apostolis koutinas, mary alexandri, chrysanthi pateraki

Valorisation of pulp and

paper industry waste streams for the production of

succinic acid

Department of Food Science and Human Nutrition

Agricultural University of Athens

Greece

Apostolis Koutinas, Mary Alexandri, Chrysanthi Pateraki,

Harris Papapostolou, Anestis Vlysidis

IWWATV, 21st – 23rd May 2015

Biorefinery development based on renewable resources

Valorisation ofrenewableresources

Industrial wastes and by‐product streams

Biorefinerydevelopment

Added‐valueproducts

Food waste and by‐products

Food

Feed

Antioxidants Chemicals

Biofuels

Biopolymers

HeatBiomaterials

White biotechnology

Bioprocess / biorefinery engineering

Bioprocess / biorefinery design

Βioprocess optimisation

Agricultural crops and residues


Worldwide production quantity of bleached sulphite pulp in the last decade

Global annual production of bleached sulphite pulp: 3.6 Mt/yr (FAO, 2012) 14% increase since 2009

− Annual production in the USA: 1 Mt/yr (FAO, 2012) 21% increase since 2009

− Annual production in South America: 0.21 Mt/yr (FAO, 2012) 74% increase since 2009

− Annual production in the European region: 2 Mt/yr (FAO,2012) 0,01% increase since 2009


SSL Characterisation

Value St Dev

pH 2.7

Density (g/mL) 1.277 0.007

Viscosity (cP) 552 167

Dry Matter (g-DM/L) 816.5 0.6

Lignosulphonates (g/L) 458.8 2.7

Ash % (g/g-DM) 8.62 0.55

Phenolics % (g/g-DM) 1.55 0.04

Carbohydrates (g/L) 176.41

Xylose (g/L) 128.08 0.59

Galactose (g/L) 21.47 5.50

Glucose (g/L) 19.27 0.39

Mannose (g/L) 7.41 1.30

Arabinose (g/L) 0.18 0.05

Acetic Acid (g/L) 6.91 0.49

SSL Production in Pulp and Paper Industry

BLOW TANK

WOOD CHIPS

WATER

CELLULOSE FIBRES

THIN LIQUOR

1 2 3 4 5 6 7

THICK LIQUOR

COOKING CHEMICALSSO2/MeHSO3

(Me: Ca, Mg, Na, NH4)

SSL

DIGESTER WASHER

SO2RECOVERY

MULTIPLE EVAPORATION PROCESSES

Sugars are destroyed during precipitation after the addition of calcium or sodium hydroxide

