U.S. patent application number 12/856566 was filed with the patent office on 2011-03-10 for apparatus and process for fermentation of biomass hydrolysate.
Invention is credited to Lisa Beckler Andersen, John H. Evans, IV, Christine A. Singer.
Application Number | 20110059497 12/856566 |
Document ID | / |
Family ID | 43586365 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059497 |
Kind Code |
A1 |
Beckler Andersen; Lisa ; et
al. |
March 10, 2011 |
APPARATUS AND PROCESS FOR FERMENTATION OF BIOMASS HYDROLYSATE
Abstract
A process for converting biomass hydrolysate into biofuel, the
process comprising the steps of: obtaining a biomass hydrolysate
solution comprising monosaccharides; immobilizing Pachysolen
tannophilus; contacting the solution with the immobilized
Pachysolen tannophilus; and recovering a fermented biofuel.
Inventors: |
Beckler Andersen; Lisa;
(Lakewood, CO) ; Evans, IV; John H.; (Superior,
CO) ; Singer; Christine A.; (Lakewood, CO) |
Family ID: |
43586365 |
Appl. No.: |
12/856566 |
Filed: |
August 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61233821 |
Aug 13, 2009 |
|
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Current U.S.
Class: |
435/161 ;
435/170; 435/171; 435/174; 435/178 |
Current CPC
Class: |
C12P 7/06 20130101; Y02E
50/17 20130101; C12P 7/14 20130101; Y02E 50/10 20130101; C12P 7/10
20130101; Y02E 50/16 20130101; C12N 11/10 20130101; C12P 7/065
20130101 |
Class at
Publication: |
435/161 ;
435/178; 435/174; 435/171; 435/170 |
International
Class: |
C12P 7/06 20060101
C12P007/06; C12N 11/10 20060101 C12N011/10; C12N 11/00 20060101
C12N011/00; C12P 1/02 20060101 C12P001/02; C12P 1/04 20060101
C12P001/04 |
Claims
1. A process for converting biomass hydrolysate into biofuel, the
process comprising the steps of: a. obtaining a biomass hydrolysate
solution comprising monosaccharides; b. immobilizing Pachysolen
tannophilus; c. contacting the biomass hydrolysate solution with
the immobilized Pachysolen tannophilus; and d. recovering a
fermented biofuel.
2. The process according to claim 1, wherein Pachysolen tannophilus
is immobilized in calcium alginate.
3. The process according to claim 2, wherein the calcium alginate
is in the form of beads ranging from 0.1 mm to 5 mm in
diameter.
4. The process according to claim 2, wherein the calcium alginate
is in the form of a coating applied to a natural matrix.
5. The process according to claim 2, wherein the calcium alginate
is in the form of a coating applied to a synthetic matrix.
6. The process according to claim 2, further comprising the step of
treating the calcium alginate immobilized Pachysolen tannophilus
with a yeast growth medium.
7. The process according to claim 3, further comprising the step of
recovering and recycling calcium alginate used to immobilize the
Pachysolen tannophilus.
8. The process according to claim 7, wherein the calcium alginate
used to immobilize the Pachysolen tannophilus is recovered and
recycled by a process comprising the steps of: a. treating the
calcium alginate with a calcium chelator and monovalent counter-ion
to thereby form a solution; and b. performing dialysis on the
solution against an inorganic salt to form sodium alginate.
9. The process according to claim 1, wherein the biomass
hydrolysate solution comprises a substantial amount of fermentation
inhibitors.
10. The process according to claim 9, wherein the solution of
monosaccharides has furfural levels in the range of about 0.01 to
10 g/L.
11. The process according to claim 9, wherein the solution of
monosaccharides has 5-hydroxymethylfurfural levels in the range of
about 0.01 to 10 g/L.
12. The process according to claim 9, wherein the solution of
monosaccharides has acetic acid levels in the range of about 0.5 to
20 g/L.
13. The process according to claim 1, wherein more than 80% of the
monosaccharides in the solution are converted to ethanol.
14. The process according to claim 1, wherein the biomass
hydrolysate is obtained by pressing a pretreated biomass.
15. The process according to claim 1, wherein the biomass
hydrolysate is obtained by pressing biomass subjected to a
pretreatment process and a saccharification process.
16. The process according to claim 2, wherein the calcium alginate
is hardened to increase structural stability.
17. A process for converting biomass hydrolysate into biofuel, the
process comprising the steps of: a. contacting the biomass
hydrolysate solution with a first immobilized microbe strain; b.
contacting the biomass hydrolysate solution with a second
immobilized microbe strain; and c. recovering a fermented
biofuel.
18. The process according to claim 17, wherein the first
immobilized microbe strain is a bacterium and the second
immobilized microbe strain is a yeast.
19. The process according to claim 17, wherein the first
immobilized microbe strain is contained in a first reactor and the
second immobilized microbe strain is contained in a second
reactor.
20. The process according to claim 17, wherein the first
immobilized microbe strain and the second immobilized microbe
strain are immobilized together within the same immobilization
medium.
21. The process according to claim 20, wherein the immobilization
medium is a calcium alginate bead.
22. The process according to claim 17, wherein the first
immobilized microbe strain is immobilized in a first immobilization
medium and the second immobilized microbe strain is immobilized in
a second immobilization medium.
23. The process according to claim 22, wherein the first
immobilization medium is a first plurality of calcium alginate
beads and the second immobilization medium is a second plurality of
calcium alginate beads.
24. The process of claim 17 wherein the second immobilized microbe
strain is capable of fermenting a hexose mannose to a biofuel.
25. A process for converting biomass hydrolysate into biofuel, the
process comprising the steps of: a. flowing a biomass hydrolysate
solution comprising monosaccharides and one or more inhibitory
secondary products through a continuous flow reactor containing an
immobilized microbe strain and contacting the immobilized microbe
strain with the biomass hydrolysate; e. recovering a fermented
biofuel.
26. The process according to claim 25, wherein the flow rate of the
biomass hydrolysate exceeds the sedimentation rate of the
immobilized microbe strain in a "free" condition.
27. The process according to claim 25, wherein the continuous flow
reactor is an upflow reactor.
28. The process according to claim 25, wherein the productivity of
the biofuel conversion process is at least 0.3 g/Lh for a flow rate
corresponding to a 10 hour retention time.
29. The process according to claim 25, wherein the productivity of
the biofuel conversion process is at least 0.42 g/Lh for a flow
rate corresponding to a 5 hour retention time.
30. A medium for fermenting biomass hydrolysate, the medium
comprising: calcium alginate beads ranging from 0.1 mm to 5 mm in
diameter; a microbe strain capable of fermenting pentoses
immobilized in the calcium alginate beads, wherein the immobilized
microbe strain is capable of converting at least 70% of available
pentoses in a biomass hydrolysate to a biofuel.
31. A medium for fermenting biomass hydrolysate, the medium
comprising: an immobilization substance capable of providing a
micro environment for a microbe strain; a microbe strain capable of
fermenting pentoses into a biofuel immobilized in the
immobilization substance, wherein the microbe strain comprises
about 5% by volume of the immobilization substance.
32. A process for converting biomass hydrolysate into biofuel the
process comprising the steps of: contacting a biomass hydrolysate
solution with an immobilized fermentative microbe strain for a
sufficient reaction time to convert monosaccharides in the biomass
hydrolysate to biofuel; and recovering biofuel from the fermented
hydrolysate.
33. The process according to claim 32, wherein the immobilized
fermentative microbe strain is a yeast and the process further
comprises the step of treating the yeast with a yeast regeneration
medium.
34. The process according to claim 32, further comprising the step
of conditioning the biomass hydrolysate by passing the hydrolysate
over activated carbon, strong acid ion exchange resin and weak base
ion exchange resin.
35. The process according to claim 32, wherein the immobilized
fermentative microbe strain is immobilized in calcium alginate and
the process further comprises the step of recovering and recycling
the calcium alginate.
36. The process according to claim 32, wherein the biomass
hydrolysate solution contains inhibitory secondary products
sufficient to prevent more than 50% conversion of pentoses by the
fermentative microbes in their "free" state.
37. The process according to claim 32, wherein the immobilized
fermentative microbe strain is at least one strain selected from
the group consisting of Pichia, Candida, Klyveromyces and Zymomonas
mobilis NREL strain 8b.
38. The process according to claim 32, wherein the immobilized
fermentative microbe strain converts about 30% more pentoses than
the same fermentative microbe strain in a "free" condition.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/233,821, filed Aug. 13, 2009, which is hereby
incorporated by reference.
FIELD
[0002] The present patent document relates to an apparatus and
process for fermentation of biomass hydrolysate.
BACKGROUND
[0003] Recently, conversion of biomass through saccharification and
fermentation into ethanol or other useful products as a replacement
for fossil fuels has garnered considerable attention. Because
biomass is a renewable resource typically rich in polymers of
hexoses and pentoses, it is a promising substrate for
fermentation.
[0004] Biomass for such conversion processes may be potentially
obtained from numerous different sources, including, for example:
wood, paper, agricultural residues, food waste, herbaceous crops,
and municipal and industrial solid wastes to name a few.
[0005] Biomass is made up primarily of cellulose and hemicellulose
bound up with lignin. The lignin inhibits the conversion of the
biomass into ethanol or other biofuels, and, as a result, typically
a pretreatment step is required to expose the polysaccharides,
cellulose and hemicellulose. Once hemicellulose and cellulose are
exposed, saccharification, either enzymatic or chemical, may be
performed to break the polysaccharides into their constituent
monosaccharide monomers. Pretreatment and saccharification are
used, therefore, to break down the long polysaccharide chains and
free the sugars before they are fermented for biofuel production.
Fermentation can begin once free sugars are present, either because
they are naturally present or because a portion of the biomass has
been reduced to its component sugars, or both.
[0006] In order to be effective, current pretreatment and
saccharification processes attempt to liberate the biomass sugars
while also minimizing the formation of secondary products from the
degradation of hemicellulose, cellulose, and lignin, because of the
inhibitory effects secondary products may have on the subsequent
fermentation processes. The presence of inhibitory secondary
products has historically complicated ethanol production and
increased the cost of production due to elaborate detoxification
steps.
[0007] Although numerous techniques for pretreatment and
saccharification exist, the most popular methods, and the most cost
effective methods, including acid hydrolysis, produce secondary
products in addition to sugars, that are inhibitory to
fermentation. Inhibitory secondary products created as a result of
the degradation of hemicellulose pentoses and hexoses include
furfural and 5-hydroxymethylfurfural (HMF), respectively. Furfural
and HMF may further be broken down into levulinic, acetic, and
formic acids. Other inhibitory secondary products include phenolic
compounds produced from the degradation of lignin and acetic acid
produced by cleavage of acetyl groups within the hemicellulose.
Concentrations of inhibitory secondary products in the hydrolysate
will vary based on the source of the biomass and the hydrolysis
method used.
[0008] Some of the secondary products formed from the breakdown of
hemicellulose, cellulose and lignin are in themselves valuable
substances. The inventors have realized that recovery of high-value
secondary products from the hydrolysate can improve the economics
of the biomass to biofuel process.
[0009] Other secondary products are not formed from chemical
decomposition, but may be extracted from the biomass during
pretreatment and hydrolysis. These extracted secondary products
include terpenes, sterols, fatty acids, and resin acids. These
extracted compounds may also be inhibitory to fermentation.
