U.S. patent application number 13/954294 was filed with the patent office on 2014-02-06 for biological/electrolytic conversion of biomass to hydrocarbons.
This patent application is currently assigned to Ion Research, Inc.. The applicant listed for this patent is Ion Research, Inc., The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture. Invention is credited to Anthony B. Kuhry, Paul J. Weimer.
Application Number | 20140038254 13/954294 |
Document ID | / |
Family ID | 43974446 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038254 |
Kind Code |
A1 |
Kuhry; Anthony B. ; et
al. |
February 6, 2014 |
Biological/Electrolytic Conversion of Biomass to Hydrocarbons
Abstract
Hydrocarbon and hydrogen fuels and other products may be
produced by a process employing a combination of fermentation and
electrochemical stages. In the process, a biomass contained within
a fermentation medium is fermented with an inoculum comprising a
mixed culture of microorganisms derived the rumen contents of a
rumen-containing animal. This inoculated medium is incubated under
anaerobic conditions and for a sufficient time to produce volatile
fatty acids. The resultant volatile fatty acids are then subjected
to electrolysis under conditions effective to convert said volatile
fatty acids to hydrocarbons and hydrogen simultaneously. The
process can convert a wide range of biomass materials to a wide
range of volatile fatty acid chain lengths and can convert these
into a wide range of biobased fuels and biobased products.
Inventors: |
Kuhry; Anthony B.; (Skokie,
IL) ; Weimer; Paul J.; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
Ion Research, Inc. |
Washington
Skokie |
DC
IL |
US
US |
|
|
Assignee: |
Ion Research, Inc.
Skokie
IL
|
Family ID: |
43974446 |
Appl. No.: |
13/954294 |
Filed: |
July 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12760911 |
Apr 15, 2010 |
8518680 |
|
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13954294 |
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Current U.S.
Class: |
435/167 |
Current CPC
Class: |
Y02E 50/30 20130101;
C25B 1/02 20130101; Y02E 50/343 20130101; C12P 5/026 20130101; C12P
7/40 20130101; C12P 5/02 20130101; C12N 1/22 20130101; C12P 7/52
20130101; C12P 3/00 20130101; C12P 39/00 20130101; C12P 5/023
20130101; C12P 7/54 20130101; C12P 7/6409 20130101; C25B 3/04
20130101 |
Class at
Publication: |
435/167 |
International
Class: |
C12P 5/02 20060101
C12P005/02 |
Claims
1. A method for producing hydrocarbon and hydrogen fuels
comprising: a. fermenting a biomass material containing
fermentation medium with an inoculum comprising a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing animal and incubating under anaerobic conditions
and for a sufficient time to produce volatile fatty acids in said
medium; b. subjecting said volatile fatty acids or their salts to
electrolysis under conditions effective to convert said volatile
fatty acids or their salts to hydrocarbons and hydrogen; and
further comprising concentrating said volatile fatty acids in said
medium prior to said electrolysis, wherein said concentrating
comprises distillation, filtration, or capacitive deionization.
2. The method of claim 1 wherein said volatile fatty acids comprise
C-2 to C-6 straight or branched chain fatty acids or salts
thereof.
3. The method of claim 2 wherein said volatile fatty acids comprise
acetic acid, propionic acid, butyric acid, isobutyric acid,
2-methyl butyric acid, valeric acid, isovaleric acid, caproic acid
or salts thereof.
4. The method of claim 1 wherein said hydrocarbons comprise
alkanes, alkenes, dienes, trienes or mixtures thereof.
5. The method of claim 4 wherein said hydrocarbons comprise
methane, ethane, propane, butane, pentane, hexane, heptane, octane,
ethylene, propylene, butene, pentene, or isomers or mixtures
thereof.
6. The method of claim 1 wherein said fermenting is conducted
without the addition of a significant amount of methane production
inhibitors.
7. The method of claim 1 wherein said time is between 1 to 4
days.
8. The method of claim 7 wherein said time is between 2 to 3
days.
9. The method of claim 1 wherein said biomass comprises a
cellulosic biomass.
10. The method of claim 1 wherein said biomass is selected from the
group consisting of plants, plant parts, plant materials,
agricultural crops, forages, grasses, aquatic plants, bagasse, corn
stover, corn cobs, dried distillers grains, hay, flax straw, oat
hulls, wood, sawdust, paper products, paper processing wastes,
cardboard, yard or landscape waste, animal wastes, animal
processing wastes, animal carcasses, and spent sausage casings.
11. The method of claim 1 wherein said fermentation medium further
comprises a glycol or alcohol.
12. The method of claim 1 wherein said fermentation medium further
comprises one or more C-8 to C-22 fatty acids, and said
electrolysis converts said fatty acids or their salts to
hydrocarbons and hydrogen.
13. The method of claim 1 wherein said biomass is not subjected to
chemical or enzymatic pretreatment prior to said fermenting which
pretreatment would be effective to substantially increase the
availability of fermentable substrates in the biomass to said
microorganisms.
14. The method of claim 1 wherein said mixed culture of
microorganisms derived from the rumen contents are produced by
adaptation to anaerobic growth in a biomass-containing fermentation
medium.
15. The method of claim 1 wherein said inoculum further comprises a
supplemental inoculum selected from the group consisting of
Clostridium kluyveri, Clostridium butyricum, Clostridium
tyrobutyricum, Butyrvibrio fibrisolvens, a mixed culture of
microorganisms derived from the rumen, sewage sludge, landfills,
soil or aquatic environments, a mixed culture of microorganisms
derived from the gut of insects, and mixtures thereof.
16. The method of claim 1 further comprising a contacting said
medium containing said volatile fatty acids with a second inoculum
selected from the group consisting of Clostridium kluyveri,
Clostridium butyricum, Clostridium tyrobutyricum, Butyrvibrio
fibrisolvens, a mixed culture of microorganisms derived from the
rumen, sewage sludge, landfills, soil or aquatic environments, a
mixed culture of microorganisms derived from the gut of insects,
and mixtures thereof, and incubating under anaerobic conditions and
for a sufficient time to further increase the production of, or
change the molar proportions of, volatile fatty acids in said
medium.
17. The method of claim 1 further comprising removing one or more
of cells of said microorganisms, lignin-containing residues, or
carbonates or other non-carboxylate anions from said medium prior
to said electrolysis.
18. The method of claim 17 wherein said cells, lignin-containing
residues, or carbonates or other non-carboxylate anions are removed
by filtration, flocculation, settling, centrifugation,
precipitation or combinations thereof.
19-41. (canceled)
42. A method for producing hydrocarbon and hydrogen fuels
comprising: a. fermenting a biomass material containing
fermentation medium with an inoculum comprising a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing animal and incubating under anaerobic conditions
and for a sufficient time to produce volatile fatty acids in said
medium; b. subjecting said volatile fatty acids or their salts to
electrolysis under conditions effective to convert said volatile
fatty acids or their salts to hydrocarbons and hydrogen; and
further comprising extracting said volatile fatty acids from said
medium prior to said electrolysis, wherein said extracting
comprises a liquid-liquid extraction of said medium with an alcohol
followed by addition of salt.
43. A method for producing hydrocarbon and hydrogen fuels
comprising: a. fermenting a biomass material containing
fermentation medium with an inoculum comprising a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing animal and incubating under anaerobic conditions
and for a sufficient time to produce volatile fatty acids in said
medium; b. subjecting said volatile fatty acids or their salts to
electrolysis under conditions effective to convert said volatile
fatty acids or their salts to hydrocarbons and hydrogen; further
comprising adding glycerol to said fermentation medium to increase
the production of propionic acid during said fermenting.
44. A method for producing hydrocarbon and hydrogen fuels
comprising: a. fermenting a biomass material containing
fermentation medium with an inoculum comprising a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing animal and incubating under anaerobic conditions
and for a sufficient time to produce volatile fatty acids in said
medium; b. subjecting said volatile fatty acids or their salts to
electrolysis under conditions effective to convert said volatile
fatty acids or their salts to hydrocarbons and hydrogen; and
further comprising adding one or more C-8 to C-22 fatty acids or
their salts to said fermentation medium, and said electrolysis
converts said fatty acids or their salts to hydrocarbons and
hydrogen.
45. A method for producing hydrocarbon and hydrogen fuels
comprising: a. fermenting a biomass material containing
fermentation medium with an inoculum comprising a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing animal and incubating under anaerobic conditions
and for a sufficient time to produce volatile fatty acids in said
medium; b. subjecting said volatile fatty acids or their salts to
electrolysis under conditions effective to convert said volatile
fatty acids or their salts to hydrocarbons and hydrogen; wherein
said electrolysis is conducted using carbon or graphite anode
electrodes.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of Ser. No. 12/760,911, filed on Apr.
15, 2010, pending, which claims the benefit under 35 U.S.C. 1.19(e)
of U.S. provisional 61/212,949, filed Apr. 17, 2009, the contents
each of which are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is drawn to a novel method to produce
hydrocarbon and hydrogen fuels simultaneously from biomass by a
combination of fermentation and electrolysis.
[0004] 2. Description of the Prior Art
[0005] The essential role of renewable fuels in fostering
economically and environmentally sustainable growth is now widely
recognized. Although technologies for providing electricity via
wind, hydropower, and direct biomass consumption are widely
employed, the production of liquid transportation fuels remains the
greatest energy security issue in the U.S. today. Corn ethanol,
once touted as a major future source of motor fuels, has succeeded
in the marketplace only through substantial government subsidies to
producers, and questions regarding its environmental
sustainability, net energy balance, and role in exacerbating food
shortages have now come to the fore.
[0006] Cellulosic ethanol is considered to be a more promising
long-term source of transportation fuels (Lynd et al., 2002).
Cellulosic materials are available in much larger quantities; can
be produced on more marginal lands; feature a much larger net
energy balance; and do not have a competing human food use.
However, despite major funding efforts, development of economically
viable cellulosic ethanol technologies have not yet attained
commercial success. Chemical pretreatment is considered necessary
to enhance the accessibility of the feedstock to enzymatic attack,
yet such pretreatment adds costs, generates a waste stream, and
produces certain chemical products that inhibit sugar fermentation.
Contaminating microbes such as lactic acid bacteria can convert
considerable amounts of the hydrolyzed sugars to products other
than ethanol, necessitating expensive control measures to permit
maintenance of the fermentative monoculture; this is already a
major problem in the corn ethanol industry, which has become one of
the major users of antibiotics in the U.S. (Olmstead, 2009).
Moreover, the most active of the ethanol producers ferment only the
hexose fraction of carbohydrate, and even the best strains that
utilize the pentose sugar fraction only ferment the carbohydrate
fraction but not the other components (proteins, nucleic acids,
lipids, organic acids and other phytochemicals) that represent a
substantial proportion of plant biomass. This greatly reduces the
yield of fuel product. Finally, no obvious use has emerged for the
unfermented residue--a critical shortcoming given its likely large
volume and the likely low profitability of the cellulosic ethanol
process.
[0007] Ethanol has a relatively low energy density (with attendant
reductions in vehicle miles-per-gallon), and for most
gasoline-powered vehicles can only be blended to a low proportion
of the total fuel mixture. These disadvantages, along with the slow
pace of development of cellulosic ethanol technology, have
stimulated a search for routes to convert biomass to hydrocarbon
fuels (Regalbuto, 2009). Several such schemes have been proposed.
Some of these rely on chemical conversion of biomass materials
under heat and pressure (often in the presence of expensive
catalysts) and yield either liquids (e.g., pyrolytic oils) or gases
that can be reformulated into liquid motor fuels. Several
biologically based processes have also been proposed. These
processes face some formidable hurdles. For example, photosynthetic
algal-based processes either require large areas for cultivation
(because of the shallow depth of the photic layer under intense
cultivation, while the "dark algal" process or processes based on
bacteria that have been genetically engineered for hydrocarbon
biosynthesis require sugars as the feedstock (which revisits the
high cost of cellulolytic enzymes that has hampered ethanol
production via simultaneous saccharification and fermentation).
Biorefinery Processes:
[0008] Biorefinery processes that produce various types of biobased
fuels from biomass are well known. It is known, for example, that
natural mixtures of anaerobic microbial cultures that work together
to digest biomass material occur in habitats such as the rumen of
ruminant animals, sewage sludge, soil, landfills, aquatic
(freshwater, marine, and brackish) sediments, and insect (e.g.,
termite) guts. These mixed microbial cultures work in concert to
provide the necessary enzymes to convert biomass into organic
acids. The organic acids are primarily "Volatile Fatty Acids" (VFA)
which includes straight and branched chain fatty acids with carbon
chain lengths from C2 to C6.
