Syamsul Falah Suryani Azmi Azhari. Department of Biochemistry Faculty of Matemathics and Natural Sciences Bogor Agricultural University

Transcription

1 Bioethanol Production from Falcata (Paraserianthes falcataria) Wood by Enzymatic Delignification and Simultaneous Saccharification Fermentation using Immobilized Cells Syamsul Falah Suryani Azmi Azhari Department of Biochemistry Faculty of Matemathics and Natural Sciences Bogor Agricultural University NO GASOLINE ALTERNATIVE FUEL 1

2 SOURCES OF BIOETHANOL SUGARS Sugarcane, sugar beets, molasses, fruits STARCHY MATERIALS Corn, wheat, sorghums, cassava, potatoes, root crops LIGNOCELLULOSIC MATERIALS Bagase, Bamboo, Forestry wastes, Agricultural residues, paper mills wastes 2

3 Introduction Bioethanol is as an alternative fuel was developed primarily from lignocellulosic biomass Lignocellulosic Materials : Cellulose Hemicellulose Lignin Delignification Bioethanol Simplified flow sheet for ethanol production from lignocellulosic biomass. LIGNOCELLULOSIC MATERIALS PRETREATMENT/ DELIGNIFICATION ENZYMATIC HYDROLYSIS FERMENTATION DISTILLATION PHYSICAL METHODS ENZYMATIC METHODS CHEMICAL METHODS Simultaneous Saccharification & Fermentation BIOETHANOL 3

4 Introduction Enzymatic delignification could be used microorganism of the class Actinoycetes and Basidiomycetes Bioethanol can be produced by simultaneous saccharification fermentation (SSF) method Bioethanol was produced by enzymatic delignification followed by SSF method using Trichoderma viride, and immobilized cells of Zymomonas mobilis The aim of Study was to carry out delignification of falcata (Paraserianthes falcataria) wood using white-rotfungi Trametes versicolor and produce ethanol by simultaneous saccharification fermentation technique using Trichoderma viridae and immobilized cells of Zymomonas mobilis. 4

5 Materials Falcata wood was collected from a 4 years old of tree, obtained from private forest in Sumedang, West Java, Other materials were Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB), Nutrient Agar, Nutrient Broth, yeast extract, H 2 SO 4, NaClO 3, NaOH, CH 3 COOH, HNO 3, Na 2 SO 3, (NH 4 ) 2 PO 4, MgSO 4.7H 2 O, 3,5-dinitrosalisylate, glucose EtOH, benzene, aquades, CaCl 2, and Na-alginate. Microorganisms used in this study were Trametes versicolor, Trichoderma viridae, and Zymomonas mobilis. Falcata Wood Delignification by Trametes versicolor Falcata wood was chopped mesh 150 g sample soaked for 24 hours in distilled water Autoclaving at 121 C for 15 minutes The samples at before and after the delignification Incubated at room temperature for 20 days Inoculated with 100 ml of a suspension of T. versicolor Extractive subtance Lignin content Hemicelulose content Cellulose content 5

6 Extractive Subtances Content Weighed 2 grams of falcata woods Jidal washed with distilled water Samples transferred into beakser glass 1000 ml and 500 ml Soxhlet jidal Solvent removed by vacuum Extracted with EtOH-benzene (1:2, v/v) for 6 hs Sampled transferred to Buchner funnel Heat distilled water Added Trasnferred Buchner funnel Washed jidal with ethanol Powered samples dreind in air and stored in container Solvent removed by rotary vacuum evaporator Samples transferred into jidal Reextracted with 95% ehtanol for 4 hs Lignin Content 15 ml H 2 SO 4 72% Transferred to the Erlenmeyer 1 gram samples Stirred with temperature maintained at 20 C for 2 hours Diluted Diluted with distilled water up to a concentration of 3% Total volume of 575 ml Samples are free of acid Lignin was filltered Samples are boiled for 4 hours Samples dried at 105 o C and then weighed Washed with hot distilled water Added with distilled water 6