[0010] Inhibitory secondary products may be detrimental to the
fermentation process, particularly as their concentration
increases. Thus, it would be advantageous if a process could be
developed that allows specific microbes, like yeast for example, to
efficiently convert biomass hydrolysate into biofuels, such as
ethanol, in the presence of inhibitory secondary products formed
during pretreatment and hydrolysis.
[0011] Many inhibitory products have compound impacts when present
with other inhibitory compounds; thus, a non-inhibitory amount of a
certain compound may become inhibitory in the presence of a second
inhibitory compound. Furthermore, even following partial recovery
and/or removal of inhibitory secondary products, the remaining
concentrations may be inhibitory to fermentation due to these
synergies. Thus, it would be advantageous if a process could be
developed that allows specific microbes, like yeast for example, to
efficiently convert biomass hydrolysate into biofuels, such as
ethanol, in the presence of inhibitory secondary products formed
during pretreatment and hydrolysis, even when the concentrations of
the individual inhibitory secondary products are below their
respective inhibitory concentration level but their combined
concentration is inhibitory.
[0012] Cellulose is a homogeneous polysaccharide composed of
linearly linked glucose units. Glucose is a hexose, which may be
readily fermented by a number of microbes including Saccharomyces
cerevisiae (traditional baker's yeast) and Kluyveromyces marxianus.
Yeast cells are especially attractive for cellulosic ethanol
processes, as they have been used in biotechnology for hundreds of
years, are tolerant to high ethanol and inhibitor concentrations,
and can grow at low pH values. A low pH value helps avoid bacterial
contamination and is therefore advantageous.
[0013] Unlike cellulose, hemicellulose is a heterogeneous polymer
of pentoses, hexoses, and uronic acids. The saccharides principally
found in hemicellulose are the pentoses xylose and arabinose and
the hexoses glucose, mannose and galactose. The relative amounts of
different pentoses and hexoses vary with the biomass type. The
hemicellulose content of some cellulosic biomass may reach as high
as 38% or more of the total dry biomass weight. Therefore,
hemicelluloses, and the pentoses and hexoses they contain, may
comprise a substantial portion of the convertible sugars available
in the biomass. As a result, in order to improve the economics of
the biomass to biofuel conversion process, much research has been
performed on identifying microorganisms that efficiently convert
pentoses and hexoses to biofuel, such as ethanol.
[0014] While numerous microbes have been found to process hexoses
into ethanol, efficiently fermenting pentoses has proven more
elusive. Some bacteria and fungi can inefficiently convert pentoses
to ethanol and many microbes can only process pentoses when
assisted by enzymes. For a long time it was thought that yeast
strains could not anaerobically ferment pentoses. However, U.S.
Pat. No. 4,359,534 to Kurtzman et al. discloses the use of
Pachysolen tannophilus to ferment pentoses. Similarly, U.S. Pat.
No. 7,344,876 to Levine discloses a pure culture of Kluyveromyces
marxianus capable of proliferation on pentoses as the sole carbon
source.
[0015] While the patents to Kurtzman and Levine disclose the use of
yeasts for fermentation of pentoses into ethanol, commercial
applications have been limited because of the detrimental effects
of inhibitory secondary products typically found in biomass
hydrolysate. Yeasts that can ferment xylose and other pentoses in
an artificial, or controlled, medium generally perform poorly in
acid hydrolysates. Challenges presented by biomass hydrolysate
include an acidic pH and a high concentration of toxic compounds,
including acetic acid, phenolic compounds, 5-hydroxymethylfurfural
(HMF) and furfural, and other inhibitory molecules produced during
hemicellulose hydrolysis.
[0016] Because of the detrimental effects of inhibitory secondary
products on the production of ethanol, biomass hydrolysate is
currently subjected to a conditioning process after pretreatment
and hydrolysis to reduce the concentration of inhibitory secondary
products. This conditioning process adds complexity and cost to the
overall process and reduces the efficiency and cost-effectiveness
of the conversion process. Furthermore, the greater the required
reduction in the concentration levels of the inhibitory secondary
products, the greater the complexity and cost. A need, therefore,
exists for a process in which microbes, such as different yeast
strains, could more effectively convert pentoses, as well as
hexoses, into ethanol and other biofuels in the presence of
inhibitors formed during the pretreatment and hydrolysis process.
In addition, it would be beneficial to develop schemes whereby
inhibitory secondary products may be partially recovered and
purified, instead of only removed and discarded, from
hydrolysate.
[0017] Furthermore, if an efficient method for converting pentoses
to ethanol existed, the discarded hemicellulose in the paper
pulping process might be converted into alcohol instead. Similarly,
sugar cane residues, referred to as bagasse, could also be
subjected to hemicellulose conversion prior to being combusted for
their fuel values. The possibility of removing hemicellulose from
the paper pulping process and converting it to ethanol was
hypothesized by the Georgia Institute of Technology in W. J.
Fredrick et al., Co production of ethanol and cellulose fiber from
Southern Pine: A technical and economic assessment, 32 Biomass and
Bioenergy 1293-1302 (2008). However, the Georgia Institute of
Technology process explicitly requires the hydrolysate to be
conditioned to remove inhibitors and noted the lack of an efficient
process to convert pentoses into ethanol. The study noted that
"Fermentation is carried out after inhibiting contaminants have
been removed from the hydrolysate." The study further notes that
the 85% conversion factor of pentoses to ethanol "is an optimistic
estimate that assumes that on-going research will make it possible
. . . ." The study concludes that ethanol production from loblolly
pine may not be competitive with ethanol from other lignocellulosic
sources when it is co-produced with cellulose fiber.
SUMMARY OF THE INVENTION
[0018] In view of the foregoing, an object according to one aspect
of the present patent document is to provide an improved apparatus
and process for converting biomass hydrolysate into ethanol or
other biofuel. Preferably the apparatus and process address, or at
least ameliorate one or more of the problems described above. To
this end, a process for converting biomass hydrolysate into biofuel
is provided; the process comprises the steps of: obtaining a
biomass hydrolysate solution comprising monosaccharides;
immobilizing a fermentative microbe contacting the solution with
the immobilized fermentative microbe; and recovering a fermented
biofuel. The recovered biofuel preferably comprises alcohol, and
more preferably comprises ethanol.
[0019] In another embodiment, a process for converting biomass
hydrolysate into biofuel is provided comprising the steps of:
contacting a biomass hydrolysate solution with immobilized
fermentative microbe strain for a sufficient reaction time to
convert monosaccharides in the biomass hydrolysate to biofuel; and
recovering biofuel from the fermented hydrolysate.
[0020] In certain implementations of the foregoing embodiments, the
fermentative microbe is Pachysolen tannophilus and Pachysolen
tannophilus is immobilized in calcium alginate. The calcium
alginate may be in the form of beads ranging from 0.1 mm to 5 mm in
diameter, and are more preferably about 2 mm to 3 mm in diameter.
The calcium alginate is not required to be in bead form and may be
in any other form that permits the Pachysolen tannophilus to be
immobilized but still allows the sugar substrates in the biomass
hydrolysate to kinetically interact with the yeast. For example,
the calcium alginate may be in a sponge or mesh form. Similarly,
the Pachysolen tannophilus/calcium alginate mixture may be applied
as a coating to a natural or synthetic matrix to increase the
surface area per mass of Pachysolen tannophilus/calcium alginate
mixture.
[0021] Preferably, the immobilized culture of Pachysolen
tannophilus is periodically treated with a yeast growth medium to
restore metabolic efficiency to the Pachysolen tannophilus. The
metabolic efficiency may be lost over long periods of use,
especially in connection with continuous flow bioreactors.
[0022] In another embodiment, the immobilized fermentative microbe
strain is at least one microbe selected from a group consisting of
Pichia, Candida, Klyveromyces and Zymomonas mobilis NREL strain
8b.
[0023] In yet another embodiment, the alginate used to immobilize
the culture of Pachysolen tannophilus is periodically recovered and
recycled by treating the Pachysolen tannophilus/calcium alginate
with a calcium chelator and monovalent counter-ion, such as sodium
citrate. The resulting dialysis of the solution with an inorganic
salt, such as sodium chloride, regenerates sodium alginate, from
which calcium alginate may be regenerated.
[0024] In yet another embodiment, the biomass hydrolysate contains
a substantial amount of secondary products that inhibit
fermentation. The hydrolysate solution may contain furfural levels
in the range of about 0.01 to 10 g/L, 5-hydroxymethylfurfural
levels in the range of about 0.01 to 10 g/L, and acetic acid levels
in the range of about 0.05 to 20 g/L, or even 0.5 to 20 g/L. In
addition, the hydrolysate solution may contain phenolic compounds
in the range of about 0.01 to 10 g/L. These levels of furfural,
HMF, phenolic compounds, and acetic acid may occur in combination
or in isolation. Other inhibitors may also be present.
[0025] In yet another embodiment, more than 80% of the
monosaccharides in the solution are converted to ethanol.
[0026] In still another embodiment, the biomass hydrolysate is
obtained from the biomass by pressing. The biomass and biomass
hydrolysate may be subjected to a high pressure press capable of
squeezing the sugar-containing liquid forming the biomass
hydrolysate out of the biomass residue.
[0027] In other embodiments, the biomass hydrolysate may be
conditioned by passing the hydrolysate over activated carbon, a
strong acid ion exchange resin and/or a weak base ion exchange
resin.
[0028] In the various embodiments described above, the biomass
hydrolysate solution may contains inhibitory secondary products
sufficient to prevent more than 50% conversion of pentoses by the
fermentative microbes in their "free" state.
[0029] In another aspect, a process for converting biomass
hydrolysate into biofuel is provided comprising the steps of:
contacting the biomass hydrolysate solution with a first
immobilized microbe strain; contacting the biomass hydrolysate
solution with a second immobilized microbe strain; and recovering a
fermented biofuel.
[0030] In one embodiment the first immobilized microbe strain is a
bacterium and the second immobilized microbe strain is a yeast.
Further, the first immobilized microbe strain may be contained in a
first reactor and the second immobilized microbe strain may be
contained in a second reactor. In an alternative embodiment, both
immobilized microbe strains may be in the same reactor. If
implemented so both strains are in the same reactor, the first
immobilized microbe strain and the second immobilized microbe
strain may also be immobilized together within the same
immobilization medium.
[0031] Preferably, the immobilization medium is a calcium alginate
bead, but other immobilization mediums may also be used. Further,
the first immobilized microbe strain may be immobilized in a first
immobilization medium and the second immobilized microbe strain may
be immobilized in a second immobilization medium.
[0032] In one embodiment, the second immobilized microbe strain is
capable of fermenting mannose to a biofuel.
[0033] In yet another aspect of the present patent document, a
process for converting biomass hydrolysate into biofuel is provide
comprising the steps of: flowing a biomass hydrolysate solution
comprising monosaccharides and one or more inhibitory secondary
products through a continuous flow reactor containing an
immobilized microbe strain and contacting the immobilized microbe
strain with the biomass hydrolysate; and recovering a fermented
biofuel.
[0034] In one embodiment, the flow rate of the biomass hydrolysate
is set to exceed the sedimentation rate of the immobilized microbe
strain in a "free" condition. Preferably, the continuous flow
reactor is an upflow reactor, but other continuous reactors may
also be used.
