BIOCATALYTIC CONVERSION OF
SHORT CHAIN FATTY ACIDS TO ALCOHOLS
VIA SYNGAS FERMENTATION
Hanno Richter, PhD
Angenent Lab
Biological and Environmental Engineering
Cornell University
Ithaca, NY
hr95@cornell.edu
SunGrant Initiative
2012 National Conference
New Orleans, LA, October 3, 2012
Carboxylic acid
Solventogenic clostridia
Energy + electrons
Alcohol
Talk Outline
1) Why carboxylic acids as substrates for biofuels: The
carboxylate platform in the Angenent lab
2) Biochemical conversion with energy and electrons
from sugar: Advantages and disadvantages
3) Biochemical conversion with syngas: Achievements
and challenges
http://angenent.bee.cornell.edu/
Carboxylate platform :
Conversion of biomass into short chain fatty acids
Lignocellulose
Carboxylic
Acids
Agler et. al. (2012).
Environmental Science & Technology
46: 10229-10238
Carboxylate platform :
n-butyric acid as the main product from corn stover
Agler et. al. (2012).
Environmental Science & Technology
46: 10229-10238
Carboxylate platform :
Caproic acid production from corn beer
Agler et. al. (2012). Energy & Environmental Science.
DOI: 10.1039/c2ee22101b
Carboxylate platform :
Caproic acid production from corn beer
Agler et. al. (2012). Energy & Environmental Science. DOI: 10.1039/c2ee22101b
Conversion of biomass into alcohols
via carboxylic acids with solventogenic clostridia
Energy, electrons
Lignocellulose
N-butyric Acid
Carboxylate
platform
N-butanol
ABE
fermentation
2-stage continuous culture
with C. saccharoperbutylacetonicum
for conversion of n-butyric acid into n-butanol
Gas
pump
pump
n-butyric acid
pH auxostat
pump
Reservoir:
Medium
+ glucose
pump
Stage 1:
Growth,
Acidogenesis
pump
Stage 2:
Solventogenesis
Condensor,
Concentrated
solvents
Richter et. al. (2012). Biotechnology and Bioengineering 109 (4); 913-921
Effluent,
Solvents
depleted
Complete bench scale setup for conversion of
n-butyric acid into n-butanol with glucose
Richter et. al. (2012). Biotechnology and Bioengineering 109 (4); 913-921
System produces butanol at good rates
Stage 1, growth
Separating funnel
Stage 2, production
Condenser
Product: 0.25 L per day (33 g butanol/L)
Large amounts of glucose required for
conversion of n-butyric acid into n-butanol
Î Costly !
Richter et. al. (2012). Biotechnology and Bioengineering 109 (4); 913-921
Substrates
Products
Glucose
n-butyrate
n-butanol
acetone
ethanol
acetate
rates (g/(L*h))
1.39
0.23
0.39
0.20
0.02
0.01
Molar ratios
3.0
1.1
2.2
1.4
0.2
0.1
Problem: formation of fermentation byproducts.
Theoretical ratio of butyrate : glucose consumed = 2:1
Achieved: only 1:3
There is a need for a more economic/efficient source of electrons and energy
for carboxylate to alcohol conversion, without mandatory formation of
energy-dense byproducts …Î syngas
Syngas as a source of energy and electrons
Gasification
Syngas (CO, H2, CO2)
CO2
Lignocellulose
N-butyric Acid
Carboxylate
platform
N-butanol
Solventogenic
Clostridium
Theoretically possible: Consumption of
two mol CO per one mol n-butyric acid.
Note that no byproducts have to be formed
2 CO + 2 H2O
2 CO2
0.5 ATP
2 Reduced ferredoxin
1 oxidized ferredoxin
1 NADH2
R-COOH
Carboxylic acid
R-COH
Aldehyde
1 oxidized ferredoxin
R-CH2OH
Alcohol
Schematic:
Syngas fermentation in batch cultures
Perez, J.M., Richter, H., Loftus, S.E., Angenent, L.T.
Biocatalytic reduction of short-chain carboxylic acids into their corresponding alcohols with syngas fermentation.
Submitted to Biotechnology and Bioengineering in August 2012, revision in progress.
