U.S. patent application number 14/342712 was filed with the patent office on 2014-11-06 for novel extreme thermophilic bacteria of the genus caldicellulosiruptor.
This patent application is currently assigned to DIREVO INDUSTRIAL BIOTECHNOLOGY GMBH. The applicant listed for this patent is DIREVO INDUSTRIAL BIOTECHNOLOGY GMBH. Invention is credited to Simon Curvers, Vitaly Svetlichnyi.
Application Number | 20140329296 14/342712 |
Document ID | / |
Family ID | 47913923 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140329296 |
Kind Code |
A1 |
Curvers; Simon ; et
al. |
November 6, 2014 |
NOVEL EXTREME THERMOPHILIC BACTERIA OF THE GENUS
CALDICELLULOSIRUPTOR
Abstract
The present disclosure pertains to novel isolated cellulolytic
extreme thermophilic bacterial cells belonging to the genus
Caldicellulosiruptor, mutants thereof, isolated strains, microbial
cultures and microbial compositions. The novel bacteria are in
particular suitable for the production of fermentation products
such as ethanol and lactic acid from lignocellulosic biomass.
Inventors: |
Curvers; Simon; (Koln,
DE) ; Svetlichnyi; Vitaly; (Koln, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DIREVO INDUSTRIAL BIOTECHNOLOGY GMBH |
Koln |
|
DE |
|
|
Assignee: |
DIREVO INDUSTRIAL BIOTECHNOLOGY
GMBH
Koln
DE
|
Family ID: |
47913923 |
Appl. No.: |
14/342712 |
Filed: |
September 21, 2012 |
PCT Filed: |
September 21, 2012 |
PCT NO: |
PCT/EP2012/068627 |
371 Date: |
March 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61537892 |
Sep 22, 2011 |
|
|
|
61669981 |
Jul 10, 2012 |
|
|
|
Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
C12P 7/40 20130101; Y02E
50/17 20130101; C12P 7/62 20130101; C12P 7/54 20130101; C12P 7/56
20130101; C12P 7/10 20130101; C12P 3/00 20130101; C12N 1/20
20130101; C12R 1/01 20130101; Y02E 50/10 20130101; C12P 2201/00
20130101; C12P 7/065 20130101; Y02E 50/16 20130101 |
Class at
Publication: |
435/252.1 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12R 1/01 20060101 C12R001/01 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2011 |
EP |
11007706.2 |
Jul 10, 2012 |
EP |
12175679.5 |
Claims
1. An isolated Caldicellulosiruptor sp. cell comprising a 16S rDNA
sequence which is at least 99%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9%
or 100% identical to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID
NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 7.
2. The isolated cell according to claim 1, wherein the cell is
capable of growing in a medium comprising a lignocellulosic biomass
material.
3. The isolated cell according to claim 2, wherein the
lignocellulosic biomass material is selected from the group
consisting of grass, switch grass, cord grass, rye grass, reed
canary grass, mixed prairie grass, miscanthus, sugar-methoding
residues, sugarcane bagasse, sugarcane straw, agricultural wastes,
rice straw, rice hulls, barley straw, corn cobs, cereal straw,
wheat straw, canola straw, oat straw, oat hulls, corn fiber,
stover, soybean stover, corn stover, cotton stalks, forestry
wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood,
and softwood.
4. The isolated cell according to claim 3, wherein the
lignocellulosic biomass material is subjected to mechanical,
thermochemical, and/or biochemical pretreatment.
5. The isolated cell according to claim 4, wherein pretreating the
lignocellulosic biomass material comprises exposing the
lignocellulosic biomass to steam treatment.
6. The isolated cell according to claim 4 wherein pretreating the
lignocellulosic biomass material comprises mechanical comminution
and a subsequent treatment with sulfurous acid or its anhydride
under heat and pressure with a sudden release of pressure.
7. The isolated cell according to claim 1, which is capable of
producing a fermentation product selected from the group consisting
of an acid, an alcohol and hydrogen.
8. The isolated cell according to claim 7, wherein the alcohol is
selected from the group consisting of ethanol, butanol, propanol,
methanol, propanediol and butanediol.