[0009] As a result of thousands of years of natural evolution of
biomass processing, the ruminal fermentation is a particularly
attractive process because it is natural, rapid, and efficient
(Hungate, 1950, 1966); it converts most biomass components to
useable products (Weimer et al., 2009); and it can readily be
conducted in a biorefinery (i.e., in vitro in bioreactors; Goering
and Van Soest, 1970).
[0010] Ruminal microbes have long been known to convert cellulosic
and other feed materials to VFA (Hungate, 1950, 1966), and have
also been used for treating organic wastes. In the RUDAD
(Rumen-Derived Anaerobic Digestion) process (Zwart et al., 1988),
mixed ruminal microbes (including both bacteria and protozoa) are
used in a primary stage fermentation to convert cellulosic and
other organic wastes to VFA that are passed to a second reactor in
which the VFA are converted by other microorganisms to methane and
carbon dioxide. The process is used exclusively for waste
treatment, although as in many other wastewater treatment plants,
the methane produced can be used as a fuel to offset the operating
energy requirement of the treatment plant. Differences in the
growth rates of microbes in the two reactors, along with problems
in maintaining flocculation in the bioreactors, have limited the
utility of the RUDAD process (Hack and Vellinga, 1995). An improved
process (described in Hack and Vellinga, 1995, U.S. Pat. No.
5,431,819) employs a three-stage process in which the solids
fraction from the primary cellulosic fermentation is further
degraded in another reactor while the liquid phase from the primary
fermentation is further treated in a third, methanogenic,
reactor.
[0011] Biorefinery-produced organic acids may be converted into
useful fuels by different methods (e.g., those of Holtzapple and of
Bradin). Holtzapple et al. (1999) describe processes that produce
biofuels of mixed alcohols (MixAlco) and other products such as
mixed ketones, by thermochemical treatment of organic acids that
are produced by the action of natural microbial mixtures on biomass
material. These processes are described in detail in U.S. Pat. Nos.
5,693,296; 5,865,898; 5,874,263; 5,962,307; 5,986,133; 5,969,189;
6,043,392; 6,262,313; 6,395,926, and 6,478,965. The natural mixed
microbial cultures used by Holtzapple are obtained primarily from
anaerobic sewage digesters comprising municipal solid waste (MSW)
and sewage sludge (SS) that transform chemically pretreated biomass
material into volatile fatty acid (VFA) mixtures as described in
U.S. Pat. No. 6,043,392 under the "Pretreatment and Fermentation"
section. The biomass components that are converted into organic
acids are: cellulose, hemicellulose, pectin, sugar, protein, and
fats. These processes are characterized by alkaline pretreatment of
biomass, followed by the fermentation process, followed by
dewatering, thermal conversion to produce ketones, and addition of
hydrogen plus catalyst, heat, and pressure, all of which are needed
to produce mixed alcohol fuel products. Total reaction processing
times are therefore, necessarily long. In order to facilitate VFA
accumulation during the fermentation stage, Holtzapple's process
typically uses a "stuck" fermentation, in which microbial methane
formation is prevented by keeping the pH low and/or by adding
specific (toxic) inhibitors of methanogenesis (e.g., bromoform
[CHBr.sub.3], or iodoform [CHI.sub.3]). This causes the
fermentation intermediates (organic acids) to accumulate, but also
leaves these toxic inhibitors of methanogenesis in the wastewater
stream necessitating further cleanup.
[0012] Biofuels can also be produced by using specific types of
microbes, as opposed to mixed microbial cultures, in order to
produce specific types of hydrocarbons exclusively from sugars. For
example, Bradin (2007; publication WO 2007/095215 A2) describes a
process that produces n-hexane from the fermentation of sugars,
using specific natural bacteria or yeast that produce specifically
butyric acid as a single product. The butyric acid is then
subjected to Kolbe dimerization electrolysis to form n-hexane.
However, the preferred microbes are selected to reduce or eliminate
acetic acid as a byproduct because it lowers butyric acid yield.
Moreover, the preferred microbes must be either naturally isolated
or genetically engineered pure cultures, and must be cultivated
under controlled conditions to prevent culture contamination, thus
reducing flexibility and increasing the cost. In addition, in order
to produce sugars from complex carbohydrates such as cellulose and
hemicellulose, specific enzymes must be added to the biomass, thus
further adding to the reaction processing time and the cost. The
Bradin process also requires the separation of lignin from
cellulose and hemicellulose, by other enzymes or oxidizing agents
to delignify the biomass prior to fermentation. The single n-hexane
product also requires further refinement in order to be used as a
transportation fuel.
[0013] Anaerobic fermentations of plant biomass yield a variety of
fermentation end products having high potential energy. Some of
these products, like ethanol or butanol, can be recovered by
distillation and used directly as motor vehicle fuels. Others, like
VFA (e.g., acetic, propionic or butyric acids) can be produced in
substantial quantities, but are not directly usable as fuels.
[0014] The literature contains a number of examples of conversions
of carboxylic acids to hydrocarbons using electrochemistry. For
example, the alkyl groups of fatty acids can be combined
tail-to-tail during anodic electrolytic decarboxylation to yield
alkanes (e.g., ethane from acetic acid, butane from propionic acid,
etc.), the so-called Kolbe reaction. The Kolbe reaction can proceed
via dimerization of similar radical species to produce single
alkanes, or cross-radical reactions with dissimilar radical species
to produce alkane mixtures (see Table 1). Moreover, fatty acids can
be partially cleaved and converted to alkenes (e.g., ethylene from
propionic acid) by the so-called Hofer-Moest reaction. The
Hofer-Moest reaction can produce alkenes via deprotonation and
alcohols via substitution. Under certain reaction conditions,
dienes and trienes can also be produced. The hydrocarbon reaction
formulas are shown in Formula 1, comparing one-electron (Kolbe) and
two-electron (Hofer-Moest) schemes for electrolytic decarboxylation
(Adapted from Lund [2001] and Seebach et al. [1995]).
Kolbe and Hofer-Moest Reactions
I. Kolbe Electrochemical Decarboxylative Radical Coupling
(Dimerization)=Single Alkanes:
[0015] Anode:
2RCO.sub.2.sup.-(aq).fwdarw.2R..fwdarw.R--R+2CO.sub.2(g)+2e.sup.-
Cathode: 2H.sup.+(aq)+2e.sup.-.fwdarw.H.sub.2(g)
II. Kolbe Electrochemical Decarboxylative Cross-Radical
Coupling=Alkane Mixtures:
[0016] Anode:
R'CO.sub.2.sup.-(aq)+RCO.sub.2.sup.-(aq).fwdarw.R'.R..fwdarw.R'--R+2CO.su-
b.2(g)+2e.sup.-
Cathode: 2H.sup.+(aq).fwdarw.H.sub.2(g)
III. Hofer-Moest Electrochemical Oxidative Decarboxylation
Deprotonation)=Alkenes:
[0017] Anode:
R'RCO.sub.2.sup.-(aq).fwdarw.R'R.sup.+.fwdarw.R'.dbd.R+CO.sub.2(g)+2e.sup-
.-
Cathode: 2H.sup.+(aq)+2e.sup.-.fwdarw.H.sub.2(g) Formula 1
[0018] The Kolbe and Hofer-Moest reactions are among the oldest
reactions described in electrochemistry. Although they can readily
convert VFA to alkanes and alkenes (or alcohols and esters), they
have generally been used commercially in synthesis of low-volume
specialty chemicals.
Electrochemical Conversion of Fatty Acids to Alkanes from
Biomass:
[0019] As a result of petroleum price increases due to increased
demand, many processes for alternatives to petroleum liquid fuels
were developed in the 1970's in the U.S. and other countries. Most
of the emphasis has been on ethanol production, and little was
devoted to the production of renewable liquid alkane hydrocarbon
fuels. Alkane liquid fuels research was performed in the late
1970's concerning the feasibility of Kolbe electrolysis of mixed
bacterial anaerobic fermentations using inocula from sewage sludge
(Levy et. al., 1983). The research disclosed that these
microorganisms converted specifically the sugar portion (hexose and
pentose sugars) from both cellulose and hemicellulose to organic
acids. The fermentation produced variable amounts of C2 to C6
carbon chain VFA including acetic, propionic, butyric, valeric, and
caproic acids. These carboxylic acids were produced by nonsterile
anaerobic fermentations. The higher chain VFA (butyric, valeric and
caproic acids) were separated and concentrated by liquid-liquid
extraction with kerosene. It was theorized that the higher chain
VFA could be treated by electrolytic oxidation (Kolbe and
Hofer-Moest electrolysis) to produce hydrocarbons, alcohols, and
esters. The lower chain VFA (acetic, propionic) remained in the
primary fermentor as a feedstock in order to produce higher chain
VFA in subsequent primary fermentations. This fermentation using
sewage sludge at long residence times (at least 5 to 18 days or
longer) necessitated the suppression of the methane-forming
microorganisms (methanogens) by the use of a chemical inhibitor
(viz. BES, 2-bromoethane sulfonic acid) similar to the Holtzapple
process mentioned above. Thus, despite these advances, the need
remains for improved techniques for producing hydrocarbon fuels
from renewable resources.
SUMMARY OF THE INVENTION
[0020] We have now discovered a novel process for producing
hydrocarbon fuels and other products by a process employing a
combination of fermentation and electrochemical stages (referred to
as herein as Biological-Electrolytic Conversion, or BEC). In this
process, a biomass-containing fermentation medium is fermented with
a mixed culture of microorganisms derived the rumen contents of a
ruminant animal, and incubated under anaerobic conditions and for a
sufficient time to produce volatile fatty acids. The resultant
volatile fatty acids (VFA) are then subjected to electrolysis under
conditions effective to convert them to gaseous and liquid
hydrocarbons and hydrogen gas (H.sub.2), with carbon dioxide
(CO.sub.2) as a co-product. The process of this invention can
produce a wide range of VFA chain lengths and can convert each of
these into biobased fuels and biobased products.
[0021] The process uses the primary ruminal fermentation, in vitro,
for a rapid, high-yield conversion, and can also use an optional
secondary fermentation with an augmented microbial inoculum to
convert lower chain volatile fatty acids to higher chain volatile
fatty acids, if desired. An additional separate fermentation can be
used to convert carbon dioxide (CO.sub.2) and hydrogen (H.sub.2)
produced from the in vitro ruminal fermentation and the
electrolysis stages, respectively, into acetic acid, which can then
be included as a feedstock in a subsequent electrolysis stage.
Alternatively, this carbon dioxide (CO.sub.2) and hydrogen
(H.sub.2) can be converted in a separate bioreactor to methane gas.
This increases the overall yield of the fuel products, and utilizes
carbon dioxide (CO.sub.2) as a fuel-producing feedstock instead of
generating a waste product. Thus, as an industrial process, BEC can
convert virtually any type of biomass into a variety of hydrocarbon
fuels and hydrogen gas (H.sub.2) without the high temperatures or
high energy requirements that are inherent in some other conversion
methods, such as pyrolysis.
[0022] In accordance with this discovery, it is an object of this
invention to provide a rapid, flexible, inexpensive, and efficient
process for the production of hydrocarbon and hydrogen fuels from
biomass.
[0023] Another object of this invention is to provide a process
that uses mixed cultures from ruminal inocula to produce volatile
fatty acids from biomass in a single-stage bioreactor, which
volatile fatty acids may then be converted into a plurality of
hydrocarbon products and hydrogen gas (H.sub.2) in a second reactor
using a non-biological process employing electrochemistry.
[0024] Another object of this invention is to provide a process for
converting mixtures of organic acids produced by microbial
anaerobic fermentation of biomass into a variety of gaseous and
liquid hydrocarbon fuels, hydrogen and other chemical products, via
electrolytic decarboxylation.
[0025] Another object of this invention is to provide a process for
combining organic acids obtained by anaerobic fermentation of
biomass with other organic acids already present in plant or animal
biomass material, and converting these combined organic acid
mixtures into a variety of useful hydrocarbon fuels, hydrogen and
other chemical products, via electrolytic decarboxylation.
