Note: Descriptions are shown in the official language in which they were submitted.
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CELLULOSE AND XYLAN FERMENTATION BY NOVEL ANAEROBIC
THERMOPHILIC CLOSTRIDIA ISOLATED FROM SELF-HEATED
BIOCOMPOST
RELATED APPLICATIONS
[0001] This application claims priority of U. S. Provisional Application
No. 61/246,440 filed on September 28, 2009, and U. S. Provisional Application
No.
61/249,102 filed on October 6, 2009, the contents of which are hereby
incorporated
into this application by reference.
GOVERNMENT INTERESTS
[0002] The United States government may have certain rights in the
present invention as research relevant to its development was funded by a
grant DE-
AC05-000822725 from the BioEnergy Science Center (BESC), a U.S. Department of
Energy (DOE) Bioenergy Research Center supported by the Office of Biological
and
Environmental Research in the DOE Office of Science and by Mascoma Corp.
SEQUENCE LISTING
[0003] This application is accompanied by a sequence listing both on
paper and in a computer readable form that accurately reproduces the sequences
described herein. These sequences have been deposited in GenBank under
accession
numbers FJ808599, FJ808600, GQ265352 and GQ265353.
BACKGROUND
[0004] The present invention pertains to the field of biomass processing to
produce ethanol and other products and more specifically, to the selection,
isolation
and use of novel anaerobic thermophilic cellulolytic and xylanolytic
organisms. The
invention relates to isolation of novel species of bacterium designated as
Clostridium
sp. 4-2a having ATCC deposit number PTA-10114. Ths Clostridium sp. strains 4-
2a
and 4-1 have been previously designated as a Clostridium polyfermentans strain
4-2a
and strain 4-1, respectively. For purpose of consistency, these two strains
have been
re-designated as Clostridium sp. strain 4-2a and strain 4-1, respectively, and
will be
referred to under the new nomenclature throughout this disclosure.
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[0005] Biomass represents an inexpensive and readily available cellulosic
feedstock from which sugars may be produced. These sugars may be recovered or
fermented to produce alcohols and/or other products. Among bioconversion
products,
interest in ethanol is high because it may be used as a renewable domestic
fuel.
[0006] Cellulose and xylan present in biomass represent an inexpensive
and readily available raw material from which sugars may be produced. These
sugars
may be used alone or fermented to produce alcohols and other products. Among
bioconversion products, interest in ethanol is high because it may be used as
a
renewable domestic fuel. Bioconversion processes are becoming economically
competitive with petroleum fuel technologies. Various reactor designs,
pretreatment
protocols, and separation technologies are known, for example, as shown in
U.S.
Patent Nos. 5,258,293 and 5,837,506.
[0007] Several anaerobic thermophiles have been shown to utilize
cellulose, including Clostridium thermocellurn, C. straminisolvens, C.
stercorarium,
C. clariflavum and Caldicellulosiruptor saccharolyticus (Freier et al 1988;
Kato et al.
2004; Madden 1983; Rainey et al. 1994; Shiratori et al. 2009).
[0008] The ultimate combination of biomass processing steps is referred to
as consolidated bioprocessing (CBP). CBP involves four biologically-mediated
events: (1) enzyme production, (2) substrate hydrolysis, (3) hexose
fermentation and
(4) pentose fermentation. These events may be performed in a single step by a
microorganism that degrades and utilizes both cellulose and hemicellulose.
Development of CBP organisms could potentially result in very large cost
reductions
as compared to the more conventional approach of producing saccharolytic
enzymes
in a dedicated process step. CBP processes that utilize more than one organism
to
accomplish the four biologically-mediated events are referred to as
consolidated
bioprocessing co-culture fermentations.
[0009] Among bacteria, Clostridia play an important role in anaerobic
cellulose fermentation. Cellulolytic clostridia have been isolated from a wide
variety
of environments that are rich in decaying plant material such as soils,
sediments,
sewage sludge, composts, etc. (Leschine 2005).
[0010] C. thermocellum exhibits a high growth rate on crystalline cellulose
(Lynd et al. 2002), but it does not utilize xylan. C. thermocellum does not
grow on
xylose or other pentoses, and grows poorly on glucose (Lynd et al. 2008).
Extremely
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thermophilic cellulolytic Caldicellulosiruptor saccharolyticus can co-utilize
glucose
and xylose (van de Werken et al. 2008), while Anaerocellum thermophilum DSM
6725 has been found to degrade xylan and xylose by Yang et al (2009). However,
the
original report on this strain by Svetlichny et al (1990) showed that it did
not utilize
xylose. A. thermophilum has recently been shown to utilize cellulose and
hemicellulose originating from lignocellulose with or without pretreatment
(Yang et
al., 2009). Cellulose conversion achieved by A. thermophilum cultures was <
20%,
although higher conversion was observed upon re-inoculation. Although several
mesophilic Clostridium species have been reported to utilize both cellulose
and xylan,
including C. phytofer mentas, C. cellulovorans (Warnick et al. 2002; Kosugi et
al.
