Integration of chemical catalysis with extractive fermentation to produce fuels

Article abstract of DOI:10.1038/nature11594

Nearly one hundred years ago, the fermentative production of acetone by Clostridium acetobutylicum provided a crucial alternative source of this solvent for manufacture of the explosive cordite. Today there is a resurgence of interest in solventogenic Clostridium species to produce n-butanol and ethanol for use as renewable alternative transportation fuels. Acetone, a product of acetone-n-butanol-ethanol (ABE) fermentation, harbours a nucleophilic α-carbon, which is amenable to C-C bond formation with the electrophilic alcohols produced in ABE fermentation. This functionality can be used to form higher-molecular-mass hydrocarbons similar to those found in current jet and diesel fuels. Here we describe the integration of biological and chemocatalytic routes to convert ABE fermentation products efficiently into ketones by a palladium-catalysed alkylation. Tuning of the reaction conditions permits the production of either petrol or jet and diesel precursors. Glyceryl tributyrate was used for the in situ selective extraction of both acetone and alcohols to enable the simple integration of ABE fermentation and chemical catalysis, while reducing the energy demand of the overall process. This process provides a means to selectively produce petrol, jet and diesel blend stocks from lignocellulosic and cane sugars at yields near their theoretical maxima.

Full text of DOI:10.1038/nature11594

LETTER
doi:10.1038/nature11594
Integration of chemical catalysis with extractive
fermentation to produce fuels
Pazhamalai Anbarasan1,2*, Zachary C. Baer2,3*, Sanil Sreekumar1,2, Elad Gross1,4, Joseph B. Binder2, Harvey W. Blanch2,3
,
Douglas S. Clark2,3 & F. Dean Toste1,2,4
Nearly one hundred years ago, the fermentative production of acet- deoxygenated to paraffins. These paraffins, from pentane to undecane,
one by Clostridium acetobutylicum provided a crucial alternative are components of petrol, diesel and jet fuel.
source of this solvent for manufacture of the explosive cordite.
Today there is a resurgence of interest in solventogenic Clostridium
species to produce n-butanol and ethanol for use as renewable
alternative transportation fuels1–3. Acetone, a product of acetone–
n-butanol–ethanol (ABE) fermentation, harbours a nucleophilic
a-carbon, which is amenable to C–C bond formation with the elec-
trophilic alcohols produced in ABE fermentation. This functionality
can be used to form higher-molecular-mass hydrocarbons similar to
those found in current jet and diesel fuels. Here we describe the
integration of biological and chemocatalytic routes to convert ABE
fermentation products efficiently into ketones by a palladium-cata-
lysed alkylation. Tuning of the reaction conditions permits the pro-
duction of either petrol or jet and diesel precursors. Glyceryl
tributyrate was used for the in situ selective extraction of both acetone
and alcohols to enable the simple integration of ABE fermentation
and chemical catalysis, while reducing the energy demand of the
overall process. This process provides a means to selectively produce
petrol, jet and diesel blend stocks from lignocellulosic and cane sugars
at yields near their theoretical maxima.
Using a synthetic ABE mixture of pure acetone, n-butanol and
ethanol, we investigated the double alkylation of acetone to obtain
heptan-4-one (B in Fig. 1) (alkylation with ethanol), nonan-4-one
(D) (alkylation with one molecule each of ethanol and butanol) and
undecan-6-one (F) (alkylation with butanol) under transition-metal-
catalysed conditions (Fig. 1)11,12. Two possible side reactions in the
transition-metal-catalysed alkylation of acetone with primary alcohols
are self-condensation of the alcohol through the corresponding alde-
hyde (Guerbet reaction)13 and oligomerization of acetone into diace-
tone alcohol, mesityl oxide, cyclohexenones and other products14. In
particular, self-condensation of acetone affects the overall efficiency of
the desired process by lowering the ratio of ketone to alcohol in the
reaction mixture. Similarly, if too much butanol reacts through the
Guerbet pathway then not all of the acetone will be consumed; how-
ever, the Guerbet reaction does form higher branched alcohols that
could themselves be valuable fuel precursors or be combined with
acetone to form up to C19 ketones (Supplementary Fig. 1). The trans-
ition metal and base catalysts should therefore be chosen to minimize
these competing processes. As previously observed15, we found that
Natural biological routes to produce alcohols (ethanol and n-buta- solid potassium phosphate tribasic (K3PO4) was the optimal base for
the transition-metal-catalysed alkylation of acetone (Supplementary
nol) from carbohydrates have been known for more than 100 years,
and these compounds have been produced in fermentations at high Table 1). Using solid K3PO4 as a heterogeneous base, we observed only
titres (,100 and 15 g l21, respectively) and at yields near their theor- minor amounts of products from acetone condensation (E) and no
Guerbet products.