[0035] In another embodiment, the productivity of the biofuel
conversion process is at least 0.3 g/Lh for a flow rate
corresponding to a 10 hour retention time. In still another
embodiment, the productivity of the biofuel conversion process is
at least 0.42 g/Lh for a flow rate corresponding to a 5 hour
retention time.
[0036] In a further aspect, a medium for fermenting biomass
hydrolysate is provided. In one embodiment, the medium comprises
calcium alginate beads ranging from 0.1 mm to 5 mm in diameter, and
a microbe strain capable of fermenting pentoses immobilized in the
calcium alginate beads, wherein the immobilized microbe strain is
capable of converting at least 70% of available pentoses in a
biomass hydrolysate to a biofuel.
[0037] In yet another aspect, a medium for fermenting biomass
hydrolysate is provided, comprising an immobilization substance
capable of providing a micro environment for a microbe strain; and
a microbe strain capable of fermenting pentoses into a biofuel
immobilized in the immobilization substance, wherein the microbe
strain comprises about 5% by volume of the immobilization
substance.
[0038] As described more fully below, the apparatus and processes
of the present patent document permit the efficient conversion of
biomass hydrolysate into ethanol, even in the presence of high
levels of inhibitory secondary products formed or extracted during
pretreatment and/or fermentation steps of the process. Further
aspects, objects, desirable features, and advantages of the methods
disclosed herein will be better understood from the detailed
description and drawings that follow in which various embodiments
are illustrated by way of example. It is to be expressly
understood, however, that the drawings are for the purpose of
illustration only and are not intended as a definition of the
limits of the claimed invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 illustrates an overview of one embodiment of a
process for the conversion of biomass into a biofuel such as
ethanol.
[0040] FIG. 2 illustrates an overview of another embodiment of a
process for the conversion of biomass into biofuels such as
ethanol.
[0041] FIG. 3 illustrates a process for recycling a calcium
alginate immobilization medium.
[0042] FIG. 4 illustrates a view of one embodiment of a bioreactor
for performing submerged fermentation of biomass hydrolysate using
immobilized microbes.
[0043] FIG. 5A illustrates a side view of another embodiment of a
bioreactor for performing submerged fermentation of biomass
hydrolysate using immobilized microbes.
[0044] FIG. 5B illustrates a front view of the bioreactor shown in
FIG. 5A.
[0045] FIG. 6 illustrates an up-flow reactor for performing
submerged fermentation of biomass hydrolysate using immobilized
microbes.
[0046] FIG. 7 is a graph illustrating ethanol yield of regenerated
calcium alginate beads with immobilized fermentative microbes over
a series of fermentations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] Consistent with its ordinary meaning as a renewable energy
source, the term "biomass" is used herein to refer to living and
recently dead biological material including carbohydrates, proteins
and/or lipids that may be converted to fuel for industrial
production. By way of non-limiting example, "biomass" refers to
plant matter, including, but not limited to switchgrass, sugarcane
bagasse, corn stover, corn cobs, alfalfa, Miscanthus, poplar, and
aspen, biodegradable solid waste such as dead trees and branches,
yard clippings, recycled paper, recycled cardboard, and wood chips,
plant or animal matter, and other biodegradable wastes.
[0048] The present patent document teaches new and improved
processes and apparatuses for fermenting biomass hydrolysate.
Processes used to convert polysaccharides in biomass into hexoses
and pentoses often create inhibitory secondary products that
prevent or hinder fermentation. Furthermore, the combinations of
inhibitory secondary products found in actual biomass hydrolysate
are more toxic to ferments than any single inhibitory secondary
product added to a defined, artificial medium. The present patent
document teaches novel processes that increase the tolerance of the
fermentative microbes to inhibitory secondary products found in
biomass hydrolysate by immobilizing the microbes. In certain
embodiments, fermentation of hemicellulose hydrolysate containing
inhibitory secondary products is carried out using immobilized
Pachysolen tannophilus. In some embodiments, fermentation of
hemicellulose hydrolysate is carried out using an immobilized
microbe, even though the concentration of an individual secondary
product or the combined concentration of secondary products in the
biomass hydrolysate would be inhibitory to the microbe in its free
state.
[0049] Immobilization confers an increased resistance on microbes
to inhibitory secondary products. For example, immobilization in a
calcium alginate greatly reduces the susceptibility of the yeast
Pachysolen tannophilus to inhibitors contained in softwood
hydrolysate. The benefits of immobilization, however, are not
limited to Pachysolen tannophilus. Indeed, numerous different
microbes may benefit from immobilization including, for example,
yeasts from the genera Pichia, Candida, and Klyveromyces. In
addition, bacterium microbes such as Zymomonas mobilis, NREL strain
8b, also show an increased resistance to inhibitory secondary
products when immobilized.
[0050] Preferably the calcium alginate, or other material used to
immobilize the microbes, is in a form with a high surface area such
as in bead, sponge, or mesh form. In addition, the immobilized
microbe or combination of microbes should also be able to ferment
monosaccharides found in hemicellulose hydrolysates--including the
hexoses mannose, galactose and glucose and the pentoses xylose and
arabinose--to biofuel with high efficiency.
[0051] FIG. 1 illustrates a general overview of one embodiment of a
process for converting biomass to ethanol or other biofuels. The
primary steps include pretreatment 100, hydrolysis 102,
fermentation 104, and biofuel recovery 106. FIG. 2 illustrates
another embodiment of a process for converting biomass to ethanol
or other biofuels. The process in FIG. 2 differs from that in FIG.
1 in that it also includes a solid/liquid separation step 108, an
optional evaporation step 112, an optional conditioning step 110,
and an optional secondary product recovery step 114. If the biomass
hydrolysate is provided from another source instead of generated on
site, the process of the present patent document may be condensed
to performing step 104 or steps 104 in combination with step
106.
[0052] Before biomass can be fermented, it often needs to undergo
some form of process to disrupt the polymer network of cellulose,
hemicellulose, and lignin forming the biomass structure so the
polysaccharides can be reduced to monosaccharides. This process is
commonly referred to as "pretreatment" and is designed to reduce
the recalcitrance of the biomass to enzymatic or chemical
saccharification of the cellulose and hemicellulose, therein. The
pretreatment step 100 may occur through a number of methods,
including for example, in a pressure reactor. Table 1 lists
appropriate ranges for temperature, dwell time, and moisture
content suitable for pretreatment in a pressure reactor. However,
other operating conditions may also be suitable.
TABLE-US-00001 TABLE 1 Pressure Reactor Pretreatment Conditions
Temperature* 105-200.degree. C. Time 1 minute-24 hours Moisture
Content 25-95% *Temperature dictates the pressure in a sealed
vessel assuming a saturated steam system
[0053] Effectiveness of the pretreatment step 100 may be increased
by adding one or more reagents. Reagents may include, but are not
limited to: nitric acid, phosphoric acid, hydrochloric acid,
sulphuric acid, sulphur dioxide, and sodium sulphite. Other
reagents that reduce the recalcitrance of the biomass to
hemicellulose removal may also be added.
[0054] In addition to performing pretreatment 100 in a pressure
reactor, pretreatment 100 may be performed using a number of other
methods, including acid prehydrolysis, steam cooking, alkaline
processing, rotating augers, steam explosion, ball milling, or any
other method that reduces the recalcitrance of the biomass to
saccharification of the cellulose and hemicellulose contained
therein.
[0055] Once the cellulose and hemicellulose are exposed through
pretreatment 100, the polysaccharides are broken down into their
monosaccharide components so they can be fermented.
[0056] The Hydrolysis step 102 is used for converting the
polysaccharides into fermentable sugars. In some of the harsher
pretreatments 100, hydrolysis 102 may occur simultaneously with the
pretreatment step 100 and a separate hydrolysis step 102 is not
required. The two basic forms of hydrolysis 102 are thermo-chemical
and enzymatic. Thermo-chemical hydrolysis is typically performed
using a concentrated acid such as sulfuric acid or hydrochloric
acid at relatively low temperatures or by using a dilute acid at
relatively high temperatures.
[0057] Once the monosaccharides have been generated through the
hydrolysis step 102, fermentation can begin. Although fermentation
can occur within the biomass residue with some fermentation
techniques, in the processes described in the present patent
document, a biomass hydrolysate solution comprising monosaccharides
will typically be obtained by pressing and/or washing the biomass
residue. The obtained biomass hydrolysate is then fermented ex-situ
in fermentation step 104.
[0058] Recovery of the sugars from the biomass residue is
preferably achieved through solid-liquid separation. For example,
as shown in FIG. 2, a solid-liquid separation step 108 may be used
to recover the sugars from the biomass residue. Solid-liquid
separation may be performed using a number of methods including,
but not limited to, centrifuging or pressing. Preferably, pressing
may be accomplished with a hydraulic press. However, numerous types
of mechanical or machine presses may be used. For example, a
mechanical press such as a conventional screw press, a
hydro-mechanical press, a pneumatic press or any other type of
press that can apply the necessary pressure to remove the
hemicellulose hydrolysate from the cellulose/lignin residue may be
used. The press may have a range of capabilities and configurations
for pressing out the hemicellulose hydrolysate. Preferably the
press can generate from at least about 10.5 kg/cm.sup.2 to about
21.1 kg/cm.sup.2. In other embodiments, it is desirable if the
press can generate at least approximately 1,410 kg/cm.sup.2.
[0059] Pressing has additional advantages because the biomass
residue (which will comprise cellulose and lignin at this point)
may be more valuable as a coal replacement if its density can be
maximized and its moisture content minimized, thereby increasing
its energy density. For pulp mill feed there are no requirements
for moisture or density but minimization of fiber damage is
important. Pulp quality is measured based on its fiber length,
among other variables, but not moisture content. However, if a high
energy density fuel replacement is made instead of paper pulp,
reducing the moisture content is an important factor.
[0060] Accordingly, the final product that the biomass residue is
to eventually be used for may determine what size and kind of press
to use for solid/liquid separation. For example, if the biomass
residue is to eventually be used to generate cellulose and/or
lignin fibers to make paper products, cardboard, or fiberboard, a
lower pressure, such as in the range of 10.5 kg/cm.sup.2 to 21.1
kg/cm.sup.2 may be advantageous to minimize damage to the cellulose
fibers. In processes that turn the biomass residue into high energy
density fuel, higher pressures may be used to minimize the moisture
content, without regard to fiber quality. As a result, it may be
desirable to employ pressures of about 1,410 kg/cm.sup.2 or even
higher. In other embodiments, however, pressures within the range
of 10.5 kg/cm.sup.2 to 21.1 kg/cm.sup.2 may still be used, as
presses generating these types of pressures are readily available
and comparatively inexpensive as compared to presses that are
capable generating about 1410 kg/cm.sup.2 of pressure. For example,
presses that generate between about 10.5 kg/cm.sup.2 and 21.1
kg/cm.sup.2 of pressure are routinely used in the wine and olive
oil industries to press grapes and olives, respectively.
[0061] When sugarcane bagasse is used as the biomass from which the
hydrolysate is pressed, fiber condition is generally unimportant.
However, when used as a high energy density fuel replacement, the
moisture content is an important factor. Therefore, higher, rather
than lower pressures, may be desirable for purposes of performing
the solid/liquid separation step 108.