2001; Sleat et al. 1984), C. stercorarium is the only cellulolytic
thermophilic
Clostridium that has been reported to utilize both xylan and cellulose. One
disadvantage of C. stercorarium is that its utilization of cellulose is modest
as
compared to C. thermocellum (Adelsberger et al. 2004; Zverlov and Schwartz
2008).
[0011] Microbial cellulose utilization is among the most promising
strategies for biofuels production (Lynd et al. 2008a). After cellulose, xylan
is the
most predominant polymer in plants (Thompson 1993). Plant biomass represent an
abundant and valuable renewable natural resource that may be put to wide range
of
uses, as a source of food, fiber chemicals, energy, etc. (Leschine 2005).
[0012] Isolation of novel microorganisms that are able to degrade major
plant cell wall polymers such as cellulose, hemicelluloses and lignin, is
essential for
overcoming the recalcitrance of cellulosic biomass (Lynd et al. 2008b).
Cellulolytic
and xylanolytic Clostridium sp. strains 4-2a and 4-1 may be useful in
processes for
bioconversion of lignocelluloses to fuels, chemicals, protein, silage, biogas,
etc.
SUMMARY
[0013] The present instrumentalities advance the art and overcome the
problems outlined above by providing methods for isolation and culture of
cellulolytic
microbes. By utilizing bacterial strains capable of metabolizing both
cellulose and
xylan containing material, these novel strains may serve as a source of
thermostable
xylanases and cellulases for industrial applications resulting in increased
bioprocessing efficiency and economy.
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[0014] More specifically, the present disclosure, provides a biologically
pure culture of the Clostridium sp. strain 4-2a. Clostridium sp. strain 4-2a
has been
deposited, under the provisions of the Budapest Treaty, in the culture
collection
American Type Culture Collection (ATCC, Manassas, VA) on June 9, 2009 and
bears
the ATCC Deposit No. PTA-10114. It is also disclosed herein a second
Clostridium
sp. strain 4-1.
[0015] In an embodiment, an isolated biologically pure culture of an
anaerobic thermophilic cellulolytic and xylanolytic bacterium bearing ATCC
Deposit
No. PTA-10114 is described.
[0016] In another embodiment, a biological material may be prepared
which comprises an isolated biologically pure culture of an anaerobic
thermophilic
cellulolytic and xylanolytic bacterium bearing ATCC Deposit No. PTA-10114.
[0017] In another embodiment, the biological material of the present
disclosure comprises an isolated biologically pure culture of an anaerobic
thermophilic cellulolytic and xylanolytic bacterium which contains an
endogenous
gene having at least 70%, 80%, 90%, 95%, 99.9%, or most preferably, having
100%
identity with SEQ ID No. 2.
[0018] In another embodiment, the biological material of the present
disclosure comprises an isolated biologically pure culture of an anaerobic
thermophilic cellulolytic and xylanolytic bacterium which contains a gene
having at
least 70%, 80%, 90%, 95%, 99%, or most preferably, having 100% identity with
SEQ
ID No. 4.
[0019] In another embodiment, the biological material of the present
disclosure comprises an isolated biologically pure culture of an anaerobic
thermophilic cellulolytic and xylanolytic bacterium which contains a
functional
exoglucanase having at least 70%, 80%, 90%, 95%, 99%, or most preferably,
having
100% identity with the enzyme encoded by the polynucleotide sequence of SEQ ID
No. 4.
[0020] In another embodiment, it is disclosed a functional exoglucanase
having at least 70%, 80%, 90%, 95%, 99% or most preferably, having 100%
sequence
identity with the enzyme encoded by the polynucleotide sequence of SEQ ID No.
4.
[0021] In another embodiment, a polynucleotide having at least 70%,
80%, 90%, 95%, 99%, or most preferably, having 100% identity with SEQ ID No. 4
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may be introduced into an organism and caused to be expressed in said organism
in
order to confer upon said organism the functionality similar to that of the
exoglucanase of the new strain disclosed herein. By way of example, the
polynucleotide may be introduced into the organism using transgenic or
conjugation
methods, among others. Such an organism may be called a transgenic organism,
and
the polynucleotide that is introduced into said organism may be called a
transgene.
[0022] In a preferred embodiment, at least 50% of the artificially cultured
biological material is the anaerobic thermophilic cellulolytic and xylanolytic
bacterium bearing ATCC Deposit No. PTA-10114. Even more preferably, the
cultured biological material contains at least 60%, 70%, 80%, 90% or 100% of
the
anaerobic thermophilic cellulolytic and xylanolytic bacterium bearing ATCC
Deposit
No. PTA-10114.