Our tests of different transition-metal catalysts (Ir, Ru, Rh, Pt, Pd
etical maxima. These low-molecular-mass compounds are primarily
suitable as additives or in certain situations (for example, E100 flex fuel
vehicles) as alternatives to petrol. Advances in metabolic engineering and Ni) with K3PO4 in toluene for the alkylation reaction revealed that
have enabled the biological production of several higher-molecular- palladium was superior to the other metals (Supplementary Table 2)15.
mass jet and diesel fuel compounds from carbohydrates, but until now Further, trials of various palladium precursors showed a marked effect
these processes have suffered from low titres and yields4–6
.
of the precursor on the outcome of the reaction (Supplementary Table
3). Although most of the palladium precursors examined catalysed the
alkylation of acetone, Pd/Cwas significantly more active than the other
palladium precursors at 110 uC in toluene.
Here we propose a chemical route to convert fermentation products
from a variety of renewable carbohydrate sources into hydrocarbons
that can be used for petrol, jet fuel and diesel. Because solventogenic
fermentation products have lower carbon numbers than are appropri-
Increasing the reaction temperature to 145 uC resulted in a minor
ate for these fuels, coupling chemistry can be used to produce mole- improvement in the selectivity for doubly alkylated products, but the
cules that are larger than these natural fermentation products, ideally overall efficiency of the reaction declined with increasing temperature
achieved byexploiting the functionalitiesinherent inthestarting mate- (Fig. 2a). Increasing the amount of base led to a marked improvement
rials7–9. The acetone, n-butanol and ethanol (ABE) mixture produced in both the selectivity for double alkylation and the overall yield. With
by Clostridium acetobutylicum in a 2.3:3.7:1 molar ratio (3:6:1 mass 1.28 molar equivalents of K3PO4 with respect to total alcohols (ethanol
ratio) provides such a system. C. acetobutylicum is able to produce and butanol) at 145 uC, an overall 86% molar yield, based on acetone as
ABE from a variety of sugars and carboxylic acids10, providing the the limiting reagent, of identified ketone products was obtained
flexibility needed to accommodate regionally specific feedstocks. The (Fig. 2b). We envisaged that tuning the reaction conditions would
ABE products harbour both the nucleophilic a-carbons of the acetone afford selective monoalkylation of acetone to obtain petrol-range
and the electrophilic a-carbon of the alcohols. These paired function- hydrocarbon chains. Consequently, alkylation was examined at vari-
alities enable us to construct higher alkanes from two-carbon, three- ous temperatures with only 0.32 molar equivalents of K3PO4. Under
carbon and four-carbon precursors by the alkylation of acetone with these conditions, pentan-2-one and heptan-2-one became the major
the electrophilic alcohols. As shown in Fig. 1, alkylation under suitable products, with C5–C7 species accounting for nearly 50% of the overall
conditions results in C5–C11 or longer-chain ketones, which may be yield, with respect to acetone as the limiting reagent. The best result
1Department of Chemistry, University of California, Berkeley, California 94720, USA. 2Energy Biosciences Institute, University of California, Berkeley, California 94720, USA. 3Department of Chemical and
Biomolecular Engineering, University of California, Berkeley, California 94720, USA. 4Chemical Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.
*These authors contributed equally to this work.
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RESEARCH LETTER
HO
OH
OH
HO
O
HO
O
O
HO
HO
O
OH
HO
HO
OH
OH
OH
Oligomerization
O
O
OH
HO
HO
R
O
Dimerization
+
R
Guerbet product
OH
R = Prn; E
O
O
C5–C7 alkanes
C
A
OH
HO
OH
HO
C7–C11 alkanes
O
O
O
B
D
F
C7 and higher alkanes
C9 and higher alkanes
C11 and higher alkanes
Figure 1
|
A general approach to the transition-metal-catalysed production of biofuels from the ABE fermentation mixture.
was obtained at 145 uC with the production of pentan-2-one and unbranched products. This combination of kinetic controls on the
heptan-2-one in 11% and 38% yield, respectively, along with 17% of alkylation reaction therefore enables the conversion of a mixture of
double-alkylated products.