[0062] Pressing is also advantageous because it reduces dilution
from wash water. Using wash water to separate the hydrolysate from
the biomass will dilute the sugar stream and thus lower the
resulting ethanol concentration in the fermented hydrolysate. If
wash water is used, however, dilution of the sugar stream may be
mitigated by the use of evaporators or similar machinery to reduce
water content in the hydrolysate through optional evaporation step
112, shown in FIG. 2. The recovered water from evaporation may be
recycled into subsequent wash processes. Addition of an evaporation
step 112 as a process step increases the sugar concentration of the
hydrolysate and thus the ethanol concentration resulting from
fermentation, which in turn reduces the costs of distillation.
[0063] Once the monosaccharides are separated from the biomass,
there are a number of microbes that may be used for converting the
monosaccharides of the biomass hydrolysate into ethanol or other
biofuels in fermentation step 104. For example, if the biomass
hydrolysate comprises a cellulose hydrolysate, so as to include
glucose (which is a hexose), the glucose in the hydrolysate may be
fermented by a number of yeast strains including Saccharomyces
cerevisiae (traditional baker's yeast) and Kluyveromyces marxianus
to name a few.
[0064] On the other hand, if the biomass hydrolysate comprises a
hemicellulose hydrolysate, the hydrolysate will include the
pentoses xylose and arabinose, and a lower concentration of
hexoses, except in the case of softwood hydrolysate. In the case of
softwood hemicellulose, the hexose mannose is the major saccharide
and the pentose xylose is the next most abundant. Microbes that can
convert the combination of pentoses and hexoses found in
hemicellulose hydrolysate into ethanol are not as abundant as those
available for cellulose hydrolysate. To convert sugars from
hemicellulose hydrolysate into ethanol, microbes that can convert
both five-carbon and six-carbon sugars are preferably utilized so
that all of the available constituent sugars of the hemicellulose
hydrolysate may be converted to ethanol or other biofuels. The same
is true if the biomass hydrolysate comprises a combination of
cellulose hydrolysate and hemicellulose hydrolysate. Microbes that
can ferment hexoses and pentoses may be derived from the genera
Pachysolen, Kluyveromyces, Pichia, and Candida. Pachysolen
tannophilus is preferably used in fermentation of a liquid
hydrolysate comprising a hemicellulose hydrolysate. In particular,
when immobilized, Pachysolen tannophilus has been found to
effectively ferment hemicellulose hydrolysate produced from
softwood.
[0065] In addition to immobilized yeasts, immobilized bacterium may
also be used to ferment hexose and pentose sugars in biomass
hydrolysate. For example, the recombinant bacterium Zymomonas
mobilis (NREL recombinant 8b) may be used to ferment hemicellulose
hydrolysate produced from softwood, hardwood, and/or herbaceous
sources.
[0066] Microbes with complementary metabolic properties may also be
combined in the same fermentation process in step 104 to allow
their complementary properties and abilities, such as complementary
hexose and pentose fermentation capabilities or complimentary
metabolic rates, to be used together. For example, recombinant
Zymomonas is unable to ferment mannose, the most prevalent sugar
contained in softwood hydrolysate, the recombinant Zymomonas
mobilis is preferably paired with a complementary yeast or
bacterium that is able to effectively ferment the hexose mannose to
ethanol or another biofuel when it used to ferment softwood
hydrolysate. On the other hand, in the case of sugarcane bagasse,
where the hydrolysate primarily comprises xylose and glucose,
another microbe is not required to assist the recombinant Zymomonas
to achieve a satisfactory fermentation of the contained sugars.
[0067] Other combinations of microbes are also possible including
pairing different bacterium together, pairing different yeasts
together, pairing various yeasts and bacterium together, or pairing
or combining any number of microbes with complimentary features
including using any number of microbes at the same time. As the
number of combined microbes increases, however, their capabilities
may begin to overlap significantly and thereby reduce the additive
value of the additional microbes.
[0068] Depending on the biomass and treatments employed, the
pretreatment step 100 and hydrolysis step 102 may yield soluble
sugars from the biomass in the form of xylose, mannose, arabinose,
galactose, and glucose ready for fermentation in step 104. However,
other secondary products, which are inhibitory to the fermentation
step 104, are also produced or extracted from the biomass. The
concentrations of fermentation inhibitors that form in converting
biomass to fermentable hexoses and pentoses will vary depending on
the source of the biomass and the methods used for the pretreatment
step 100 and the hydrolysis step 102. For example acetic acid is
produced by cleavage of acetyl groups from hemicellulose. In
addition, some of the pentoses and hexoses are degraded due to
dehydration into furfural and HMF. Phenolic and polyphenolic
compounds (collectively "Phenolic Compounds") are also formed from
the degradation of lignin. While the generated Phenolic Compounds,
furfural, HMF, and acetic acid are all potentially valuable
compounds, they are also fermentation inhibitors, and may prevent
or inhibit fermentation, particularly as their concentrations
increase.
[0069] In addition, Furfural and HMF degrades to produce levulinic
acid, acetic acid, and formic acid, which are even more potent
fermentation inhibitors. Phenolic and polyphenolic compounds
produced from hydrolysis of wood hemicellulose and the concomitant
lignin degradation include guaiacol, vanillin, phenol, vanillic
acid, syringic acid, salicylic acid, gentisic acid, and others.
Many of these compounds, for instance vanillin and vanillic acid,
are known to inhibit the growth of and/or fermentation with
microbial yeasts, such as Pachysolen and Saccharomyces.
[0070] In addition to secondary products made from the degradation
of hemicellulose components, other molecules may be extracted from
biomass by the pretreatment and/or saccharification conditions
during the pretreatment step 100 and/or hydrolysis step 102. These
extracted compounds may include terpenes, sterols, fatty acids, and
resin acids. These extracted compounds can also be inhibitory to
metabolic processes, including fermentation, in yeast and other
microbes, such as bacteria.
[0071] Furthermore, metal cations including calcium, aluminum,
potassium, and sodium are found in hemicellulose hydrolysate and
heavy metals may be present from degradation of the metal vessels
due to hydrolysis. The presence of such metal cations may also be
inhibitory above certain concentrations.
[0072] As made clear from the foregoing discussion, the environment
experienced by microbes in biomass hydrolysate is in stark contrast
to a defined, artificial medium where all or most of these
additional inhibitors are not present or are added experimentally
one at a time to study their effects. Indeed, in a biomass
hydrolysate the various inhibitory compounds discussed above, as
well as others, may work synergistically with one another so that a
non-inhibitory amount of a certain compound may become inhibitory
in the presence of one or more additional compounds that are also
below their respective individual inhibitory concentrations.
[0073] Because many secondary products can degrade the fermentation
process as their concentrations increase, prior methods for
conversion of biomass into ethanol have employed a costly
conditioning step to remove or reduce the concentration of
inhibitors from the hydrolysate prior to fermentation. Furfural,
HMF, and acetic acid, as well as phenolics are the most commonly
found inhibitors in biomass hydrolysate. Levels in the range of
0.2-5.0 g/L furfural, 0.2-6.0 g/L HMF, and 3.0-10.9 g/L acetic acid
are considered common and may greatly reduce fermentation or
prevent it all together. Likewise, concentrations of phenolics in
the range of 0.1-10 g/L are common and may be inhibitory. A method
commonly used to ameliorate the toxicity of hydrolysates by
reducing HMF and furfural concentration is pH adjustment through
"overliming" with calcium hydroxide. Overliming is the process
whereby lime is added beyond that necessary for pH adjustment. Even
after overliming, however, high levels of inhibitors may still
exist. In addition, overliming precludes recovery of secondary
products that have high value from the hydrolysate.
[0074] In order to deal with the potential for high levels of
inhibitory secondary products often found in biomass
hydrolysate--for example, levels that would inhibit the
fermentation microbes in their free state--during the fermentation
step 104, the present patent document teaches processes to protect
the fermentation microbes from the degradation effects of the
inhibitors by immobilizing the microbes, and more preferably
immobilizing the microbes in calcium alginate. Immobilization of
microbes is the attachment or inclusion in a distinct solid phase,
such as calcium alginate, that permits exchange of substrates,
products, inhibitors, etc. with the microbe, but at the same time
separates the microbes from the bulk biomass hydrolysate
environment. Therefore, the microenvironment surrounding the
immobilized microbes is not necessarily the same as that which
would be experienced by their free-cell counterparts. As a result,
for example, the present patent document teaches processes for
immobilizing Pachysolen tannophilus and for fermenting pentoses and
hexoses in the presence of inhibitors found in hemicellulose
hydrolysate, even at concentrations that would inhibit the
fermentative microbe in its free state.
[0075] By immobilizing the fermentative microbe(s) during the
fermentation step 104, the need for conditioning the biomass
hydrolysate to reduce the concentration of, or possibly even
completely remove, inhibitory secondary products is significantly
ameliorated. This is because the need to lower the concentration of
inhibitory secondary products to the levels necessary for
fermentation using free microbes is eliminated. Thus, as reflected
in FIG. 1, conditioning to reduce the concentration of inhibitors
may be omitted in some embodiments, or, as shown in FIG. 2,
included as an optional conditioning step 110.
[0076] Conditioning the biomass hydrolysate in conditioning step
110 to reduce the concentration of inhibitory secondary products
may still be desirable where, for example, the concentration of the
secondary products (either individually or in combination) is
sufficiently high to interfere with the fermentation of sugars even
by the immobilized microbe(s). In such cases, however, the
concentration of the inhibitory secondary products will generally
not need to be reduced to the same levels as necessary for
fermentation using free microbes and thus a less severe and less
costly conditioning process may be employed. To offset the costs
associated with the overall fermentation process, it may also be
desirable to recover secondary products having a high value through
an optional high value secondary product recovery step 114 shown in
FIG. 2. Following partial removal (and possible recovery) of many
secondary products from the biomass hydrolysate, however, the
concentrations of these products may remain sufficiently elevated
within the hydrolysate, particularly considering the synergistic
nature of the inhibitors, to interfere with fermentation of sugars
to ethanol or other biofuel by the fermentative microbe(s) in their
free state. Accordingly, the use of immobilized fermentative
microbe(s) in fermentation step 104 is an important aspect of the
processes described herein, even when the optional conditioning
step 110 is employed to reduce the concentration of secondary
products contained in the biomass hydrolysate.
[0077] In some instances, it may also be desirable to perform
conditioning step 110 even when the concentration of inhibitory
secondary products is insufficient to inhibit fermentation by the
immobilized microbe(s) where, for example, the secondary products
have high value and thus it is desirable to separately recover the
high value secondary products through high value secondary product
recovery step 114. This may be desirable, for example, where the
net value of the recovered high value secondary products may be
used to offset, and hence lower, the costs associated with the
overall fermentation process.
[0078] There are numerous methods of performing the conditioning
step 110 to reduce the concentrations of inhibitory secondary
products. Employing different conditioning methods for conditioning
step 110 will result in different concentration levels of
inhibitory secondary products remaining in the hydrolysate. The
method of conditioning chosen for conditioning step 110 may depend
on a variety of factors, including the sensitivity of the microbe
used during fermentation to inhibitory secondary products, costs,
and whether there is a desire to recover high value secondary
products during a recovery step 114. The more sensitive the
microbe, the more desirable it will be to reduce the concentration
of the inhibitory products from the biomass hydrolysate during
conditioning of the hydrolysate in step 110. Immobilization of the
fermentative microbe(s), however, will decrease the sensitivity of
the microbe to inhibitory secondary products and thus may reduce
the complexity and costs incurred during conditioning step 110.