[0026] Another object of this invention is to provide a process for
combining organic acids obtained by anaerobic fermentation of
biomass with other industrial organic chemicals and converting
these combined organic mixtures into a variety of useful
hydrocarbon fuels, hydrogen and other chemical products, via
electrolytic decarboxylation.
[0027] Another object of this invention is to provide a process
that will offer a solution to the ever-growing environmental
problem of landfilling cellulosic biomass waste, by economically
converting this waste into hydrocarbon fuels, hydrogen and other
chemical products, eliminating the need for landfilling of this
material.
[0028] Another object of this invention is to provide a process
that will offer an economic solution to biomass waste disposal by
utilizing damaged, spoiled, or spent cellulosic commodities to
produce hydrocarbon fuels, hydrogen and other chemical products by
reprocessing them instead of discarding in landfills.
[0029] Other objects and advantages of this invention will become
readily apparent from the ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows a flow diagram describing a preferred
embodiment of the invention for producing hydrocarbon fuels,
hydrogen, and other biobased products from biomass.
DEFINITIONS
[0031] The following terms are employed herein:
[0032] Biomass can be any plant or animal material containing
carbohydrate (including cellulose, hemicelluloses, starch, pectins,
and fructans), protein, nucleic acid, organic acid, or fat. The
term biomass refers to any organic matter that is available on a
renewable or recurring basis, such as, but not limited to
agricultural crops and trees, wood and wood wastes and residues,
plants (including aquatic plants), grasses, residues, fibers, and
animal wastes, food wastes, municipal wastes, and other waste
materials.
[0033] The term biobased fuel or biofuel refers to any
transportation fuel produced from biomass.
[0034] The term biobased product refers to an industrial product
(including chemicals, materials, and polymers) produced from
biomass, or a commercial or industrial product (including animal
feed and electric power) derived in connection with the conversion
of biomass to fuel.
[0035] Hydrocarbon biobased fuels consist exclusively of carbon and
hydrogen, such as alkanes, alkenes, dienes, and trienes. Other
chemical biobased products include carbon and hydrogen components
combined with other chemical elements such as oxygen, nitrogen,
etc.
[0036] The term Biological-Electrolytic Conversion or "BEC" is used
herein to describe the process of the invention disclosed herein.
The BEC is a biorefinery process that converts biomass into
biobased fuels and biobased products.
[0037] The conversion of biomass into organic acids refers to the
conversion to volatile fatty acids (VFA), which includes straight
and branched chain fatty acids with carbon chain lengths from C2 to
C6, including but not limited to acetic, propionic, butyric,
isobutyric, 2-methyl butyric, valeric, isovaleric, and caproic
acids. Medium Chain Fatty Acids (MCFAs) and Long Chain Fatty Acids
(LCFAs) are naturally produced from certain plant and animal
products and together span the range of carbon chain lengths from
C8 to C22. This invention describes a process that converts VFA, by
itself, into biobased fuels and biobased products. Additionally,
this invention describes a process that converts combinations of
both VFA and VFA/MCFA/LCFA into many different biobased fuels and
biobased products (see Table 1). As used herein, MCFA refers to
both MCFA and LCFA.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The preferred inoculum for use herein in the fermentation to
produce the volatile fatty acids from biomass is a mixed culture of
microorganisms derived from the rumen contents of a
rumen-containing (ruminant) animal. We have discovered that the use
of a ruminal inoculum significantly reduces the length of the
fermentation and increases yields in comparison to inocula prepared
from other sources such as sewage sludge. Moreover, use of the
ruminal inoculum also eliminates the need to suppress methane
production during the primary fermentation with chemical
inhibitors; the primary fermentation using ruminal inocula to
produce volatile fatty acids from biomass as described in this
invention is preferably conducted without the addition of methane
production inhibitors. The time course for the ruminal fermentation
without methanogenic inhibitors is typically 2-3 days, as opposed
to 5 to 20 days or longer for anaerobic digestion with sewage
sludge inocula and methanogenic inhibitors. The fermentation time
course is affected by biomass loading where higher concentrations
of biomass solids require longer residence times. As a comparison,
fermentation time course for human and animal sewage conversion to
methane without inhibitors normally takes about 3 weeks. Another
measure of the fermentation time course is the first-order rate
constant (k) for conversion of particular biomass components:
TABLE-US-00001 Rumen Sewage Sludge k for cellulose
0.06-0.1/hr.sup.a 0.09/day (0.0038/hr).sup.c k for starch
0.384/hr.sup.b 0.34/day (0.014/hr).sup.c .sup.aWeimer, et al.,
1990. .sup.bWeimer and Abrams, 2001. .sup.cLevy et al., 1983.
The use of the ruminal inocula provides other advantages as well.
The microorganisms of this mixed culture produce their own enzymes
for hydrolyzing and fermenting complex substrates such as
cellulose, hemicelluloses, pectins, starches, sugars, proteins,
nucleic acids, and dicarboxylic and tricarboxylic acids to the
high-energy end product volatile fatty acids. Thus, the biomass
substrate does not need to be chemically or enzymatically
hydrolyzed to low molecular weight sugars prior to the
fermentation, and minimal additional nutrients are used.
Furthermore, unlike processes using other inocula, the ruminal
inoculum produces volatile fatty acids rather than ethanol or
methane as the primary fermentation products. The microorganisms of
the ruminal inoculum also convert not only the carbohydrate
(hexoses, pentoses, and their polysaccharides) portion of the
biomass, but also the protein, nucleic acid, dicarboxylic and
tricarboxylic acid fractions, and some of the lipid fraction to
volatile fatty acids. Therefore, yields of volatile fatty acids
from these mixed culture fermentations are much higher than in
conventional biomass fermentations employing
carbohydrate-fermenting microbes. The protein fermentation by the
rumen-derived mixed culture is particularly beneficial, in that
some of the volatile fatty acids produced (in addition to acetic,
propionic, butyric and valeric acids) include 2-methylpropionic
(isobutyric), 2-methylbutyric, and 3-methylbutyric (isovaleric)
acids. These branched-chain volatile fatty acids, upon subsequent
electrolytic conversion, will yield branched-chain hydrocarbons
that may have improved fuel performance properties.
[0039] The ruminal inoculum for use herein may be obtained from any
ruminant animal, although caprine (goat), ovine (sheep) and bovine
(cattle) species are preferred, and bovine is particularly
preferred. The ruminal contents, primarily liquid although solids
may be included, can be collected from the ruminant animal through
an implanted rumen fistula, or through stomach tube,
ruminocentesis, or directly from rumen contents at slaughter. Once
collected the ruminal contents may be used directly as the inoculum
in the fermentation herein. However, in a preferred embodiment, the
mixed populations of microorganisms in the contents are first
subjected to one or a plurality of successive, small-scale
anaerobic fermentations with the desired biomass substrate prior to
their use in a larger-scale fermentation. This adaptation of the
rumen microorganisms increases their tolerance to the volatile
fatty acids, and increases the molar concentration of volatile
fatty acids in the fermentation. Although the rumen contents used
herein contain a diverse mixed population of microorganisms, the
microbial consortia are relatively stable in culture, and can be
maintained by sequential transfer without sterilization of the
substrate or reactor vessels. The ruminal inocula from these
enrichment cultures are also essentially devoid of the protozoa
that reduce the overall efficiency of the fermentation by consuming
the fermentative bacteria.
[0040] An undefined, mixed, bacterial composition of a preferred
adapted ruminal inoculum of this invention, designated RCBP, has
been deposited under the provisions of the Budapest Treaty in the
Agricultural Research Service Culture Collection in Peoria,
Illinois, on May 6, 2010, and has been assigned deposit accession
number NRRL B-50366. The composition is composed of an undefined,
yet stable mixture of anaerobic bacteria.
[0041] The ruminal inoculum may be used alone or optionally may be
augmented by addition of one or more supplemental pure or mixed
cultures of microorganisms. Anaerobic fermentation of biomass with
the microorganisms of the ruminal inoculum produce primarily lower
chain volatile fatty acids (i.e., propionic and acetic with
relatively less butyric and isobutyric) with low amounts of higher
chain C5 and C6 volatile fatty acids (valeric and caproic).
However, addition of supplemental inocula can increase the
production of the C4, C5, and C6 higher chain volatile fatty acids.
As an alternative to adding supplemental microorganisms to the
primary fermentation of the biomass with the ruminant inoculum,
these supplemental microorganisms may be used as the inocula for a
secondary fermentation of the biomass, following the primary
fermentation with the ruminal inoculum. For instance, an
additional, separate fermentation can be used to convert carbon
dioxide (CO.sub.2) and hydrogen (H.sub.2) produced from the
ruminant inoculum fermentation and the electrolysis stages,
respectively, into acetate, which can then be included as a
feedstock in a subsequent electrolysis stage. Alternatively, this
carbon dioxide (CO.sub.2) and hydrogen (H.sub.2) can be converted
in a separate fermentation to methane gas for use as a fuel.
Without being limited thereto, examples of supplemental
microorganisms include one or more of the following: Clostridium
kluyveri, Clostridium butyricum, Clostridium tyrobutyricum,
Butyrvibrio fibrisolvens, mixed culture of microorganisms derived
from sewage sludge, landfills, soil or aquatic (freshwater, marine,
brackish) environments, or mixed cultures of microorganisms derived
from the gut of termites or other insects. Supplementation with the
butyric acid producing B. fibrisolvens is preferred, particularly
as an augment with the ruminant inoculum in the primary
fermentation. B. fibrisolvens is capable of utilizing
hemicelluloses, and it is envisioned that its use should increase
the production of butyric acid. Due to its relatively slow growth
rate, C. kluyveri is preferably employed in a secondary
fermentation after the ruminant inoculum fermentation. While the
organism produces butyric and caproic acids, a suitable electron
donor such as ethanol (Kenealy et al., 1995), or hydrogen gas
(H.sub.2) (Kenealy and Waselefsky, 1985), can be added to this
secondary fermentation to increase the yield of the longer chain
VFA (Kenealy et al., 1995). The hydrogen gas (H.sub.2) can be
supplied as the hydrogen produced by the cathodic reaction in the
BEC electrolytic stage.
[0042] The production of the volatile fatty acids may be effected
by fermentation of the biomass with the aforementioned ruminal
inoculum in an aqueous medium, using conventional fermentation
techniques under anaerobic conditions. Use of a CO.sub.2 atmosphere
is preferred, although it is envisioned that other gases such as
N.sub.2 may be used. Suitable pH and temperature ranges of the
fermentation may typically range between about 4.5 to 7.5 and 30 to
50.degree. C., respectively, with a pH and temperature between
about 5.5 to 7.0 and 35 to 45.degree. C., respectively, being
preferred. The fermentation may be conducted as a batch, fed-batch
or continuous process, in a single- or multi-stage reactor. The
fermentation does not require, and is preferably conducted in the
absence of, aseptic conditions, without sterilization of the
biomass substrate, reactor, or any other components. The
fermentation is incubated for a sufficient time to produce volatile
fatty acids therein. The precise incubation period for the
fermentation may vary somewhat with the biomass substrate and
conditions, but the fermentation may be discontinued after about 1
to 4 days, preferably after 2 to 3 days, as the time for completion
of the ruminal fermentation is typically 2 to 3 days. Depending
upon the particular biomass used, volatile fatty acid
concentrations in the fermentation broth from about 0.1 M to 0.2 M,
up to about 0.3 M, can be obtained by the fermentation process
herein. As noted hereinabove, the primary fermentation is
preferably conducted without the addition of an effective amount of
methane production inhibitors (inhibitors of methanogenesis or
methanogenic microorganisms), such as, but not limited to
2-bromoethane sulfonic acid (BES).
[0043] A variety of biomass substrates are suitable for use herein,
including plants or parts, residue or waste material thereof.
Without being limited thereto, biomass sources include agricultural
crops (including fruits and vegetables), trees, forages, grasses,
aquatic plants, bagasse, corn stover, corn cobs, hay, flax straw,
oat hulls, wood (including timber, forestry slash, and other wood
waste), sawdust, paper products, paper processing wastes (including
fines from paper recycling), cardboard, and yard or landscape
waste, spent sausage casings, mixed stands of vegetation, rotting,
spoiled or devalued plant materials, and certain food processing
wastes. Although cellulosic biomass substrates are preferred,
animal wastes and waste products, including animal carcasses, may
also be used. In an optional embodiment, the biomass fermentations
may also include plant material (including plants or parts or
materials thereof) that produce organic acids and/or medium chain
fatty acids (MCFA) some of which are relatively inert to anaerobic
degradation and do not need prior separation processing, which
would carry through the fermentation, intact, to serve as
precursors for liquid hydrocarbons such as octane and kerosene in
the electrolysis stage.
