Screening of lignocellulolytic fungi for hydrolyzation of lignocellulosic materials in paddy straw for bioethanol production

Aims: Paddy straw is known to have lignocellulosic materials such as cellulose and hemicellulose which can be readily converted into fermentable sugar for production of bioethanol via simultaneous saccharification and fermentation (SSF). In order to produce ethanol competently, the degradationof biomass by cellulase and highly ethanol-producing microorganism in fermentation process are necessarily needed. However, there is lacking in cellulose degrading organism in producing adequate amount of lignocellulosic enzyme. Therefore, the screening and selection for the best fungi to hydrolyze the lignocellulosic materials as well as forming consortium between two species of fungi has become the main focus. Methodology and results:Thirteen strains of fast-growing fungi were tested qualitatively forcellulase (congo red staining) and polyphenol oxidase (Bavendamm test). All tested strains displayed lignocellulolytic fungi characteristics. The selection was narrowed down by quantitative assay on endoglucanase, exoglucanase, β-glucosidase and xylanase and the highest cellulases enzyme producer were Trichoderma asperellumB1581 (3.93 U/mL endoglucanase; 2.37 U/mL exoglucanase; 3.00 IU/mL β-glucosidase; 54.87 U/mL xylanase), followed by Aspergillus nigerB2484 (5.60 U/mL endoglucanase; 1.08 U/mL exoglucanase; 1.57 IU/mL β-glucosidase; 56.85 U/mL xylanase). In compatibility test, both T. asperellumB1581 and A. nigerB2484 were inoculated on the same Petri dish for 4 days and the interaction showed by the two species was mutual intermingling.Conclusions, significance and impact of study:Both T.asperellumB1581 and A.nigerB2484 produced the highest cellulase enzyme. Since both strainscan co-exist and produce enzymes that complete each other, a fungal consortiumwas suggested to increase the yield of sugars in saccharification process

Improvement of delignification, desilication and cellulosic content availability in paddy straw via physico-chemical pretreatments

Aim: Paddy straw consists of cellulose and hemicellulose as their plant materials leading to their potential to produce bioethanol through several processes such as pretreatment, enzymatic hydrolysis and ethanol fermentation. Among these processes, pretreatment of paddy straw is particularly important for enzymatic hydrolysis process as they are being limited by the presence of ash and silica content. This study was set to observe the effect of different pretreatments on cellulose, hemicellulose, lignin and ash content of paddy straw. Place and Duration of Study: This study was conducted in Department of Biology, Faculty of Science, Universiti Putra Malaysia, between October 2015 and June 2016. Methodology: Pretreatments comprises the combination of physical (mechanical) and chemical treatments to modify the lignocellulosic structure while reduce lignin and separate silica content in paddy straw fibre. Paddy straw was prepared into three different sizes (2mm, 5mm and 8 mm) for physical treatment. Autoclave, boiled and four different concentrations (0.5%, 1%, 2% and 5% (v/v) and (w/v) respectively) of nitric acid and sodium hydroxide, respectively for chemical treatment were used on paddy straw. Results: Size five millimeter paddy straw showed the highest cellulose content (35.61%) compared to the other sizes and when the paddy pretreated with 2% (w/v) sodium hydroxide (NaOH), the percentage of cellulose content escalated to 72.47%. Pretreatment of 2% (w/v) NaOH have performed the most efficient delignification and desilication process (1.02% lignin; 5.44 ash content); and the performance was supported with SEM images on surface area of the paddy straw with large distortion caused by the treatment. Conclusion: Therefore, a physico-chemical pretreatment of size 5 mm and 2% (w/v) NaOH was found to be the most suitable condition to break the cellulose-lignin complex and make the paddy straw becomes feasible for biofuel production

Conversion of paddy straw to bioethanol through consolidated bioprocessing using lignocellulolytic fungi