Some of the conditioning methods that may be employed in
conditioning step 110 to reduce the concentration of secondary
products include, but are not limited to: 1) overliming of
hydrolysate; 2) activated carbon (AC) treatment followed by pH
adjustment; 3) ion exchange followed by overliming; 4) AC treatment
followed by ion exchange; and 5) AC treatment followed by
nanofiltration.
[0079] When hydrolysate from solid-liquid separation step 108
contains one or more high value secondary products, the secondary
products may be recovered in step 114 from the hydrolysate and
subsequently used for other purposes. Some of the high-value
secondary products that may be recovered in step 114 include, but
are not limited to, the mineral acid used in the pretreatment
process 100, such as sulfuric acid, acetic acid hydrolyzed from
hemicellulose polymers, anti-oxidant molecules (phenolic and
polyphenolic compounds) liberated from the partial hydrolysis of
lignin during hydrolysis step 102, other organic acids,
nutraceutical, cosmeceutical, or pharmaceutical products, and
different furans and furan derivatives, such as
5-hydroxymethylfurfural and furfural. High value secondary product
recovery step 114 may be accomplished by adsorption of the
secondary products to different matrices, including activated
carbon, ion exchange resin, ion exchange membrane, organic molecule
"scavenging" resins, polystyrene beads, or any other similar type
medium with a high surface area. High value secondary product
recovery step 114 may also be accomplished by separating the
secondary product(s) from the soluble hexoses and pentoses through
ion exclusion chromatography, pseudo-moving bed chromatography,
high performance liquid chromatography or by filtration methods
including micro-, nano-, and ultrafiltration using hollow fiber or
membrane technologies. High value secondary product recovery step
114 may include several of the aforementioned processes in series
to recover different molecular species. Furthermore, the recovery
process(es) employed in step 114 may be tailored to recover
specific secondary products according to the nature of the starting
biomass. Because many of the recovered secondary products (acetic
acid, furans and their derivatives, phenolic and polyphenolic
compounds, levulinic acid, formic acid, and others) are inhibitory
to yeast and bacterial fermentation of sugars to ethanol, recovery
of high value secondary products in step 114 may both increase the
economics of the entire process and allow for more efficient
fermentation in step 104 of the pentoses and hexoses.
[0080] In general, microbes may be immobilized for fermentation 104
of biomass hydrolysate in step 104 using a number of different
methods. Microbes may be bound to a matrix material or, more
preferably, immobilized by entrapment in the matrix material. For
example, microbes may be immobilized by entrapment using a
drop-forming procedure. The resultant beads may be of different
size and possess different pore sizes. For example, the beads may
range in size from 0.1 mm to 5 mm in diameter, more preferably the
beads may range from 2 mm to 3 mm in diameter, and more preferably
the beads are about 3 mm in diameter.
[0081] The drop-forming procedure may be enhanced through a number
of processes. The beads, may be hardened to different degrees and
may have coatings applied to withstand shear forces in a reactor
and to reduce cell loss. For example, if calcium alginate is used,
the beads may be dried to increase compression stress. The beads
may also be hardened by glutaraldehyde treatment or coated with
catalyst-free polymer to enhance their stability. The beads may be
recoated with plain alginate as a double layer to enhance their gel
stability. Furthermore, the beads may have a polyacrylamide coating
to enhance their structural stability. The beads may also be coated
with a copolymer acrylic resin to increase diffusion and reduce
cell leakage. Similarly, other additions to the drop forming
procedure may be incorporated to enhance the effectiveness of the
matrix.
[0082] Other techniques for improving the efficiency of immobilized
microbes include increasing the surface area of the
microbe/immobilization medium mixture once it is formed. For
example, a Pachysolen tannophilus/calcium alginate or other
microbe/calcium alginate mixture may be applied as a coating to a
natural or synthetic, high surface area, support structure. In one
implementation, the support structure only need be able to support
the microbe/immobilization medium and itself. For example, the
support structure may comprise a ceramic sponge, honeycomb, reactor
packing material or other support structure to increase the surface
area per mass of the microbe/immobilization medium when it is
applied. The mixture may also, or in the alternative, be applied to
parts of the reactor surfaces, such as, the walls or the surface of
the mixing devices.
[0083] In addition to immobilization by entrapment, the microbes
may be immobilized by other methods including adsorption,
cross-linking, or immobilized by any other means capable of
providing a micro-environment for the microbe.
[0084] A variety of different materials may be used to immobilize
microbes. If the microbes are immobilized using entrapment calcium
alginate, a natural product from brown algae (seaweed) may be
preferably used. However, other materials, both natural and
synthetic, may also be used to immobilize microbes using entrapment
including carrageenan, xanthan gums, agarose, agar and luffa,
cellulose and its derivatives, collagen, gelatin, epoxy resin,
photo cross-linkable resins, polyacrylamide, polyester, polystyrene
and polyurethane.
[0085] Other materials that may be used to immobilize microbes
using adsorption or other immobilization methods include
kieselguhr, wood, glass ceramic, plastic materials, polyvinyl
acetate, and glass wool.
[0086] When combining microbes with complimentary properties, the
microbes may be combined within the same immobilization vehicle, or
the microbes may be immobilized separately and the separately
immobilized microbes combined in the same fermentation reactor. For
example, if calcium alginate beads are used as the immobilization
vehicle, different complimentary microbes may be combined within
the same bead. As one example, to effectively ferment softwood
hydrolysate, which contains the sugars mannose, galactose, glucose
and xylose, to ethanol, one may combine Zymomonas mobilis, NREL
strain 8b, which ferments glucose and xylose to ethanol, with
Saccharomyces cerevisiae, which ferments mannose and galactose,
into a single bead product. In this way advantageous fermentative
properties of different microbial species are combined in a single
bead product.
[0087] Alternatively, separate beads can be made containing each
microbe and then the beads may be combined in the fermentation
reactor. For example, the fermentation of the hexoses and pentoses
to fuel may be performed by combining beads composed of different
microbial species with complementary hexose and pentose
specificities, metabolic rates, or the like. In yet another
example, different microbes are immobilized in separate reactors
and the biomass hydrolysate is then run through each reactor to
expose the biomass hydrolysate to each microbe. In addition,
different immobilization methods may be combined with different
microbes.
[0088] One of the many advantages of immobilizing the microbes is
that the microbes become more stable and bioreactors may be run in
a continuous mode instead of batch mode. Running the bioreactor in
a continuous mode is advantageous for efficiency reasons but the
microbes may begin to lose metabolic efficiencies after long
periods of use. In order to restore metabolic efficiency,
immobilized microbes may be periodically treated with yeast growth
medium. For example, Pachysolen tannophilus and other fermentative
microbes immobilized in calcium alginate may be periodically
treated with a yeast growth medium to restore metabolic
efficiency.
[0089] Another advantage of microbe immobilization is that the
microbe biomass may be better retained within a continuous
fermentation reactor. In a continuous fermentation process
involving a high flow rate, such as that which may be experienced
during the continuous running of a columnar up-flow reactor, free
cells will tend to wash out. Wash out reduces the number of cells
in the reactor and thus lowers the rate of the fermentation
reaction. To maintain the rate of fermentation, new cells must be
propagated and added to the reactor, increasing costs. The examples
associated with Table 2 below demonstrate the advantages of using
immobilized microbes in a continuous fermentation process under
wash out conditions (i.e., under a flow rate that would cause wash
out of more than 5% of the free cells.)
TABLE-US-00002 TABLE 2 Effect of cell washout on ethanol
concentration and productivity in a continuous reactor. Retention
time Productivity (h) Cells Ethanol (g/L) (g/L h) 10 Imm 3.03 0.30
Free 1.84 0.18 5 Imm 2.08 0.42 Free 0.68 0.14 Imm--immobilized
[0090] The data in Table 2 illustrates the benefits of
immobilization to prevent wash out for one particular fermentative
microbe. Specifically, the example in Table 2 demonstrates the
improvement of biofuel (e.g., ethanol) yield for immobilized
Pachysolen tannophilus (NRRL Y2460) over free cells of the same
microbe during continuous fermentation in a column up-flow reactor.
However, immobilization can be used to prevent wash out for any
type of fermentative microbe in any continuous flow bioreactor and
thereby increase ethanol or other biofuel yield.
[0091] The data presented in Table 2 was generated by adding
8.38.times.10.sup.11 cells of Pachysolen tannophilus to two
identical up-flow reactors. In the first reactor, the cells were
immobilized in 2-3 mm calcium alginate beads. In the second
reactor, the cells were added free in solution. Both reactors, were
connected to the same reservoir of artificial medium and the same
peristaltic pump was used to pump the artificial medium through the
reactors during the continuous fermentation process. The artificial
medium within the reservoir contained 10 g/L yeast extract, 20 g/L
peptone, 7.2 g/L glucose, and 42.5 g/L xylose. The artificial
medium was pumped into the bottom of both reactors simultaneously
at the same rate and both reactors were incubated at 30.degree.
C.
[0092] In a first test, the two reactors were each run at a flow
rate corresponding to a retention time of 10 hours. The reactors
were each run for a total of 20 hours or for a total of 2.times.
the retention time. In a second test, set up as indicated above,
the two reactors were each run at a flow rate corresponding to a
retention time of 5 hours. In the second test, the reactors were
run for a total of 10 hours, or again for a total of 2.times. the
retention time. The ethanol content of the first and second
reactor's effluent was analyzed for ethanol content at the end of
the 2.times. retention time period for each test. Hence, ethanol
content of effluent was determined for each reactor at two separate
flow conditions. The productivity (ethanol production per hour) was
also determined for each flow condition. The results are reported
in Table 2.
[0093] Table 2 reveals that the ethanol concentration at the end of
20 hours for the 10 hour retention time flow rate was much greater
for the reactors containing immobilized Pachysolen than free
Pachysolen, 3.03 versus 1.84 g/L, respectively. The corresponding
productivity was also greater for the immobilized Pachysolen. For
the second test, which employed a flow rate that resulted in a 5
hour retention time, the ethanol concentration in the effluent of
the reactor containing immobilized cells was 2.08 g/L at the end of
10 hours or 2.times. the retention time, but the productivity
increased by 40% over that in the first test due to the faster flow
rate.
[0094] In contrast, at the flow rate that resulted in a 5 hour
retention time, the ethanol concentration in the reactor containing
free cells decreased from 1.84 to 0.68 g/L and the productivity
experienced a 23% decrease, from 0.18 to 0.14 g/L*hour.
[0095] The examples of Table 2 illustrate that immobilizing
fermentative microbes decreases wash out and increases biofuel,
such as ethanol, productivity in the reactor. When the cells were
not immobilized, the flow rate of the medium exceeded the
sedimentation rate of the free Pachysolen tannophilus (at both flow
rates tested) and the concentration of the cells in the free state
reactor decreased to a low level causing the ethanol concentration
and ethanol productivity to also decrease. By contrast, the
Pachysolen tannophilus that was immobilized in the calcium alginate
beads remained in the reactor and the reactor was able to increase
the ethanol productivity with the increased flow rate.
