Post on 22-May-2022
MEMBRANE ASSISTED PROCESS INTENSIFICATION PAVES THE WAY FOR THE APPLICATION OF BIOCATALYSIS IN INDUSTRIAL PROCESSES.
Yamini Satyawali, Claudia Matassa, Wouter Van Hecke, Heleen De Wever, Marzio Monagheddu, Winnie DejongheYamini.Satyawali@vito.be
7/02/2020
©VITO – Not for distribution 1
VALUE CHAIN OF ENZYMATIC PROCESSES
7/02/2020
©VITO – Not for distribution 2Reference: Ferrer et al., 2015, Microbial biotechnology, 9, 22-34
ESTERIFICATION CATALYZED BY LIPASE TO SYNTHESIZE FATTY ACID ESTERS
3
Enzymatic Eco-friendly
• Solvent free
• Milder process conditions
Applications
• Food, cosmetics, personal care products, plasticizers, pharmaceuticals
Ref: Schumacher and Thum, Chem.Soc.Rev., 2013, 42, 6475
• Chemical catalyst• High temperature (150°C-
250°C (Unwanted) side reactions
resulting in intensive DSP
• Deodorization• Decoloring• Catalyst neutralization
(unstable product)• Distillation to purify product
INDUSTRIALLY DRIVEN ESTER RESEARCH AT VITO: THE PROCESS
4
• Technical aspects
• Sustainability analysis
• CO2 emission• Energy usage
ProcessConceptualization
Lipase selection
Process in mLscale
Processdevelopment (1-5 L scale)
Techno-economicevaluation
Pilot testing
▪ Free / Immobilized
▪ Reaction conditions
▪ Enzyme kinetics
▪ Pure & technical grade substrates
▪ Definitions of figures of merit▪ Yield▪ Productivity▪ Specific & total
productivity
▪ Process conditions
▪ Process intensification: In situ water removal
▪ Enzyme stability & Re-use
▪ Use of model to combine technical and economic data
▪ Identification of most influential parameter(s) on process economics
▪ At own or industrial site
▪ Mobile Pilot equipment
• Lipase used as biocatalyst for solvent free synthesis of esters using fatty acid and alcohol as substrates
• Infrastructure for batch and (semi) continuous processes (upscaling from 100 mL to approximately 5 L)
• Water removal approaches e.g. pervaporation for process intensification/zeolites
• Process up-scale to pilot scale with coupled hydrophilic pervaporation
INDUSTRIALLY DRIVEN ESTER RESEARCH AT VITO: IN PICTURES
5
Initial experiments in mL scale
Lab scale 3L reactor Lab scale PV set-up Pilot scale PV set-up
1/ EMOLLIENTS: UPSCALED PROCESS WITH MEMBRANE ASSISTED WATER REMOVAL
Confidential 6
Solvent free Kg scale ester production process
Coupled with PV
Various Kg scale batches tested
Enzyme reusability and membrane stability
Product purification and application testing
0
20
40
60
80
100
Fatt
y ac
id c
on
vers
ion
(%
)
0
0,5
1
1,5
2
2,5
3
Wat
er (
%)
• High conversion with 99% fatty acid conversion
• <20 wt% residual alcohol (in the end product)
• 1-2 wt% residual acid (in the end product)
• No loss of substrates or products during water removal
• Very stable enzyme and membrane performance
2/ MONO (DI)ACYLGLYCEROLS
Voettekst invulling 7
0
20
40
60
80
100
0 5 10 15 20 25 30
Fatt
y ac
id c
on
vers
ion
(%
)
Time (h)
7
4234
512
7
4234
612
Lauric acid Monolaurin Dilaurin Trilaurin Glycerol
We
igh
t (%
)
65
32
3
65
31
4
MAG DAG TAG
Sele
ctiv
ity
(%)
3/POLYGLYCEROL ESTERS
Confidential 8
Polyglycerol + fatty acid → polyglycerol mono ester + water
Tests conducted at various polyglycerol-10: fatty acid weight ratios
Weight ratio(polyglycerol/fattyacid)
Finalconversion(%)
Acid value (mg KOH/g) Average degree ofesterification
1:1 99 0,64 3,73
1,5:1 99 0,43 3,09
2:1 99 0,5 2,82
3:1 100 0 2,78
Image source: whattech.comImage source:chemicalsinourlife.echa.europa.eu
Chem. Biochem. Eng. Q., 33 (4) 501–509 (2019)
0
20
40
60
80
100
0 20 40 60 80 100
Fatt
y ac
id c
on
vers
ion
(%
)
Time (h)
1:1 weight ratio
4/ ACETATE ESTERS
Many interesting acetate esters in flavour and fragrance industry
Figure 1: Acetate esters used as flavours and/or fragrances and investigated in VITO
DETERMINATION OF REACTION KINETICS : CITRONELLYL ACETATE AS AN EXAMPLE …
10
Experimentally determined methyl acetate (▲ ) and citronellol (■) concentrations and the corresponding model results (…).