Current usesPlasticizers for concrete production among others

+NaOH+Ca(OH)2


Formation of Sugars & Inhibitors During the Process

LigninPhenolic

compounds

Lignosulphonates

Cellulose

Hemicellulose

glucose

xylose

arabinose

galactose

mannose

Furfural

5‐HMF

Acetic acid

Formic acid

Levulinic acid


Bioprocess optimisationTechno-economic evaluation

Life Cycle Analysis

Integrated biorefinery based on current pulp and paper mills

SSLFermentation

Succinic acid productionSuccinic acid Separation

and PurificationPolybutylene

succinate

Phenolic Extract

Phenolic Extraction with Ethyl acetate

Ultrafiltration

N2

LS

Fireproof biopolymers


Succinic acid applications

Plasticizers

PolyesterPolyols • Among top‐10 most promising

platform chemicals

• Can be produced via bioprocesses

• Utilisation of renewable resources

Allied market research, 2013


Pyruvate Lactate

Formate

Ac-coA

EthanolAcetate

Glucose 6P

Fructose 6P

Fructose 1,6 BP

Glyceraldheyde3P

Glycerate 2P

Phosphoenolpyru vate

Ribulose5P

Sedoheptulose 7P Ribose 5P

Erythrose 4P

Xylulose5P

AcP Ald

Galactose

Gal1P

XyloseXylXylu

RiboseRib

Fructose F1PDHAP

Sorbitol

Mannose

Mannitol MOH1P

Man6P

SOH6P

Sucrose

β -Glucoside

Maltose

LactoseGluconate

Idonate

Arabinose

Glycerol

5-Ketogluconate

Glucarate

Galactarate

Ascorbate

Glucose

PectinPecKDPG

SucCoA

Isocitrate

Citrate

Malate

Fumarate

Succinate

α-Ketoglutarate

Oxaloacetate

Gly

S6P

Frc

βG6P

Lac Gal

5K4DG

Gt6PA6P

5KGGte AraRibu

Ido

Glucose1P

Glucose

Mal

Glcte

Galte

membrane

PTS Facillitated transporter ATP dependent transporter Antiporter

CO2


0 10 20 30 40 50 60 70 800

10

20

30

40

50

60

70

80 Initial sugar concentration (g/L) Consumed sugars (g/L) Final succinic acid concentration (g/L)

Tota

l sug

ars,

suc

cini

c ac

id (g

/L)

Initial sugar concentration (g/L)

0 10 20 30 40 50 60 70 800,0

0,2

0,4

0,6

0,8

1,0Productivity (g/L/h)Yield (g/g)

Prod

uctiv

ity (g

/L/h

), Yi

eld

(g/g

)

Initial sugar concentration (g/L)

Initial substrate tolerance and initial product inhibition

Actinobacillus succinogenes


0 40 80 120 160 200 2400,00

0,05

0,10

0,15Sp

ecifi

c gr

owth

rate

(h-)

Initial concentration (g/L)

0 40 80 120 160 200 2400,00

0,05

0,10

0,15

Spec

ific

grow

th ra

te (h

-)


0 40 80 120 160 200 2400,00

0,05

0,10

0,15

Spec

ific

grow

th ra

te (h

-)


0 20 40 60 800,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 20 40 60 800,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 20 40 60 800,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 5 10 15 200,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 5 10 15 200,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 5 10 15 200,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


A. su

ccinog

enes 130

ZA. su

ccinog

enes CCU

G 334

3B. su

cciniciprodu

cens JF

401

6

Initial substrate Initial succinate Initial formate

65 g/L

58 g/L

69 g/L

23 g/L

21 g/L

25 g/L


0 10 20 30 40 500,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 40 500,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 40 500,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 40 50 600,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 40 50 600,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 40 50 600,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 400,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 400,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


0 10 20 30 400,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


A. su

ccinog

enes 130

ZA. su

ccinog

enes CCU

G 334

3B. su

cciniciprodu

cens JF

4016

Initial acetate Initial lactate Initial mixed acids

51 g/L

72 g/L

48 g/L

75 g/L

54 g/L

62 g/L

33 g/L

19 g/L

35 g/L


0

5

10

15

20

0 20 40 60 80 100 120

Fermentation with LS additionFermentation with SSL

Succ

inic

Aci

d C

once

ntra

tion

(g/L

)

LS Concentration (g/L)

Actinobacillus succinogenes

Basfia succiniciproducens

Effect of SSL and extracted lignosulphonate (LS) concentrationon succinic acid production

0

5

10

15

20

0 20 40 60 80 100 120

Fermentation with LS additionFermentation with SSL

Succ

inic

Aci

d C

once

ntra

tion

(g/L

)

LS Concentration (g/L)

Inhibitionbegins

Inhibitionbegins


Pretreatment of SSL

SSLUltrafiltration

N2

Immobilisation on alginate beads


Fermentations



0

1

2

3

4

0

10

20

30

40

0 20 40 60 80

DCW

(g/L)

Concen

tration (g/L)

Time (h)

Total sugarsSuccinic acid

Fed-batch fermentations in bench-top bioreactorB.

succ

inic

ipro

duce

nsPu

re su

gars

Filtr

ated

SSL

0

5

10

15

20

0 20 40 60 80

Concen

tration (g/L)