We explored the mechanism of the reaction by following the reac-
renewable metabolites into a well-defined range of ketone products.
One important aspect of any catalytic process is the lifetime of the
tion progress. As shown in Fig. 2c, monoalkylation of acetone with catalyst, which we investigated by adding additional starting material
butanol and ethanol occurred quickly within the first 1.0–1.5 h of the after the reaction had been allowed to proceed to near completion. In a
reaction to produce pentan-2-one and heptan-2-one. These species typical experiment using Pd/C and 0.96 molar equivalents of K3PO4 at
underwent further reaction to form double-alkylated products. No 145 uC, we added an additional ABE mixture equivalent to the initial
aldehydes were observed during the reaction, suggesting that the charge at 10 and 20 h after the beginning of the reaction and measured
aldehyde intermediates were present in very low concentrations and the overall yield at 10, 20 and 30 h. In this case, the overall yield based
reacted rapidly with acetone and other ketones. Hence, the forma- on the total acetone feed for each time point was only slightly
tion of Guerbet products was minimized, and acetone alkylation decreased: 80% at 10 h, 72% at 20 h, and 61% at 30 h, demonstrating
predominated.
that the catalysts remained active and continued to convert each new
amount of starting material. Furthermore, catalytic activity was main-
tained for 25 h under flow reaction conditions (Supplementary Fig. 4).
The catalyst also retained activity in the absence of added solvent,
achieving catalytic turnover numbers of more than 3,000 (moles of
product per mole of palladium; Supplementary Tables 4 and 6).
Although wet organic solvents could be used for the alkylation reac-
tion, no reaction was observed in water. The addition of water was
found to slow the overall reaction, resulting in lower double alkylation
and overall yields (Supplementary Fig. 2).
On the basis of our experiments and previous studies of the alkyla-
tion of N-nucleophiles and C-nucleophiles11,16,17, we propose a mech-
anism for the double alkylation of acetone with ethanol and butanol
(Fig. 3). The alcohols (butanol and ethanol) are dehydrogenated by the
palladium catalyst, generating the reactive aldehydes and hydrogen. At
this stage the aldehydes undergo either a self-aldol reaction to form the
Guerbet product precursor, or an aldol reaction with acetone. Under
the conditions employed here, condensation with acetone seems to be
favoured, because acetone is present at much higher concentrations
than the transient aldehydes. Subsequent dehydration of the aldol
To overcome the sensitivity of the catalysts to water, it was of inter-
product under the reaction conditions furnishes a,b-unsaturated est to investigate highly selective water-immiscible extractants to
ketones that undergo palladium-catalysed hydrogenation with the remove acetone, n-butanol and ethanol in situ. In situ removal of
hydrogen generated in the first step. Completing the same cycle with the inhibitory product n-butanol during fermentation has been shown
monoalkylated products affords the expected double alkylation pro- to increase solvent titres and yields, decrease distillation costs signifi-
ducts. The relative ease of attack by the unsubstituted versus the sub- cantly, and decrease water use and reactor sizes18–21. However, the
stituted a-carbon of the monoalkylated products leads primarily to extractants typically employed are only capable of efficiently removing
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LETTER RESEARCH
Figure 2
|
Product distribution in Pd-catalysed alkylation as a function of
a
b
c
reaction parameters and time course. a, Effects of temperature on product
yields and distribution. Red, pentan-2-one; dark blue, heptan-4-one; green,
heptan-2-one; orange, nonan-4-one; purple, 2-methylnonan-4-one; light blue,
undecan-6-one. Yields based on acetone. Reaction conditions: acetone
(2.3 mmol), ethanol (1 mmol), butanol (3.7 mmol), 5% Pd/C (0.01mmol),
K3PO4 (3 mmol), toluene (1.5 ml), temperature (x axis), 20 h. b, Effect of K3PO4
loading on product yields. K3PO4 loading is represented as molar equivalents
with respect to total alcohol loading (ethanol 1 butanol). Reaction conditions
were identical to the temperature studies with the exception of K3PO4 loading
(x equivalents) and T 5 145uC. c, Time course of product distributions during
the palladium-catalysed alkylation reaction. Colours as in b. Reaction
conditions: acetone (4.6 mmol), ethanol (2 mmol), butanol (7.4 mmol), 5%
Pd/C (0.02 mmol), K3PO4 (9 mmol), toluene (3 ml), 145uC.