7 Cellulose Content 2 gram ofthe sample Sample free acid Samples dried at 105 o C and then weighed 125 ml HNO 3 added Washed with distiled water Added 100 ml CH 3 COOH 10% Heated for 12 hours at 80 C Filtered and rinsed with distiled water until colorless Added 50 ml Na 2 ClO 3 10% Filtered and washed with distiled water until colorless Air dried Sample was poored into the Erlenmeyer Heated for 2 hours at 50 C Added 125 ml of mixed solution of NaOH and Na 2 SO 3 Hemicellulose Content 2 grams holocellulose samples into flask 250 L Samples dried at 105 o C for 24 hours 10 ml 17.5 % NaOH Added at 20 o C Stirred slowly Cooled in desicator weighed to constant weight Every 5 minutes, Add 5 ml 17.5 % NaOH until total volume 25 ml Flushing continued with distilled water Added distilled water 33 ml, stirred and left for 1 hour at 20oC Sample left for 30 minutes Samples filtered by filter cup and rinsed with 100 ml 8.3% NaOH. 7

8 Cells Immobilization Gel beads made by dripping the cell suspension in Naalginate into a solution of 0.2 M CaCl 2 sterile Stiring 100 rpm for 2 hours at 4 C Filtered Transferred into a sterile Erlenmeyer Washed with sterile distilled water 3 times Bioethanol Fermentation 3 gram delignified sengon put into fermentation media 5% of isolates Trichoderma viride and 10% of liquid inoculum immobilized Zymomonas mobilis Incubation 5 days Every 0, 24,48, 72, 96, 120 hours samples were taken Analyzed the levels of reducing sugars and ethanol productivity 8

9 Measurement of Reducing Sugar Levels used DNS method Standard curve was made glucose value (0.011, 0.022, 0.033, 0.044, and 0.056%) 3 ml DNS reagent added Heated for 5 minutes Measured absorbance at 550 nm Plotted in the graph liniear 1 ml sample 3 ml DNS reagent added Heated for 5 minutes Measured absorbance at 550 nm Ethanol concentration measurement Measurement of ethanol samples immobilized and without immobilized performed using GC (Gas Chromatography) Pye Unicam PU4500. The column used was a column OV-17. The carrier gas was nitrogen. Flow rate of ml / min with FID (Flame Ionization Detector). Determination was done by comparing the retention time of the sample with a standard retention time of ethanol. Standard ethanol was injected with a concentration of 1% (v/v) 9

10 Ethanol Concentration (g/l) 23/10/2015 CHEMICAL DEGRADATION OF FALCATA WOOD BY Trametes versicolor Components Extractive Substance Pre Delignification (%) Post Delignification (%) %Total Reduction Lignin Holocellulose Cellulose Hemicellulose Results and Discussion ,91 9,96 10,06 10,4 5,49 7,37 5,87 2,02 2,62 0 1, Incubation Time (Hours) Figure 1 Ethanol production from sengon wood by T. viride and immobilized Z. mobilis cells ( ) and free cells ( ) on simoultaneous fermentation saccharification 10

11 Sugar Concentration (g/l) 23/10/2015 Results and Discussion 0, ,0004 0, ,0003 0, , Incubation Time (Hours) Figure 2 Sugar productivity from sengon wood by immobilized Z. mobilis cells ( ) and free cells ( ) on simoultaneous fermentation saccharification Conclusion Ethanol can be produced from woody biomass falcata as a results of delignification followed with the technique of simultaneous saccharification and fermentation using Trichoderma viride and immobilized cells of Zymomonas mobilis. Production of ethanol from falcata wood with immobilized Zymomonas mobilis was higher than the free cells in a simultaneous saccharification and fermentation. Falcata wood has potential as a source of biomass in the production of ethanol. However, it need to explore the other fungus which more specific to degrade lignin without losing excessive content of cellulose and hemicellulose. 11

12 THANK YOU Enzymatic fermentation of sugars 12