This document summarizes enzyme catalysis for biodiesel production. It discusses the advantages of using enzymes over chemical catalysts, including improved sustainability and reduced hazardous waste. It provides an overview of the enzymatic biodiesel production process involving transesterification and esterification reactions. The document also reviews the history of enzymatic biodiesel research and commercialization efforts. It discusses various approaches to overcome challenges such as enzyme deactivation, including the use of cosolvents and multi-stage methanol addition. Commercial enzymatic biodiesel facilities are achieving ASTM fuel specifications using real-world feedstocks.

1. Enzyme Catalysis
for Biomass Based Diesel Fuels
Rachel Burton
February 20, 2014
Department of Chemical Engineering,
North Carolina State University
CHE 596-16 Biodiesel Production Technology
February 20, 2014

2. “People
don’t
buy
what
you
do,
they
buy
why
you
do
it.”
–Simon
Sinek

3. Outline
• Why
use
an
enzyma:c
approach
to
biodiesel?
• -­‐Benefit
over
chemical
catalysts
• Overview
of
enzyma:c
biodiesel
• -­‐Enzymes
for
commercial
produc:on
• -­‐Transesterifica:on
• -­‐Esterifica:on
• Enzyme
Reuse

4. Quick Definitions
TAG = Triglycerides (fat/oil)
DAG = Diglycerides
FFA = Free fatty acids
FAME (or ME) = Fatty Acid Methyl Esters (biodiesel made using methanol)
Immobilized vs. Liquid Enzymes
CALB = Candida Antarctica Lipase B
Novozym 435 = CALB immobilized on a plastic support (.5mm beads)
TL = Thermomyces Lanuginosa lipase

5. Advantages of Enzymes for Biodiesel

6. Sustainability Profile
• National Research Council s Committee on
Water Implications of Biofuels Production: 1
gallon of wastewater: gallon of biodiesel
produced. (Oct. 2007)
• 2007, Harding et al, LCA analysis between
enzymatic & chemical catalysis for biodiesel
• Flammable and hazardous substance exposure
reduction

7. Improved
performance
linked
to
lower
energy
requirements
for
hea:ng
in
the
process
Terrestrial
ecotoxicity
levels
are
reduced
by
40%
with
the
removal
of
mineral
acid
from
the
process

8. Hurdles to Enzymes for Biodiesel
• 15 years of research
• Hundreds of articles
• Many different
enzymes, reaction
conditions, etc.
• Overall conclusion in
most cases – too
expense

9. Why Now?
Chicken or the egg problem:
enzyme development vs. market
development.
Confluence of events:
• Biodiesel industry is more mature
and more secure
• Drive for lower cost feedstocks,
presence of high FFA virgin oils
• Demand for increased fuel quality
• Competition for fats/oil from
renewable diesel will push efficiency
• Industry recognition of problems
with soaps, low quality glycerin, and
difficulty of acid esterification
Causes process development:
• Commercial enzyme reuse trials
• Lower cost enzyme production
http://www.channelshirt.com/product/13/208/Chicken-Or-Egg-
Tshirt.html
Commercial Viability!

10. Access to affordable
feedstocks
Yellow Grease
Tallow
Soy oil
Feedstock prices rising for the 2nd time;
now in accordance to petrol market

11. Reusability
Enzymes used for biodiesel production lose
activity by:
High heat (>50C or >122F)
Excess alcohol
In addition, Immobilized enzymes can lose
activity by:
Any alcohol out of phase
Large excess of alcohol in phase (~5% or more)
Glycerol out of phase
High shear can cause immobilized enzymes to come
off of the carrier
Certain polar contaminants
Mineral acids
Temporary vs. Permanent activity loss
Temporary: physical blocking of active sites
Permanent: denaturing due to excess methanol
Reusability of
enzymes is the key
to commercial
viability!

12. A brief history
Early Work (1987 - 1995)
• Studied cosolvents like iso-octane, hexane, diesel fuel.
• Evaluated aqueous, non aqueous, and solvent-free systems, different types of
alcohols, impact of water
• Studied a wide variety of liquid enzymes
• Mike Haas, Tom Foglia, Martin Mittelbach, and many others
• No real biodiesel production, so these were interesting but not practical
Resurgence of interest (1999 - 2008)
• Better immobilized enzymes
• Solvent free systems, good enzyme reuse
• First large scale plant in China using cosolvents ( tert-butanol )
Current Developments
• Novozymes developing enzymes, immobilization techniques, lowering costs
• Piedmont Biofuels
• Transbiodiesel in Israel
• Sunho in Taiwan
• Blue Sun & Viesel
• Renewed interest from universities and private biodiesel producers for lab and
pilot sized reactors