[0096] Certain microbes that can be used in conversion of sugars to
biofuels are motile; that is, they possess cilia and/or flagella
and swim in fermentation medium. Another advantage of
immobilization is that the motile microbe biomass may be better
retained within a continuous fermentation reactor, even in
fermentation process involving a low flow rate. Motile cells in the
free state will tend to wash out in all flow conditions. Wash out
reduces the number of cells in the reactor and thus lowers the rate
of the fermentation reaction. To maintain the rate of fermentation,
new cells must be propagated and added to the reactor, increasing
costs.
[0097] Another advantage of immobilizing microbes is the ability to
obtain a high biomass concentration in a continuous fermentation
process. In a column upflow reactor, as a non-limiting example,
more than half, preferably about two thirds to about three quarters
of the reactor volume will be composed of the bead material and the
rest will be inter particle void volume when the fermentative
microbes are immobilized in beads of about 2 mm to 3 mm in
diameter. In the case of using yeast as the fermenting microbe,
where 5% of the volume of the bead is yeast biomass, the reactor
will effectively contains about 3.3 to 3.75% by volume yeast
biomass, which is a relatively high yeast concentration for a
fermentor.
[0098] Other benefits of yeast and bacteria immobilization by
entrapment in calcium alginate over free cells in suspension
include greater ethanol tolerance, possibly due to changes in cell
membrane composition; greater specific ethanol production,
increased rate of ethanol production due to increased glucose
uptake and lower dissolved CO.sub.2 in solution, and increased
thermo-stability of bacteria.
[0099] As described above, there are numerous methods of actually
immobilizing the microbes. In one preferred embodiment for
immobilizing Pachysolen tannophilus in calcium alginate, the
microbes are initially immobilized in sodium alginate which is then
converted to calcium alginate. Sodium alginate can have different
viscosities when a given amount is dissolved in an aqueous
solution. Viscosities for different sodium alginate products range
from 100 or 200 mPa, to even as much as 1236 mPa. In a preferred
embodiment, alginate with medium-low viscosity of about 324 mPa is
used to produce beads, although alginates with different
viscosities may be used for different biomass hydrolysates or for
solid-state ferments.
[0100] The sodium alginate is prepared by adding from 0.05 to 10%,
or preferably about 3.5% (w/v) sodium alginate to deionized water.
Alternatively, the sodium alginate can be dissolved into growth
medium, into a mixture of vitamins, including biotin, or into
growth medium supplemented with vitamins, or into a natural
solution containing biotin. The initial sodium alginate
concentration will depend on the final concentration desired to
produce beads and on the volume added by mixing with a concentrated
microbe slurry.
[0101] In order to get some sodium alginate preparations into
solution, the mixture may be heated and stirred on a stir plate.
However, heating alginate polymers may cause some amount of
hydrolysis of the alginate and thereby change the properties of the
alginate solution, including its viscosity. As a result, it may be
desirable to use a sodium alginate preparation that does not
require heating in order to go into solution. In embodiments where
the alginate may not be heated for solubilization nor autoclaved
for sterilization, it may be desirable to treat the alginate with a
chemical sterilizer or it may be desirable to irradiate the
alginate with ultraviolet light for sterilization.
[0102] Cells may be cultivated in their respective media, and
pelleted by centrifugation. Alternatively, a mass of Pachysolen or
other in fermentative microbe may be propagated in at least a 10 L,
or more preferably at least a 200 L, or even more preferably at
least a 2000 L bioreactor to a concentration of about 1 to about 20
grams wet mass per liter growth medium. The resulting biomass may
then be concentrated using, for example, a tangential flow
filtration device to produce a 20-70% wet mass slurry of Pachysolen
cells. This technique is particularly well suited for the
production of large volumes of calcium alginate beads having one or
fermentative microbes, such as Pachysolen, immobilized therein.
[0103] Following concentration, the concentrated cells are then
recovered and thoroughly mixed with the sodium alginate medium.
Mixing the alginate with the microbial cells can occur in the same
device as is used for the resuspension of the alginate or in a
separate device. The mixing continues to homogenity of the mixture.
Mixing of the microbes with the highly viscous sodium alginate
solution requires a mixing method that does not shear the microbes,
such as a reciprocating disc mixer. The cell loading into the
sodium alginate medium is both organism and substrate dependent.
For example, a suitable target loading for Pachysolen tannophilus
in hydrolysate is at least 5 g cells/100 mL sodium alginate
medium.
[0104] Calcium alginate beads are produced by extruding the sodium
alginate medium/cells into a sterile calcium chloride solution. A
peristaltic pump and sterilized Master-flex Bulk-Packed Silicone
Tubing that has an attached sterile 18 G needle may be used in the
extruding process. The entire process is preferably done
aseptically. In an alternative embodiment that is more suitable
where large amounts of immobilized microbe beads are desired to be
produced, a sterile 96 hollow 19 gauge pin device may be used in
place of an 18 gauge needle. The beads may then be produced by
extrusion and gravity dropping. Other methods may include a
so-called Jet Cutter to produce beads from a continuous stream of
an alginate/microbe slurry. Other modifications of producing beads
from a continuous stream include using electrostatic attraction to
produce droplets, using vibration to produce droplets, using air to
produce droplets, and using a rotating disk atomizer, to name a
few.
[0105] In order to exchange sodium ions with calcium ions to effect
polymerization of the alginate, beads are dropped in a solution
containing calcium chloride. In one method, a 0.22M solution of
calcium chloride dihydrate is also prepared in deionized water to
receive sodium alginate/microbe mixture. The sodium alginate medium
and calcium chloride solution may both be autoclaved for
sterilization purposes. The beads may be kept at 4.degree. C. in
the calcium chloride solution for about 60 minutes to harden. Once
the beads have hardened, they are preferably rinsed several times
with sterile deionized water. In a preferred embodiment, the beads
are dropped into sterile growth medium containing 0.1 to 0.25 M
calcium chloride. The growth medium may also contain different
vitamins or biotin. After about 30 minutes of hardening, the beads
may be either used immediately in a fermentation or may be stored
at 4.degree. C. until use. There is no need to rinse beads prior to
use or prior to storage when hardening is carried out in such a
growth medium.
[0106] In certain implementations, it may also be desirable to
recycle components of the immobilization processes. The solid
calcium alginate used to immobilize microbes in beads or on a
support structure may delaminate, break-up, shear, or otherwise
physically degrade after prolonged use. In addition, the
microbe/calcium alginate mixture may also become degraded and
discolored through repeated use due to the trapping of contaminants
such as extractives, microbial inhibitors, and other materials.
Degradation of the structure, whether due to physical and/or
chemical degradation affects the performance of the fermentation
process. To overcome deleterious effects of this degradation, new
or fresh microbe/calcium alginate mixture may be used in the
bioreactor to improve the reactors performance. However, the
frequent replacement of the mixture may be uneconomical both in
terms of the material costs associated with production of the
calcium alginate, but also due to the cost of the lost
microbes.
[0107] FIG. 3 illustrates a process 140 for recycling calcium
alginate used in the microbe immobilization process. For example,
in the case of Pachysolen tannophilus immobilized in calcium
alginate beads, the calcium alginate from the beads used to
immobilize the microbes may be recovered and recycled using process
140. In process 140, the degraded microbe/calcium alginate mixture
148, is dissociated with a calcium chelator complexed with a
monovalent ion 150, such as sodium citrate or potassium citrate.
Step 150 of process 140 dissociates the alginate and liberates the
microbes (bacteria or yeast cells). In one preferred embodiment of
process 140, step 150 is accomplished by stirring the
microbe/calcium alginate mixture in 20 g/L sodium citrate or
potassium citrate with a pH 8.2. at room temperature for 15
minutes.
[0108] Once the microbes have been liberated and the alginate
dissociated, the solution is filtered to remove the large
particulate and microbes (bacteria or yeast cells) in step 152. The
filtered solution is then dialyzed, step 154, against a sodium salt
156, such as sodium chloride, to remove the calcium citrate,
extractives, and soluble microbial inhibitors 158. The resulting
dialysis of the filtered solution with an inorganic salt, such as
sodium chloride, regenerates sodium alginate. The toxic materials
are removed as waste stream 160. The sodium alginate is
concentrated during dialysis and then used again to produce calcium
alginate in steps 142, 144, and 146 as described above. In one
preferred embodiment, the sodium alginate is used to immobilize
Pachysolen tannophilus in calcium alginate beads as taught in the
above process.
[0109] In addition to the use in processes specifically designed to
produce an alcohol, such as ethanol, the processes of the present
patent document may be used in conjunction with other processes.
For example, the paper-pulping process usually burns or discards
the hemicellulose portion of the biomass. Using the processes
taught herein, however, the hemicellulose may be separated and
removed from the biomass and processed into ethanol, or other
biofuel. Accordingly, the processes of the present patent document
provide an efficient, cost-effective means for converting
hemicellulose into ethanol, or other biofuels, in the
paper-pulping, and other, industries. As a further non-limiting
example, the processes disclosed in the present patent document may
also be used to ferment monosaccharides, both hexose and pentose,
obtained from the saccharification of sugarcane bagasse.
[0110] The following discussion will now be directed to bioreactors
designed for use with immobilized microbes and in particular with
immobilized Pachysolen tannophilus.
[0111] Fermentation may occur using a number of methods. Preferably
the biomass hydrolysate is removed and fermented ex-situ. A variety
of bioreactor designs, including a traditional non-stirred
fermenter or stirred fermenter, may be used for the fermentation of
the biomass hydrolysate using immobilized microbes. The reactor may
be a submerged reactor or other type of liquid reactor. In order to
provide the highest yield, a submerged reactor is preferable to
ferment five-carbon sugars.
[0112] In the case of microbes that are immobilized, a packed bed
reactor could be utilized, or a tankage system similar to that
employed for carbon-in-pulp processes in the gold mining industry
could be used. In the latter, beads would be moved counter-current
to the solution flow and could be easily recovered for
regeneration. Thin film reactors may also work well with
immobilized microbes.
[0113] In addition, solid/liquid contactors may be used with
immobilized microbes. These types of reactors include ion exchange
columns, packed bed reactors, trickle flow reactors, and rotating
contactors. Other reactors that may be used are fluidized-bed and
upflow type reactors.
[0114] If the entrapment method of immobilization is used, the
microbes may be incorporated into a bioreactor using a number of
different methods. In addition to beads, the matrix/microbe gel may
be applied to a support structures to increase the effective
surface area. These configurations may include coating paddle
structures, used in stirred tank reactors, rotating contactors, and
thin film reactors. The microbes could also be incorporated in
large three-dimensional open-cell supports for use in trickle flow
reactors or fluidized-bed and upflow reactors.
[0115] FIG. 4 illustrates a view of one embodiment of a bioreactor
for performing submerged fermentation of biomass hydrolysate using
immobilized microbes. Bioreactor 200, which may be referred to as a
rotating disk contactor, comprises vessel 202, input 204, rotating
stir stick 206, outputs 208 and 210, stators 212, and rotors
214.
[0116] Although vessel 202 is shown in a vertical configuration it
may also be horizontal or in some other orientation. Vessel 202,
preferably includes a large opening. For example, vessel 202 may be
made of two separable halves in order to facilitate maintenance
access to the stators 212 or rotors 214 located within vessel
202.