Normally, paddy straw was disposed of via open burning even though it contains valuable lignocellulosic materials which can be readily converted into fermentable sugar for bioethanol production. The second-generation of bioethanol production utilizes useful lignocellulosic substrates especially cellulose for bioconversion process. However, this material is enclosed within hemicellulose and lignin matrix in the cell wall, making the accessibility of cellulose become the major problem in bioethanol production from such sources in consolidate bioprocessing (CBP). The CBP is preferable as it produces faster saccharification result, low risk of contamination and cost-effective. Nevertheless, finding an optimize condition for efficient bioethanol production in CBP is still ambiguous as a different strain of lignocellulolytic fungi has their own environment preferences. Therefore, the main aim of this study is to explore a new approach in converting paddy straw into bioethanol using only filamentous fungi throughout the entire CBP process, thus eliminating the use of yeast as a fermenter organism. In this study, the research objectives involves the pretreatment method of paddy straw, selecting the best lignocellulolytic agent for hydrolyzation, optimizing all factors influencing the bioethanol production via one-factor-at-a-time (OFAT) as well as Response Surface Methodology analysis (RSM) and evaluating the final CBP set-up. Paddy straw sieved into three different sizes; 2 mm, 5 mm and 8 mm were prepared and underwent several physical pretreatment (autoclave, boil) and chemical pretreatment (HNO3 and NaOH). Size five millimeter paddy straw showed the highest cellulose content (35.61%) and the percentage of cellulose content went escalated to 72.47% when pretreated with 2% (w/v) sodium hydroxide (NaOH). Pretreatment of 2% (w/v) NaOH also shown the most efficient delignification and desilication process (1.02% lignin; 5.44% ash content) compared to others. All strains of fast-growing fungi were quantitatively assayed and the results indicate that the highest cellulases enzyme producer were Trichoderma asperellum B1581 (3.93 U/mL endoglucanase; 2.37 U/mL exoglucanase; 3.00 U/mL β-glucosidase; 54.87 U/mL xylanase), followed by Aspergillus niger B2484 (5.60 U/mL endoglucanase; 1.08 U/mL exoglucanase; 1.57 U/mL β-glucosidase; 56.85 U/mL xylanase). A further test on compatibility test revealed mutual intermingling between both T. asperellum B1581 and A. niger B2484. Six single factors that are crucial for bioethanol production were tested in one-factor-at-a-time (OFAT) analysis for both selected strains of lignocellulolytic fungi. With all factors combined, T. asperellum B1581 prefers 2 days of both saccharification and fermentation process at 30°C with an amount of 3% substrate level and 10% of media level. While A. niger B2482 prefers 3 days of saccharification, 1 day of fermentation; at 30°C with an amount of 2% substrate level and 20% of media level. The results produced by OFAT were used as the centre point in the Central Composite Design (CCD) through Response Surface Methodology (RSM) software. However, comparison between the actual and the predicted value of ethanol produced in RSM’s recommended CBP set-up for both T. asperellum B1581 and A. niger B2484 showed no significant difference, thus proving the model’s stability to navigate experiment. In order to test effectiveness T. asperellum B1581 and A. niger B2484 as a fungi consortium, several combination of consortia concentrations (spore/mL) were tested and the amount of ethanol was quantified. However, a single strain of T. asperellum B1581 (6:0) was able to match the amount of ethanol produced by consortia of T. asperellum B1581 and A. niger B2484 (5:1, 4:2, 3:3, 2:4 and1:5) by producing the highest total amount of ethanol (1.11 g/L). The final amount of ethanol detected by GC-FID was 1.25 g/L; which was not significantly different from the ethanol assayed spectrometrically (1.11 g/L). As a conclusion, a pretreatment of size 5 mm using 2% (w/v) NaOH had enhanced the breaking of cellulose-lignin complex, delignification, and desilication. Thus making the paddy straw becomes feasible for biofuel production. Both T. asperellum B1581 and A. niger B2484 were found to produce the highest cellulase enzyme and displayed mutual intermingling relationship suggesting the possibility of fungal consortium formation between these two species. Even though the recommended model for CBP set-up by RSM showed no significant differences between an actual and predicted value of ethanol produced, both species unable to improve the value of ethanol produced as consortia compare to single T. asperellum B1581 culture set-up. Thus, indicating that the potential of T. asperellum B1581 as single culture for bioethanol production in consolidated bioprocessing (CBP)

Improvement of bioethanol production in consolidated bioprocessing (CBP) via Consortium of Aspergillus niger B2484 and Trichoderma asperellum B1581

Consolidated bioprocessing (CBP) in bioethanol production involves the combination of four essential biological procedures in a single bioreactor, using a mixture of organisms with favourable cellulolytic ability without the addition of exogenous enzymes. However, the main disadvantage of this process is the complexity to optimise all factors considering both enzymes and microbial activity at the same time. Hence, this study aimed to optimise suitable culture conditions for both organisms to work efficiently. Six single factors that are considered crucial for bioethanol production were tested in one-factor-at-a-time (OFAT) analysis and analysed using Response Surface Methodology (RSM) software for Aspergillus niger B2484 and Trichoderma asperellum B1581 strains. The formulation of a new consortia setting was developed based on the average of two settings generated from RSM testing several combinations of consortia concentrations (5:1, 2:4, 3:3, 4:2, and 1:5). The combination of 5:1 Aspergillus niger B2484 and Trichoderma asperellum B1581 produced the most ethanol with 1.03 g/L, more than A. niger B2484, alone with 0.34 g/L of ethanol, indicating the potential of the combination of A. niger B2484 and T. asperellum B1581 co-culture for bioethanol production in CBP