[0044] Although the ruminal microorganisms readily ferment
chemically or enzymatically pretreated feedstocks (that is,
pretreatments which function to increase the availability of the
fermentable substrates in the feedstock to the microorganisms,
e.g., pretreatments with acid, base, oxidizing agents, or enzymes,
and including but not limited to hydrolysis), no such chemical or
enzymatic pretreatments are necessary in the process of the
invention, and are preferably omitted. However, for large biomass
materials, the materials should be reduced in size using
conventional techniques such as simple mechanical grinding and/or
rolling (e.g., burr milling). This physical pretreatment is
preferred in order to decrease feedstock particle size, increase
the aggregate surface area, and thus increase the fermentation
rate. Any large feedstock particles that remain in the fermentation
broth will settle out, and may be separated and re-ground, which
mimics the characteristic chewing habits of ruminant animals
(chewing the cud). In a preferred embodiment, a small amount of
nutrients may be added to the fermentation medium to enhance
microbial growth, with distillers dried grains (DDGs) or other
low-cost nutrients being preferred. Other adjuvants which may be
added to the fermentation include small amounts of caustic, such as
NaOH, KOH or Ca(OH).sub.2 to produce salts of the volatile fatty
acids and adjust pH. Other mineral salts may also be needed in
small amounts to provide inorganic components of cell material, or
osmotic balance for the microbial cells in the in vitro
fermentation process.
[0045] In a particularly preferred embodiment, glycerol, a common
byproduct of biodiesel production, may also be added to the biomass
for fermentation by the ruminant inoculum. We have unexpectedly
discovered that the addition of glycerol not only increases the
production of propionic acid, but also increases the production of
butyric acid during the fermentation. Other optional adjuvants
include alcohols such as methanol, a common contaminant of the
glycerol produced by biodiesel manufacture, and which is converted
by the rumen microorganisms to a mixture of methane and CO.sub.2 in
a ratio of approximately 3:1. Yet other adjuvants which may be
added include organic acids such as those naturally produced and
available from natural sources. For example, fruits and fruit
processing waste contain hydroxy-, dicarboxylic, and tricarboxylic
acids such as malic acid and citric acid. These acids can be
included in the process of the invention by addition of the fruit
materials or separated acids to the fermentation medium, whereupon
the hydroxy-, dicarboxylic, and tricarboxylic acids may be
converted by fermentation to volatile fatty acids. Alternatively,
the hydroxy-, dicarboxylic, and tricarboxylic acids from the fruit
materials may be added after the anaerobic fermentation, directly
to the volatile fatty acid salt containing solution, to obtain
different electrolytic products.
[0046] As described above, the anaerobic fermentation of many
biomass materials with the ruminant inoculum will typically produce
C2 to C6 volatile fatty acids. In an optional yet preferred
embodiment, the process may be modified by addition of one or more
C8 to C22 MCFA, which will therefore produce even longer chain
hydrocarbon products from the subsequent electrolysis, including
octane and kerosene. Supplementation with MCFA may be effected by
direct addition of MCFA, or by addition of materials incorporating
MCFA such as materials from plants that produce MCFA, particularly
oilseeds. In this embodiment, oilseed oils contain a variety of
MCFA, are globally abundant, and may be obtained in large
quantities. Common fatty acids available in large supply from
various plant sources include, but are not limited to, coconut,
palm kernel, cuphea, soybean, rapeseed, peanut, sunflower, and
jatropha (see Tables 3, 4 and 5). Coconut oil, for example,
contains a range of fatty acids with a characteristic profile that
includes C8 to C18 carbon chain atoms. As with the organic acid
containing fruit materials, these MCFA-containing plants or plant
materials (e.g., seeds per se or the oil recovered from oilseeds)
may be added to the anaerobic primary fermentation of the biomass
materials by the ruminant inoculum. Most of the MCFA will pass
through the fermentation unchanged, and the resulting mixtures of
volatile fatty acids and MCFAs can then be pH-adjusted and
subjected to the subsequent anodic electrolytic decarboxylation.
Alternatively, the MCFA-containing plant materials may be processed
separately from other biomass materials to recover the oilseed
oils, which are converted to MCFA salts, and added to the volatile
fatty acid-containing fermentation broth solution. The oilseed oils
should first be treated with base (caustic pH adjustment) to make
carboxylate salts, which can then be subjected to the anodic
electrolytic decarboxylation. In accordance with this embodiment, a
large variety of hydrocarbon products may then be produced on an
industrial scale, limited only to the amount of feedstock.
[0047] In accordance with another alternative embodiment, by
limiting the MCFA content of the fermentation broth to the lower
carbon chain fatty acids (C8 to C10), liquid hydrocarbon products
may be produced by electrolysis in relatively pure form. This can
be readily performed by selecting specific fatty acid profiles from
specific sources. For example, seed oil from certain species of
Cuphea (e.g., C. painteri, C. hookeriana) may contain predominant
amounts of C8 (caprylic acid) and C10 (capric acid); these two
acids together can comprise up to 88.degree.-930% of the total
fatty acids in the oil (Table 4). Under Kolbe electrolysis
conditions, C8 fatty acids react with VFA to produce liquid
n-octane and higher carbon chain liquid hydrocarbons like kerosene
(see Table 1). Many oilseed plants produce a large percentage of
fatty acids in the C12-C18 range, which could be electrolytically
converted with VFA into other higher kerosene-type fuels. Some
oilseed plants produce even higher chain fatty acids and fats, for
example, C20 (peanut oil, fish oil), C22 (rapeseed oil). The
resulting hydrocarbons can be readily broken down into more
desirable shorter chain alkanes with existing catalytic crackers.
Combined with volatile fatty acid salt containing solutions, these
new mixtures of organic acids (volatile fatty acid/MCFA) allow a
large variety of hydrocarbon fuels and chemical products to be
produced by cross-linking the radical intermediates through the
anodic electrolytic decarboxylation process. Using this method,
relatively pure liquid, as well as pure gas products can be
produced directly, including high-quality transportation fuels such
as n-octane or kerosene. These are all produced relatively pure
without large quantities of contaminants such as nitrogen, sulfur,
mercury, or particulates. Any traces of contaminating gases
(ammonia, hydrogen sulfide) can be either recycled to the
fermentation broth as nutrients for the microbes, or removed via
adsorption to wood chips, or other adsorbent materials, or via
reaction with iron or iron salts.
[0048] At the conclusion of the primary fermentation of the biomass
with the ruminal inoculum, the volatile fatty acid mixtures from
the fermentation broth typically yield an aqueous solution ratio of
approximately 6:2:1 of acetic, propionic, and butyric acids at a
total VFA concentration of about 0.1 M to 0.2 M within a pH range
of about 5 to 6.5, although total volatile fatty acid
concentrations up to 0.3 M may be produced. In one embodiment, the
electrolysis of the volatile fatty acids to produce hydrocarbons
may be performed directly upon the fermentation broth without
further treatment or extraction of the volatile fatty acids
therefrom. However, in a first preferred embodiment, the microbial
cells, lignin-containing residues, and non-carboxylate anions
(e.g., bicarbonates and carbonates) are removed from the broth
(containing the carboxylate anions or salts of the VFA) prior to
electrolysis. The cells, lignin-containing residues and
non-carboxylate anions may be removed by one or more of a variety
of techniques, such as filtration, flocculation, settling,
centrifugation or precipitation. Soluble proteins may also be
removed such as by an additional ultrafiltration step, as some
dissolved proteins may decrease current flow to the anode and slow
the electrolysis process when carried out in fermented broth.
Non-carboxylate anions will also inhibit the electrolysis. In
another preferred embodiment, the volatile fatty acids are also
concentrated or extracted to a higher molar concentration or
separated into individual volatile fatty acids, thereby improving
yields or allowing the production of specific products. In a
particularly preferred embodiment, the VFA are separated from the
fermentation broth, and concentrated to improve electrolytic
efficiency. The volatile fatty acids can be extracted efficiently
by liquid-liquid extraction of the fermentation broth with alcohols
such as butanol or isopropanol, or with other polar and non-polar
organic solvents. Isopropanol can extract volatile fatty acids and
is miscible with water, but is insoluble in highly saline
solutions. Additions of salt (e.g., NaCl) to the aqueous fermented
broth-isopropanol solution allow for an aqueous/alcohol separation
layer to form, which can then be recovered. Alternatively, the
volatile fatty acids may be concentrated by distillation,
capacitive deionization (CDI), evaporation, ultrafiltration,
reverse osmosis, forward osmosis, carbon nanotubes, or biomimetics.
The recovered volatile fatty acid solution may also be sequestered
stored and processed electrochemically at a later time.
[0049] Although filtering the fermentation broth is a useful method
to improve electrolysis conditions within the fermented broths,
concentrating the VFA from the fermentation broth is preferred
because it eliminates or minimizes unwanted anodic electrolytic
products. Concentrated organic acid substrates maximize the
efficiency and effectiveness of the electrolytic process. It is
envisioned and anticipated that the preferred electrolysis
conditions include a concentrated carboxylate (VFA salts, VFA-MCFA
salts) substrate solution relatively free of dissolved proteins and
other unwanted anions. In this embodiment, the VFA are separated
from the fermented broth and concentrated. This can be achieved by
several known methods including distillation and liquid-liquid
solvent extraction. Although VFA solvent extraction is the
preferred concentration method over distillation due to cost
considerations, other methods can be used that do not require heat
or solvent processing that would otherwise increase the total BEC
processing cost.
[0050] It is envisioned and anticipated that another concentration
method will be preferred due to its low cost and effectiveness on
an industrial scale. Capacitive Deionization (CDI) is an economical
process that has been used for water purification and can be
adapted to concentrate carboxylate (VFA salts, VFA-MCFA salts)
anions within aqueous solutions. CDI uses very low voltages and
very high surface area electrodes to electrostatically attract and
hold anions and cations in aqueous solutions to their respective
anode and cathode. High surface area electrodes can be any material
but mainly include carbon aerogels, carbon nanofoams, and lower
cost carbon electrodes obtained from pyrolysis of papers. The
voltages must be below certain oxidation potential and chemical
reaction thresholds. These anions and cations are then sequestered
into concentrated brine streams. Voltages used are generally about
0.5 to 1 volt, since at voltages less than that of about 1.23 volts
(see Formula 2), no water oxidation occurs and no oxygen is formed
at the anode. CDI can be operated at DC power levels as low as 0.5
Volts and 100 mA. However, at these low voltages, the solvated
anions and cations migrate to the high surface area electrodes due
to the capacitive ion effect, are adsorbed onto their respective
electrode surfaces, and held until a polarity reversal releases
them, thus separating anions from cations without distillation,
reagents, or electrolysis. By adapting this technology to BEC,
lower concentrations of carboxylates (VFA salts, VFA-MCFA salts)
can be concentrated at high surface area anodes and then
electrolytically separated from other anions in aqueous solutions
due to the inherently high discharge potentials of
carboxylates.
[0051] It is also envisioned that an adaptation of a Flow-Through
Capacitor (FTC) can be used as a preferred CDI concentration
process. Flow-through capacitors use supercapacitors and are
specifically designed to separate anions and cations from flowing
liquids in an efficient manner at a specific flow rate. An example
of a FTC is described in U.S. Pat. No. 6,462,935 (the contents of
which are incorporated by reference herein) that possesses
conically wound supercapacitor electrode surfaces using ferric
oxide and carbon powders.