[0117] In a preferred embodiment, microbes immobilized in a matrix
substance, such as calcium alginate, are applied to the stators 212
and the rotors 214. With this structure biomass hydrolysate flows
through the vessel 202 from input 204 and through outputs 208 and
210. While the biomass is flowing, the rotating stir stick 206 may
be rotated to provide agitation to the biomass hydrolysate as it
flows through the bioreactor 200. Preferably the bioreactor 200 is
designed for continuous flow fermentation.
[0118] FIG. 5A and FIG. 5B illustrates a side and front view of
another embodiment of a bioreactor for performing submerged
fermentation of biomass hydrolysate using immobilized microbes.
Bioreactor 300, comprises motor 302, rotating shaft 304, media disk
panels 306, biomass hydrolysate 308, vessel 310, and optional air
tube 312. Biomass hydrolysate 308, is added to the bioreactor 300
for fermentation.
[0119] Vessel 302 of bioreactor 300 is shown as only a bottom half,
but vessel 302 may completely encapsulate the rotating media disks
306. In a preferred embodiment, microbes immobilized in a matrix
substance may be applied to the media disk panels 306. Motor 302
rotates the media disks 306 through the biomass hydrolysate
308.
[0120] In one embodiment, bioreactor 300 includes an optional air
tube 312 that may be used to further agitate the biomass
hydrolysate 308 and increase fermentation by injecting air below
the rotating media disk panels 306.
[0121] FIG. 6 illustrates an upflow reactor. Upflow reactor 400
contains sludge bed or sludge blanket 402. For use to ferment
biomass hydrolysate, sludge bed 402 comprises immobilized microbes.
Sludge bed 402 may be comprised of one or more fermented microbes
immobilized in any of the various medium described above. For
example, sludge bed 402 may be comprised of Pachysolen tannophilus
immobilized in calcium alginate beads. Upflow reactor 402 further
comprises inlet(s) 404 for influent. Inlet(s) 404 may be a single
inlet or more preferably a plurality of inlets across the bottom of
the upflow reactor 400 to distribute the influent evenly underneath
the sludge bed 402. Inlet(s) 404 allow the biomass hydrolysate to
enter the upflow reactor from beneath the sludge bed 402. As the
biomass hydrolysate is fermented, biogas 406 rises to the surface
of the reactor and is collected at the top 408 of the upflow
reactor 400. Effluent 410 is removed from the reactor and recycled
through the inlet(s) 404.
[0122] Preferably the upflow reactor 400 is a columnar upflow
reactor with a low aspect ratio between the range of about 1:1 to
2:1 height to width. Carbon dioxide gas produced by the
fermentation process disrupts the packing of the beads loaded in
the column and promotes a `self-fluidizing` bed, similar to the
effect achieved by a gas-lift type of reactor.
[0123] In a preferred embodiment, two or more `self-fluidizing` bed
columnar upflow reactors 400 can be run in series. The beads in
each reactors may contain the same or different microbes, so as to
ferment different sugars in different reactor stages. An increase
in the number of reactors placed in series will reduce the
sugar/ethanol variation within any given reactor, which in turn
will promote better microbe performance.
[0124] In addition to the bioreactor designs shown in FIG. 4, FIG.
5A, 5B, and FIG. 6, it is to be understood that numerous other
submerged or contact bioreactor designs may be used with the
processes taught herein.
[0125] Bioreactors based on immobilized microbes offer several
advantages over `free cell` systems. One advantage is the increased
feasibility to employ a continuous fermentation system.
Immobilization ensures no loss of cell mass, such as occurs with
batch fermentation and with continuous fermentation where the flow
rate is such that the free cells are washed out of the reactor with
the product. Continuous fermentation also decreases production
down-time compared to batch fermentation. Continuous fermentation
using microbes immobilized in beads increases the flow rate and the
ethanol productivity possible with, for example, an upflow reactor.
Immobilization also ensures no loss of cell mass of motile cells,
where the flow rate is either high or low, where the inherent
motility of the cell leads to loss of cell mass.
[0126] The following example demonstrates the application of one
embodiment of the present patent document applied to beetle-killed
pine. For the purposes of the present example Pachysolen
tannophilus was either immobilized in calcium alginate beads with
about a 3 mm diameter (generated using the method describe above)
or was in a free cell state. Tables 3 and 4 below summarize the
improvement of ethanol yield, and in glucose and xylose conversion
resulting from the reactor design employed according to the present
example.
[0127] The present example demonstrates the improvement of ethanol
yield, and in glucose and xylose conversion, for calcium
alginate-immobilized Pachysolen tannophilus in two different
softwood hydrolysates (`A` and `B`) over free (i.e. unrestricted)
Pachysolen tannophilus. The hydrolysates were pH adjusted or
overlimed and pH adjusted. The Pachysolen tannophilus strain NRRL
Y2460 was used in carrying out the experiment; however, other
adapted or mutated strains of Pachysolen tannophilus may also be
immobilized in calcium alginate and used in processes according to
the present patent document.
[0128] The pine was transformed into a softwood hydrolysate by
dilute acid hydrolysis. The hydrolysate was either simply pH
adjusted with sodium hydroxide or `overlimed`. As mentioned above
overliming with calcium hydroxide is commonly used to ameliorate
the toxicity of hydrolysates. The resulting solutions were
fermented using Pachysolen tannophilus immobilized in 3 mm calcium
alginate beads.
[0129] The beads were incubated in a flask of Yeast Peptone
Dextrose (YPD) broth for 22 hours at 30.degree. C. and 75 rpm. YPD
is a standard yeast medium containing 10 g/L yeast extract, 20 g/L
peptone, and 20 g/L dextrose. Similarly, the free cells were
cultured from a working slant into a flask of YPD broth and
incubated for 24 hours at 30.degree. C. and 75 rpm.
[0130] To prepare the pH adjusted hydrolysate, the solution was
adjusted to pH 6.0 with 8M potassium hydroxide, followed by filter
sterilization. Preparation of overlimed and pH adjusted hydrolysate
required overliming to pH 10.0 with calcium oxide, followed by a 30
minute hold at 50.degree. C. under stirring conditions. The
overlimed hydrolysate was then filtered to remove the solids.
Following re-acidification to pH 6.0, the hydrolysate was filter
sterilized.
[0131] Serum vials were aseptically prepared to obtain a final
concentration of 95% hydrolysate with the following nutrient
additions: 0.2% urea w/v, 0.2% yeast extract, and 0.05% potassium
dihydrogen phosphate. The inoculation rate for immobilized beads
was 0.2 g beads per mL. Following rinsing and re-suspension in
sterile buffer, the free cells were inoculated at a rate of 0.3
OD.sub.600 nm per mL. All experimental conditions were set up in
triplicate serum vials. The vials were aseptically vented and
incubated for 72 hours at 30.degree. C. and 75 rpm prior to
sampling for analysis.
[0132] In pH adjusted hydrolysate "A", as shown in Table 3, `free`
Pachysolen was unable to convert sugars to ethanol and no xylose
was utilized. Immobilized Pachysolen converted most of the sugars
(81%) to ethanol and converted 51% of the xylose. The data shows
that immobilization greatly increased the ability of Pachysolen to
overcome the inhibitory effects of the toxic compounds contained in
the pH adjusted hydrolysate.
[0133] In overlimed hydrolysate "A", as reflected in Table 3,
`free` Pachysolen converted 60% of sugars to ethanol, and
immobilized Pachysolen 86% of sugars. Xylose utilization was 0% for
free cells. This is a surprising result with respect to reports in
the current literature that Pachysolen tannophilus will ferment
pentoses, and particularly xylose, in a defined medium. It is the
inventors' hypothesis that despite removal of detectable levels of
HMF and furfural by overliming, significant amounts of other
inhibitors, discussed above, or combinations thereof still remain
in the hydrolysate thus preventing fermentation. When the
Pachysolen tannophilus was immobilized xylose utilization jumped to
76%. Immobilization thus enhances the benefit of overliming and
greatly increases xylose utilization.
[0134] Table 4 shows similar results to Table 3. In pH adjusted
hydrolysate "B", as shown in Table 4, `free` Pachysolen was unable
to convert sugars to ethanol and no xylose was utilized.
Immobilized Pachysolen converted a majority of the sugars (57%) to
ethanol.
[0135] Moreover, as reflected in Table 4, in overlimed hydrolysate
"B" that contained very high inhibitor concentrations, `free`
Pachysolen was unable to ferment available sugars, while
immobilized Pachysolen fermented 83% of available sugars, including
xylose, to ethanol.
TABLE-US-00003 TABLE 3 Softwood hydrolysate `A` fermentation
characteristics with Pachysolen tannophilus pH adjusted Overlimed
and pH adjusted Ethanol yield Sugar Utilization Solution Ethanol
yield Sugar Utilization Solution (% (%) Inhibitors (g/L) (% (%)
Inhibitors (g/L) Cells theoretical).dagger. glucose xylose furfural
HMF theoretical).dagger. glucose xylose furfural HMF Free 0.0% 0.0%
0.0% 0.42 4.03 61.8% 72.2% 0.0% <DL <DL 57.4% 75.8% 0.0% Imm.
81.3% 65.2% 51.1% 0.42 4.03 79.7% 61.6% 79.3% <DL <DL 92.7%
67.9% 73.4% .dagger.Glucose concentration: 13.5 g/L; Xylose
concentration: 3.4 g/L DL = Detectable Limit; Imm. =
Immobilized
TABLE-US-00004 TABLE 4 Softwood hydrolysate `B` fermentation
characteristics with Pachysolen tannophilus pH adjusted Overlimed
and pH adjusted Ethanol yield Sugar Utilization Solution Ethanol
yield Sugar Utilization Solution (% (%) Inhibitors (g/L) (% (%)
Inhibitors (g/L) Cells theoretical).dagger. glucose xylose furfural
HMF theoretical).dagger. glucose xylose furfural HMF Free 0.0% 0.0%
0.0% 5.91 1.32 0.0% 0.0% 0.0% 1.04 0.79 Imm 56.9% 35.7% 15.9% 5.91
1.32 83.3% 53.8% 73.0% 1.04 0.79 .dagger.Glucose concentration: 4.7
g/L; Xylose concentration: 3.2 g/L Imm. = Immobilized
[0136] In the preceding example summarized in Tables 3 and 4, and
the subsequent examples in Tables 5-7 below, ethanol yield (%
theoretical) is based on glucose and xylose only and is calculated
from total glucose and xylose concentrations before treatment.
Other monosaccharides are not considered. All sugar utilization
data is calculated using YSI results for glucose and xylose. Sugar
utilization calculations do not differentiate between end products
(i.e., includes ethanol, xylitol, biomass) and is calculated as
follows (accounting for lost sugars after treatment like
overliming, autoclaving, etc.):
For Hydrolysate Calculations:
[0137] % Sugar .times. Conversion = NS - RS TS * 95 ##EQU00001## NS
= Sugar .times. Concentration after Treatment ( i . e . , Negative
Control ) ##EQU00001.2## RS = Residual Sugar .times. Concentration
after Fermentation ##EQU00001.3## TS = Total Sugar .times.
