1. Cross-border network for knowledge transfer and innovative development in wastewater treatment WATERFRIEND HUSRB/1203/221/196 1st HUSRB Students Meeting

2. .. 1st Students Meeting Waterfriend Bioethanol production from cellulosic and hemicellulosic agricultural waste Dóra Vitay Bioengineering Bsc. student Supervisor: prof. Dr. Cecília Hodúr Co-supervisor: Marietta Ábel

3. .. 1st Students Meeting Waterfriend Objectives How tobacco can be used as a feedstock for bioethanol production Cellulose and hemicellulose enzymatic degradation: With cellulase and cellobiase mixture on different pH With xylanase Possibility of enzyme recycling Membrane separation Monitoring the activity of used and separated enzymes Tobacco plant

4. .. 1st Students Meeting Waterfriend Materials and methods Tobacco: (Nicotiana rustica, Nicotiana tabacum) Usually lives in tropic or subtropical zone, angiosperm plant 1-2 meters high, good adaptibility Used samples: Experimental  leaves + vines By-product  remaining vines after manufacturing process Grinding Fermentation in shaking water bath with cellulase (Asp. niger), cellobiase (Trichoderma reesei) and xylanase enzymes Reducing sugar analysis with 3,5-dinitrosalicylic acid (DNSA) Sample before grinding Sample after grinding

5. .. 1st Students Meeting Waterfriend Materials and methods Ultrafiltration: (Millipore) 5 KD, PES (poliethersulfone) membrane 3.5 bar Ultrasonic wave generator Reused enzyme activity inspection „filter paper” / avicell test Shaking water bathUltrafilrtation equipment with ultrasonic wave generator

6. .. 1st Students Meeting Waterfriend pH comparison tobacco [g]H 2 O [cm 3 ] cellulase [cm 3 ] cellulase [Unit] cellobiase [cm 3 ] Cellobiase [Unit] BP04500000 BP14500.466391.440.386115.8 EX04500000 EX14500.372312.480.458137.4 Circumstances: pH = 5.0 and 4.8 T = 50°C Sampling and pH setting in every 24 hours Reducing sugar analysis on every sample Experimental tobacco samples after fermentation

7. .. 1st Students Meeting Waterfriend pH comparison

8. .. 1st Students Meeting Waterfriend Results of degradation with xylanase Sample Xylanase enzym [mg] Xylanase enzym [Unit] Tobacco [g] Water [cm 3 ] 0 00 450 1 200 2 100 3 50 4 25 5 12,5 Circumstances: pH = 4.5 T = 30°C Sampling and pH setting in every 24 hours Reducing sugar analisys on every sample ( Experimental tobacco samples after fermentation Experimental tobacco samples during reducing sugar analysis

9. .. 1st Students Meeting Waterfriend Results of degradation with xylanase

10. .. 1st Students Meeting Waterfriend Results of membrane separation Circumstances: 3.5 bar 350 rpm Membrane: 5KD PES Ultrasound occasionally By product samples: feed, concentratum, permeatum Flux equation Resistance equation

11. .. 1st Students Meeting Waterfriend Results of membrane separation

12. .. 1st Students Meeting Waterfriend Results of membrane separation

13. .. 1st Students Meeting Waterfriend Results of enzyme recycling Circumstances: pH = 5.0 T = 50°C Sampling and pH setting in every 24 hours Reducing sugar analisys on every sample

14. .. 1st Students Meeting Waterfriend Conclusion The main goals of our experiments were to study the possibilities of the by-product tobacco usage As a feedstock for bioethanol production First, we used cellulase and cellobiase mixture and xylanase enzymes to hidrolyze the samples Both enzymatic ways are able to degrade the tobacco There was no significant difference between pH 4.8 and 5.0 Xylanase was more effective than cellulase-cellobiase mixture Further studies showed, that membrane separation is a possible method to regain the enzymes After separation the enzymes keeped their activity, even after the ultrasound treatment, but with lesser activity

15. .. 1st Students Meeting Waterfriend Thank you for your kind attention!

16. .. 1st Students Meeting Waterfriend Presentation/lecture has been produced with the financial assistance of the European Union. The content of the presentation/lecture is the sole responsibility of University of Novi Sad, Faculty of Technology and can under no circumstances be regarded as reflecting the position of the European Union and/or the Managing Authority. Acknowledgements Jelen eredmények megjelenését „Zöld Energia Felsőoktatási Együttműködés (ZENFE)” című, TÁMOP- 4.1.1.C-12/1/KONV-2012-0012 azonosítószámú projekt támogatja. A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. A szerzők köszönetet mondanak az Országos Tudományos Kutatási Alapprogram pénzügyi támogatásáért. A projekt azonosító száma: K 105021. I would like to thank to my supervisor prof. dr. Cecília Hodúr and my co-supervisor Marietta Ábel.