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Bioethanol production from cellulosic and hemicellulosic agricultural waste
Dóra VitayBioengineering Bsc. student
Supervisor: prof. Dr. Cecília HodúrCo-supervisor: Marietta Ábel
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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
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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
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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 bath Ultrafilrtation equipment with ultrasonic wave generator
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pH comparisontobacco [g] H2O [cm3] cellulase
[cm3] cellulase [Unit] cellobiase [cm3]
Cellobiase [Unit]
BP0 4 50 0 0 0 0BP1 4 50 0.466 391.44 0.386 115.8EX0 4 50 0 0 0 0EX1 4 50 0.372 312.48 0.458 137.4
• 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
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pH comparison
24 48 72 96
-0.050
0.000
0.050
0.100
0.150
0.200
0.250
0.300Experimental pH=5.0 Experimental pH=4.8 By-product pH=5.0By-product pH=4.8
Fermentation time [h]
Suga
r coc
nent
ratio
n [m
g/cm
3Uni
t]
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Results of degradation with xylanaseSample
Xylanase enzym [mg]
Xylanase enzym [Unit]
Tobacco [g]
Water [cm3]
0 0 0
4 50
1 200 200
2 100 100
3 50 50
4 25 25
5 12,5 12,5
Circumstances:• pH = 4.5• T = 30°CSampling and pH setting in every 24 hoursReducing sugar analisys on every sample (
Experimental tobacco samples after fermentation
Experimental tobacco samples during reducing sugar analysis
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Results of degradation with xylanase
24 48 72 960.000
0.200
0.400
0.600
0.800
1.000
1.200
EX1EX2EX3EX4EX5
Fermentation time [h]
Spec
ific
suga
r con
cent
ratio
n [m
g/cm
3Uni
t]
24 48 72 960.000
0.200
0.400
0.600
0.800
1.000
1.200
BP1BP2BP3BP4BP5
Fermentation time [h]
Spec
ific
suga
r con
cent
ratio
n [m
g/cm
3Uni
t]
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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
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Results of membrane separation
0 500 1000 1500 2000 250010.000
15.000
20.000
25.000
30.000
35.000
40.000
45.000
50.000
ExperimentalBy-productExperimental + USBy-product + US
Time [s]
J [L/
m2
h]
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Results of membrane separation
RGRF
RM
0.0E+005.0E+121.0E+131.5E+132.0E+132.5E+13
By-product
Experimental
Experimental + US
By-product + USRe
sist
ance
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Results of enzyme recycling
0 240.000
0.500
1.000
1.500
2.000
2.500
f(x) = 0.067635784971823 ln(x) + 0.294056703447632
f(x) = 0.150499357223816 ln(x) + 1.84252082693657
f(x) = 0.017688843782107 ln(x) + 0.0264798921287508
f(x) = 0.116282035373111 ln(x) + 0.932924082372535
ExperimentalLogarithmic (Experimental)By-productLogarithmic (By-product)Experimental + USLogarithmic (Experimental + US)time [h]
mg
sug
ar/g
dry
mas
s
• Circumstances: • pH = 5.0• T = 50°C
• Sampling and pH setting in every 24 hours
• Reducing sugar analisys on every sample
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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
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Thank you for your kind attention!
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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.
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