Concentration before Treatment ##EQU00001.4##
[0138] Other embodiments of the processes taught in the present
patent document will include using different microbes and different
conditioning methods. For example, Tables 5 and 6 illustrate the
improvement in ethanol yield, and in glucose and xylose conversion,
for calcium alginate-immobilized Zymomonas mobilis NREL strain 8b,
Pachysolen tannophilus (NRRL Y2460), and Pichia stipitis (NRRL
Y7124) in sugarcane hydrolysate over free cells of the same.
Similar to the examples in tables 3 and 4, pH adjusted hydrolysate
was compared against another conditioning method for both free and
immobilized microbes. In contrast to the examples illustrated in
tables 3 and 4, the hydrolysate used for the examples shown in
tables 5-7 used hydrolysate derived from sugarcane bagasse instead
of hydrolysate derived from softwood. Tables 5 and 6 illustrate the
benefit of immobilization on a variety of microbes including both
yeasts and bacterium.
[0139] The effects of the different conditioning steps on the
concentrations of secondary inhibitory products are shown in Table
7. As shown in Table 7, the hydrolysates were conditioned by pH
adjustment or by passing the hydrolysate over activated carbon
(AC), strong acid ion exchange (IE) resin and weak base ion
exchange resin, a treatment hereafter termed AC/IE.
TABLE-US-00005 TABLE 5 Percent conversion of glucose and xylose to
ethanol. Strain Cells pH adjustment AC/IE Z. mobilis, NREL 8b Imm.
31.4 .+-. 0.9 71.4 .+-. 1.0 Free 22.1 .+-. 0.7 32.4 .+-. 1.4 P.
tannophilus Imm. 24.8 .+-. 0.6 63.6 .+-. 0.4 Free 5.7 .+-. 0.3 50.0
.+-. 0.2 P. stipitis Imm. 11.9 .+-. 0.3 54.4 .+-. 0.7 Free 4.1 .+-.
0.1 56.4 .+-. 2.1 Imm. = Immobilized
TABLE-US-00006 TABLE 6 Percent xylose utilized in 6 day
fermentation. Strain Cells pH adjustment AC/IE Z. mobilis, NREL 8b
Imm. 30.8 .+-. 0.7 75.1 .+-. 0.4 Free 17.5 .+-. 0.0 17.6 .+-. 3.2
P. tannophilus Imm. 23.7 .+-. 1.7 95.8 .+-. 0.5 Free 11.5 .+-. 0.7
55.9 .+-. 4.3 P. stipitis Imm. 16.4 .+-. 3.1 67.3 .+-. 1.9 Free
N.D. 61.7 .+-. 3.0 N.D.--not detected; Imm.--Immobilized;
TABLE-US-00007 TABLE 7 Inhibitor Concentrations in differently
conditioned hydrolysates. Acetic Formic acid acid 5-HMF Furfural
Conditioning (g/L) (g/L) (g/L) (g/L) pH adjustment 10.7 3.8 1.1 3.5
AC/ion exchange 0.1 0.4 N.D. N.D. N.D.--Not Detected.
[0140] The examples of Table 5-7 were conducted by transforming
sugarcane bagasse into a bagasse hydrolysate by dilute acid
hydrolysis. The hydrolysate was conditioned by either simply pH
adjusting with sodium hydroxide or by treating the hydrolysate with
activated carbon and the two ion exchange resins mentioned above.
Namely, the bagasse hydrolysate was passed over a column containing
activated carbon, over a column containing a strong acid cation
exchange column, and a weak base anion exchange column. The
resulting solutions were further separated into three separate
solutions each to be fermented by three different microbes,
Zymomonas mobilis NREL strain 8b, Pachysolen tannophilus (NRRL
Y2460), and Pichia stipitis (NRRL Y7124) respectively. For each of
the microbe solutions, two separate examples were performed, one
with the microbe immobilized in 2-3 mm calcium alginate beads, and
the other using free microbes. Consequently, there were four
different fermentations for each microbe resulting in 12 total
fermentations. Two fermentations with the microbe immobilized, one
with a pH adjusted solution and one with an AC/IE conditioned
solution and two fermentations using free microbes, one with a pH
adjusted solution and one with an AC/IE conditioned solution.
[0141] The two differently-conditioned bagasse hydrolysates
contained different amounts of the inhibitors acetic acid, formic
acid, 5-hydroxyfurfural (5-HMF), and furfural. The measured values
are reported in Table 7. These inhibitor levels are for the
particular batch of sugarcane bagasse hydrolysate used in the
experiments summarized above for which the results are reported in
Tables 5 and 6.
[0142] The beads used for immobilizing the different microbes were
incubated in a flask of Yeast Peptone Dextrose (YPD) broth for 22
hours at 30.degree. C. and 75 rpm. Similarly, the free cells were
cultured from a working slant into a flask of YPD broth and
incubated for 24 hours at 30.degree. C. and 175 rpm.
[0143] Serum vials were aseptically prepared to obtain a final
concentration of 95% hydrolysate with the following nutrient
additions: 0.2% urea w/v, 0.2% yeast extract, and 0.05% potassium
dihydrogen phosphate. The inoculation rate for beads was 0.2 g
beads per mL. Following rinsing and re-suspension in sterile
buffer, the free cells were inoculated at a rate of 0.01 g (wet
weight) per mL for P. tannophilus and P. stipitis, and 0.006 g (wet
weight) per mL for Z. mobilis 8b. All experimental conditions were
set up in triplicate serum vials. The vials were aseptically vented
and incubated for 6 days at 30.degree. C. and 75 rpm prior to
sampling for analysis.
[0144] For sugarcane bagasse hydrolysate conditioned by pH
adjustment, `free` Zymomonas was able to convert 22% of the glucose
and xylose to ethanol, while immobilized Zymomonas converted 31%
(Table 5). Similarly, `free` Pachysolen was able to convert 6% of
the glucose and xylose to ethanol, while immobilized Pachysolen
converted 25%, and `free` Pichia was able to convert 4% of the
glucose and xylose to ethanol, while immobilized Pichia converted
12% (Table 5). The data shows that immobilization greatly increased
the ability of Zymomonas, Pachysolen, and Pichia to overcome the
inhibitory effects of the toxic compounds contained in the pH
adjusted bagasse hydrolysate (Table 5).
[0145] In AC/IE conditioned bagasse hydrolysate, as reflected in
Table 5, `free` Zymomonas was able to convert 32% of the glucose
and xylose to ethanol, while immobilized Zymomonas converted 71%.
Similarly, `free` Pachysolen was able to convert 50% of the glucose
and xylose to ethanol, while immobilized Pachysolen converted 64%.
Unlike Zymomonas and Pachysolen, immobilized Pichia was actually
less effective at converting glucose and xylose to ethanol than
`free` Pichia. As shown in Table 5, `free` Pichia was able to
convert 56% of the glucose and xylose to ethanol, while immobilized
Pichia converted 54%. The data shows that immobilization greatly
increased the ability of Zymomonas and Pachysolen to overcome the
inhibitory effects of the toxic compounds contained in the AC/IE
conditioned bagasse hydrolysate.
[0146] Xylose utilization in the fermentations generally mirrored
the extent of fermentation of glucose and xylose to ethanol.
Immobilized Zymomonas utilized 31% of xylose in pH adjusted
hydrolysate and 75% in AC/IE conditioned hydrolysate, while the
free cells utilized only 18% of the xylose in both conditions
(Table 6). Immobilized Pachysolen utilized 24% of xylose in pH
adjusted hydrolysate and 96% in AC/IE conditioned hydrolysate,
while the free cells utilized only 12% and 56% of the xylose,
respectively (Table 6). Immobilized Pachysolen utilized 25% of
xylose in pH adjusted hydrolysate and 64% in AC/IE conditioned
hydrolysate, while the free cells utilized only 6% and 50%,
respectively. Immobilized Pichia utilized 16% of xylose in pH
adjusted hydrolysate and 67% in AC/IE conditioned hydrolysate,
while the free cells utilized no xylose in pH adjusted hydrolysate,
but 62% in AC/IE conditioned hydrolysate (Table 6).
[0147] It is the inventors' hypothesis that despite removal of
detectable levels of HMF and furfural and a great decrease in
acetic and formic acids by AC/IE conditioning, significant amounts
of other inhibitors, discussed above, and the remaining formic and
acetic acids, or combinations thereof still remain in the
hydrolysate thus interfering with fermentation. For Zymomonas and
Pachysolen, immobilization increased xylose utilization
significantly. Immobilization thus enhances the benefits of
conditioning and greatly increases xylose utilization.
[0148] In another example of the processes taught in the present
patent document, the microbe/calcium alginate beads were re-used in
sequential fermentations and the microbes in the beads were
metabolically `regenerated` between fermentations to increase
ethanol yield.
[0149] For the present example, fermentations using 2 g
Pachysolen/calcium alginate beads per 10 ml softwood hydrolysate
supplemented with 0.2% Urea, 0.2% Yeast Extract, and 0.05%
KH.sub.2PO.sub.4 were performed at 30.degree. C. and 75 rpm for 72
hours. After the fermentation reaction (Fermentation 1), the liquid
was aseptically removed and analyzed for ethanol content, and the
beads were aseptically rinsed several times with sterile deionized
water. The same Pachysolen/calcium alginate beads were used in a
second fermentation (Fermentation 2), in the same conditions, as
Fermentation 1. Similarly, the fermentation liquid was subsequently
analyzed and the beads rinsed. This was repeated for Fermentation
3. FIG. 7 illustrates the decreased ethanol yield in Fermentations
2 and 3 compared to Fermentation 1.
[0150] Next, the same Pachysolen/calcium alginate beads were
regenerated between Fermentations 3 and 4 (shown as a dotted line
in FIG. 7 between Fermentations 3 and 4) by incubating for 22 hours
in a shaking incubator at 30.degree. C. and 100 rpm in a yeast
culture medium, Yeast Peptone Dextrose (YPD), after washing. The
YPD was then aseptically removed and the beads were used in yet
another fermentation (Fermentation 4). FIG. 7 illustrates that the
regeneration of the Pachysolen/calcium alginate in culture medium
restored the fermentative ability of the Pachysolen to produce
ethanol.
[0151] Similar washes, fermentations, and a second regeneration
(shown as a dotted line between fermentations 7 and 8) were
performed using the same beads in another 6 fermentations. The
results are shown in FIG. 7. FIG. 7 illustrates that immobilized
microbes may be used in sequential fermentations and that the
Pachysolen in the beads can be metabolically regenerated. Although
the present example employs a regeneration step after 3 or 4
consecutive uses of the immobilized microbes, it is possible to
regenerate the microbes more or less often. It is expected that if
a greater number of beads are used in sequential fermentations
(i.e. fermenting under conditions of a saturating yeast
concentration), the ethanol yields would remain at a higher level
in successive fermentations before requiring metabolic
regeneration.
[0152] As discussed above, the immobilization medium, for example
calcium alginate, can degrade due to use. If the microbes are
regenerated and re-used according to the present example, it may be
necessary to recycle the immobilization medium as taught above.
[0153] Although the invention has been described with reference to
preferred embodiments and specific examples, it will readily be
appreciated by those skilled in the art that many modifications and
adaptations of the methods and bioreactors described herein are
possible without departure from the spirit and scope of the
invention as claimed hereinafter. Thus, it is to be clearly
understood that this description is made only by way of example and
not as a limitation on the scope of the invention as claimed
below.
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