[0052] As a preferred embodiment of the electrolytic stage of the
BEC process, it is also envisioned that the CDI concentration and
electrolytic processes can be combined together into a
CDI/electrolytic process. The CDI process can be adapted to
integrate with the electrolytic stage by using lower voltages first
to concentrate the carboxylates, and then by using higher voltages,
above the critical potential, to perform the electrolysis. This
would involve a simple voltage timing-cycle which would allow
sufficient time for the carboxylates to concentrate at the anode
before performing the actual electrolytic stage. Then the
electrolysis would be performed for a time necessary for
hydrocarbon conversion until the carboxylate concentration drops
(at the anode) to a lower level, which would start the CDI
concentration process again. For example, the potential can be held
at 1 volt for a sufficient time to concentrate carboxylates (at the
anode) at a desired level, and then the voltage can be increased
quickly to over 3 volts for a sufficient time to convert
carboxylates to hydrocarbons, and then decreased quickly to 1 volt
to concentrate carboxylates. These cycles would be repeated until
majorities of the carboxylates within a batch are converted to
hydrocarbons. In this embodiment, high surface area electrodes are
inserted into the fermentation broth with a low applied voltage for
a period of time necessary to adsorb and concentrate carboxylate
anions at the anode at which point the voltage is increased to
commence the electrolysis reaction. This thereby allows the
carboxylate concentration process to occur within the VFA
fermentation broth and the decarboxylation electrolysis process to
be performed in the same vessel. This CDI/electrolysis process may
operate at lower current densities (less than 1 mA/cm.sup.2) due to
the high electrode surface area for a given applied voltage
(although higher electrolysis voltages can be used to offset this).
However, due to the high carboxylate concentration and the high
discharge potentials of carboxylates, decarboxylation electrolysis
still occurs even at low current densities. Additionally, the CDI
concentration process can include a CDI filter membrane which
entirely surrounds the anode within the fermentation broth and
separates the anode from the cathode. The CDI filter membrane must
be composed of appropriately sized pores which will prevent
non-carboxylate anions (larger in size than carboxylate anions of
the VFA) from adsorbing onto the anode surface during the CDI
concentration process. In this way, many larger sized negatively
charged anions within the fermentation broth will be effectively
separated from the carboxylate anions during the CDI concentration
process. The use of a CDI filter membrane improves carboxylate
concentration by removing some non-carboxylate anions that may
otherwise inhibit the electrolysis process.
[0053] In another embodiment, the CDI method can be used in a
separate process to remove VFA (as carboxylate anions) from the
fermentation broth during (concurrent with) the primary rumen
fermentation (CDI/Fermentation process). In this embodiment, the
CDI process is used to decrease the VFA concentration of the
fermentation broth and can be used at any time during the primary
fermentation process. As VFA concentrations increase within the
fermentation broth, the fermentation rate typically decreases.
However, use of the CDI/Fermentation process described herein
allows the speed of the primary fermentation process to be
maximized by removing the VFA during the fermentation. The VFA may
be removed continuously or periodically during the fermentation. In
a biorefinery process, CDI can be integrated with the fermentation
stage in order to continuously or periodically remove VFA from the
fermentation broth while separating the fermentation broth from the
vessel containing the CDI apparatus. An adaptation of a flow
through capacitor (FTC) is preferably used for this purpose. In
this embodiment, the electrodes of the CDI or FTC are positioned
within the fermentation medium and a low voltage is applied as
described above. Again, the carboxylates of the VFA are attracted
to and sequestered at the surface of the anode, effectively
concentrating the VFA at the locality of the anode and thereby
decreasing the VFA concentration in the remainder of the
fermentation medium (away from the anode). The VFA adsorbed at the
anode may then be converted to hydrocarbons by electrolysis in situ
by increasing the voltage in the same manner as described for
CDI/electrolysis, or alternatively, the VFA may be recovered for
subsequent electrolysis by simply separating the electrodes from
the fermentation broth. The electrodes may then be placed in the
same or different electrolyte solution-containing vessel and
electrolysis conducted to produce hydrocarbons as described above.
Alternatively, the VFA may be released from the anode into the
electrolyte solution-containing vessel by reversing the polarity of
the anode, and the electrolysis performed as described above at a
later time. In a particularly preferred configuration, the
fermentation medium may be continuously passed across or past the
electrodes in a single vessel or multiple vessels connected in
series. In this way the rumen microorganisms will be allowed to
work at peak efficiency and effectiveness and further decrease
total fermentation time.
[0054] In yet another embodiment, the CDI/Fermentation process can
include a semi-porous membrane that entirely surrounds the CDI
anode similar to that used in the CDI concentration process. The
purpose of this membrane is to prevent non-carboxylate anions
(larger in size than carboxylate anions of the VFA), and other
contaminants such as lignins, cells, and proteins from interfering
with or adsorbing onto the anode surface during the
CDI/Fermentation process.
[0055] The electrolysis stage of the process of the invention
converts the complete range of the volatile fatty acid products
(including any added MCFA) to hydrocarbon and hydrogen fuels. The
volatile fatty acids may be converted into large quantities of gas
and liquid hydrocarbons and hydrogen gas (H.sub.2) using the Kolbe
and/or Hofer-Moest reactions of electrochemical decarboxylation. As
described above, the Kolbe Reaction is a decarboxylative coupling
(dimerization, radical cross coupling), which yields alkanes such
as ethane and propane, while the Hofer-Moest Reaction is an
oxidative decarboxylation (deprotonation), which yields alkenes
such as ethylene and propylene. Both of these reactions occur
simultaneously during electrolysis but can be adjusted to favor one
reaction or the other by changing several easily controlled
variables as described herein below. Different products can be
produced by changing these variables, thus allowing a very flexible
process.
[0056] In accordance with this invention, the electrolysis converts
the volatile fatty acids to mixtures of alkanes, alkenes, H.sub.2
and CO.sub.2 in water, under very mild reaction conditions. For
instance, predominant hydrocarbon products of the electrolytic
conversion typically include methane and ethane from acetic acid,
propane, butane and ethylene from propionic acid, and butane,
pentane, hexane and propylene from butyric acid. Mixtures having
the same proportions of VFA as the major VFA in the typical in
vitro ruminal fermentation noted above (acetate: propionate:
butyrate, 6:2:1 molar basis) will yield mixtures of the above
products. All reactions will also yield substantial amounts of
H.sub.2 at the cathode. When other naturally produced organic acids
are added to VFA solutions, the number of different hydrocarbon
products increases (see Table 1). Hydrocarbon products derived from
VFA-MCFA mixtures can include alkanes and alkenes with carbon
numbers C5 to C22, including n-octane and kerosene. Likewise, when
other organic chemicals such as glycerol, alcohols, and other
readily available organic compounds are added to VFA solutions, the
number of electrochemical products increases further.
[0057] Surprisingly, the electrolysis reactions may be conducted in
a simple undivided electrochemical cell under mild electrolysis
conditions, at or above 3 volts DC (VDC) and at or above 1
mA/cm.sup.2 anode current density, using low-cost carbon or
graphite electrodes, at room temperature and ambient pressure,
under aqueous conditions. However, when using an integrated
CDI/Electrolysis method as described above, the anode current
density may be much lower due to the high electrode surface area.
For electrolysis conditions, the pH of the aqueous volatile fatty
acid solution may range between 4.5 to 11, preferably between about
5.5 to 8.0. At acidic or neutral pH, Kolbe dimerization to alkanes
is favored, while at alkaline pH ranges, Hofer-Moest oxidative
deprotonation to alkenes is favored. However, the volatile fatty
acids and MCFA need to be in the salt form to be useful for
electrolysis. Therefore low pH will tend to decrease volatile fatty
acid anion concentration and MCFA solubility. The pH may be
adjusted with caustic to maintain a high concentration of acid
salts to be electrolyzed. In general, no substrate solvent is
needed other than water and no additional organic co-solvents or
reagents are required. Moreover, because a major component of the
volatile fatty acid produced from biomass is acetic acid, it is
itself a useful solvent for anodic electrolytic decarboxylation.
Non-aqueous solvents are only required for poorly water soluble
reactants (e.g., higher chain MCFA). Methanol, ethanol or
isopropanol additions can be used as a substrate solvent for these
higher-chain fatty acids (MCFA). Alternatively, due to their
relative aqueous insolubilities, these higher-chain fatty acids can
be easily separated and processed separately at very high
concentrations and therefore can give very high yields of
electrolytic products.
[0058] The anodic products can be separated from the cathodic
products easily during electrolysis by segregating the product
receiving vessels from one another. Alternatively, all electrolytic
products can be combined easily into a single receiving vessel for
further separation or processing. Once collected, the gaseous
products (hydrocarbons, H.sub.2 and CO.sub.2) may be compressed
into high pressure tanks and can be further separated into their
component products via gas liquefaction. In this manner, carbon
dioxide CO.sub.2 can be removed easily and sequestered from the
hydrocarbon and hydrogen fuel products. In the easiest case
possible, all electrolysis products (including CO.sub.2) can be
combined and used as a fuel in a most field-expedient manner
without any further processing.
[0059] In contrast to previous studies, the effective conversion of
the volatile fatty acids to hydrocarbons by the Kolbe electrolytic
reaction conducted in an aqueous medium, using carbon or graphite
electrodes at a low current density as described herein, is
unexpected. Platinum electrodes and high current densities although
desirable, are not required although under certain combinations of
reaction conditions they can be used.
[0060] In accordance with a preferred embodiment, semi-permeable
membranes are provided within the electrolysis cell between
(separating) the anode and cathode electrodes. The use of
semi-porous membranes allow the current density to increase by
decreasing electrode spacing and therefore cell resistance. The
membranes permit electrolytes to carry the current while offering
good separation of gas products from anode and cathode. Either
semi-permeable membranes or salt additions (to provide
electrolytes) may be used to decrease cell resistance between the
anode and cathode while electronically insulating them from each
other, thereby allowing a lower voltage to be used while increasing
current density, and improve the product performance. Other
semi-porous membranes may be used. The use of a membrane in this
manner also allows the electrodes to be positioned near one
another, allowing current to flow without arcing. Power for the
electrolytic cell may be provided from any convenient source.
However, because any voltage above about 3 VDC is adequate for the
electrolysis, alternative sources of electricity such as solar
cells, wind generators and even fuel cells may be used as power
sources to generate hydrocarbons in rural areas or in
field-expedient military situations.
[0061] For both the Kolbe and the Hofer-Moest decarboxylation
reactions, at critical potentials above about 2.0 volts to 2.8
volts for individual carboxylic acids and about 3 volts for
carboxylic acid mixtures, and at current densities above 1.0
mA/cm.sup.2, no hydroxyl anions are oxidized at the anode and no
oxygen gas (O.sub.2) is formed (Torii and Tanaka, 2001). This is
due to the high discharge potential of carboxylates and because
hydroxyl ions are formed at the cathode at the same rate as
carboxylate ions are consumed at the anode. Moreover, because the
organic acids themselves possess good solvation properties,
electrolysis can be carried out in low-cost aqueous solutions
without any solvent additions. The products are hydrocarbon
mixtures, hydrogen (H.sub.2), and carbon dioxide (CO.sub.2) gases,
along with several other products including alcohols, but without
contaminants (e.g., H.sub.2S) normally found in natural gas liquids
(NGL) exclusively obtained from the petroleum industry.
[0062] The selection of the particular electrode material for use
in the electrolytic cell is a significant factor in determining
which kinds of products will be produced within the electrochemical
cell (Torii and Tanaka, 2001, Table 2, p. 505). For instance,
electrodes constructed from platinum or porous (amorphous) carbon,
favor the Kolbe or Hofer-Moest reactions, respectively. Carbon and
graphite electrodes are preferred for use herein, and specific
electrode materials that may be used herein include but are not
limited to platinum, diamond, vitreous (glassy) carbon, carbon
aerogel, carbon nanofoam, graphite, and others. In an industrial
application, the preferred electrode material is either platinum or
graphite. In order to use graphite electrodes and increase Kolbe
products, lower reaction temperatures may be used (Levy et al.,
1983). Industrially, platinum coated electrodes are used in PEM
cells for the production of hydrogen (H.sub.2). These cells may
also be used.
[0063] Other conditions may also effect the electrolysis reactions
and product yields. As noted above, membranes may be used to
separate the anode and cathode, and help to decrease or control the
electrochemical cell resistance similar to the membranes used in
PEM electrolytic cells that produce hydrogen fuels. Additionally,
other reaction variables which can be used to enhance product
yields and variability within the electrolytic cell include
alternating current (waveforms), magnetic fields, and ultrasonic
energy, to name a few.