Citronellol
Confidential
SUGAR ESTERS
11
0
20
40
60
80
100
0 20 40 60 80
Fatt
y ac
id c
on
vers
ion
(%
)
Time (h)
Glucose
Xylose
73
52
Glucose Xylose
Sugar conversion
• Lauric acid as acyl donor equimolar• Lipozyme 435• 2-methyl 2-butanol as solvent• Solvent recycling
7/02/2020
©VITO – Not for distribution 12
SOLVENT RECYCLING BY NF – NANOFILTRATION TESTS
Membrane Glucose Lauric acidGlucose
mono laurateGlucose di
laurate
Polymeric membrane 1
21 % 19 % 7 % 26 %
Ceramic membrane
72 % 64 % 70 % 77 %
Polymeric membrane 2
72 % 65 % 80 % 87 %
Polymeric membrane 3
100 % 82 % 95 % 92 %
Membrane rejections
Cross-flow velocity = 2 m/s (polymeric mem.), 0.3 m/s (ceramic mem.)Pressure = 20 bar (polymeric mem.), 10 bar (ceramic mem.)Temperature = room temp (22˚C)
INnovative Chemoenzymatic InTEgrated processes – fosters competitiveness of the European green chemistry industry
A MULTI-STAKEHOLDERS PROJECT
3 universities and research organizations 2 SMEs 2 large
industries 1 innovation cluster
PROJECT DURATION:
48 months, from September 2019 to August 2023
INCITE aims to prompt a transition to a more flexible and sustainable chemistry by taking novel integrated upstream and downstream processing paths involving flow chemistry and membrane technology in two chemo-enzymatic processes to an industrial level
FOLLOW US
BUDGET:
Total cost: € 17.4 MEuropean Union’s Horizon 2020 Research and
Innovation Programme contribution: € 13.3 MOBJECTIVES
Contact: adouard@iar-pole.com
ASYMMETRIC SYNTHESIS OF CHIRAL AMINES FROM ω-TRANSAMINASE
7/02/2020
©VITO – Not for distribution 14
• Pros:
• Ketones are readily available pro-chiral building blocks
• Very high regio & stereoselectivity of biocatalyst
• Engineered ω-transaminases are available (increasing substrate scope and stability)
• Cons:
• Unfavorable thermodynamic equilibrium →excess of the donor amine often required
• Product and co-product ketone inhibit the enzyme
• To achieve high yield in-situ product and/or co-product removal is often required
APPLICATION OF HIGH MOLECULAR WEIGHT AMINE DONORS
7/02/2020
©VITO – Not for distribution 15
Development of an innovative process for chiral amine production and separation
NF Membrane
IPA
Jeffamine(400 & 600
g/mol)
HMW amine donors
Enzymatic reaction for chiral amine synthesiscombined with membrane ISPR for productrecovery and TD equilibrium shifting
Reaction optimization
Membrane filtration
ISPR proof of concept
APPLICATION OF HIGH MOLECULAR WEIGHT AMINE DONORS
7/02/2020
©VITO – Not for distribution 16
HMW AD 6 (Jeffamine 600 g/mol) LMW AD 8
Filtration
Wild type
Reaction
Engineered
HMW AD 3 (Jeffamine 400 g/mol)
APPLICATION OF HIGH MOLECULAR WEIGHT AMINE DONORS
7/02/2020
©VITO – Not for distribution 17
• 25% additional conversion → ISPR proof of concept
• ~85% of HMW amino donors retained by NF
• Low product concentration • Membrane stability • Loss of unreacted ketone substrate
Process Biochemistry, Volume 80, 2019, Pages 17-25
ENZYMATIC TRANSAMINATION IN ORGANIC SOLVENT/ SOLVENT-FREE MEDIUM AND MEMBRANE ASSISTED PRODUCT EXTRACTION
7/02/2020
©VITO – Not for distribution 18
N-heptane phase
Amine donor phase
BA
TA-v2
N-heptane phase
Amine donor phase
MPPA(BA)
Unreacted BAATA-v2(MPPA)
TA reaction
BA
Jeffamine ED-600
MPPA
TA in organic solvent
PIPI
Reaction Extraction
Heptane
Jeff amine ED-600 Enzyme
Aqueous extracting buffer pH 3
Membrane assisted product extraction
MEMBRANE ASSISTED PRODUCT EXTRACTION IN ORGANIC SOLVENT
7/02/2020
©VITO – Not for distribution 19
Membrane contactor screening with synthetic solutions
0
20
40
60
80
100
0 1 2 3 4 5 6 7
MP
PA
EE
(%)
Time (h)
Puramem Selective
Puramem Performance
Hollow Fiber
Hollow fiber (HF) contactor with modified housing and the flat sheet (FS) membrane contactor
High product
purity (>97%)
PIPI
Reaction Extraction
Heptane
