biorefinery development based on valorization of ... · l-lysine l-threonine l-methionine...

1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy

Apostolis Koutinas

Department of Food Science and Human Nutrition

Agricultural University of Athens

Greece

Biorefinery development based on

valorization of industrial by-product

streams


Founded in 1920 as Superior Agricultural School of Athens, it became Agricultural

University (AUA) in 1990 and from 2013 it will have two major schools:

1) School of Agricultural Production, Infrastructure and Environment

2) School of Food, Biotechnology and Development

Department of Food Science and Human Nutrition

The 3rd oldest University in Greece

(Location: Athens)

Website: www.aua.gr


Apostolis Koutinas, Agricultural University of Athens

C5, C6 Sugars

Pyruvate

Acetyl-CoA

Citrate

Oxaloacetate

cis-Aconitate

Isocitrate

α-Ketoglutarate Succinate

30-50 kT

Fumarate 90 kt

Malate 200 kt

1,2-propanediol 1,3-propanediol 45 kt

3-Hydroxypropionate

L-Lactate 450 kt

Acetaldehyde

Acetate

Ethanol

L-Alanine

Acetoacetyl-CoA

Acetolactate

2,3-Butanediol

L-Aspartate

L-Lysine

L-Threonine

L-Methionine

L-Isoleucine

Cadaverine 1,600 kt

Caprolactam

Adipic acid 2,200 kt

Itaconate 80 kt

L-Glutamate Putrescine 10 kt

PHB

Glycerol

Phosphoenolpyruvate

Butanol (840 kt)

Acetone

Butyrate

Adipic acid

Xylitol

Glucaric acid 42 kt

Sustainable production of basic

(platform) chemicals via microbial

bioconversion


Valorisation of renewable resources

Industrial wastes and by-product streams

Biorefinery development

Added-value products

Food waste and by-products

Food

Feed

Antioxidants Chemicals

Biofuels

Biopolymers

Heat Biomaterials

Efficient pretreatment and fractionation

Industrial biotechnology

Green chemical conversion / separation

Sustainability analysis

Agricultural crops and residues

Biorefinery development based on renewable resources


Biorefinery development based on the

valorisation of by-products generated by

sunflower-based biodiesel plants

Restructuring 1st generation biorefineries



Screw press and

solvent extraction Oil

Seed

residues Transesterification

Biodiesel

Crude glycerol (35-50% glycerol)

Oilseeds

Crush Hull

Separation for

value-added

products

Antioxidants

Phytic acid (2-4%)

Protein (20-40%)

Dietary fibre

SSF Fungal

fermentation;

Hydrolysis

Fermentation

nutrients (amino acids, peptides

minerals, vitamins)

Biorefinery development from current biodiesel

production from oilseeds

Restructure

refining to levels

tolerable by the

bacterium

Microbial

fermentation



SFM composition Content

Moisture (%, wet basis) 3.76

Total Kjeldahl Nitrogen (TKN)

(mg/g, db) 41.18

Protein (%, 6.25 × TKN, db) 26.62

Oil (%, db) 0.93

Ash (%, db) 6.83

Dietary Fiber (%, db) 19.5

In 2012, the production of sunflower meal (SFM) was estimated at

about 15 million tonnes.

Conventional uses

Animal feed Food additives

Sunflower meal



Crude glycerol represents 10% (v/v) of the ester produced after the transesterification of vegetable oils. The increased production capacity of biodiesel could render crude glycerol an abudant platform chemical for the future bio-economy era.

Applications of crude glycerol in microbial bioconversions:

1,3-propanediol Dihydroxyacetone Succinic acid Propionic acid Ethanol

Citric acid Pigments Biosurfactants Polyhydroxyalkanoates

Glycerol



Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are produced as intracellular granules via

fermentation of renewable feedstocks by various microorganisms.

PHAs are biodegradable polymers that could substitute for petroleum-derived

plastics and poly(3-hydroxybutyrate) (PHB) is the most common PHA produced

from various carbon sources.

P(3HB-co-3HV) is a copolymer that contains 3-hydroxybutyric acid and 3-

hydroxyvaleric acid units with improved properties as compared to PHB.

3HV

O

O

O

O

O

O

O

O

O

O

O

O

3HB 3HO 3HHx 3HDD 3HD

Short chain length PHA Medium chain length PHA nCH2 CΟ

O

CH

CH3

Generic formula of hydroxy alkanoates (HA) PHB

P(3HB-co-3HA)

P(3HB-co-4HB)

(CH2)m C ΟH

O

CH

R

ΟH

x

CH2 CΟ

O

CH

(CH2)y

CH3

n

CH2 CΟ

O

CH

CH3

x

CH2 CΟ

O

CH2 CH2

n

CH2 CΟ

O

CH

CH3

Generic

formula


Sunflower-based biorefinery

Sunflower seed

Sunflower meal

Solid state

fermentation

Mechanical pressing and

hexane extraction Sunflower oil

Carbon source

Transesterification

Biodiesel Crude glycerol

Nutrient-rich

supplement

Enzymatic hydrolysis

Aspergillus

oryzae

Microbial

fermentation

Polyhydroxybutyrate



Hydrolysis of sunflower meal with crude enzyme consortia produced

by Aspergillus oryzae cultivated in solid state fermentation

Sunflower meal concentration

(g/L)