[0023] In an embodiment, a method for isolating a biologically pure
culture of an anaerobic thermophilic cellulolytic and xylanolytic bacterium
bearing
ATCC Deposit No. PTA-10114 is described.
[0024] In another embodiment, a method for culturing an anaerobic
thermophilic cellulolytic and xylanolytic bacterium bearing ATCC Deposit No.
PTA-
10114 is described.
[0025] It is also provided herein a method for conversion of a biomass to
at least one bioconversion product. The method may include a step contacting
the
biomass with an isolated thermophilic cellulolytic and xylanolytic bacterium.
In a
preferred embodiment, the bacterium to be used contains an endogenous gene
having
at least 99.9% sequence identity with SEQ ID No. 2, or even more preferably,
the
bacterium is identical to the strain bearing ATCC Deposit No. PTA-10114. The
biomass may be caused to be in contact with the disclosed bacterium in
conjunction
with at least one other bacterium. Alternatively, the contact between the
biomass and
the disclosed bacterium may be preceded and/or followed by another contacting
step
wherein the biomass is caused to be in contact with at least one other
bacterium. The
biomass may or may not have been pretreated before being caused to be in
contact
with the disclosed bacterium.
[0026] In another aspect, the biomass may be converted to the at least one
bioconversion product by batch simultaneous saccharification and fermentation,
by
continuous culture, or by semi-continuous culture.
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[0027] The biomass may contains a cellulosic material, a a xylanosic
material, a lignocellulosic material, or combination thereof. The bioconverion
products may include but are not limited to lactic acid, formic acid, acetic
acid,
ethanol or mixture or salt thereof. In a preferred embodiment, the acetic
acid/ethanol
ratio in the final bioconverion products is at least 13.2.
BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 shows a diversity of colonies isolated and grown on
Avicel-agar medium.
[0029] Figure 2 is a phylogenetic tree of anaerobic thermophilic
cellulolytic bacteria based on 16S rRNA gene sequence comparisons.
[0030] Figure 3 is a phylogenetic tree of anaerobic thermophilic
cellulolytic bacteria based on GHF48 gene sequence comparisons.
[0031] Figure 4 is a graph depicting the dynamics of Avicel degradation
and bacterial biomass growth in batch culture of strain 4-2a.
[0032] Figure 5 is a graph depicting product formation of Avicel
degradation in a batch culture of strain 4-2a.
[0033] Figure 6 is a graph depicting the dynamics of xylan degradation
and bacterial biomass growth in batch culture of strain 4-2a.
[0034] Figure 7 is a graph depicting product formation of xylan
degradation in a batch culture of strain 4-2a.
DETAILED DESCRIPTION
[0035] There will now be shown and described a method for the isolation
of novel cellulolytic and xylanolytic microbes.
[0036] As used herein, "cellulolytic" means capable of hydrolyzing
cellulose.
[0037] As used herein, "xylanolytic" means capable of hydrolyzing xylan.
[0038] A biologically pure culture of an organism contains 100% of cells
from said organism. As used herein, a "biologically pure culture" of bacteria
is a
genetically uniform culture of bacterial cells derived from a single colony.
Such a
culture contains 100% of cells that are progeny of the single colony. As used
herein a
culture may be a solid culture, or a liquid culture, such as but not limited
to solid
medium and liquid medium respectively. When referring to biological material
or
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culture, the term "isolated" means the biological material or culture is
prepared with
some modification or the biological material or culture is purified from its
naturally
occuring sources.
[0039] As used herein, the term "biological material(s)" refers to bacteria,
viruses, fungi, plants, animals or any other living organisms. For purpose of
this
disclosure, the biological material may contain a single biologically pure
culture, or it
may contain at least two genetically different cells from different strains
that belong
to the same or different species. For instance, the artificially cultured
biological
material of the present disclosure may be a mixture of a bacterial strain and
a fungal
strain. The biological material may be in a variety of forms, including but
not limited
to, liquid culture, solid culture, frozen culture, dry spores, live or dormant
bacteria,
etc. The term "artificially cultured" means that the biological material is
grown for at
least one cell cycle in a man-made environment, such as an incubator. The man-
made
environment may also be based on the natural environment of said biological
material
which has been modified to some degree to optimize the growth, reproduction
and/or
metabolism of the organism(s). It is to be recognized that the artificially
cultured
biological material may contain cells that are originally isolated from their
natural
environment.
[0040] As used herein, a biologically pure culture of Clostridium sp. 4-2a
may be derived from Clostridium sp. strain 4-2a. Strains 4-2a may be purified
via
single colony isolation method.
[0041] As used herein, an organism is in "a native state" if it is has not
been genetically engineered or otherwise manipulated by the hand of man in a
manner
that intentionally alters the genetic and/or phenotypic constitution of the
organism.
For example, wild-type organisms may be considered to be in a native state.