Jeff amine ED-600 Enzyme
Aqueous extracting buffer pH 3
4-fold higher product yieldJ Chem Technol Biotechnol 2020; 95: 604–613
SPINNING THREE-LIQUID-PHASE REACTOR
7/02/2020
©VITO – Not for distribution 20
Configuration 1 Configuration 2
M
A
B
C
M
A
B
CBA
N-heptane phase
Amine donor phase
MPPA(BA)
Unreacted BAATA-v2(MPPA)
• Feasibility test with synthetic solutions • Enzymatic reaction with product recovery
SPINNING THREE-LIQUID-PHASE REACTOR
7/02/2020
©VITO – Not for distribution 21
3
4
5
6
7
8
9
10
11
12
0
10
20
30
40
50
60
70
80
90
100
0 2 3,5 5,5 6 8,5 24 25
pH
ext
ract
ing
bu
ffe
r
MP
PA
dis
trib
uti
on
(%
) Time (h)
Buffer heptane Jeffamine ED-600 pH
• After 5h operation • 71% MPPA extracted • 9,7% AD loss• No BA detected
150 rpm 200 rpm
ENZYMATIC PRODUCTION OF OLIGOSACCHARIDES FROM POLYSACCHARIDES
CONFIDENTIAL 22
Oligo’s from algae
POS
COS
ChitinChitosan
Starch
Ulvan, carrageenan, laminarin, fucoidan, β-glucan
Lactose
Mannan
MOS
XOS
GOS
i
i
Studiedby VITO
• Properties▪ Prebiotic (FOS, GOS, POS, MOS)▪ Antioxidant (XOS)▪ Antimicrobial (COS) ▪ Anti-coagulant (COS)▪ Plant elicitors (POS, COS,
carrageenan)▪ Plant biostimulant (COS,
carrageenan)
ENZYMATIC PRODUCTION OF OLIGOSACCHARIDES FROM POLYSACCHARIDES
Short term research
Offline fractionation by cascade of membranesConventional batch process
CONFIDENTIAL
• Disadvantages:• Steered by reaction time:▪ Products with broad range of dp▪ Monosaccharides
• Enzyme inhibition
• Continuous processing:• Enzyme membrane reactor• combining hydrolysis with
separation
Longer term research
ENZYMATIC PRODUCTION OF CARRAGEENAN OLIGOSACCHARIDES
OLIGOCAR
7/02/2020
©VITO – Not for distribution 24
• Polysaccharide found in red seaweed• Structure▪ Linear sulfated polysaccharide (> 100-
1000 kDa)▪ Repeating D-Galactose and 3,6
anhydrogalactose units▪ Different types but mainly κ, ι, and λ
carrageenan with▪ 1, 2 or 3 ester sulphate groups
• Thickener in food
D-galactose
3,6 anhydrogalactose
Hydrolysed by carrageenase
Cloning and production of carrageenase
enzymes
Enzymatic and chemical
hydrolysis of carrageenan
Functionality testing:
Plant elicitor
Plant biostimulant
Prebiotic
OLIGOCAR
CARRAGEENAN HYDROLYSIS AND BIOACTIVITY OLIGOSACCHARIDES
7/02/2020
©VITO – Not for distribution 25
pBACT5.0: Syngulon expression vector
0 h: starting kappa carrageenan in all 3 bottles has an Mn of approx. 440 kDa
BIOWOOD
ENZYMATIC PRODUCTION OF XYLAN AND MANNAN OLIGOSACCHARIDES FROM HEMICELLULOSE
7/02/2020
©VITO – Not for distribution 26
▪ Structure hemicellulose: difference hard- and softwood
Poplar
Birch
Pauly et al., 2008; Malgas et al., 2015; Bajpai, 2016
ENZYMATIC PRODUCTION OF XYLAN OLIGOSACCHARIDES FROM XYLAN BIRCHWOOD
7/02/2020
©VITO – Not for distribution 27
0
500
1000
1500
2000
2500
3000
3500
xylose XOS2 XOS3 XOS4 XOS5 XOS6 glucose
suga
r [m
g/L]
Xylan (7%) + Viscozyme (12 U/g)
0 h 1 h 2 h 4 h 6 h 24 h
0
1000
2000
3000
4000
5000
6000
7000
8000
xylose XOS2 XOS3 XOS4 XOS5 XOS6 glucose
suga
r [m
g/L]
Xylan (7%) + CellicCTec2 (12 U/g)
0 h 1 h 2 h 4 h 6 h 24 h
Mainly glucose producedLow conversion xylan
Less glucose producedHigher conversion xylanMainly xylose and DP2 produced
PRODUCTS
CONTINUOUS ENZYMATIC PRODUCTION OF XYLAN AND MANNAN OLIGOSACCHARIDES IN EMR
7/02/2020
©VITO – Not for distribution 28
▪ Enzymatic hydrolysis of xylan and mannan: Continuous enzyme membrane reactor
• Control degree of polymerization OS• Decrease enzyme inhibition • Enzymes can be recycled and re-used:▪ Cost for hydrolysis▪ Total productivity (g product/g enzyme)
OS = oligosaccharide
7/02/2020
©VITO – Not for distribution 30
HEALTH
MATERIALS
ENERGY
CHEMISTRY
LAND USE
New value chainsfrom alternative
feedstock
Biomass
CO2
Sustainableindustrialprocesses
ProcessIntensification
New synthesisroutes
Reuse/valorisation
process streams
FROM SCENCETO APPLICATION