Biorefinery development based on valorization of ... · L-Lysine L-Threonine L-Methionine...
Transcript of 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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
Biorefinery development based on the
valorisation of by-products generated by
sunflower-based biodiesel plants
Restructuring 1st generation biorefineries
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
PHB production from whole sunflower meal hydrolysate
(SFM) and crude glycerol
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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%
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
)
Fermentation time (h)
PHB fermentation using the hydrolysate produced when the
lignocellulose fraction was used
FAN (△)
IP (■)
Glycerol ()
Total Dry Weight (▲)
PHB (●)
NOT efficient medium for PHB production
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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 ()
Total Dry Weight (▲)
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
Biorefinery development based on the
valorisation of by-products generated by
wineries
Valorisation of wine lees
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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)
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
• “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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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
Apostolis Koutinas, Agricultural University of Athens
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
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)
Apostolis Koutinas, Agricultural University of Athens
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
)
Fermentation time (h)
Glycerol Total Dry Weight PHB FAN
1st EUBis Action TD1203 workshop, 29-30/10/2013, Torino, Italy
The AUA team
Thank you for your attention
The AUA team