[0042] As used herein, thermophilic means capable of survival, growth
and reproduction at temperatures greater than about 50 C.
[0043] Clostridium sp. strain 4-2a is an anaerobic thermophilic cellulolytic
and xylanolytic gram positive bacterium.
[0044] Cellulase refers to a class of enzymes produced chiefly by fungi,
bacteria, and protozoa that catalyze the cellulolysis (or hydrolysis) of
cellulose.
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[0045] As used herein, bioconversion products are the products that are
generated by the breakdown of biomass. These products include, but are not
limited
to, ethanol, lactate, formate and acetate.
EXAMPLE 1
ISOLATION OF CLOSTRIDIUMsp. 4-2a
Materials and Methods
[0046] Compost samples were collected at Middlebury College compost
facilities in Middlebury Vermont, USA. Samples were collected between 40 cm to
50
cm below the surface of the compost pile. The compost temperature varied
between
52 C and 72 C.
[0047] In contrast to previous studies, strictly anaerobic conditions were
employed starting from primary sampling. Compost samples of between 8g and 15g
were inoculated into bottles containing 100 ml of mineral medium, pH 7. One
gram
of Avicel (PHI 05; FMC Corp., Philadelphia, Pa.) was added to each bottle as a
carbon source and flashed with nitrogen.
[0048] The primary mineral medium was formulated as follows: KH2PO4,
2.08 g/L; K2HP04, 2.22 g/L; MgC12x6H2O, 0.1 g/L; NH4C1, 0.4 g/L; CaC12x2H2O,
0.05 g/L.
[0049] Upon arriving at the laboratory, the primary enrichments were
brought to a temperature of 55 C and incubated for 4 to 6 days. For
consecutive
transfers, defined minimal medium was prepared: Avicel, 3; KH2PO4, 1.04;
K2HPO4,
1.11; NaHCO3, 2.5; MgC12x6H2O, 0.2; NH4C1, 0.4; CaC12x2H2O, 0.05; FeC12x4H2O,
0.05; L-cysteine HC1, 0.5; resazurin 0.0025. SL10-trace element, 1 ml/L
(Atlas, 1996)
and vitamin, 4 ml/L, solutions were added as concentrated solutions. The
vitamin
solution contained (g/1): pyridoxamine dihydrochloride, 0.2; PABA, 0.1; D
biotin,
0.05; vitamin B 12, 0.05; thiamine-HC1, 0.0125; folic acid, 0.5; Ca-
pantothenate,
0.125; nicotinic acid, 0.125; pyridoxine-HC1, 0.025; thioctic acid, 0.125;
riboflavin,
0.0125.
[0050] Phosphates and other minerals were prepared and autoclaved
separately to avoid precipitation and unwanted chemical interactions during
autoclaving. Vitamins were sterilized by filtration. Stock solutions (x 100)
of L-
cysteine HCl, FeC12x4H2O, MgC12x6H2O; NH4C1; CaC12x2H2O were flashed with N2
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immediately after dissolving and autoclaved. Serum bottles with sterile medium
were
placed into an anaerobic glove box, cooled down, mixed with reducing agent
solution,
closed with sterile rubber stoppers and caped with aluminum seals. To avoid
contamination due to gas exchange during loading inside an airlock, all serum
bottles
were closed with sterile cotton balls and aluminum foil caps or rubber
stoppers.
[0051] Descriptive statistics of primary data, including mean, confidence
interval and standard deviation were done with MS Excel. 2-5 replicates were
used for
all analytical measurements (HPLC and TOCN) and relative error did not exceed
5%.
The growth batch experiments were done at least twice with two replicate
bottles. The
time series data were used to calculate maximal specific growth rate and yield
by
using linear and non-linear regression with the Solver, MS Excel.
[0052] Phylogenetic trees were assembled using a bootstrap test with 1000
replicates to evaluate robustness.
[0053] To analyze Avicel, xylan, xylose and pretreated wood utilization
products, anaerobic cellulolytic thermophilic strains were transferred into
fresh
defined medium with 3g/l of related substrate. Batch cultures were incubated
at 55 C
on shaker at 180 rpm for 2-7 days. Fermentation products were analyzed by HPLC
at
zero point and at the end of incubation.
Isolation of pure cultures
[0054] Isolation of pure cultures of cellulose degrading bacteria was
performed on agar-Avicel and agar-cellobiose media after 10 consecutive
transfers of
primary enrichments. The mineral composition was the same as described above.
Vitamins were substituted with 2.0 g/l of yeast extract. Avicel was added at
concentration 20 g/l, cellobiose at 10 g/l, and agar at 15 g/l. Cellulolytic
consortium
grown on defined Avicel medium was serially diluted into melted and cooled
agar-
Avicel medium (55 C to 60 C) and plated into Petri dishes inside an anaerobic
glove
box. After solidifying, the plates were incubated inside anaerobic jars at 55
C.
