Technologies for Valorization of Bio-wastes_pptn.

8/10/2019 Technologies for Valorization of Bio-wastes_pptn.

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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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Technologies for Valorization of Bio-wastes_pptn.

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