9. The isolated cell according to claim 7, wherein the acid is
selected from the group consisting of lactic acid, propionic acid,
acetic acid, succinic acid, malic acid, butyric acid and formic
acid.
10. The isolated cell according to claim 7, wherein the acid is
lactic acid or a salt or ester thereof.
11. The isolated cell according to claim 1, wherein one or more
genes have been inserted, deleted or substantially inactivated.
12. The isolated cell according to claim 1, which is DIB041C (DSMZ
Accession number 25771) or a mutant thereof.
13. The isolated cell according to claim 1, which is DIB087C (DSMZ
Accession number 25772) or a mutant thereof.
14. The isolated cell according to claim 1, which is DIB103C (DSMZ
Accession number 25773) or a mutant thereof.
15. The isolated cell according to claim 1, which is DIB104C (DSMZ
Accession number 25774) or a mutant thereof.
16. The isolated cell according to claim 1, which is DIB107C (DSMZ
Accession number 25775) or a mutant thereof.
17. The isolated cell according to claim 1, which is DIB004C (DSMZ
Accession number 25177) or a mutant thereof.
18. The isolated cell according to claim 1, which is DIB101C (DSMZ
Accession number 25178) or a mutant thereof.
19. An isolated strain of the genus Caldicellulosiruptor, wherein
the strain is selected from the group consisting of
Caldicellulosiruptor sp. DIB041C, deposited as DSM 25771,
Caldicellulosiruptor sp. DIB087C, deposited as DSM 25772,
Caldicellulosiruptor sp. DIB103C, deposited as DSM 25773,
Caldicellulosiruptor sp. DIB104C, deposited as DSM 25774,
Caldicellulosiruptor sp. DIB107C, deposited as DSM 25775,
Caldicellulosiruptor sp. DIB 101 C, deposited as DSM 25178 and
Caldicellulosiruptor sp. DIB004C, deposited as DSM 25177,
microorganism derived therefrom, progenies or mutants thereof.
20. The isolated strain of claim 19, having one or more of the
following characteristics: a) it is a microorganism of the genus
Caldicellulosiruptor; and/or b) it is a microorganism of the
species Caldicellulosiruptor saccharolyticus; and/or c) in a
DNA-DNA hybridization assay, it shows a DNA-DNA relatedness of at
least 80%, optionally at least 90%, at least 95%, optionally at
least 98%, optionally at least 99%, and optionally at least 99.9%
with one of the strains of claim 19; and/or c) it displays a level
of 16S rDNA gene sequence similarity of at least 98%, optionally at
least 99%, at least 99.5% or at least 99.7%, optionally 99.99% with
one of the strains listed in claim 19; and/or d) it is capable of
surviving and/or growing and/or producing a fermentation product
selected from the group consisting of acids and alcohols at
temperature conditions above 70.degree. C., in particular of above
72.degree. C.
21-25. (canceled)
Description
FIELD OF THE DISCLOSURE
[0001] The present disclosure pertains to novel isolated
cellulolytic extreme thermophilic bacterial cells belonging to the
genus Caldicellulosiruptor, mutants thereof, isolated strains,
microbial cultures and microbial compositions. The novel bacteria
are in particular suitable for the production of fermentation
products such as ethanol and lactic acid from lignocellulosic
biomass.
BACKGROUND
[0002] In general, fermentation products are produced by
degradation of starch-containing material into fermentable sugars
by liquefaction and saccharification followed by conversion of the
sugars directly or indirectly into the desired fermentation product
using a fermenting organism.
[0003] However, the industry of producing fermentation products
such as ethanol and lactic acid is facing the challenge of
redirecting the production process from fermentation of relatively
easily convertible but expensive starchy materials, to the complex
but inexpensive lignocellulosic biomass such as plant biomass.
[0004] Unlike starch, which contains homogenous and easily
hydrolysed polymers, lignocellulosic biomass contains variable
amounts of cellulose, hemicellulose, lignin and small amounts of
protein, pectin, wax and other organic compounds. Lignocellulosic
biomass should be understood in its broadest sense, so that it
apart from wood, agricultural residues, energy crops also comprises
different types of waste from both industry and households.
