Utilization of Trichoderma harzianum SNRS3 for sugar recovery from rice straw for acetone-butanol-ethanol production by Clostridium acetobutylicum ATCC 824

Acetone-Butanol-Ethanol ﴾ABE﴿ production from various agricultural residues has been a recent issue of interest for many scientists. However, research carried out on ABE production from rice straw is limited. Pretreatment and hydrolysis required prior to fermentation are costly. Hence, the aims of the study were to produce cellulolytic enzyme complex ﴾FPase, CMCase, and β-glucosidase﴿, and xylanolytic enzyme ﴾xylanase﴿ from untreated rice straw by local Trichoderma harzianum SNRS3 and to utilize the crude cellulolytic enzyme complex for sugar production through enzymatic hydrolysis of alkali-pretreated rice straw which was finally used as the substrate for ABE production. The use of untreated rice straw as the substrate for enzyme production by T. harzianum SNRS3 under solid state fermentation resulted in the production of cellulase and xylanase at a higher activity (FPase 6.25 U/g substrate, CMCase 111.31 U/g substrate, β-glucosidase 173.71 U/g substrate, and xylanase 433.75 U/g substrate), as compared to when alkali-pretreated rice straw was used as fermentation substrate (FPase 1.72 U/g substrate, CMCase 23.01 U/g substrate, β-glucosidase 2.18 U/g substrate, and xylanase 45.46 U/g substrate). The results of XRD analysis indicated an increase in relative crystallinity of cellulose due to the hydrolyzation and peeling of amorphous regions during pretreatment. The SEM images showed that the pretreatment process disrupted the hemicelluloses and lignin, which might have caused the changes in the structure of cellulose. According to the results of FTIR analysis, alkali pretreatment caused lignin removal and the changes in the structure of cellulose. In fact, alkali pretreatment of the substrate caused the crystallinity of cellulose to decrease. Absolute crystallinity could most impact cellulase production. However, the overall complexity of the untreated substrate might have actually induced greater enzyme production. The crude cellulase enzyme produced by T. harzianum SNRS3 via solid state fermentation was then characterized. At 60 ºC, β-glucosidase activity was still above 70% of its maximum activity and FPase and CMCase remained active almost up to 100% that could be an advantage for cellulases. FPase and CMCase retained their highest activity in the acidic region over an almost broad pH range that is considered an advantage for industrial enzymes. At room temperature, FPase almost retained 60% of its original activity at the end of week 3 of storage. Whereas, CMCase retained 60% of its original activity at the end of the 2nd week of storage at room temperature. β-glucosidase activity only decreased to above 80% of its original activity at the end of the 3rd week of storage at room temperature. Saccharification of alkali-pretreated rice straw was conducted to obtain reducing sugar for use as fermentation substrate. The use of rice straw pretreated with 2% (w/v) NaOH resulted in the production of 5.82 g/L reducing sugar after 72 h of enzymatic hydrolysis. At 5% (w/v) substrate, a reducing sugar yield of 0.6 g/g substrate was obtained that was equivalent to 60.75% saccharification. Rice straw hydrolysate containing 10 g/L glucose was then utilized as the substrate for ABE production by Clostridium acetobutylicum ATCC 824. Rice straw was shown to be a potential substrate for ABE production and a maximum total ABE of 2.73 g/L (0.82 g/L acetone, 1.62 g/L butanol, and 0.29 g/L ethanol) at 72 h was obtained. Fermentation of rice straw hydrolysate resulted in an ABE yield of 0.27 g/ g glucose. Therefore, untreated rice straw serves as a better substrate than alkali-pretreated rice straw for cellulase production and produced a higher cellulase enzyme activity for the subsequent use in saccharification for production of glucose which finally can be utilized as the substrate for ABE production. The results of this study could contribute to future research on production of cellulolytic enzymes from lignocellulosic agricultural waste including rice straw for their various industrial applications such as their use in biomass-biofuel conversion

Production and characterisation of cellulase from solid state fermentation of rice straw by Trichoderma harzianum SNRS3

Research on production and the use of cellulase and xylanase by commercial microbial strains is widely reported. However, research on production of cellulase and xylanase by local isolates of Trichoderma harzianum known as potential cellulase producers is still very limited. T. harzianum SNRS3 was used for cellulase and xylanase production from rice straw under solid state fermentation. Our study revealed that unlike Trichoderma sp. that is normally associated with low amounts of β-glucosidase, insufficient to perform an efficient hydrolysis, T. harzianum SNRS3 could be considered as a potential β-glucosidase producer, but not an efficient xylanase producer. As a result of storage of the crude cellulase at room temperature, β-glucosidase activity only decreased to above 80% of its original activity at the end of the 3rd week of storage. The crude cellulase produced by T. harzianum SNRS3 could be industrially applied as the enzyme is still highly active at 60°C and over a wide range of acidic pH

Effect of alkali pretreatment of rice straw on cellulase and xylanase production by local Trichoderma harzianum SNRS3 under solid state fermentation

Use of alkali-pretreated rice straw and untreated rice straw as substrates for enzyme production under solid-state cultivation was investigated. Cellulase produced from untreated rice straw showed higher activity of FPase, CMCase, β-glucosidase, and xylanase at 6.25 U/g substrate, 111.31 U/g substrate, 173.71 U/g substrate, and 433.75 U/g substrate respectively, as compared to 1.72 U/g substrate, 23.01 U/g substrate, 2.18 U/g substrate, and 45.46 U/g substrate for FPase, CMCase, β-glucosidase, and xylanase, respectively, when alkali-pretreated substrate was used. The results of the X-ray diffractogram analysis showed an increase in relative crystallinity of cellulose in alkali-pretreated rice straw (62.41%) compared to 50.81% in untreated rice straw. However, the crystalline structure of cellulose was partially disrupted after alkali pretreatment, resulting in a decrease in absolute crystallinity of cellulose. The higher the crystallinity of cellulose, the more cellulase production was induced. The structural changes of rice straw before and after alkali pretreatment were compared by using Scanning Electron Microscopy. Fungal mycelial growth was also observed for both untreated and alkali-pretreated substrates. The results of this study indicated that untreated rice straw is a better substrate for cellulase and xylanase production under solid-state fermentation with low environmental impact

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