Hydrolysis Time (h)

Free Amino Nitrogen

(mg/L)

Inorganic Phosphorus

(mg/L)

45 48 685 108

68 48 1144 162

90 48 1533 245

Production of a nutrient rich supplement that can be employed as

fermentation feedstock in microbial bioconversions



0

100

200

300

400

500

0

5

10

15

20

25

0 20 40 60 80

FAN

, IP (m

g/L)

Gly

cero

l, T

DW

, PH

B (

g/L

)

Fermentation time (h)

FAN (△)

In. Phosphorus (■)

Glycerol ()

Total Dry Weight (▲)

PHB (●)

PHB production stage

Crude glycerol

Sunflower meal

hydrolysate

Microbial

fermentation Polyhydroxybutyrate

Microbial growth stage


PHB production from whole sunflower meal hydrolysate

(SFM) and crude glycerol


Fed-batch fermentations for PHB production carried out on whole

sunflower meal hydrolysate (SFM) and crude glycerol

Initial FAN

(mg/L)

Tf (h)

TDW g/L

RCM g/L

PHA g/L

3HB mol %

3HV mol %

PHA content

%

412 78 21.6 6.5 15.1 99.66 0.34 69.8

585 98 37 10.02 26.98 99.84 0.16 72.91

710 75 33 8.42 24.57 99.83 0.17 74.46

809 76 29.8 9.27 20.53 99.87 0.13 68.89

1114 82 19.8 13.88 5.92 99.5 0.5 29.89



Effect of levulinic acid addition on P(3HB-co-3HV) production in shake

flask cultures of Cupriavidus necator DSM 7237

Initial FAN

(mg/L)

Tf (h)

TDW (g/L)

RCM (g/L)

PHA (g/L)

Final Glycerol

(g/L)

Levulinic acid* (g/L)

3HB (mol %)

3HV (mol %)

PHA content

(%)

365 73 11.35 4.08 7.27 0.92 0 99.14 0.86 64.1

353 77 13.45 4.77 8.68 1.35 2.56 89.4 10.6 64.5

382 73 14.55 4.98 9.57 0.94 5.75 86.2 13.8 65.8

364 79 15.5 4.12 11.38 0.97 10.85 78.5 21.5 73.4

* Represents cumulative levulinic acid consumption during fermentation



Effect of levulinic acid addition on P(3HB-co-3HV) production in

bioreactor fermentation of C. necator DSM 7237

Glycerol (■)

FAN (○)

Total Dry Weight (●)

Inorg. Phosphorus (△)

P(3HB-co-3HV) (◊)

3HV (▲)

Levulinic acid ()

3HV content reached a maximum of 27%



Sunflower seeds

Partial Dehulling

Solid state fermentation

Mechanical pressing and hexane extraction

Oil

Partly dehulled or undehulled sunflower

meal

Aqueous extraction Protein-rich fraction

Lignocellulosic fraction

Liquid fraction

Antioxidants

Protein isolate

Microbial fermentation

Nutrient supplement

Fermentation Products

Biodiesel

Crude glycerol

Transesterification

Enzymatic hydrolysis

Advanced sunflower-based biorefinery



Exploitation of on-site solid state fermentation

Development of on-site solid

state fermentation using

industrial by-products

Protein hydrolysate

production with improved

properties

Fermentation feedstock

production to substitute for

commercial nutrient

supplements

Utilisation of crude enzymes

for lysis of microbial cells to

release intracellular products



Soluble fraction

Protein-rich fraction (PF)

Lignocellulose-rich fraction (LF)

Sunflower meal (SFM)

Solid Residue (SR)

Extracted proteins

Alκaline extraction

Supernatant Protein Isolate

Acid precipitation

Sunflower meal fractionation



Samples Total phenolic content

(mg of GAE/100g, db)

Total phenolic content

(mg chlorogenic acid /100g, db)

Sunflower meal (SFM) 10.5 ± 1.1 11.7 ± 0.9

Protein rich fraction (PF) 10.2 ± 0.9 11.3 ± 0.8

Lignocellulosic fraction (LF) 4.9 ± 0.5 5.5 ± 0.5

Phenolic content of SFM, PF and LF as measured by the Folin–Ciocalteu method

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

SFM PF LF Ascorbic

acid

BHT

IC50 (

mg/m

L)

Antioxidant activity of

SFM fractions using the

DPPH (1,1-diphenyl-2-

picryl-hydrazyl) method

Total phenolic content and antioxidant activity



0

200

400

600

800

1000

0 10 20 30 40 50

Fre

e A

min

o N

itro

gen

(m

g/L

)