Cellulose degrading bacteria formed zones of clearing in the Avicel-agar layer
during
incubation. Colonies were picked with a syringe needle and inoculated into
defined
Avicel and cellobiose liquid media. Isolates, primarily grown on cellobiose
medium,
were transferred onto Avicel-defined medium to assess their ability to degrade
cellulose.
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[0055] Two active cellulolytic strains 4-2a and 4-1 able to degrade
cellulose, xylan and xylose were isolated from biocompost
DNA extraction, PCR amplification, sequencing and alignment
[0056] Genomic DNA was extracted from microbial biomass with the
GenElute Genomic DNA Kit (Sigma) according to manufactures instructions. PCR
amplification of the 16s rRNA gene and sequencing was done as described before
(Sizova et al. 2003). Amplification of GHF48 genes was performed with GH48F
and
GH48R degenerate primers (Izquierdo et al., 2010). Amplified PCR products were
sequenced at Agencourt Bioscience Corporation, MA. Nucleotide sequences were
aligned with sequences from GenBank using BioEdit v.7Ø5 (Hall 1999) and
CLUSTALW (Thompson et al. 1994).
Phylogenetic analysis of bacterial isolates
[0057] Phylogenetic trees were reconstructed using the ME-algorithm
(Rzhetsky and Nei 1992) via the MEGA4 program package (Tamura et al. 2007).
Screening for similarity was carried out with BLAST.
[0058] Figure 2 shows a phylogenetic tree of anaerobic thermophilic
cellulolytic bacteria based on 16S rRNA gene sequence comparisons.
Phylogenetic
analysis revealed that isolated strains 4-1 and 4-2a are most closely related
to novel
Clostridium clariflavum that actively fermented paper waste in thermophilic
methanogenic reactor (Shiratori et al. 2006; Shiratori et al. 2009). The
sequences of
16S rRNA from 4-1 (SEQ ID No. 1) and 4-2a (SEQ ID No. 2) have been deposited
with GenBank and have been assigned accession numbers FJ808599 and FJ808600,
respectively.
[0059] Figure 3 is a phylogenetic tree of anaerobic thermophilic
cellulolytic bacteria based on GHF48 gene sequence comparisons.
[0060] Glycoside hydrolases (GHs) (EC 3.2.1.) are a widespread group of
enzymes which hydrolyze the glycosidic bond between two or more carbohydrates
or
between a carbohydrate and a non-carbohydrate moiety. The IIJB-MB enzyme
nomenclature of glycoside hydrolases is based on their substrate specificity
and
occasionally on their molecular mechanism; such a classification does not
reflect the
structural features of these enzymes.
[0061] In most cases, the hydrolysis of the glycosidic bond is performed
by two catalytic residues of the enzyme: a general acid (proton donor) and a
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nucleophile/base. Depending on the spatial position of these catalytic
residues,
hydrolysis occurs via overall retention or overall inversion of the anomeric
configuration.
[0062] Phylogenetic analysis was also carried out with respect to
exocellulases of glycosyl hydrolase family 48 (GHF48), a major enzyme of
interest
within cellulolytic microorganisms. Clostridium sp. strains 4-1 and 4-2a,
formed a
distinct cluster of identical nucleotide sequences with no known sequences
closely
related to them. The closest matches were C. thermocellum CelY (74.1%
similarity in
nucleotide sequence, 87% translated amino acid sequence similarity) and C.
straminisolvens (73.4% similarity in nucleotide, 87% translated amino acid
sequence
similarity). The translated amino acid sequence of GHF48 enzymes from 4-1 or 4-
2a
may be obtained by translating the GHF48 gene sequences from 4-1 (SEQ ID No 3)
or from 4-2a (SEQ ID No 4) using standard genetic codes.
[0063] GHF48 genes in Clostridium sp. strains 4-2a and 4-1 displayed a
very similar grouping as observed in 16S rRNA gene analyses, suggesting a very
strict conservation of this particular family of glycosyl hydrolases within
cellulolytic
Clostridia. GHF48 sequences isolated from strains 4-1 (SEQ ID No. 3, GenBank
Accession # GQ265352) and 4-2a (SEQ ID No. 4, GenBank Accession # GQ265353)
have been deposited with GenBank. These GHF48 genes encode proteins which
represent novel exoglucanases that may be useful in the biofuel industry.
Fermentation Physiology
[0064] To analyze Avicel, xylan, xylose and pretreated wood utilization
products, anaerobic cellulolytic thermophilic strains were transferred into
fresh
defined medium with 3g/l of related substrate. Batch cultures were incubated
at 55 C
on a shaker at 180 rpm. Fermentation products were analyzed by HPLC with an
Aminex HPX-87H column (Bio-Rad Laboratories) at zero point and at the end of
incubation. Major products of Avicel, xylan, xylose and pretreated wood
fermentation are shown in Table 1. Major fermentation products of Avicel were
acetate and formate, with lactate accumulating at the late stage of
fermentation (Fig.