Consolidated bioethanol production using Trichoderma asperellum B1581

Consolidated bioprocessing (CBP) is an alternative commercial process that combines all essential processes in a single bioreactor for the conversion of lignocellulosic biomass into ethanol. The challenge in the development of CBP is to find microorganisms with crucial properties for the utilisation of lignocellulosic materials to produce bioethanol. Also, it can be difficult to determine the optimal culture conditions for all processes to occur simultaneously. Therefore, this study focused on the potential of Trichoderma asperellum B1581 to produce ethanol and optimised the physicochemical parameters required for paddy straw waste conversion via CBP. Six parameters (days of saccharification, saccharification temperature (°C), days of fermentation, fermentation temperature (°C), medium level (%, v/v), and substrates loading (%, w/v)) were optimised in one-factor-at-a-time (OFAT) analysis via Response Surface Methodology (RSM). The numerical optimisation was statistically validated by comparing the volume of ethanol produced to the volume predicted by the RSM. T. asperellum B1581 produced 0.94 g/L bioethanol in CBP and is a more convenient, manageable and cost-effective process as all the crucial steps were performed by only one organism

A simple method for the determination of bioethanol from lignocellulosic materials using gas chromatography-flame ionisation detector (GC-FID)

Aims: The utilisation of lignocellulosic biomass for bioethanol production reduces the dependency on fossil fuels as a source of energy and emission of greenhouse gas (GHG). However, studies in this emerging field are hampered by the cost of ethanol quantification methods. Due to the volatile nature of ethanol, the method for the quantification of bioethanol production should be reproducible and rapid to avoid any evaporation loss to the surroundings. Therefore, this study aimed to develop a simple, rapid and precise bioethanol quantification method using a gas chromatography- flame ionisation detector (GC-FID) without having to go through distillation process for ethanol purification. Methodology and results: The bioethanol was produced via consolidated bioprocessing (CBP) using Trichoderma asperellum B1581 and paddy straw. The peak corresponding to ethanol was obtained at 2.347 min with a peak area of 189.66, equating to 0.159% (v/v) or 1.25 g/L ethanol. A comparison between the quantity of ethanol detected by GC-FID and spectrophotometric analysis (340 nm) showed no significant difference (p>0.05) in the amount of ethanol detected by GC analysis, thus validating the accuracy of the GC method. Conclusion, significance and impact of study: This work presents a simple, precise and reliable method to determine the amount of bioethanol in the sample using a GC-FID. Currently, there are many GC-FID methods available for the determination of ethanol/alcohol in a human blood samples or in beverages but not in bioethanol samples. Thus, this method was developed to facilitate the determination of bioethanol in the samples produced from lignocellulosic materials

Improvement of bioethanol production in Consolidated Bioprocessing (CBP) via consortium of Aspergillus niger B2484 and Trichoderma asperellum B1581

Consolidated bioprocessing (CBP) in bioethanol production involves the combination of four essential biological procedures in a single bioreactor, using a mixture of organisms with favourable cellulolytic ability without the addition of exogenous enzymes. However, the main disadvantage of this process is the complexity to optimise all factors considering both enzymes and microbial activity at the same time. Hence, this study aimed to optimise suitable culture conditions for both organisms to work efficiently. Six single factors that are considered crucial for bioethanol production were tested in one-factor-at-a-time (OFAT) analysis and analysed using Response Surface Methodology (RSM) software for Aspergillus niger B2484 and Trichoderma asperellum B1581 strains. The formulation of a new consortia setting was developed based on the average of two settings generated from RSM testing several combinations of consortia concentrations (5:1, 2:4, 3:3, 4:2, and 1:5). The combination of 5:1 Aspergillus niger B2484 and Trichoderma asperellum B1581 produced the most ethanol with 1.03 g/L, more than A. niger B2484, alone with 0.34 g/L of ethanol, indicating the potential of the combination of A. niger B2484 and T. asperellum B1581 co-culture for bioethanol production in CBP