[0064] The hydrocarbon products of the electrolysis are
spontaneously evolved from solution, and once recovered can be
converted to gasoline fractions using well-known and widely
practiced industrial chemistry methods (e.g., thermal
polymerization), which converts lighter hydrocarbon gases into
liquid hydrocarbon fuels. Thermal Polymerization, for example, is a
well-known petroleum refining process that converts lighter
hydrocarbon gases into liquid hydrocarbon fuels. This involves
cracking feedstocks of saturated hydrocarbons (alkanes) to produce
unsaturated hydrocarbons (alkenes). Heat and pressure are applied
to the alkane feedstocks at the same time, which produces an end
product of "Polymer Gasoline" (Speight, 2006). Another refining
method that may be used is the Shell Higher Olefin Process (SHOP)
of producing desired higher carbon number chain lengths from lower
carbon number hydrocarbons. The SHOP process requires ethylene as
the feedstock. Ethylene represents a major alkene product that is
derived from the VFA salt containing solutions in the invention
process. All alkene products are generated by the Hofer-Moest
reaction of oxidative electrolytic decarboxylation. Other
well-known refining methods such as the Ziegler-Natta reaction and
the Wurtz reaction may be also be used to the same effect as well
as other known methods. The various products of this invention may
be separated either by well-known methods such as fractionally
compressing the gaseous products, distilling the liquid products,
or filtering the solid products. This is not necessary in all
cases, however, because the gaseous products from VFA-salts
electrolysis alone will yield NGL (natural gas liquids) when
compressed and liquefied. NGL may not need to be separated in order
to be valuable, since they can also be used as mixed products.
[0065] In addition to the volatile fatty acid products, the
fermentation with the ruminal inoculum produces roughly about 10%
methane and 20% carbon dioxide as byproducts (based on mass of
biomass feedstock) that may be sequestered and reused. The methane
may be used to power generators to produce the energy needed in the
electrolysis step, or combined with the other hydrocarbon products
and refined further. The carbon dioxide produced may be used to
deoxygenate the biomass and fermentation medium by displacing air,
thereby making it ready for anaerobic fermentation, and may be
further sequestered and reused. Alternatively, the H.sub.2 and
CO.sub.2 formed at the cathode and anode electrodes, respectively,
can be combined and converted by methanogenic microbes to produce
methane gas, or by acetogenic microbes to produce acetic acid. The
leftover inorganic salts after electrolysis may be returned to the
bioreactor and combined with the next fermentation batch, which may
be a continuous process. The solid fermentation residue, including
cells and lignin, can be processed into wood-adhesive products
(Weimer, U.S. Pat. No. 7,651,582), or used as an animal feed, or
used as a fuel to generate electricity for the electrolysis step.
The hydrogen (H.sub.2) may be used as a fuel or used as a reactant
in refining hydrocarbons or other chemicals.
[0066] As described herein, nearly all of the conversion products
and byproducts produced by the process may be used. There is very
little net carbon footprint, because those reactants that come from
plant material have already removed the carbon from the atmosphere
as opposed to petroleum obtained from beneath the earth. Even the
carbon dioxide produced in the conversion process is sequestered,
reused and converted to useful products making this invention a
truly "Green Process".
Water Electrolysis Reactions
[0067] Anode:
2H.sub.2O(l).fwdarw.O.sub.2(g)+4ll.sup.+(aq)+4e.sup.-K.sup.0.sub.on=-1.23
V
Cathode 2H(aq)+2e.sup.-.fwdarw.H.sub.2(g)E.sup.0.sub.red=0.00 V
Formula 2
[0068] A significant advantage of the process of this invention is
evidenced by a comparison of electrical power needed to produce
hydrocarbons from VFA-carboxylic acid solutions (see Formula 1)
versus producing hydrogen from water (see Formula 2). Potential
energy as stated here is bond dissociation energy (BDE). As
indicated in Table 2, over 8 times more potential energy is
obtained using the same amount (2e.sup.-) of electrical power, to
produce hydrocarbon-hydrogen products from VFA-electrolysis over
hydrogen-water electrolysis. For example, ethane and propane
produced electrochemically from acetic and propionic acids contain
6 and 9 times, respectively, the potential energy of hydrogen
(H.sub.2) as a fuel. VFA-MCFA mixtures produce even larger chain
hydrocarbon products providing up to 50 times the potential fuel
energy of hydrogen-water electrolysis using the same amount of
electric power. For example, a MCFA like C16 palmitic acid, mixed
with VFAs in an electrolytic cell will produce up to C18
hydrocarbons (n-octadecane) in carbon chain length through Kolbe
cross-radical coupling, which yields a BDE 50-times that of
hydrogen using the same amount of electrical power.
[0069] The following examples are intended only to further
illustrate the invention and are not intended to limit the scope of
the invention that is defined by the claims.
Example 1
[0070] A mixed microbial consortium obtained from bovine ruminal
fluid was fermented in vitro with eastern gamagrass (a harvested
native grass) or alfalfa (harvested legume crop). Both are
representative of a perennial cellulosic biomass feedstock.
Separation of the resulting fermentation broth containing VFAs,
from the solid residue (including cells and lignin) was
accomplished by filtration and/or centrifugation.
[0071] The electrolytic apparatus was generally comprised of a
simple undivided cell with carbon or graphite plates or rods used
for both anode and cathode electrodes. Platinum anodes were also
used for comparison purposes. Stainless steel and other metal
electrode material were also used for cathodes. Although many anode
materials can be used, carbon/graphite electrodes were selected in
addition to platinum anodes, for example, due to their low cost and
effectiveness on an industrial scale. The electrodes were immersed
directly into the fermentation broth which generally had a pH range
of about 5.5-6.5 at room temperature. pH adjustment was performed
in some cases to bring the pH range to about 6.0-6.5 to increase
the carboxylic salt concentration. Electrical potentials were kept
above 3 volts DC so that oxygen production at the anode was
eliminated. It is known that aqueous solutions of salts of
carboxylic acids, that is, carboxylate anions, form a film on the
anode electrode surface at a specific critical voltage potential
that prevents any oxidation of the hydroxyl ion from the aqueous
solution. The critical potentials (from the Tafel equation and
Tafel plots) for decarboxylation of a variety of carboxylic acids
are within a range of about 2.0 volts to 2.8 volts (Torii and
Tanaka, 2001, FIG. 1, p. 502). In practice, the electrolysis can be
performed at any voltages that exceed these critical potentials for
the respective individual carboxylic acids. As a general rule for
various aqueous carboxylic acid mixtures, it is practical to keep
electrical potentials above about 3 volts DC which exceeds the 2.8
volts higher threshold critical potential for carboxylates.
[0072] Fine mesh porous nylon membranes (to minimize electrode
spacing) and salts additions (to provide electrolytes) were used to
decrease cell resistance, which allows a lower voltage to be used
while increasing current density, and improved the product
performance. Other semi-porous membranes may be used. Membranes
permit electrolytes to carry the current while offering good
separation of gas products from anode and cathode. Gases were
collected above the anode by liquid displacement of the fermented
broth samples from Balch tubes (Balch and Wolfe, 1986), which were
positioned over the respective anode and cathode. The tubes were
then sealed with butyl rubber stoppers while still immersed in the
liquid of the electrolysis cell, after which the stoppers were
sealed with aluminum crimp seals.
[0073] Electrolytic cell resistance can be higher without the use
of porous or semi-porous membranes or electrolytes because
electrodes need to be separated at greater distances to prevent
anode/cathode gas products from mixing. Anode current density can
be increased to higher levels by decreasing electrode spacing with
porous or semi-porous membranes, or by adding MCFA salts or NaCl to
the substrate. The electrolytic cell can be adjusted to deliver any
current level desired by varying the applied potential, electrode
surface area, electrode spacing, substrate concentration,
electrolytes and other factors. Current densities at the electrode
surfaces are a key factor in electrochemical cell performance. The
current density may be balanced equally between anode and cathode
or may be designed to be unbalanced. For example, decreasing the
anode surface area or increasing the cathode surface area for the
same cell current may increase anode current density. Products and
yields can differ and can be controlled using different anode
current densities. For example, at lower current densities and
higher voltage potentials, alkenes (Hofer-Moest products) are
favored over alkanes. At higher anode current densities and lower
voltage potentials, alkanes (Kolbe products) are favored over
alkenes. There is an inherent flexibility in this invention to
tailor products by varying fermentation and electrolytic
conditions.
[0074] For experimental purposes, voltages of above 3 VDC with 1
mA/cm.sup.2-100 mA/cm.sup.2 anode current density were used
depending on the cell resistance due to electrode spacing within
the fermented broth. The literature (Torii and Tanaka, 2001)
suggests that higher anode current densities of about 250
mA/cm.sup.2 and higher, produce more Kolbe products. However, it
was the intention of this research to run electrolyses on the
primary fermentation broths with very simple equipment in order to
prove process efficacy with minimum possible costs.
[0075] Hydrocarbon products derived from VFA mixtures alone
include: ethane, propane, butane, pentane, hexane, ethylene, and
propylene. In addition, pure hydrogen gas (H.sub.2) is produced in
large quantities, as well as carbon dioxide, which can be
sequestered and reused in the deoxygenation/air displacement step.
Hydrocarbon products derived from VFA-MCFA mixtures can include
alkanes and alkenes with carbon numbers C5 to C20, including
n-octane and kerosene (see Table 1).
I. Biological Stage (Fermentation):
Example Bio-1
Fermentation of Eastern Gamagrass to VFA
[0076] A flask containing 600 mL of Goering-Van Soest medium
(Goering and Van Soest, 1970; contents per liter: 8.75 g
NaHCO.sub.3, 1.0 g NH.sub.4HCO.sub.3, 1.55 g KH.sub.2PO.sub.4, 1.43
g Na.sub.2HPO.sub.4, 0.15 g MgSO.sub.4), 16.4 g of air-dried
Eastern gamagrass (ground in a Wiley mill having a 1 mm screen),
1.0 g of Trypticase, and 0.002 g of resazurin, was gassed under a
stream of CO.sub.2, after which 0.6 g of cysteine HCl and 0.05 g of
Na.sub.2S.9H.sub.2O was added. The flask was inoculated with rumen
contents (.about.80 mL of liquid and 20 g squeezed solids) prepared
by mixing similar amounts from two rumen-fistulated cows. The flask
was incubated at 39.degree. C. without shaking. After 54 h
incubation, samples were removed for fermentation product analysis.
Samples were centrifuged at 10,000.times.g for 10 min, and 600 uL
of the supernatant liquid was combined with 600 uL of CHS (26.45 g
Ca(OH).sub.2 added to 100 mL H.sub.2O) and 300 uL of CSR (10 g
CuSO.sub.4+0.4 g crotonic acid per 100 mL aqueous solution). The
mixture was frozen, thawed and centrifuged. The supernatant liquid
was transferred to tubes containing 28 uL of H.sub.2SO.sub.4, and
this solution, frozen and thawed twice, then centrifuged. The
supernatant liquid was analyzed by high performance liquid
chromatography (HPLC), using a 250 mm.times.4.6 mm Bio-Rad Aminex
HPX-87H analytical column maintained at 45.degree. C. Samples (50
uL) were eluted with a mobile phase of 0.015 N
H.sub.2SO.sub.4/0.0034 M ethylenediaminetetracetic acid (EDTA) at a
flow rate of 0.7 mL/min, and separated peaks were detected with a
refractive index detector. Quantification was achieved by
comparison to standard curves, with crotonic acid as internal
standard. After incubation the culture contained 77.5 mM acetic,
21.7 mM propionic, 8.8 mM butyric, 1.4 mM isobutyric, 2.7 mM
valeric, and 2.4 mM of a combination of 2-methylbutyric and
3-methylbutyric acids.
Example Bio-2
Production of VFA in Stable Enrichment Cultures
[0077] The culture from Example Bio-1 was sequentially transferred
for 7 successive transfers of .about.50 g of liquids and solids at
2 to 3 d. The culture medium was the same as described in Example
Bio-1, except incubations were conducted at one-half volumetric
scale (300 mL medium), and the Trypticase was replaced by dried
distillers grains (DDG). After 28 h incubation at 39.degree. C.
without shaking, fermentation broth samples were collected and
analyzed as described in Example Bio-1. The amounts of fermentation
products in the cultures were 113.9 mM acetic, 33.6 mM propionic,
10.3 mM butyric, 0.6 mM isobutyric, 2.0 mM valeric, and 1.0 mM of a
combination of 2-methylbutyric and 3-methylbutyric (isovaleric)
acids.