13. Summary Articles
• Haas, 2002 (review of early literature)
• Fjerbaek, 2009
• Ranganathan, 2007
• Basic components of reviews
• enzyme type
• liquid vs immobilized
• support type
• reaction conditions
• conversion
• number of reuses
• tolerance to water, methanol

14. Fjerbaek et al.: Biodiesel Production
Using Enzymatic Transesterification.
2009.

15. Cosolvents – Li (2006)
Tert-butanol as
cosolvent
Advantages
• Can use more excess
methanol without
deactivation
• Deactivation due to
glycerol (clogging) does
not occur
• Impressive enzyme reuse
Built full scale
plant based on
this process
in China
Disadvantages
• Large amount of co-solvent required
which must be removed at the end via
distillation
• Still had high FFA at end of process
• Reaction rate still slow

16. Cosolvents – Zheng (2008)
• Tert-Amyl Alcohol as cosolvent
Batch Reactions
• 1.78g soybean oil
• 2 – 6ml amyl alcohol
• 2% Novozym 435 (CALB)
• reaction time ~ 15 hours
• Add graph here
Excellent reuse, but same
problems as Li

17. Watanabe and Shimada (2001, 2005)
No cosolvent
Use multi-stage methanol addition
to avoid deactivation
Tested batch and continuous
Advantages
• No cosolvents
• Minimize methanol use
• Excellent conversion (98%+)
• Good enzyme reuse
Disadvantages
• Still has long reaction times
• Still had high FFA at end of
process
• Enzyme reuse still not
commercially viable

18. Watanabe and Shimada (2001, 2005)
Packed
Bed
Reactors

19. Watanabe and Shimada (2001, 2005)
Esterification – very fast!
Determined maximum methanol
allowable before deactivation
• 6.3% by weight methanol
Performed Enzyme Reuse Trials
• Excellent conversion (98%+)
• Good enzyme reuse

20. Novozymes (2009)
Longevity trials: Esterification
Reaction Conditions
• 20% by volume methanol, 2 stage
reaction, 45C,
• Majority of reaction finished in 60
minutes
• Blended FAME, MeOH, and PFAD to
address the high melting point
Still has deactivation
Relatively large excess methanol
use (~2:1)
Still high FFA content (3 – 5%) after
2nd stage

21. U.S. Dept. of Energy: Piedmont Biofuels –
Commercial Focus
Esterification
Achieving ASTM specifications
Commercial viability (enzyme reuse)
Real World Feedstocks
• Yellow Grease (15% FFA)
• Brown Grease (80%+FFA)
• Palm, soy, and others

22. Enzymatic approach for Waste-Water Reduction
• Began investigation of enzyme catalysis for
biodiesel, 2009
• Focus on esterification first
• 2010---Lab working on Pilot scale (35 gal.)
• 2011/2--- Pilot moving to Commercial
• 2013---on-going Commercial Integration
• Validation for multi-feedstock production
scheme
• First to demonstrate enzyme biodiesel to
meet full ASTM specification.
• Economic analysis & Enzyme reuse
CSTR system with alcohol metering,
patent pending

23. Enzymes For Biodiesel Production
Commercial
formulations:
Callera series

24. Commercial Build

25. Feed Tanks

26. How can you use this process?
• Enzyma:c
Esterifica:on:
• A.
Pretreatment
for
exis:ng
chemical
plants
• B.
Full
conversion-­‐-­‐Esterifica:on
for
High
FFA
feedstocks
• C.
Enzyma:c
Polishing
for
TRANSESTERIFICATION
followed
by
the
enzyma:c
esterifica:on
PROCESS
•
-­‐-­‐100%
enzyme-­‐based
process
in
2
steps

27. Replace Sulfuric Acid Pretreatment
• Esterification using Callera Ultra/L
Immobilized CALB or Liquid CALB
• Replaces traditional sulfuric acid
technology
• Incoming Feedstocks: 2-100% FFA
• Patent Pending, continuous process
• Low temperature process
• Maintains water balance
• 6-12 times less methanol than acid
esterification
• No acidic methanol sidestream
FAeSTER Process:
Fatty Acid
esterification

28. Feedstock Pretreatment
Esterification occurs quickly within 30 minutes
Achieves acid value specification by 90 minutes