Syngas fermentation in the lab
Reduction of n-butyric acid
with syngas and C. ljungdahlii ERI-2
fed with 15 mM n-butyric acid*
● butyrate
□ acetate
● butanol
▪ ethanol
100
200
300
Time (h)
400
Perez et. al. (2012) Submitted to Biotechnology and Bioengineering.
- Conversion ability spread among carboxidotrophs
- Broad range of carboxylates; longer side-chain more
toxic, but also facilitates product stripping
Perez et. al. (2012) Submitted to Biotechnology and Bioengineering.
Closed system
(batch serum bottles)
- Quantitative conversion of
n-butyric acid into n-butanol
achieved.
- Unfavorable
Substrate: product ratio
- Formation of byproducts
- this system was not optimized
for efficient reduction of
n-butyric acid.
1 n-butyric acid
+ 67 CO
+ 20 H2
1 n-butanol
+ 10 ethanol
+ 1 acetic acid
+ 47 CO2
+ 4 Cell carbon
Increasing the process Efficiency:
Continuous 2-stage syngas fermentation
-Separated
cell growth
and alcohol
production
-Improved mass transfer
-Continuous feed of
nutrients
and removal of
products, while
retaining biocatalyst
-improved ethanol:
acetate ratio through
media optimization
Richter, H., Martin, M.E., Angenent, L.T. (2012). Submitted for publication in SunGrant conference proceedings .
Continuous 2-stage syngas fermentation
in our lab
Parameters for fermentation in stage 2,
normalized to 1L reactor volume
Rates of
mMol/(L*min)
g/(L*h)
CO in
0.808
1.357
CO out
0.110
0.185
CO consumption
0.698
1.172
H2 in
0.471
0.057
H2 out
0.085
0.010
H2 consumption
0.386
0.046
CO2 in
0.067
0.177
CO2 out
0.371
0.979
CO2 production
0.304
0.803
Ethanol
production
0.136
0.375
Acetic acid
production
0.025
0.090
Ca. 10% of the productivity
of yeast in sugar fermentation
Parameters for fermentation in stage 2,
normalized to 1L reactor volume
Species
Concentration exiting stage 2
CO (g)
19 vol%
H2 (g)
14 vol%
CO2 (g)
63 vol%
Ethanol (l)
428 mM
19.7 g/L
Acetic acid (l)
143 mM
8.6 g/L
Efficiency (%)
Stage 2
CO consumption
86
H2 consumption
82
CO recovery in etOH
28
H2 recovery in etOH
74
Economic disadvantage of
synthetic growth medium*
Component
Cost per L Medium
MES (buffer)
$5.18
Mineral solution
$0.53
Yeast Extract
$0.17
L-cysteine
$0.03
Trace metal solution
$0.01
Vitamin solution
$0.01
Total
$5.83
We are currently developing an alternative growth medium that does not contain
the major cost factors (MES, Mineral solution, yeast extract).
First results are promising (next slide)
* For composition of synthetic medium see: Datar et Al (2004). Biotechnology and Bioengineering, Vol 86, No. 5, 587-594
Batch cultures with alternative medium
sparged with syngas
Upcoming: Scale-up to pilot plant for the
Cornell village-Scale Pyrolysis Project
http://www.css.cornell.edu/faculty/lehmann/village_pyrolysis/index.html
distillation
Stage 2
Stage 1
media
storage tank
compressor
Gas purification
Summary
Carboxylic acids can be produced from lignocellulose and organic waste materials
using anaerobic digester technology.
Biochemical conversion into alcohols with bacteria is feasible.
Conversion into alcohols requires electrons and energy.
Sugar can deliver these, but un-efficient, due to formation of undesired byproducts
by solventogenic clostridia.
Syngas can also be an energy and electron source .
Although byproduct formation is not mandatory, acetate and ethanol are produced.
The conversion process is currently being optimized towards
- more efficient use of syngas (mass transfer)
- higher product concentration, yields, rates of formation)
- minimizing byproducts
- reducing operational costs (growth medium).
Acknowledgements
Angenent Lab
USDA collaborators
Lars T. Angenent
Sarah E.
Loftus
Michael E.
