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Technologies for valorization ofBio-wastes
Sami SAYADI
Laboratory of Bioprocesses
Regional Excellency Pole AUFCentre of Biotechnology at Sfax, Tunisia
e-mail: [email protected]
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FeedstocksFeedstocks//solidsolidagroagro--wasteswastes
A feedstock is the general name given to materials that can be
transformed. Feedstocks can vary tremendously, but are normallyderived from living organisms and are often termed as organicmaterials.
The most common and abundant form of feedstock is lignocellulosic
wastes, (more often described as green waste or yard waste). Otherfeedstocks include household kitchen waste, commercial waste fromsupermarkets, restaurants, kitchens and food processors,
There are also industrial feedstocks such as paper pulp, some
biodegradable plastics and sewage sludge.
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ForesterForester domaindomain
SaharienSaharien domaindomain
CulturesCultures
domaindomain
BioBio--fuelfuel??
BioBio--fertilizerfertilizer??BioBio--basedbased chemicalschemicals??
--Small land areaSmall land area withwith EnergyEnergy,,
fertilizerfertilizerdeficienciesdeficiencies--BioBio--wasteswastes nonnon valorizedvalorized
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Available bioAvailable bio--waste materials as possiblewaste materials as possiblerenewable resources and biorenewable resources and bio--basedbased coumpoundscoumpounds
Agro-industrial wastes such as
Agricultural and foresterie solid wastes (pruning )
Fish conditonning wastes
Slaughterhouses wastes
Dairy wastes and whey
Olive mill wastes (Olive wastewaters, leaves, husks)
Dates wastes and Grapes husks
ssame wastes commonly used in making sweets
Sugar Beets
Grual waste (wheat) as a source of glucose syrup
Sewage sludge (more than 90 WWTP in Tunisia)
Solid wastes (9 landfills are under construction plus that of J. Chakir which is
operating since1999)
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MunicipalMunicipal SolidSolid WastesWastes
MarketsMarkets wasteswastes
AgriculturalAgricultural WastesWastes (Green(Green wasteswastes))TrimmingTrimming,, pruningpruning ofofpalmpalm andand oliveolive
treestrees,, forestforest ....
AgroAgro
foodfood
wasteswastes
(olive(olive
wasteswastes
grapegrape marcmarc
WWTPWWTP sludgesludge
FarmerFarmerwasteswastes ((manuremanure,..),..)
RefRef. PR. PR KraussKrauss Tunis, 6Tunis, 6--77 decemberdecember 20062006
BioBio--WastesWastes,, FeedstocksFeedstocks inin TunisiaTunisia
2 000 000 t/an2 000 000 t/an
100 000 t/an100 000 t/an
5 000 000 t/an5 000 000 t/an
300 000 t/an300 000 t/an
5 400 000 t/an
1 250 000 t/an1 250 000 t/an
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Technologies forTechnologies forbiobio--wastewaste treatmenttreatment andand
Valorisation (Valorisation (mostmost adaptedadaptedtoto thethe TunisianTunisian contextcontext))
A broad variety of technologies for conversion of biomassand/or waste to valuable compounds, The treatment and
uti lisation of these residues and waste for energy, recyclingand added value compounds can contribute significantly togreenhouse gas emission reduction.
Anaerobic digestion: biomethanisation
Composting
Biogaz from landfills Solid state fermentation
New molecular processes and technologies for conversion of biomass and bio-waste into added value chemicals should emerge
UNDER EXPLORATION
Fermentation of lignocellulosic material and production of ethanol (Norh part)
Fermentation of dates residues in ethnol (South part)
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OleaOlea
europaeaeuropaea
L.: olive oil treeL.: olive oil tree
Economic, cultural, historicalEconomic, cultural, historical .importance in Tunisia.importance in Tunisia
Olea europaea L., which
belongs to the Oleaceae is
considered a drought-resistantspecies because it thrives in
areas where water stress is
frequent such as arid and semi-
arid regions. It has been postulated that the
mean water requirement for
olive is 2000 m3/ha year (Ryan
and Robards, 1998).
In desert climates, olives are
frequently used as yard trees. OleaOlea europaeaeuropaea L.L.
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Schematic Diagram of Olive Oil Production Processes
Selection & WeighingSelection & Weighing
Air Suction UnitAir Suction Unit
Leaves & DustLeaves & Dust
WaterWater
WashingWashing
Washing WastewaterWashing Wastewater
CrushingCrushingWater
Water
Temperature adjustmentTemperature adjustment
Horizontal CentrifugationHorizontal Centrifugation
Vertical CentrifugationVertical Centrifugation
Solid residueSolid residue
WastewaterWastewater
Oil
Oil
Storage TankStorage Tank
Micro-FiltrationMicro-Filtration
BottlingBottling
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II--CoCo--composting of the olive processingcomposting of the olive processing
solid waste (Husks): agronomic testssolid waste (Husks): agronomic tests((potatoepotatoe))
Industrial test of co-composting olive husks (C) with chicken manure (N) Agronomic tests using
potatos were realized Contact: [email protected].