5)
[0065] It was observed that xylan was degraded during the first day of
incubation while accumulation of pretreated wood and xylose fermentation
products
took between 5-7 days. In contrast to the fermentation products formed from
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pretreated wood, i.e. acetate and lactate, the major fermentation products of
xylan
were acetate and formate. Ethanol concentrations varied from 0.6 to 1.1 mM
with the
acetate to ethanol ratio being 10.9 - 19.3. Both the 4-1 and 4-2a isolates
were able to
use xylose as a single source of carbon. Microbial growth on xylose was much
slower
than on Avicel, xylan and pretreated wood. Only -50% of xylose was fermented
during 10 days of incubation. The major fermentation product of xylose was
acetate
and lactate, no ethanol was detected
Table 1. Fermentation products formed by isolates 4-1 and 4-2a from Avicel,
xylan,
pretreated wood and xylose (3 g11).
Substrate Isolate Lactate Formate Acetate Ethanol Acetate/Ethanol
mm ratio
Avicel 4-1 0.2 2.7 7.8 0.3 22.2
4-2a 1.0 3.5 9.2 0.9 10.3
Xylan 4-1 0.3 3.6 12.8 0.7 18.6
4-2a 0.5 3.0 12.2 0.6 19.3
Pretreated wood 4-1 2.1 0.7 11.6 1.1 10.9
4-2a 1.0 0.1 10.4 0.8 13.2
Xylose 4-1 0.1 2.1
4-2a 0.5 2.7
[0066] Two isolated strains, 4-1 and 4-2a, were able to degrade cellulose,
xylan and xylose. These two cellulolytic and xylanolytic strains were related
to
Clostridium clariflavum.
Dynamics of cellulose and xylan utilization
[0067] One percent of freshly grown culture was used as inoculums.
Degradation of Avicel began after a lag period of about 11-15 hr. Figure 4
shows that
about 60% of Avicel was utilized during 10-15 hrs of exponential growth of
strain 4-
2a (symbols: o, concentration of Avicel; A, cells biomass). Bacterial biomass
accumulated exponentially during first 21 hrs. Approximate biomass yield was
about
0.13 mg C-biomass/mg C-Avicel. The degradation process abruptly ceased as the
pH
of the culture medium dropped from pH 8 to pH 6. pH was measured using an
Ultra
Basic Bench top pH meter UB-10 (Denver Instrument).
[0068] The major fermentation products were acetate, formate, lactate and
ethanol. As shown in Figure 5, acetate, formate and ethanol were formed
exponentially in parallel with bacterial growth (symbols: =, acetate; ^,
formate; A,
ethanol; =, lactate; o, xylose; ^, cellobiose; A, glucose; 0, glycerol). It
was observed
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that, as pH declined, lactate, cellobiose, glucose, glycerol and xylose
accumulated in
the cultural medium. At the end of incubation the acetate/ethanol ratio was
about
12:1.
[0069] Figure 6 is a graph illustrating the dynamics of xylan degradation
in batch cultures of strain 4-2a (symbols: o, concentration of xylan; A, cells
biomass). Degradation of xylan began immediately after inoculation. During the
first
21 hrs of incubation about 75% of xylan was degraded, while bacterial biomass
and
accumulation of fermentation products and intermediates increased (Fig. 7;
symbols:
=, acetate; ^, formate; A, ethanol; =, lactate; o, xylose; A, glucose; 0,
glycerol).
During incubation, pH declined (data not shown).
[0070] Approximate biomass yield on xylan was 0.14 mg C-biomass/mg
C-xylan, comparable to biomass yield on Avicel. The degradation process
stopped as
pH decreased from about pH 8 to about pH 6.3. The major fermentation products
acetate, formate, lactate as well as the xylose, glucose and glycerol
intermediates
accumulated over time. The concentration of intermediate xylose reached 3.5
mM,
while ethanol concentration reached only 0.6 mM during 60 hrs of incubation.
The
acetate/ethanol ratio was about 22:1.
[0071] Clostridium sp, strains 4-2a and 4-1 represent a new anaerobic,
thermophilic and cellulolytic organism within the Clostridium genus, besides
C.
stercorarium (Adelsberger et al. 2004) that is capable of degrading cellulose,
xylan
and xylose.
Description of Clostridium sp. strains 4-2a and 4-1.
