SSMSF). In SSMSF with L. brevis, sugars present in rice straw hydrolysate were completely utilized while the cellulase maintained its maximum activity due to the lack of feedback inhibition from glucose and/or cellobiose. By comparison to a sequential hydrolysis and fermentation process, SSMSF reduced operation time and the amount of cellulase enzyme necessary to produce the same amount of lactic acid

Biological and Non-Biological Methods for Lignocellulosic Biomass Deconstruction

Owing to their abundance and cost-effectiveness, lignocellulosic materials have attracted increasing attention in clean energy technologies over the last decade. However, the complex polymer structure in these residues makes it difficult to extract the fermentable sugars. Therefore, various pretreatment regimes have been used resulting in the breaking of lignocelluloses’ physical and chemical structures, thereby enhancing the availability of the polysaccharides which are subsequently hydrolysed into different biocommodities. This chapter provides an evaluation of some of the latest exploited methodologies that are used in the pretreatment of lignocellulosic materials. Moreover, the chapter discusses the advantages and disadvantages of each method

Response of cellulase activity in pH-controlled cultures of the filamentous fungus Acremonium cellulolyticus

Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and beta-glucosidase. Avicelase and beta-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5-6. The CMCase activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity defined by filter paper unit were high, but at a neutral pH, the beta-glucosidase ratio was high. The pH 6.0 culture showed the highest cellulase activity within the range of pH 3.5-6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80% after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is a step toward the establishment of an efficient system to reutilize cellulolytic biomass.publishe

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production

Background: Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Results: Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme. Conclusion: Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification