1. “Energy Access and Energy Efficiency Towards Sustainable Energy Ecosystems for Urban, Rural, and Island Communities”

2. BIOETHANOL PRODUCTION FROM SUGARCANE MOLASSES WITH SEPARATE HYDROLYSIS AND FERMENTATION (SHF) METHOD USING Saccaromyces Cerevisiae- Pichia stipitis CONSORTIUM Audiananti M. Kartini, Yeny Dhokhikah Study Program of Environmental Engineering, Faculty of Engineering, Universitas Jember Jl. Kalimantan No. 37, Sumbersari Jember Regency, East Java 68121, Indonesia

3. 1. Introduction Sugar-making IndustriesMolasses WASTE ETHANOL PRODUCTION USING HYDROLYSIS AND FERMENTATION (SHF) METHOD

4. Aim of the research To synthesize the bio-ethanol from sugarcane molasses using Saccaromyces cerevisiae-Pichia stipitis consortium To determine the optimal condition based on yield.

5. METHODS Tools and Materials Molasses, aquades, acetic acid buffer, sodium acetate, Potato Dextrose Broth (GDP), Potato Dextrose Agar (PDA), A. niger culture, T. viride, S. cerevisiae and P stipitis. Reactor, erlenmeyer, petridish, shaker, analytic balance sheet, test tube, thermometer, indicator pH paper, spectrophotometer, gas chromatography-mass spectrometry (GC-MS).

6. Preparation A. niger and T. Viride were bred at the Microbiology Laboratory of FMIPA, University of Jember. S. cerevisiae CC 3012 has been bred in the Food And Nutrition Culture Collection (FNCC), PAU Food and Nutrition, Gajah Mada University. P. stipitis has been bred in the Laboratory of Microbiology, Department of Biology, Airlangga University. Breeding in Potato Dextrose Broth (PDB), agitation of 180 rpm shakers and incubated based on the growth velocity of ½ of Vmax (log phase) A. niger and T. viride which were 3 days, ½ of Vmax S. cerevisiae CC 3012 and P. stipitis is 24 hours. Sugarcane molasses 100g/L 200g/L REACTOR: JAR GLASS VOLUME 2L

7. Hydrolysis INOCULUM A. NIGER AND T. VIRIDE (1: 2) ARE PUT INTO THE MOLASSES SUBSTRATE THEN INCUBATED FOR 72 HOURS. MEASUREMENT OF REDUCING SUGAR LEVELS IS CARRIED OUT EVERY 24 HOURS FOR 72 HOURS OF HYDROLYSIS. PH OF THE MEDIUM WAS MEASURED ± 5 WITH THE ADDITION OF 0.2 M ACETATE BUFFER PH ± 5 THE REDUCTION SUGAR WAS TESTED USING THE NELSON SAMOGYI METHOD. INOCULUM A. NIGER AND T. VIRIDE (1: 2) ARE PUT INTO THE MOLASSES SUBSTRATE THEN INCUBATED FOR 72 HOURS. MEASUREMENT OF REDUCING SUGAR LEVELS IS CARRIED OUT EVERY 24 HOURS FOR 72 HOURS OF HYDROLYSIS. PH OF THE MEDIUM WAS MEASURED ± 5 WITH THE ADDITION OF 0.2 M ACETATE BUFFER PH ± 5 THE REDUCTION SUGAR WAS TESTED USING THE NELSON SAMOGYI METHOD. S. CEREVISIAE CC3012-PICHIA STIPITIS (2: 1). OPTICAL DENSITY (OD) OF YEAST OD 600 = 1 = 0.33 X 10^8 CFU PH OF THE MEDIUM WAS MEASURED ± 5 WITH THE ADDITION OF 0.2 M ACETATE BUFFER PH ± 5 MICROBIAL GROWTH IS OBSERVED EVERY 24 HOURS. REDUCTION SUGAR TEST WAS CARRIED OUT BY THE NELSON SAMOGYI METHOD MEASURED FOR BIOETHANOL AFTER ADDING S. CEREVISIAE CC 3012 AND P. STIPITIS, STARTING FROM 72, 96 AND 120 HOURS HOURS WITH THE GAS CHROMATOGRAPHY METHOD. S. CEREVISIAE CC3012-PICHIA STIPITIS (2: 1). OPTICAL DENSITY (OD) OF YEAST OD 600 = 1 = 0.33 X 10^8 CFU PH OF THE MEDIUM WAS MEASURED ± 5 WITH THE ADDITION OF 0.2 M ACETATE BUFFER PH ± 5 MICROBIAL GROWTH IS OBSERVED EVERY 24 HOURS. REDUCTION SUGAR TEST WAS CARRIED OUT BY THE NELSON SAMOGYI METHOD MEASURED FOR BIOETHANOL AFTER ADDING S. CEREVISIAE CC 3012 AND P. STIPITIS, STARTING FROM 72, 96 AND 120 HOURS HOURS WITH THE GAS CHROMATOGRAPHY METHOD. Fermentation

8. Results And Discussion Table 3.1 C: N: P Mollases C: N: P FOR BIOMASS FOR MICROORGANISM GROWTH IS 100: 5: 1 (%). C / N RATIO AFTER HYDROLYSIS WAS 21.41% - ideal ratio of 20-30% (Napon et al., 2013)

9. Reduction Sugar Levels in Hydrolysis Stage of SHF Figure 1. Reduction Sugar in Hydrolysis Stage of SHF. Description: M1 = 10% substrate concentration (100 g / L) M2 = 20% substrate concentration (200 gr / L) K = Control

10. Reduction Sugar Levels in Fermentation with 8% Consortium Figure 2. Reduction Sugar Levels in SHF using 8% consortium. Description: M1 = 10% substrate concentration (100 g / L) M2 = 20% substrate concentration (200 gr / L) K = Control

11. Reduction Sugar Levels in SHF with 10% Consortium Figure 3. Reduction Sugar Levels in SHF using 10% consortium. Description: M1 = 10% substrate concentration (100 g / L) M2 = 20% substrate concentration (200 gr / L) K = Control

12. Microbial Growth During Hydrolysis Figure 4. Microbial Growth During Hydrolysis with 10% and 20% Substrate. Description: TV = Trichoderma viride AN = Aspergillus niger

13. Microbial Growth During Fermentation Figure 5. Microbial Growth During Fermentation Description: M1 = 10% substrate concentration (100 g / L) M2 = 20% substrate concentration (200 gr / L)

14. Ethanol Yield in the SHF Method Figure 6. Ethanol Yield of SHF using 8% and 10% Consortium Description: M1 = 10% substrate concentration (100 g / L) M2 = 20% substrate concentration (200 gr / L) K = Control

15. RESPONSE SURFACE

16. GRAPHIC RESPONSE

17. CONTOUR PLOT

18. Conclusion Ethanol yield from sugarcane molasses using the SHF methods has the highest bioethanol concentration of 7.83% (v / v) with 10% consortium treatment and 20% substrate (200g/L) usage at 48 hours fermentation. Optimum ethanol yield using the response surface method is 6,99 % (v/v) for the 20% substrate (200g/L) using 10% consortium with 29 hours incubation time.

19. Thank you for your attention