80
60
40
20
0
110
130
145
160
180
Temperature (°C)
acetone (Kd 5 1.1) and n-butanol (Kd 5 2.6) from aqueous solution22.
Ethanol, however, remains preferentially in the aqueous phase
(Kd 5 0.2). In addition, glyceryl tributyrate efficiently removed several
of the inhibitors of biofuel fermentation (for example furfural, p-cou-
maric acid and ferulic acid) found in acid-pretreated lignocellulosic
biomass (Supplementary Table 5). This affords the simultaneous
removal of residual inhibitors and the desired product during biofuel
fermentation, a key advantage over other recovery technologies23.
Studies of growth inhibition and cell viability with the use of up to
1:1 volume ratios of extractant to media showed glyceryl tributyrate to
be non-toxic to C. acetobutylicum (data not shown). A 60-h 2-l fer-
mentation of C. acetobutylicum on glucose with a 1:1 volume ratio of
medium and glyceryl tributyrate produced 40.8 g of solvents with
16.4 g of n-butanol, 3.7 g of acetone and 0.8 g of ethanol, partitioning
into the extractant phase (Fig. 4). These solvents were produced from
105 g of glucose and 1.6 g of acetate, achieving an overall ABE weight
yield of 90% of the theoretical maximum. Similar results were observed
with sucrose, the main carbohydrate of sugar cane, as the primary
carbon source (Supplementary Fig. 5).
80
60
40
20
0
0.32
0.64
0.98
1.28
Molar equivalents of K3PO4
40
30
20
10
0
The in situ removal of ABE from the aqueous phase by a high-
boiling-point extractant such as glyceryl tributyrate decreases energy
requirements for product distillation24 and allows integration of the
biological and catalytic processes. Furthermore, any high-boiling
impurities present in the extractant (for example furfural, p-coumaric
acid or ferulic acid) would remain in the distilled extractant upstream
of the catalyst, thus having no effect on catalyst performance. Solvents
distilled from the extractant phase of the 2-l fermentation were as
reactive as the pure chemicals, achieving an overall molar yield of
0
100
200
1,200
Time (min)
n-butanol from the aqueous phase, leaving most of the acetone pro-
duced in the aqueous phase. Glyceryl tributyrate was identified as a
water-immiscible solvent capable of efficiently recovering both
O
HO
Oxidation–aldol–
Oxidation–aldol–
HO
O
dehydration–reduction
dehydration–reduction
HO
Pd
Pd
Pd + H
2
Pd + H
2
O
O
O
O
O
(Fast)
+ Base
(Slow)
O
OH
O
Base
O
H2O
HO
H2O
O
OH
Oligomerization
Figure 3
|
Possible mechanism for the palladium-catalysed alkylation of acetone.
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RESEARCH LETTER
ABE fermentation
Solvent extraction
Distillation
Catalytic upgrade by dehydrogenation
and aldol condensation
O
O
CO2
1.7%
93%
49.8%
O
ABE
ABE +
extractant
O
Extractant
phase
Glucose
Acetate
ABE + CO2
+ acetate
H2O
ABE
Sugars
< 0.5%
8.3%
O
Pd/C–K3PO4 (s)
145 °C
ABE + CO2
O
Extractant
Aqueous
phase
Y
overall = 90%
4.2%
2.8%
Extractant
Higher alkylated products: 17%*
Alcohol and related products: 9%
ABE + water/cells
Water/cells
(waste)
Figure 4
|
Block flow diagram for integration of ABE fermentation with
drying over molecular sieves; they were performed for 20 h in toluene at 145uC
chemical catalysis. Dashed lines represent proposed recycle streams for
with 1.28 molar equivalents of K3PO4. Yields are based on acetone. Asterisk,
continuous operation. The results correspond to a 2-l 60-h fed-batch extractive higher alkylated products and alcohol and related product yields were
fermentationwith glyceryl tributyrateto produce ABEfrom glucose. Alkylation approximated by FID response factor and were assumed to have incorporated a
reactions were performed on distilled solvents from the extractant phase after single equivalent of acetone.
gas chromatography–flame ionization detection, with dodecane as an internal
standard. Reactions conditions (including temperature, catalyst and base loading)
for individual alkylation reactions are described in Supplementary Methods.