Time (h)

Lactic acid Formic acid Acetic acid

0

10

20

30

40

0 20 40 60 80

Concen

tration (g/L)

Time (h)

Total sugars

Succinic acid

0

5

10

15

20

0 20 40 60 80

Concen

tration (g/L)

Time (h)

Lactic acid Formic acid Acetic acid

Final SA concentration: 34.3 g/L at 58 h

Yield: 0.54 g/g at 58 hProductivity: 0.57 g/L/h


Yield: 0.54 g/g at 58 hProductivity: 0.57 g/L/h


Yield: 0.59 g/g at 68 hProductivity: 0.46 g/L/h at 56 h


Yield: 0.59 g/g at 68 hProductivity: 0.46 g/L/h at 56 h

Puresugars

Filtrated SSL with 3 kDa

MWCO

LA:SA Ratio FA:SA Ratio AA:SA Ratio Total by‐product:SARatio

Pure sugars 0.13 0.15 0.15 0.43

Filtrated SSL 0.14 0.09 0.20 0.43

0 10 20 30 400,00

0,05

0,10

Spec

ific

grow

th ra

te (h

-)


35 g/L31,5


Pretreatment of SSL

SSLUltrafiltration

N2


FermentationsImmobilisation on alginate beads



• Extraction in 4 different pH values (SSL-to-solvent ratio 1:3 (v/v), 30 min extraction time)pH =1pH = 2pH = 3.4pH = 5

Optimisation of solvent extraction - Effect of pH

0

1

2

3

4

5

6

7

pH=1 pH=2 pH=3,4 pH=5

TPC

C (g GAE/L)

Folin-Ciocalteu- TPC

DPPH - AAI

Poor antioxidant activity : AAI < 0.5 Moderate antioxidant activity : 0.5 < AAI < 1.0 Strong antioxidant activity : 1.0 < AAI < 2.0 Very strong antioxidant activity : AAI > 2.0

8,4

8,6

8,8

9

9,2

9,4

9,6

9,8

10

10,2

pH=1 pH=2 pH=3.4 pH=5

AAI


Filtrated-Extracted SSL - Batch Fermentations

0

5

10

15

20

25

30

0 10 20 30 40 50 60

Concen

tration (g/L)

Time (h)


0

5

10

15

20

25

30

0 10 20 30 40 50 60

Concen

tration (g/L)

Time (h)


0

5

10

15

20

25

30

0 10 20 30 40 50 60

Concen

tration (g/L)

Time (h)

Lactic acidFormic acidAcetic acid

0

5

10

15

20

25

30

0 10 20 30 40 50 60

Concen

tration (g/L)

Time (h)

Lactic acidFormic acidAcetic acid

18.2 g/L SASA yield 0.71 g/g

28 hProductivity 0.65 g/L/h

LA:SA = 0.07FA:SA = 0.15AA:SA = 0.21

14.2 g/L SASA yield 0.60 g/g

49 hProductivity 0.29 g/L/h

FA:SA = 0.27AA:SA = 0.28

Basfia succiniciproducens Actinobacillus succinogenes


Filtrated-Extracted SSL in fed-batch fermentation

0

5

10

15

20

25

30

35

40

45

0 20 40 60 80 100 120 140

Concen

tration (g/L)

Time (h)


After the batch culture with filtrated‐extracted SSL, the fermentation was continued by feeding with pure sugars

39 g/L final SA concentration achieved with 0.63 g/g SA yield

Low by‐product formation SA productivity 0.31 g/L/h

SA LA FA AATotal

product‐yield

Produced (g/L) 39 10.7 2 3.5 ‐

Yield (g/g TS) 0.63 0.17 0.03 0.06 0.89

Ratio LA:SA = 0.27Ratio FA:SA = 0.05Ratio AA:SA = 0.09Ratio Total by‐prod:SA = 0.41