[0072] Clostridiuim sp. strains 4-2a and 4-1 cells are straight and slightly
curved rods 3-12 x 0.1-0.3 m when grown on Avicel and straight rods 3-5 x 0.2-
0.3
m when grown on xylan. Clostridiuirn sp. strain 4-2a and 4-1 forms terminal
spores. Surface colonies (in agar-cellobiose medium) are extremely slimy and
light
cream colored. Colonies grown in agar-Avicel medium produce 5-10 mm zones of
clearing during 7 days of incubation. Clostridiuim sp. strain 4-2a and 4-1 is
an
obligate anaerobe. Bacterial cultures of Clostridiuim sp, strain 4-2a and 4-1
robustly
grow on Avicel or xylan as a single carbon source. Biomass yield is 0.13 mg C-
biomass/mg C-Avicel with N/C ratio 0.27. Major fermentation products were
acetate,
formate, lactate and ethanol. Clostridiuim sp. strain 4-2a and 4-1 grows on
cellobiose
and partially ferments xylose. Growth occurs at temperature 55-60 C and pH 6.0-
8Ø
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[0073] Adaptation of traditional plating techniques allowed for the
isolation of new anaerobic thermophilic bacteria that utilize cellulose.
[0074] Microbial culture purification and identification requires the
isolation of a single colony. Consistent results were observed when consortia
grown
in cellulose liquid medium till the middle of log phase were plated within
agar layer.
It was important to make all manipulations inside of anaerobic glove box and
prepare
serial dilutions in nutrient medium but not sterile water.
[0075] The major methodological principle was to mimic natural
conditions of anaerobic cellulose degradation in situ. Conditions that were
crucial in
this process were: a) strictly anaerobic conditions starting from primary
sampling; b)
cellulose (Avicel or filter paper) as the only source of carbon and energy (no
yeast
extract or vitamins were added); c) enrichment incubation temperature was the
same
as in situ; d) nitrates, sulfates, sulfides were excluded to avoid the
development of
competitive microorganisms.
[0076] Thus, anaerobic sampling procedures in combination with adapted
plating techniques allows for the isolation of novel cellulolytic
microorganisms even
from very well studied environments like biocompost piles. Biocompost remains
one
of the most promising natural environments for isolation of active plant
biomass
degraders.
[0077] Microbial cellulose utilization is among the most promising
strategies for biofuels production (Lynd et al. 2008). Plant biomass
represents an
abundant and valuable renewable natural resource that may be put to wide range
of
uses, as a source of food, fiber chemicals, energy, etc (Leschine 2005). Novel
cellulolytic and xylanolytic strains described in this study can serve as
potential
source of previously unknown thermo stable xylanases and cellulases for plant
biomass conversion and other industrial applications. After cellulose, xylan
is the
most predominant polymer in plants (Thompson 1993). Microorganisms and
enzymes actively fermented plant polymers are extremely useful for a broad
range of
environmentally friendly industrial processes. Microbial xylanases assume
special
importance in the paper and pulp industry as they help to minimize the use of
toxic
chemicals (Kulkami et al. 1999). Xylanases are also used as nutritional
additives to
silage and grain feed, for the extraction of coffee and plant oils and in
combination
with pectinases and cellulases for clarification of fruit juices (Beg et al.
2001).
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[0078] Therefore, cellulolytic and xylanolytic strains described above are
useful for further characterizing cellulase and xylanase diversity as well as
in
processes for bioconversion of lignocelluloses to fuels, chemicals, protein,
silage,
biogas, etc.
EXAMPLE 2
PREPARATION OF CULTIVATION MEDIUM
[0079] Two different solutions of chemicals were prepared separately in
order to avoid precipitation and chemical interactions during autoclaving.
Vitamins
were sterilized by filtration.
[0080] Preparation of a 1000X solution of trace elements SL-10 is
described in Table 2.
Table 2. Trace element solution SL-10 1000X)
Component Amount
HCl (25% 10 ml
FeC12x4H2O 1.5 /l
CoC12x6H2O 0.19 g/l
MnCl2x4H2O 0.1 /l
ZnC12 0.07 g/l
Na2MoO4x2H2O 0.036 g/l
NiC12x6H2O 0.024 g/l
H3BO3 0.006 g/l
CuCl2x2H2O 0.002 g/l
[0081] Preparation of a 250X solution vitamins is described in Table 3.
Table 3. Vitamin solution (250X)
Component Amount g/l
Pyridoxamine Dihydrochloride 0.2
Para-aminobenzoic acid (PABA) 0.1
D Biotin 0.05
Vitamin B 12 0.05
Thiamine HC1 0.0125
Folic Acid 0.05
Pantotenic acid-Ca++ salt 0.125
Nicotinic acid 0.125
P ridoxine-HC1 0.025
Thioctic acid 0.125
Riboflavin 0.0125
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[0082] Preparation of solution A is described in Table 4.
Table 4. Solution A
Components Final Amount
Avicel 3.0 /l
KH2PO4 1.04 /l
K2HPO4 1.11 /l
Trace Elements SL-10 1 ml
NaHCO3 2.0 g/l
Resazurin 0.025% 0.01 g11
[0083] Preparation of a 100X stock solution B is described in Table 5.