Time (h)

6.6 U/mL

13.2 U/mL

19.8 U/mL

0

10

20

30

40

0 10 20 30 40 50

FA

N/T

KN

h

yd

roly

sis

yie

ld (

%)

Time (h)

6.6 U/mL

13.2 U/mL

19.8 U/mL

Protein isolates (PI) could be hydrolysed to produce free amino nitrogen (FAN) to total

kjeldahl nitrogen (TKN) conversion yields higher than 35%. Proteolytic enzymes are

produced as crude enzyme-rich extracts via solid state fermentation (SSF) by Aspergillus

oryzae cultivated on the lignocellulosic fraction extracted from sunflower meal

Production of hydrolysate from protein isolate



Solid State Fermentation with LF stream

Hydrolysis of solid residue stream suspended in

soluble fraction

Fermentation nutrient supplements

from remaining streams

0

50

100

150

200

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

IP (m

g/L) FA

N (

mg

/L)

Hydrolysis time (h)

0

50

100

150

200

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

IP (m

g/L) FA

N (

mg

/L)

Hydrolysis time (h)

FAN and IP production during

enzymatic hydrolysis of 50 g/L (□, FAN;

△, IP) and 100 g/L (■, FAN; ▲, IP)

initial LF concentrations.

FAN and IP production during enzymatic

hydrolysis of 50 g/L (□, FAN; △, IP) and

100 g/L (■, FAN; ▲, IP) initial SR

concentrations.

Use of only lignocellulose-rich fraction (LF) for nutrient production



0

100

200

300

400

500

0

5

10

15

20

25

0 10 20 30 40 50 60

FAN

, IP (m

g/L)

Gly

cero

l, T

DW

, PH

B (

g/L

)


PHB fermentation using the hydrolysate produced when the

lignocellulose fraction was used

FAN (△)

IP (■)

Glycerol ()


PHB (●)

NOT efficient medium for PHB production



0

100

200

300

400

500

600

700

800

0

10

20

30

40

50

60

70

0 30 60 90 120 150

FAN

and

IP (m

g/L)

Gly

cero

l, T

DW

an

d P

HB

(g

/L)

Fermentation Time (h)

FAN (△)

IP (■)

Glycerol ()


PHB (●)

PHB fermentation using the hydrolysate produced when all residual

streams are employed

This medium is efficient for enhanced PHB production

PHB production stage

Microbial growth stage



Advanced sunflower-based biorefinery

Evaluate the potential to produce various combinations of products from biodiesel

industry by-products

Development of glycerol

bioprocessing

Planned/future projects to

follow


Biorefinery development based on the

valorisation of by-products generated by

wineries

Valorisation of wine lees



Wineries

‘‘Wine lees is the residue that forms at the bottom of recipients containing wine,

after fermentation, during storage or after authorized treatments, as well as the

residue obtained following the filtration or centrifugation of this product” (EEC

regulation No. 337/79)



• “WL are considered as by-products that must be

sent to alcohol distilleries, producing a solid waste (namely exhausted grape marc, EG) and a liquid waste (namely vinasse, V)”.

(European Council Regulation (EC) No 1493/1999)

• However, small wineries do not abide by this law. • Hence, in 2008, waste generation by wineries in

EU-27 could be estimated for a wine production of 15.93×106 m3 in approximately 1.4×106 tons/year of wine lees.

Wine Lees



Wine Lees

Centrifugation

Liquid Solids

Distillation

Ethanol Alcohol-free

nutrient rich

liquid

1. Potable alcohol

2. Biofuel

3. Platform chemical

Enzymatic lysis

of yeast cells

Crude enzymes produced

through Solid State

Fermentation (SSF)

Nutrient rich medium for

microbial fermentations

Extraction

Residual solids 1 Antioxidants

Added Value

product Acidification with HCl

and centrifugation

Residual solids 2

(rich in yeast cells) Liquid containing

tartaric acid

Tartaric salts

Tartaric acid


Free amino nitrogen production during yeast lysis

It is worth noting that FAN production was equal to a 24 g/L liquid medium of

yeast extract, which contains about 50 mg FAN per g of yeast extract

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50 60

FA

N p

rod

ucti

on

(m

g/L)

Hydrolysis Time (h)

100 g/L, 12 U/mL

50 g/L, 12 U/mL

100 g/L, 24 U/mL



PHB fermentation supplemented with wine lees hydrolysate

Wine lees hydrolysate was supplemented with minerals and the final PHB

concentration reached 30 g/L with a PHB content of 71% (w/w)


0

250

500

750

0

10

20

30

40

50

0 10 20 30 40 50 60 70 80

FAN

(m

g/L

)

Gly

cero

l, T

DW

, PH

B (

g/L

)


Glycerol Total Dry Weight PHB FAN


The AUA team

Thank you for your attention

The AUA team

biorefinery development based on valorization of ... · l-lysine l-threonine l-methionine...

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