Pretreatment of SSL

SSLUltrafiltration

N2


FermentationsImmobilisation on alginate beads



Immobilisation

A. succinogenes immobilized cells on alginate beads

Microorganism: Actinobacillus succinogenes

Dilution of A. succinogenes cells in ringer solution (0.9% NaCl) Mixture of the ringer solution with sodium alginate solution

(Sigma Aldrich) Addition of this solution in 2% CaCl2 solution drop by drop in

order to form beads

Fermentation substrate:

Repeated batch fermentations were carried out with 7 times diluted SSL

Fed‐batch fermentations were carried out with ultrafiltratedSSL using 5 kDa MWCO membranes and nanofiltrated SSL from 800 Da MWCO membranes

In all cases, CO2 flow was 1 vvm and pH was controlled by the addition of 5M NaOH solution


Immobilization: Repeated Batch

SA produced (g/L)

SA yield (g/g TS) Productivity (g/L/h)

Batch 1 9 0.58 0.49

Batch 2 10.8 0.63 1.13

Batch 3 10.9 0.61 0.56

Batch 4 11.2 0.64 0.43

Batch 5 12.3 0.71 0.51

Fed batch 39.1 0.71 0.33

Higher SA productivity in 2nd

batch

After 5 batches the immobilized cells are still able to ferment SSL


Immobilisation: Fed-Batch in ultrafiltrated SSL

7 times diluted SSL filtrated with Ultrafiltration membrane (~25 g/L TS and 35 g/L LS)

Feeding: concentrated filtrated SSL (~200 g/L TS)

39.1 g/L SA 0.71 g/g yield 0.4 g/L/h productivity

Very low by‐product production (by‐products yield 0.2 g/g TS, mainly AA)

0

5

10

15

20

25

30

35

40

45

0 20 40 60 80 100 120 140

Con

cent

ratio

n (g

/L)

Time (h)



Immobilisation: Fed-Batch in nanofiltrated SSL with Actinobacillus succinogenes

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100

Con

cent

ratio

n (g

/L)

Time (h)

Total sugars

Succinic acid

Final SA : 35.4 g/L SA yield : 0.62 g/g SA productivity : 0.38 g/L/h Total by-products : 13.1 g/L Total by-product yield : 0.23 g/L/h

• Nanofiltrated SSL from 800Da membranes (initial LS ~ 5 g/L)

• Continuous feeding of concentrated nanofiltrated SSL ( ~ 400 g/L TS)


Immobilisation: Fed-Batch in nanofiltrated SSL with Basfia succiniciproducens

• Nanofiltrated SSL from 800Da membranes (initial LS ~ 5 g/L)

• Continuous feeding of concentrated nanofiltrated SSL ( ~ 400 g/L TS)

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70 80 90

Con

cent

ratio

n (g

/L)

Time (h)

Total sugars

Succinic acid

Final SA : 45 g/L SA yield : 0.66 g/g SA productivity : 0.58 g/L/h Total by-products : 14.9 g/L Total by-product yield : 0.22 g/g


Conclusions• SSL contains phenolic compounds with strong antioxidant activity that could be considered as

value-added co-product• Ultrafiltration of SSL enhance both microbial growth and SA yield.

Basfia succiniciproducens Inhibitors removal is necessary Filtration - Phenolic Extraction resulted in higher final succinic acid concentration and

yield, with similar by-product formation as in the case of pure sugar utilisation Immobilization in alginate beads led to the highest SA production

Actinobacillus succinogenes Lower succinic acid production than Basfia succiniciproducens in pure sugars Filtration - Phenolic Extraction enhance succinic acid yield with lower by-product

formation Immobilisation increased significantly the tolerance of the biocatalyst to inhibitors


The AUA teamThank you for

your attention

The research leading to these results has received funding fromthe European Union’s Seventh Framework Program for research,technological development and demonstration under grantagreement no 311935

apostolis koutinas, mary alexandri, chrysanthi pateraki

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