Example Bio-3
VFA Production from Biomass Feedstocks by Stabilized Mixed Cultures
of Ruminal Microorganisms
[0078] Fermentations were conducted at 39.degree. C. in unshaken
Erlenmeyer flasks in 150 mL of a Goering/Van Soest medium
supplemented with 4 g dried biomass feedstock (Eastern gamagrass or
alfalfa, ground through a 1 mm Wiley mill screen but otherwise not
pretreated) and 0.5 g of DDG. Flasks were gassed with CO.sub.2
prior to inoculation but were incubated with vented closures,
without additional gas sparging, during the fermentation. Cultures
were transferred at intervals of 2 to 4 days by pouring .about.20%
by volume of culture from the previous culture to a flask
containing fresh medium. VFA concentrations were determined in
culture supernatants obtained by centrifugation of 2- to 4-d old
cultures at 12,000.times.g for 10 min. Results are shown in Table
B-1.
TABLE-US-00002 TABLE B-1 VFA concentrations in stabilized
enrichment cultures or mixed ruminal microflora grown on Eastern
gamagrass (EGG) or alfalfa (Alf). The number code for the Eastern
gamagrass (EGG) and alfalfa (Alf) cultures corresponds to the
sequential transfer number of the culture after the original
ruminal inoculation. net mM VFA produced Additions Acetic Propionic
Butyric IB.sup.a IV + 2MB.sup.b Valeric Total EGG-32 102.6 32.8
14.8 0.5 0.5 2.0 153.2 EGG-57 111.8 32.4 13.7 0.4 0.7 4.8 163.8
EGG-97 110.8 38.7 12.2 0.6 0.7 3.6 166.7 EGG-141 93.9 25.0 10.6 1.4
5.0 2.5 138.3 Alf-11 71.1 31.2 7.6 1.8 4.0 3.3 118.8 Alf-23 73.5
23.9 9.2 1.0 3.3 2.1 113.0 .sup.aIB = isobutyric .sup.bIV + 2MB =
isovaleric (3-methylbutyric) acid plus 2-methylbutyric
Example Bio-4
In Vitro VFA Production from Fermentation of Biopolymers by Mixed
Ruminal Microorganisms Collected from Ruminally-Fistulated Cows
[0079] Fermentations were conducted under a CO.sub.2 gas phase in
sealed vials containing 100 mg of substrate in 8.5 mL of a
Goering/Van Soest medium (except for DNA, 7.2 mg in 1.9 mL medium)
supplemented with 1 g of Trypticase per liter plus 1.5 mL of
freshly-collected ruminal inoculum (0.3 mL for DNA) squeezed
through cheesecloth to remove large feed particles. Results are
shown in Tables B-2 and B-3.
TABLE-US-00003 TABLE B-2 Net VFA production from ~0.10 g of various
biopolymers by mixed ruminal microorganisms sampled directly from
ruminally fistulated cows. Values corrected for VFA production in
blank vials containing ruminal inoculum but lacking added
substrate. Fermentations of the same substrates with adapted
ruminal enrichment cultures gave substantially similar results. net
mM VFA produced Substrate Acetic Propionic Butyric IB.sup.b IV +
2MB.sup.c Valeric Total Cellulose 41.0 43.5 4.3 0.6 0.5 1.4 91.3
Tobacco stalk xylan 43.1 33.5 3.1 0 0 0.8 80.5 Birch xylan 50.9
34.8 3.8 0.1 0 0.7 90.2 CS hemicellulose 39.1 31.5 3.1 0 0 0.8 74.5
Corn starch 37.4 32.9 6.8 0 0.2 2.0 79.2 Fructan 16.7 13.2 3.5 0 0
0.8 34.1 Alfalfa pectin 17.5 5.9 1.2 0.2 0.3 0.5 25.5 Microbial
cells 16.9 3.5 1.9 0.9 2.7 1.3 27.7 Soybean peptone 22.6 7.8 8.4
3.5 8.7 7.1 58.0 Distillers grains 26.7 19.4 4.5 0.6 1.5 2.5 55.2
DNA (7.2 mg) 0.78 0.4 0.93 0.08 0 0 1.9 .sup.aSubstrates: CS
hemicellulose purified from stalks of cicer milkvetch (Astragalus
cicer). Fructan from orchardgrass (Dactylis glomerata); Microbial
cells from ruminal contents. .sup.bIB = isobutyric .sup.cIV + 2MB =
isovaleric (3-methylbutyric) acid plus 2-methylbutyric
TABLE-US-00004 TABLE B-3 Fractional yield (g VFA per g dry weight
of added substrate) from fermentation of various biopolymers by
mixed ruminal microorganisms sampled from ruminally fistulated
cows. Total VFA yield on a weight basis varied from 14.5 to 66.1%,
depending on substrate. g VFA per g dry weight of added substrate
Substrate Acetic Propionic Butyric IB.sup.b IV + 2MB.sup.c Valeric
Total Cellulose 0.258 0.337 0.040 0.006 0.005 0.015 0.661 Tobacco
stalk xylan 0.297 0.285 0.031 0 0 0.003 0.615 Birch xylan 0.334
0.282 0.036 0.001 0 0.008 0.656 CS hemicellulose 0.253 0.251 0.029
0 0 0.008 0.534 Corn starch 0.250 0.271 0.066 0 0.002 0.023 0.612
Fructan 0.115 0.112 0.035 0 0 0.009 0.266 Alfalfa pectin 0.337
0.140 0.034 0.006 0.010 0.015 0.542 Microbial cells 0.104 0.027
0.017 0.008 0.028 0.014 0.198 Soybean peptone 0.154 0.065 0.084
0.035 0.101 0.082 0.520 Distillers grains 0.182 0.162 0.044 0.006
0.018 0.029 0.441 DNA 0.052 0.033 0.090 0.008 0 0 0.145
.sup.aSubstrates: CS = hemicellulose purified from stalks of cicer
milkvetch (Astragalus cicer). Fructan from orchardgrass (Dactylis
glomerata); Microbial cells from ruminal contents. .sup.bIB =
isobutyric .sup.cIV + 2MB = isovaleric (3-methylbutyric) acid plus
2-methylbutyric
Example Bio-5
Fermentation of Other Polysaccharides
[0080] Polysaccharides (100 mg fresh weight) were fermented for 24
h at 39.degree. C. under a CO.sub.2 atmosphere in sealed, unshaken
vials that contained 8.5 mL of Goering/Van Soest buffer and 1.5 mL
of squeezed ruminal fluid. Subsamples of culture were centrifuged
and the supernatant assayed for VFA by HPLC. Results are shown in
Table B-4. Total VFA yield on a weight basis varied from 50.9 to
78.7%, depending on substrate.
TABLE-US-00005 TABLE B-4 Fermentation of carbohydrates by mixed
ruminal microflora. Results are corrected for VFA produced in vials
containing ruminal inocula but lacking added carbohydrate. g of VFA
per g dry weight of added polysaccharide Carbo- IV + hydrate Acetic
Propionic Butyric IB.sup.a 2MB.sup.b Valeric Total Cellu- 0.317
0.347 0.034 0.007 0.003 0.013 0.722 lose Chitin 0.417 0.063 0.015
0.009 0.001 0.004 0.509 Fructan 0.265 0.326 0.146 0.001 0 0.062
0.787 Fruc- 0.227 0.245 0.119 0.002 0 0.047 0.631 tose Sucrose
0.236 0.285 0.124 0 0 0.059 0.691 Gluco- 0.210 0.303 0.088 0.004 0
0.018 0.620 samine .sup.aIB = isobutyric .sup.bIV + 2MB =
isovaleric (3-methylbutyric) acid plus 2-methylbutyric
Example Bio-6
Augmentation of Mixed Ruminal Microflora Cultures with Ethanol or
Additional Bacteria to Shift VFA Product Ratios
[0081] Sealed vials containing 8.5 mL or Modified Dehority medium
and microcrystalline cellulose (95 mg dry weight) under a CO.sub.2
atmosphere were amended with additional substrate or an enrichment
culture of Clostridium kluyveri-like bacteria (Ckb). Vials were
incubated at 39.degree. C. without shaking for 72 h, after which
subsamples of the gas phase were withdrawn for analysis by gas
chromatography, and subsamples of liquid phase were withdrawn and
centrifuged, and the supernatant phase analyzed for VFA by HPLC.
Results are shown in Table B-5 and B-6.
TABLE-US-00006 TABLE B-5 VFA production from cellulose
fermentations amended with ethanol and/or enrichment cultures of
Clostridium kluyveri-like bacteria. Results are corrected for VFA
produced in vials containing ruminal inocula but lacking added
cellulose, ethanol or Ckb.sup.a. net mM VFA produced Addi- IV + Ca-
tions Acetic Propionic Butyric IB.sup.b 2MB.sup.c Valeric proic
None 41.3 41.1 3.0 0.7 0.4 1.2 0 Ethanol 38.6 34.8 3.1 0.7 0 1.9 0
(0.1 ml) Ckb.sup.a 38.7 40. 2.0 0.4 0.5 1.9 0.5 Ethanol + 39.5 31.9
1.1 0.4 0 4.7 1.3 Ckb.sup.a .sup.aCkb = Enrichment culture
containing Clostridium kluyveri-like bacteria. Enrichment culture
was prepared by inoculation of ruminal contents from lactating
dairy cows into a modified Dehority medium (Weimer et al., 1991)
supplemented with ethanol, acetic acid and succinic acid; these
culture was transferred at 2- to 4-week intervals of incubation at
39.degree. C. .sup.bIsobutyric .sup.cIV + 2MB = isovaleric
(3-methylbutyric) acid plus 2-methylbutyric
TABLE-US-00007 TABLE B-6 Gas production in cellulose fermentations
amended with ethanol and/or cultures of Clostridium kluyveri-like
bacteria. Results are corrected for gas produced in vials
containing ruminal inocula but lacking added cellulose, ethanol or
Ckb.sup.a mmoles gas per mol anhydroglucose added Addition H.sub.2
CH.sub.4 None 0.85 232 Ethanol (0.1 mL) 1.19 264 Ckb.sup.a 0.49 525
Ethanol + Ckb.sup.a 0.88 333 .sup.aCkb = Enrichment culture
containing Clostridium kluyveri-like bacteria.
Example Bio-7
Shift to Longer Chain VFAs Resulting of Co-Fermentation with
Glycerol
[0082] Sealed vials containing 8.5 mL of Goering and Van Soest
medium plus microcrystalline cellulose (95 mg dry weight) under a
CO.sub.2 atmosphere were amended with additions as indicated. Vials
were incubated at 39.degree. C. without shaking for 24 h, after
which subsamples of liquid phase were withdrawn for analysis of VFA
by HPLC. Results are shown in Table B-7.
TABLE-US-00008 TABLE B-7 VFA production from cellulose
fermentations amended with glycerol and/or ethanol. Results are
corrected for VFA produced in vials containing ruminal inoculum but
lacking added cellulose, glycerol or ethanol. net mM VFA produced
Addi- IV + tions Acetic Propionic Butyric IB.sup.a 2MB.sup.b
Valeric Total None 34.7 36.5 2.8 0.2 0.4 1.0 75.5 Glycerol 27.7
64.2 7.4 0 0 2.6 101.9 (0.1 mL) Ethanol 43.8 34.0 1.4 0 0 0.7 165.6
(0.1 mL) Glycerol 31.5 52.8 9.7 0 0 2.0 162.4 + Ethanol .sup.aIB =
isobutyric .sup.bIV + 2MB = isovaleric (3-methylbutyric) acid plus
2- methylbutyric
II. Electrolytic Stage (Electrochemical):
[0083] The electrolytic conditions used for examples Elec-1 and
Elec-2 below, were a high-temperature graphite electrode plate (2
cm.times.3 cm) for the anode, and a stainless steel plate (2
cm.times.3 cm) for the cathode. Electrode spacing was 1 mm using a
nylon mesh membrane. Substrate volume was 250 mL, with no pH
adjustment. In these examples, CO.sub.2 was produced at the anode
along with the hydrocarbon products noted, and H.sub.2 was produced
at the cathode.
Example Elec-1
Electrolysis of Centrifuged (Unfiltered) Fermentation Broth
[0084] Centrifuged (11,300.times.g, 30 min) samples from the broth
of an in vitro fermentation of eastern gamagrass were pooled from
several separate fermentations using adapted ruminal inocula, to
provide enough volume to nearly fill the electrolysis cell. These
supernatants were subjected to the following electrolysis
conditions:
TABLE-US-00009 Current Density 60 mA/cm.sup.2 pH 5.5-5.7
Temperature 25.degree. C.