Abid N. and Sayadi S (2006). Detrimental Effects of Olive Mill Wastewater on the Composting Process
-of Agricultural Wastes. Waste Management, 26: 1099-1107.
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Olive mill effluents and leaves should be considered asOlive mill effluents and leaves should be considered as
wastes to be valorized rather than pollutantswastes to be valorized rather than pollutants
Minutes
0 5 10 15 20 25 30 35
Volts
0,00
0,02
0,04
0,06
0,08
Volts
0,00
0,02
0,04
0,06
0,08
F3
F2
F1
SEC-HPLC of polyphenols in OMWW
OH
OH
HO O
OH
HO
OO
OH
OH
O
O
O
OH
O
O OH
O O
O
Clean Technology Processes were used to produce high added value
chemicals such as hydroxytyrosol, oleuropein, polyhydroxybutyrate,
and phenolic polymers (dimmers, trimers, oligomers) of phenolic
3 to 5 Kg of antioxidants could be recovered /ton of OMW making value
addition to OMW an attractive enterprise (Food additives)
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IIII-- Production of High added valueProduction of High added value
compounds from olive mill bycompounds from olive mill by--productsproducts
Development of recovery processes: liquid-liquid extrcation,
membrane filtration, hydrolysis,
0
100
200
300
400
500
600
700
0 40 80 120
Temps (min)
Indiceperoxide(mq/kg)
Temoin 200 ppm 500 ppm
Use of recovered compounds for fats
stabilization
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Olive leaves and branches are the byproducts of
farming of the olive grove and can be found in
high amounts in the olive oil industries (10% of
the total weight of the olives) and during pruning
of olive trees
Extract and
pur compounds
IC50
[g/ml]
BHT 0 .87
Hydroxytyrosol 0.58
Enzymatic
hydrolysis extract
0.65
Oleuropein 1.19
Ethyl acetate
extract
1.25
CH3OH/H
2O
extract
1.57
IIIIII-- Highly added value products from olive leavesHighly added value products from olive leaves
and branchesand branches
Bouaziz M, Fki I, Jemai H. Ayadi M. Sayadi S. Food Chem. (2007), in press
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Bouaziz M., Bouallagui Z., Sayadi S. J. Arid Land Studies, 2006, 54, p 435-438
3,4-dihydroxyphenyl ethanol production through
enzymatic hydrolysis of olive leaf extract
Destabil isat ion of the molecule by the cleavage of the osidic bound
(Feuilles dolivier)
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Ar.HydroxylaseCH2
CH2OH
OH
CH2
CH2OH
OH
OH
3,4-di-hydroxyphnyl thanolYield 85 %
ORAC
ORAC
4-hydroxyphnyl thanol
Ex 1Ex 1-- hydroxylation ofhydroxylation of tyrosoltyrosol by resting cellsby resting cells
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18
Time (h)
Yield(%)
0,8
0,9
1
1,1
1,2
1,3
1,4
1,5
OD(600nm)
Bouallagui, Z. and Sayadi, S. J. Agric. Food Chem. (2006)
L L
OH
N. Allouche, M. Damak, R. Ellouz, and S. Sayadi :App. Environ. Microbiol., 2004, p. 21052109
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natural labelled products
avoid purification steps of intracellular enzymes
reuse of the cellular catalyst;
stabilized enzyme activities and protection against toxicity
Reaction implicating a cascade of enzyme
Cofactors (NADH, NADPH,); later on regenerated
during the metabolism
OH
OH OH
OH
OH
OH
OH
OHp - h y d r o x y p h e n y l a c e t i c
a c i d
T y r o s o l H y d r o x y t y r o s o l 3 , 4 - d i h y d r o x y p h e n y l -
a ce ti c a c i d
OH
R i n g c l e a v a g e
O
O
OH
EX.2 Hydroxylation of OMW phenolic precursors by immobilized cells
NADH,H+ + O2 NAD++H2O
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m-tyrosol (3-hydroxyphenylethanol)
and o-tyrosol (2-hydroxyphenylethanol)
were completely transformed into 3-hydroxyphenylacetic acid and 2-
hydroxyphenylacetic acid respectively
via the oxidation of the side chain
carbon of the treated substrates
4-hydroxyphenyethanol (p-tyrosol) and
4-hydroxyphenylacetic acid were
respectively bio-converted into 3,4dihydroxyphnyl thanol and 3,4-
dihydroxyphnyl acetic acid,
respectively .