Martin
Michael Cotta
Nasib Qureshi
Bruce Dien
Cornell Village Scale Pyrolysis Project
Jose M. Perez
Viktor Hällman
Sebastian Heger
Matthew T. Agler
Funding
SunGrant; USDA NIFA; Yossie Hollander
Hyung Mo Kang
Divya Vasudevan
Matt Williams
End
Following: supplementary slides
Results for production stage (2)
of cont. syngas fermentation
Theoretical co-fermentation of
glucose/butyrate by C. saccharoperbutylacetonicum
0.5 Glucose
0.5 Acetone + 1.5 CO2
1 NADH2
1 ATP
1 R-COOH
Carboxylic
acid
1 R-CO-O-P
1 R-CO-S-CoA
1 NADH2
1 R-COH
1 R-CH2OH
Alcohol
1 HS-CoA
1 HS-CoA
Comparison of
lignocellulose to ethanol technologies
Enzymatic hydrolysis of lignocellulose
and sugar fermentation
Gasification of lignocellulose
and syngas fermentation
Energy and/or chemicals
required for pretreatment
Energy required for gasification,
(consumes part of the substrate)
Necessary removal of toxic byproducts
from pretreatment (furfural etc.)
Necessary removal of toxic byproducts
from pyrolysis
Requires enzymes for hydrolysis of (hemi) cellulose into sugars (costly)
No additional conversion of product
required
Only (hemi-) cellulose moiety converted
into substrate
All biomass can be converted into
substrate
Theoretical ethanol yields*
Enzymatic hydrolysis of lignocellulose and
sugar fermentation
Gasification of lignocellulose and syngas
fermentation
0.51 g ethanol / g sugar *
0.27-41 g ethanol / g CO *,
depending on hydrogen content
0.26 g ethanol / g biomass
Assuming 50 wt% Biomass carbon recovered in
sugar
0.19-0.29 g ethanol / g biomass,
depending on hydrogen content,
Assuming 70 wt% Biomass carbon recovered in
CO**
* Calculated from sugar or CO yields from lignocellulosic biomass and fermentation stoichiometries
** http://www.treepower.org/fuels/biomasssyngas.html HMI International. Data derived from a fixed bed updraft gasifier design.
Composition of lignocellulose
DoKyoung Lee et al. (2007). SunGrant report. http://ncsungrant.sdstate.org/uploads/publications/SGINC1-07.pdf
Composition of syngas from biomass gasification
24.0% Carbon monoxide (CO)
= 73% of total carbon in biomass
18.0% Hydrogen (H2)
6.0% Carbon dioxide (CO2)
0.4% Oxygen (O2)
3.0% Methane (CH4)
48.6% Nitrogen (N2)
http://www.treepower.org/fuels/biomasssyngas.html
HMI International. Data derived from a fixed bed updraft gasifier design.
Composition of syngas
http://www.treepower.org/fuels/biomasssyngas.html
(1) Steam - Its generation and use, Babcock and Wilcox, pp. 5-20 and 5-21 discussion of coal producer gas.
(2) HMI International. Data derived from a fixed bed updraft gasifier design.
(3) Steam -- Babcock and Wilcox, p. 5-19.
Ljungdahl-Wood-Pathway (acetogenesis)
2[H]
CO2
2[H]
CHO-THF
THF
ATP
CH-THF
Fdred
CH2-THF
CH3-THF
Formyl
tetrahydrofolate
Methyl
tetrahydrofolate
CO2
ATP generation
via CO-DH, Rnf complex,
and ATP-synthase
B12
CH3-B12
Methyl
B12
CO
‫ו‬
CO
‫ו‬
Fe
Fe
2[H]
‫ו‬
‫ו‬
-Ni
Fe
‫ו‬
-Ni
-Ni-CH3
CO dehydrogenase
CoA
H 2O
CO
CO2
Fdox
Fdred
NADH2
NAD
H+out
ADP+Pi
O
‫װ‬
CH3-C-O
Acetate
H+in
ATP
O
‫װ‬
CH3-C~SCoA
-
ATP
Acetyl-CoA
According to Brock (1997). Biology of Microorganisms. Prentice Hall
and (Koepke et. al. (2010). PNAS 107, 29: 13087-13092)