[0085] Samples of the gas phase were withdrawn using a hypodermic
syringe and analyzed by gas chromatography. In the gas
chromatograph helium carrier gas (18 mL/min) was used to separate
the analytes through a Restek Q-BOND capillary column (30
m.times.0.25 mm) prior to detection by a flame ionization detector
operated at 240.degree. C. The chromatographic conditions included
an injector split ratio of 30:1, a temperature program of the
column oven (50.degree. C. to 240.degree. C. at 8.degree. C./min,
with a final hold at 240.degree. C. for 10 min). Chromatographic
peaks were compared to those of known standards. Identified gaseous
anodic products included n-alkanes (methane, ethane, propane,
pentane, hexane), branched alkanes (2-methylpentane,
3-methylpentane) and alkenes (ethylene, propylene,
cis-2-pentene).
Example Elec-2
Electrolysis of Filtered Fermentation Broth
[0086] Fermentation broth obtained by in vitro fermentation of
eastern gamagrass was subjected to filtration through nylon mesh
screen, followed by hollow fiber filtration through a 0.2 um pore
size cartridge. The permeate was further subjected to
ultrafiltration through a nominal 10,000 Da-molecular weight cutoff
(10 kDa NMWCO) cartridge to produce a liquid permeate containing
VFAs, and a concentrated protein retentate fraction. The liquid
permeate (VFA concentration=135 mM) was subjected to the following
electrolysis conditions:
TABLE-US-00010 Current Density 70-80 mA/cm.sup.2 pH 5.9-6.1
Temperature 20.degree. C.
Gases were collected and analyzed as described in Example Elec-1.
Identified gaseous anodic products included n-alkanes (methane,
ethane, propane, hexane), branched alkanes (2-methylpentane) and
alkenes (ethylene, propylene, cis-2-pentene).
[0087] The electrolytic conditions used for example Elec-3 below,
were a coiled, fine platinum electrode wire (0.033 cm
dia..times.120 cm long) for the anode, and a stainless steel plate
(2.5 cm.times.5 cm) for the cathode. Electrode spacing was 3 mm
using a nylon mesh membrane. Substrate (butanol extract) volume was
250 mL, with KOH pH adjustment and a cooler starting/ending
temperature. In this example, CO.sub.2 was produced at the anode
along with the hydrocarbon products noted, and H.sub.2 was produced
at the cathode.
Example Elec-3
Electrolysis of a Butanol Extract of Filtered Fermentation
Broth
[0088] Ultrafiltered fermentation broth (3.00 liters) from Example
Elec-1 above was adjusted to pH 3.25 with concentrated HCl, flushed
with N.sub.2 gas, then extracted with 788 g of n-butanol. The
butanol phase (713 g) was recovered and amended with 200 mL of
H.sub.2O. After adjusting pH to 8.2 with 10 N NaOH, 200 mL of
additional H.sub.2O was added. The butanol and water phases were
allowed to separate, and the aqueous phase (containing most of the
VFAs) was recovered. [0089] The butanol extract was subjected to
the following electrolysis conditions:
TABLE-US-00011 [0089] Current Density 50 mA/cm.sup.2 pH 6.7
Temperature 10-17.degree. C.
Gases were collected and analyzed as described in Example Elec-1.
Identified gaseous anodic products included n-alkanes (methane,
ethane, propane, butane, pentane, hexane), branched alkanes
(2-methylpentane, 3-methylpentane) and alkenes (ethylene,
propylene, cis-2-pentene).
[0090] It is understood that the foregoing detailed description is
given merely by way of illustration and that modifications and
variations may be made therein without departing from the spirit
and scope of the invention.
TABLE-US-00012 TABLE 1 Hydrocarbon Products from Electrochemical
Oxidative Decarboxylation of VFA and MCFA Mixtures Physical
Electrochemical Physical Carboxylic Carbon- Electrochemical Carbon-
Properties Hofer-Moest.sup.2 Carbon- Properties (Fatty) Chain
Kolbe.sup.1 Products Chain @ Room Products Chain @ Room Acids
Length (Alkanes) Length Temp. (Alkenes) Length Temp. VFA* Mixtures
Acetic C2 Methane C1 Gas -- -- -- Ethane C2 Gas -- -- -- Propionic
C3 Propane C3 Gas Ethylene C2 Gas n-Butane C4 Gas Butyric C4
n-Butane C4 Gas Propylene C3 Gas n-Pentane C5 Liquid -- -- --
n-Hexane C6 Liquid -- -- -- VFA-M CFA** Mixtures Valeric.sup.3 C5
n-Pentane C5 Liquid 1-Butene C4 Liquid n-Hexane C6 Liquid -- -- --
n-Heptane C7 Liquid -- -- -- Caproic C6 n-Hexane C6 Liquid
1-Pentene C5 Liquid n-Heptane C7 Liquid -- -- -- n-Octane C8 Liquid
-- -- -- Caprylic C8 n-Octane C8 Liquid 1-Heptene C7 Liquid
n-Nonane C9 Liquid -- -- -- n-Decane C10 Liquid -- -- -- Capric C10
n-Decane C10 Liquid 1-Nonene C9 Liquid n-Undecane C11 Liquid -- --
-- n-Dodecane C12 Liquid -- -- -- Lauric C12 n-Dodecane C12 Liquid
Higher .alpha.-Alkenes C11 Liquid n-Tridecane C13 Liquid -- -- --
n-Tetradecane C14 Liquid -- -- -- Myristic C14 n-Tetradecane C14
Liquid Higher .alpha.-Alkenes C13 Liquid n-Pentadecane C15 Liquid
-- -- -- n-Hexadecane C16 Liquid -- -- -- Palmitic C16 n-Hexadecane
C16 Liquid Higher .alpha.-Alkenes C15 Liquid n-Heptadecane C17
Solid -- -- -- n-Octadecane C18 Solid -- -- -- Stearic, C18:0
Higher Alkanes C18 Solid Higher Alkenes C17 Solid Oleic, C18:1 C19
Dienes, and Linoleic C18:2 C20 Trienes *Volatile Fatty Acids-From
Ruminal Fermentation Contain Acetic, Propionic, and Butyric
Carboxylic Acids **Medium Chain Fatty Acids-Additions of MCFA to
VFA Yield Higher Carbon-Chain Hydrocarbons .sup.1Kolbe Reaction is
One-Electron Oxidation of Carboxylic Acids Yielding Alkanes
(Dimers, Cross-Radicals) .sup.2Hofer-Moest Reaction is Two-Electron
Oxidation of Carboxylic Acids Yielding Alkenes (Deprotonation)
.sup.3Valeric, Iso-Valeric, 2-Methyl Butyric, and Iso-Butyric Acids
are also produced in Ruminal Fermentation and represent 3% to 5% of
total VFA. Note: Higher carbon-chain fatty acids may require
solvent additions, such as methanol, instead of carrying out
electrolysis in aqueous solutions due to there relative
insolubility in water.
TABLE-US-00013 TABLE 2 Potential Energy - Bond Dissociation Energy
(BDE) Fuel Product Comparison - Electrolysis of Water to Hydrogen
vs. VFA-Carboxylic Acids to Hydrocarbons (Using the Same Amount of
Electrical Energy): BDE/mol - Hydrogen Electrolysis from Water:
Cathode: 2H.sup.+(aq) + 2e.sup.- .fwdarw. H.sub.2 (gas) 436 kJ/mol
BDE BDE/mol - Kolbe Electrolysis Products from VFA Mixture:
(Assuming R--R = 6:2:1 VFA molar Ratio of Acetic, Propionic,
Butyric Acids) Anode: 2RCO.sub.2.sup.-(aq) .fwdarw. R--R (gas) +
2e.sup.- 3346 kJ/mol BDE Cathode: 2H.sup.+(aq) + 2e.sup.- .fwdarw.
H.sub.2 (gas) 436 kJ/mol BDE Total 3782 kJ/mol BDE BDE = (8.7 Times
H.sub.2) Energy Value (kJ/mol) - Electrolysis from Volatile Fatty
Acids (VFA) Alone: Molar Total Total VFA to Fuel Ratio BDE BDE/mol
Ethane 6 2825 1883 Propane 2 3998 888 Butane 1 5171 575 Total
Hydrocarbons 3346 Hydrogen 1 436 436 Total Fuel 3782 (8.7 Times
H.sub.2) Energy Value (kJ/mol) - Electrolysis from Mixtures of
Volatile Fatty Acids (VFA) And Medium Chain Fatty Acids (MCFA)
Compared to Hydrogen: BDE VFA/MCFA Carbon Total Ratio to to Fuel
Chain BDE Hydrogen Ethane C2 2825 6:1 Propane C3 3998 9:1 Butane C4
5171 12:1 Pentane C5 6344 15:1 Hexane C6 7517 17:1 Heptane C7 8690
20:1 Octane C8 9863 23:1 Nonane C9 11036 25:1 Decane C10 12209 28:1
Dodecane C12 14555 33:1 Tetradecane C14 16901 39:1 Hexaadecane C16
19247 44:1 Octadecane C18 21593 50:1 Hydrogen 0 436 1:1
TABLE-US-00014 TABLE 3 Fatty acid composition of some plant oils
and animal fats of commercial importance..sup.1 Alpha Linoleic
Linolenic Capric Lauric Myristic Palmitic Stearic Oleic Acid Acid
Acid Acid Acid Acid Acid Acid (.omega.6) (.omega.3) Oil or Fat
C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Beef Tallow -- -- 3
24 19 43 3 1 Canola Oil -- -- -- 4 2 62 22 10 Cocoa Butter -- -- --
25 38 32 3 -- Cod Liver Oil -- -- 8 17 -- 22 5 -- Coconut Oil 6 47
18 9 3 6 2 -- Corn Oil (Maize Oil) -- -- -- 11 2 28 58 1 Cottonseed
Oil -- -- 1 22 3 19 54 1 Flaxseed Oil -- -- -- 3 7 21 16 53 Lard
(Pork fat) -- -- 2 26 14 44 10 -- Olive Oil -- -- -- 13 3 71 10 1
Palm Oil -- -- 1 45 4 40 10 -- Palm Kernel Oil 4 48 16 8 3 15 2 --
Peanut Oil -- -- -- 11 2 48 32 -- Safflower Oil -- -- -- 7 2 13 78
-- Sesame Oil -- -- -- 9 4 41 45 -- Soybean Oil -- -- -- 11 4 24 54
7 Sunflower Oil -- -- -- 7 5 19 68 1 .sup.1Adapted from Zamora
(2005). Composition varies slightly with varietal source and growth
conditions.
TABLE-US-00015 TABLE 4 Fatty acid composition of some Cuphea seed
oils and of coconut oil..sup.1 Distribution (% of total fatty
acids) C8 C10 C12 C14 Species (Caprylic) (Capric) (Lauric)
(Myristic) Others C. carthagenensis 5.3 81.4 4.7 8.6 C.
epilobilfolia 0.3 19.6 67.9 12.2 C. hookeriana 65.1 23.7 0.1 0.2
10.9 C. laminuligera 17.1 62.6 9.5 10.8 C. lanceolata 87.5 2.1 1.4
9 C. lutea 0.4 29.4 37.7 11.1 21.4 C. koehneana 0.2 95.3 1 0.3 3.2
C. painteri 73 20.4 0.2 0.3 6.1 C. stigulosa 0.9 18.3 13.8 45.2
21.8 C. viscosissima 9.1 75.5 3 1.3 11.1 C. wrightii 29.4 53.9 5.1
11.6 Coconut oil 8 7 48 18 19 .sup.1Adapted from Kleiman, 1990.
TABLE-US-00016 TABLE 5 Chemical analysis of oil from Jatropha
curcas L..sup.1 Type I Type II Type III Free fatty acid content
0.03% 0.18% 3.69% Color (51/4'' Lovibond) 17 yellow; 1, red 14
yellow; 1, 4 red Viscosity @ 100.degree. F. 38.8 CST 37 CST
Saponification number 195.5 193.6 192 Iodine number 94.9 105.2 96
Fatty Acid Profile in % Palmitic (C16:0) 14.6 3.45 15.6 Stearic
(C18:0) 7.15 7.46 6.7 Oleic (C18:1) 46.27 34.3 42.6 Linoleic
(C18:2) 30.8 43.12 33.9 Others 0.2 0.2 0.2 .sup.1Adapted from Lele,
2006.
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References