ExampleExample 3: BIOCONVERSION OF 2 PRECURSORS P3: BIOCONVERSION OF 2 PRECURSORS P--TYROSOLTYROSOL
AND PAND P--HYDROXYPHENYL ACETIC ACIDHYDROXYPHENYL ACETIC ACID
-- STEREOSPECIFICITY OF ACTIONSTEREOSPECIFICITY OF ACTION
Minutes
0 10 20 30 40 50
Volts
0,0
0,5
1,0
1,5
Volts
0,0
0,5
1,0
1,5
Channel A
EX T0
EX T0
Minutes
0 10 20 30 40 50
Volts
0,0
0,2
0,4
0,6
Volts
0,0
0,2
0,4
0,6
Channel A
EX Tf
EX Tf
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Aromatic Enr ichment condi tions Isolated strains
Tyrosol
50 g/l NaCl
37 C
150 r.p.m5 mM tyrosol
12 strains
YAS1,2,3
FE1,2,3IM1,2,3
SL1,2,3
p-coumaric acid
100 g/l NaCl
37 C
150 r.p.m
5 mM p-coumaric
4 strains
IMPA, IMPB
IMPC, IMPD
Ferulic acid
100 g/l NaCl
37 C
150 r.p.m
5 mM ferulic
2 strains
MAR
MBR
Example 4: Bioconversion of aromatic structures in extreme
conditionsBy-products of olive fermentation/brine
Abdelkafi S, Sayadi S, Ben Ali Gam Z, Casalot L, Labat M (2006) FEMS Microbiol.
Lett.,
Abdelkafi S, M. Labat, M. Chamkha, Casalot L, Sayadi S (2005) FEMS Microbiol.
Lett.
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Degradation pathway of
egradation pathway of
ferulic
erulic
acid by
cid by
Halomonas
alomonas
elongatalongata strain M Rtrain M R
Metabolis of ferulic acid
Oxidation C3-aliphatic chain
0
20
40
60
80
100
1 2 3 4 5
Repeated time of biotransformation
Yieldofv
anillicacid(%
)
Accumulation of vanillic acid by resting cells
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Ex 5: Degradation of tyrosol by the use of catalyst (Al-
Fe)PILC in the presence of UV and Hydrogenperoxide
0
20
40
60
80
100
0 5 10 15 20 25
Temps (h)
Conversiondutyrosol(%
UV/H2O2/(Al-Fe)PILC
UV/H2O2
H2O2/(Al-Fe)PILC
Conditions [tyrosol] = 500 ppm
[H2O2] = 2.10-2
M[Catalyst] = 500 ppm
= 254 nm
Najjar W., Azabou S., Sayadi S. and Ghorbel A. Appl. Cat: B Env. (2007), 74:11-18
Azabou S., Najjar W., Gargoubi A., Ghorbel A. and Sayadi S. App. Cat. B Env (2007), 77:166-174
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CH2OH
HO
Tyrosol
CH 2OH
HO
HO
Hydroxytyrosol
0
0,1
0,2
0,3
0,4
0,5
0,6
0 5 10 15 20 25 30
Time(h)
tyrosolHydroxytyrosol3,4-DHPA
t = 0 h
t = 4 h
Ex 5Ex 5-- hydroxylation ofhydroxylation of tyrosoltyrosol by (Alby (Al--Fe)PILCFe)PILC
Azabou S., Najjar W., Ghorbel A. and Sayadi S. J.Agric. Food. Chem(2007), 55:4877-4882.
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Ex: 6 Enzymatic synthesis of lipophilic
compound from p-hydroxyphenyl ethanol
The hydrophilic character of some polyphenols limits their application
Synthesis of lipophilic derived products
Lipase;
acylant;40C
Aissa I., Bouaziz M., Ghamgui H., Kamoun A.,Miled N.,Sayadi S.,Gargouri Y.
J. Agric. Food Chem.2007, 55,p 1029810305
monoacetylated compound (+95%)
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Peroxide values of refined husk oils stored at 50C and supplemented
with: (x) BHT at 200 ppm, () OMW extract at 200 ppm, () OMW
extract at 500 ppm, () hydroxytyrosol at 200 ppm, () control.
I. FKI, N. ALLOUCHE AND S. SAYADI,Food Chem., 2005, 93:197-204.
Some activities of the obtained antioxidants
Refined husk oil stabilization
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Peroxide values of refined
olive oils stored at 50C and
supplemented with: (x) BHT
at 200 ppm, () OMW extract
at 200 ppm, (
) OMWextract at 500 ppm, ()
hydroxytyrosol at 200 ppm,
() control.