Table 5. Solution B 100X
Components Final Amount Stock solution, g11
NH4C1 0.4 g1l 4.0
M Cl2x6H2O 0.1 g1l 1.0
CaC12xH2O 0.05 /l 0.5
L-cysteine HCI: 0.5 g11 5.0
C3HNO2SxHHC1xH20
FeC12x4H2O 0.05 g1l 0.5
[0084] Medium was prepared by preparing solution A and distributing
solution A into serum bottles. Serum bottles were closed with rubber stoppers
and
sealed with aluminum caps. Bottles were then flashed with nitrogen. L-cysteine
HCL
and FeC12x4H2O were dissolved and mixed with the additional components of
solution B in a serum bottle. The bottle was closed with a rubber stopper and
sealed
with an aluminum cap. The serum bottle was immediately flashed with nitrogen.
All
serum bottles were then autoclaved for 20-25 min. Sterile anaerobic stock
solution B
and vitamin solution was then aseptically transferred to serum bottles
containing
solution A using a sterile needle and syringe. After about 10-20 minutes the
combined solutions became colorless.
[0085] The disclosed microbes may be utilized in a consolidated
bioprocessing (CBP) process with no added enzymes. Methods of utilizing
cellulolytic microbes for the conversion of cellulosic material into ethanol
are known.
Cellulosic materials that may be converted by the presently described microbes
include any feedstock that contains cellulose, such as wood, corn, corn
stover,
sawdust, bark, leaves, agricultural and forestry residues, grasses such as
switchgrass
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or miscanthus or mixed prairie grasses, ruminant digestion products, municipal
wastes, paper mill effluent, newspaper, cardboard or combinations thereof.
EXAMPLE 3
SIMULTANEOUS SACCHARIFICATION AND FERMENTATION
[0086] As discussed above, the thermophilic organism Clostridium sp.
strain 4-2a and 4-1 has the potential to contribute significant savings in
lignocellulosic
biomass to ethanol conversion due to their ability to utilize cellulose,
xylose and
xylan.
[0087] Clostridium sp. strains 4-2a and 4-1 are used to produce ethanol
and other products in the bioconversion processes of consolidated
bioprocessing
(CBP)
[0088] It will be appreciated that Clostridium sp. strain 4-2a and 4-1 can
ferment both pentose and hexose sugars..
Batch SSF and relevant enzyme controls.
[0089] Five ml of a Clostridium sp.4-2a (ATCC Deposit No. PTA-10114)
stock culture is inoculated into 100 ml medium containing a 3 grams of a
carbon
source and under a N2 atmosphere. The carbon source may be Avicel, xylan,
pretreated wood, or xylose or a combination thereof. Cultures are incubated at
55 C
in a temperature controlled water bath with rotary shaking at 180 rpm. pH is
adjusted
to 8.
Continuous culture.
[0090] The reaction vessel was a modified 1L fermentor (Applikon,
Dependable Instruments, Foster City, CA, modified by NDS) with an overflow
sidearm (i.d. 0.38") and 0.5 L working volume is used for both microbial
fermentation
by Clostridium sp.4-2a (ATCC Deposit No. PTA-10114) and for SSF carried out in
continuous mode. pH was controlled by a Delta V process control system (New
England Controls Inc., Mansfield, MA) with addition of 4M NaOH, the fermentor
was stirred at between 180 rpm and 250 rpm, and temperature was controlled at
55 C
by circulating hot water through the fermentor j acket. Medium containing 3
g/L
Avicel, xylan, pretreated wood, or xylose or a combination thereof is fed by a
peristaltic pump to achieve the desired residence times. SSF experiments are
initiated
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by inoculating 50 ml of a late-exponential phase culture of Clostridium sp.4-
2a
(ATCC Deposit No. PTA-10114) into medium containing 3 g/L Avicel, xylan,
pretreated wood, or xylose or a combination thereof. Samples used to calculate
steady-state values for continuous fermentations are taken at intervals of at
least one
residence.
Strain Deposit
[0091] Clostridium sp. strain 4-2a has been deposited with the American
Type Culture Collection, Manassas, VA 20110-2209. The deposit was made on June
9, 2009 and received Patent Deposit Designation Number PTA-10114. This deposit
was made in compliance with the Budapest Treaty requirements that the duration
of
the deposit should be for thirty (30) years from the date of deposit or for
five (5) years
after the last request for the deposit at the depository or for the
enforceable life of a
U.S. Patent that matures from this application, whichever is longer.
Clostridium sp.4-
2a will be replenished should it become non-viable at the depository.
[0092] The description of the specific embodiments reveals general
concepts that others can modify and/or adapt for various applications or uses
that do
not depart from the general concepts. Therefore, such adaptations and
modifications
should and are intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood that the
phraseology
or terminology employed herein is for the purpose of description and not
limitation.
All references mentioned in this application are incorporated.
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