29. Liquid enzyme transesterification
• Heat/Circulate Feedstock (42 gallons, 40ºC)
• Initial Batches
– 1%-2% enzyme by feedstock weight
– Aqueous Phase of 20% by feedstock weight
• 40% water
• 60% glycerol
• Methanol Dosing
– 1.7 molar excess of methanol to feedstock
– 25% initial
– Remainder metered in over 5 hours
• Sampling
– 1, 2, 4, 6, 8, 10hr
• FAME Phase: Bound Glycerine & FFA
– 24 hr
• Aq. Phase: Glycerol, Methanol, H2O, Enzyme
– Determine Aq. Phase Removal & Methanol Dosing
– Part II

30. Pilot Scale Results

31. Commercial scale
Results
Reaction complete in 6-8 hours
In-spec Bound Glycerin 0.15%
1.5FFA%
2700 gallon reactor, coil-heated,
SS, cone-bottom,
35-40C/95-104F,
20% water/glycerol,
1.7:1 methanol:FA
Reaction monitored:
1, 2, 4, 6, 8 hrs.

32. NOVOZYMES PRESENTATION
2/19/14
32

33. Alkaline wash reduce free fatty acids and glycerides
and make final biodiesel meet specifications.
The soap stock is acidified and sent back
NOVOZYMES PRESENTATION
2/19/14
33
0
0.5
1
1.5
2
2.5
3
Before
CW
AXer
CW
%
in
FAME
TG
DG
MG
FFA
BG = 0.17%
BG = 0.10%

34. The enzyme works at the oil/water interface
Mixing is important – we need a an emulsion with a large
surface area between oil and glycerin/water phases
NOVOZYMES PRESENTATION
2/19/14
34
Oil/FAME
Glycerin/water
Mixing

35. NOVOZYMES PRESENTATION
2/19/14
35
Glycerides + MeOH
glycerides/glycerin + FAME
Glycerides + H2O
glycerides/glycerin + FFA
FFA + MeOH
FAME + H2O
Driving process by
increasing Methanol and
decreasing Glycerol and Water
Reaction at interface
Enzyme process works well with any content
of free fatty acids

36.
Affect of Water:
Too
much
water
leads
to
reac:on
with
FAME
back
to
FFA.
Too
lile
water
leads
to
enzyme
inac:vity.
Affect of Methanol:
Excess
methanol
inhibits
the
enzyme.
Too
lile
methanol
slows
reac:on.
Loss of Active Enzyme:
Loss
of
Enzyme
=
slower
reac:on
rate.
Balancing Act

37. –
2010: ASTM D6751
achieved using only
immobilized enzymes
2011: ASTM D6751
achieved using Liquid
TL CALB
2012 + :
Commercial Scale
Integration
100% Enzyme-Based Fuel meets
ASTM D6751

38. Glycerol from Enzymatic
Process
• High purity Glycerol
99.6%
• Economic Impact to
the producer:
• 40-50 cents/lb.
•
Chemical glycerol vs. enzymatic glycerol
Chemical FAME/glycerol vs. FAME/
enzymatic glycerol
YELLOW GREASE FEEDSTOCK

39. 2/19/14
39
Tsinghua China – Hunan plant
Lipase-mediated industrial scale production of biodiesel (20,000t/y) put
into operation Dec 8, 2006. Initially in a process with t-butanol;
Now restarting in solvent free process

40. NOVOZYMES PRESENTATION
2/19/14
40
Final product
Blue Sun - USA.
3 enzyme reactors of 300 m3 each
Started early 2013

41. NOVOZYMES PRESENTATION
2/19/14
41
Viesel Biofuels – Florida USA

42. Summary
Variables
Methanol use (minimize)
Cosolvents (minimize)
Reaction time (minimize)
Presence of water (minimize)
Ester conversion per minute (minimize)
Final ester conversion (minimum 98% esters)
Final FFA content (maximum 0.25% FFA)
Temperature (35 – 50)
System design
PBR
CSTR
Continuous
Batch
System robustness
multi-feedstock
tolerance to impurities
Enzymes as a biodiesel catalyst
are extremely well studied
but also very complex.
Still, probably worth the effort...
Type of Enzyme
Candida antarctica lipase B
Thermomyces lanuginosa
Pseudomonas cepacia
Rhizomucor miehei
Carrier
Polypropylene
Polyethylene
Activated Carbon
Silica
Form of enzyme
Liquid
Immobilized
Lower Energy Use
No Soap Formation
High Quality Glycerin
Non-Toxic
Low Methanol Use
Scale Neutral
More Complete
Separations Esterify and Transesterify
A more competitive biodiesel industry