The oxidation of refined husk and olive oils stored at 50 after the addition of
OMWW extract was measured by peroxide values (PV) and conjugated diene
formation CD (data not shown).
Both parameters CD and PV measure the primary product of lipid oxidation.
Oils treated with purified hydroxytyrosol and BHT exhibited the lowest
peroxide values as compared with the control sample.
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0
1,5
3
1 2 3 4 5 6
Treatment
AI
Effect of HCD and OMW phenolic extracts on
serum lipids levels in rats
HCD diet increase of AI compared with the rats fed normal
diet.
HCD diet + MeOH extracts, purified compounds and OMW
decrease of AI.
I. FKI, M. BOUAZIZ, Z. SAHNOUN AND S. SAYADI, 2005,Bioorganic and Medicinal Chem., 13: 5362 5370.
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ABTS
Poly R-478
RBBRNegative strain Positive strain
Precoce (24h)
Amongst 400 fungal strains isolated
on solide medium, 67 strains areable to oxidyze ABTS and
decolorize the chromogenes
Screening from local biotopes of fungal strains producing
laccases and peroxidases on solid medium + chromogene
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N CBS LiP MnP Lac
Collection Strain (U/l) (U/l) (U/l)
CTM 10125 Phlebia Sp - 182 (8) 19 (7)
CTM 10133 Oxyporus latemarginatus 4 (7) 108 (8) -
CTM 10136 Oxyporus latemarginatus 6,5 (2) 202 (11) -
CTM 10154 Trametes trogii 25 (5) 67,2 (6) 8990 (10)
CTM 10155 Polyporus Sp 35 (4) 96,7 (2) 7393 (10)
CTM 10156 Trametes trogii 5,5 (1) 56,5 (7) 9956 (7)CTM 10313 Stereum annosum - - 97 (9)
CTM 10476 Trichoderma atroviride - - 9005 (6)
Phylogenic characterization of novel strains able to produce Lac, MnP
and/or LiP
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0
10
20
30
40
50
60
0 1 2 3 4 5 6 7 8 9 10
0
5000
10000
15000
20000
25000
30000Residual BOD5
Re sidual COD
Biomass
Laccase activity
Time (days)Biomass;Residu
alBOD
5;ResidualC
OD
(gl-1)
Laccaseactivity(U
l-1)
Time course of growth and laccases production by Trametes trogii DSM 17786cultured in 7-litres bioreactor on OMW:water (80:20)-based media supplemented with urea
(2 g L-1) and Residual BOD5
and COD concentrations.
Chakroun H., Sayadi S., Machichi T and Dhouib H. Journal of chemical technology
and biotechnology (2009), in press.
Laccases production on OMW in 7litres fermenter by Trametes trogii
for further polymerization/oxidation reactions
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-- LaccaseLaccase mediatedmediatedpolymerspolymers synthesissynthesis laccases catalyze the oxidative coupling of phenolic
compounds.
Formation of polymerized products ranging from dimers topentamers (NMR), during the oxidation of several olivephenolics by T. togii laccases.
These proposed procedures are alternative approaches toobtain valuable chemicals such polymers of catechol andtyrosol in an environmentally friendly way which could
encourage their use in medical and food industries .
Minutes0 5 10 15 20 25 30 35 40 45 50
Volts
0,0
0,5
1,0
Detector A (280nm)
10 0,5h
10 0,5h
Minutes
0 5 10 15 20 25 30 35 40 45 50
Volts
0,0
0,2
0,4
0,6
Detector A (280nm)
15 18h
15 18h
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0
20
40
60
80
100
0 2 4 6 8
Inhibitionof
bioluminescenc
e
ofVibriofisheriIB(%)
Time
Fraction S+ LaccaseFraction S+ Laccase ofofTrametesTrametes
versdicolorversdicolor
AfterAfter30 min30 min
2,5 g/l of phenolic monomers polymerized
Formation of insoluble matter
Minutes0 10 20 30 40 50
Volts
0.0
0.2
0.4
0.6
0.8
Volts
0.0
0.2
0.4
0.6
0.8
Channel ALaccase 28h119Laccase 20028h123
HPLC after enzymatic reaction
Polymres
Laccases for the synthesis of polymers fromolive phenolic wastes
Minutes
0 10 20 30 40 50
Volts
0.0
0.5
1.0
1.5
Volts
0.0
0.5
1.0
1.5Channel A
Laccase To' 119
Laccase To' 119
1
7 9
2 3 4 5
6 810
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Thank you for your
attention
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