Inhibitor extraction. Simulated clostridia fermentation medium as described in
Supplementary Methods was used to calculate glyceryl tributyrate distribution
coefficients. Acid-pretreated lignocellulosic inhibitors were produced from
Miscanthus 3 giganteus as described in Supplementary Methods. The liquid
hydrolysate was pH adjusted and inhibitor concentrations were analysed by gas
chromatography–mass spectrometry.
Fermentation conditions. Clostridium acetobutylicum ATCC824 was routinely
grown in clostridial growth medium (CGM) as described previously1. Fed-batch
fermentations were conducted in 3-l bioreactors (Bioengineering AG) with a 2-l
working volume. Additionalglucose and yeastextractwere added intermittentlyto
the cultureusing a concentrated solution of 450and 50 g l21, respectively. Cultures
were grown anaerobically at 37 uC under pH control as described in Sup-
plementary Methods. Sugars and major metabolites were measured in the aqueous
phase by high-pressure liquid chromatography as described previously
(Supplementary Fig. 5). Acetone, n-butanol and ethanol concentrations in the
extractant phase were measured by gas chromatography–flame ionization detec-
tion (Supplementary Fig. 5).
93% and 97% conversion of acetone (Fig. 4). Assuming complete
recovery of ABE products from both the extractant and aqueous
phases, ,20 g of C7–C15 products would be formed, which represents
,38% of the carbon contained in the glucose feed (Supplementary
Table 6). The combined selectivity of the fermentation and the alkyla-
tion results in this particularly high-yielding transformation of carbo-
hydrates into fuel ketones. In comparison, a previous study25 used
similar aldol reactions to convert the alcohols and ketones in an aque-
ous-phase reformate into C7 and higher species with a carbon yield of
42% from the intermediate oil feed and an overall carbon yield of 21%
from the initial sorbitol feed. The lower yield of higher ketones resulted
from the non-selective production of oxygenates that could not be
upgraded in the aldol reaction. The 38% carbon yield in the present
work does not account for the 15.1 g of lower-molecular-mass (C4–C6)
petrol precursors also produced, which would then raise the overall
carbon yield to ,58%. Further deoxygenation (Supplementary Fig. 6)
of the products obtained from alkylation of the ABE mixture by using
standard hydrotreating chemistry25–27 would yield alkanes compatible
with refinery infrastructure and suitable for blending with petrol, die-
sel and jet fuels.
Received 9 February; accepted 14 September 2012.
Using this controlled alkylation of acetone, n-butanol and ethanol,
we developed a high-yield method for transforming readily accessible
fermentation products from a variety of carbohydrates into precursors
for petrol, diesel and jet fuels. By catalytically upgrading low-carbon-
number fermentation products we are able to exploit highly efficient
metabolic pathways and achieve near theoretical yields28. Combined
with the near theoretical yields attained during the alkylation reaction
these higher-molecular-mass fuel precursors can be produced at rela-
tively high titre. The tunability of this reaction to produce predomi-
nantly petrol or jet and diesel blend stocks is a significant advantage
over other methods, and aligns well with current refining processes.
Although further improvements will be required for commercial
implementation, the results demonstrate that in situ extraction of the
productsfromtheABEfermentationcoupledwithcatalyticconversion
of these products can provide hydrocarbon fuel blend stocks at high
yields from biomass. The integration of extractive fermentation with
chemical catalysis is thus a novel and potentially enabling route for the
economical conversion of biomass into liquid transportation fuels.
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Supplementary Information is available in the online version of the paper.
Acknowledgements We are grateful to H.-J. Song for performing initial experiments on
the catalytic alkylation of acetone, and V. Mitchell for analysing acid pretreatment
hydrolysate inhibitors present in Miscanthus giganteus. F.D.T. and E.G. acknowledge
funding from the Director, Office of Science of the US Department of Energy, under
contract no. DE-AC02-05CH11231. This work was funded by the Energy Biosciences
Institute.
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Author Contributions Z.C.B. and P.A. contributed equally to this work. P.A. and S.S.
performed experiments on the chemical catalysts, and E.G. performed experiments in
the flow reactor. Z.C.B. optimized the fermentation andextractive processes. All authors
contributed to the conception of the experiments, discussion of the results and
preparation of manuscript.
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and butanol. Biotechnol. Bioeng. 31, 135–143 (1988).
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Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper. Correspondence
and requests for materials should be addressed to F.D.T. (fdtoste@berkeley.edu).
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