Cellulosic biomass is a vast poorly exploited resource, and in some
cases a waste problem. However, hexoses from cellulose can be
converted by yeast to fuel ethanol for which there is a growing
demand. Pentoses from hemicellulose cannot yet be converted to
ethanol commercially but several promising ethanologenic
microorganisms with the capacity to convert pentoses and hexoses
are under development.
[0005] Typically, the first step in utilization of lignocellulosic
biomass is a pre-treatment step, in order to fractionate the
components of lignocellulosic material and increase their surface
area.
[0006] The pre-treatment method most often used is steam
pretreatment, a process comprising heating of the lignocellulosic
material by steam injection to a temperature of 130-230.degree. C.
Prior to or during steam pretreatment, a catalyst like mineral or
organic acid or a caustic agent facilitating disintegration of the
biomass structure can be added optionally.
[0007] Another type of lignocellulose hydrolysis is acid
hydrolysis, where the lignocellulosic material is subjected to an
acid such as sulphuric acid whereby the sugar polymers cellulose
and hemicellulose are partly or completely hydrolysed to their
constituent sugar monomers and the structure of the biomass is
destroyed facilitating access of hydrolytic enzymes in subsequent
processing steps.
[0008] A further method is wet oxidation wherein the material is
treated with oxygen at 150-185.degree. C. Either pretreatment can
be followed by enzymatic hydrolysis to complete the release of
sugar monomers. This pre-treatment step results in the hydrolysis
of cellulose into glucose while hemicellulose is transformed into
the pentoses xylose and arabinose and the hexoses glucose, mannose
and galactose. Thus, in contrast to starch, the hydrolysis of
lignocellulosic biomass results in the release of pentose sugars in
addition to hexose sugars. This implies that useful fermenting
organisms need to be able to convert both hexose and pentose sugars
to desired fermentation products such as ethanol.
[0009] After the pre-treatment the lignocellulosic biomass
processing schemes involving enzymatic or microbial hydrolysis
commonly involve four biologically mediated transformations: (1)
the production of saccharolytic enzymes (cellulases and
hemicellulases); (2) the hydrolysis of carbohydrate components
present in pretreated biomass to sugars; (3) the fermentation of
hexose sugars (e.g. glucose, mannose, and galactose); and (4) the
fermentation of pentose sugars (e.g., xylose and arabinose).
[0010] Each processing step can make the overall process more
costly and, therefore, decrease the economic feasibility of
producing biofuel or carbon-based chemicals from cellulosic
biological material. Thus, there is a need to develop methods that
reduce the number of processing steps needed to convert cellulosic
biological material to biofuel and other commercially desirable
materials.
[0011] The four biologically mediated transformations may occur in
a single step in a process configuration called consolidated
bioprocessing (CBP), which is distinguished from other less highly
integrated configurations in that CBP does not involve a dedicated
process step for cellulase and/or hemicellulase production. CBP
offers the potential for higher efficiency than a processes
requiring dedicated cellulase production in a distinct unit
operation.
[0012] Therefore, the availability of novel microorganisms for
converting lignocellulosic biomass material to carbon-based
chemicals would be highly advantageous.
SUMMARY OF THE DISCLOSURE
[0013] The present invention relates to novel microorganisms, and
compositions useful for processing lignocellulosic biomass.
[0014] In a first aspect, embodiments of the disclosure provide
novel isolated cellulolytic thermophilic bacterial cells belonging
to the genus Caldicellulosiruptor, in particular capable of
producing high levels of lactic acid and/or ethanol from
lignocellulosic biomass material.
[0015] Embodiments of this disclosure relate to an isolated
Caldicellulosiruptor sp. cell comprising a 16S rDNA with a sequence
selected form the group consisting of SEQ ID NO. 1, SEQ ID NO. 2,
SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO
7, or homolgues thereof.
[0016] In one aspect, embodiments of this disclosure relate to an
isolated Caldicellulosiruptor sp. DIB004C, Caldicellulosiruptor sp.
DIB041C, Caldicellulosiruptor sp. DIB087C, Caldicellulosiruptor sp.
DIB101C, Caldicellulosiruptor sp. DIB103C, Caldicellulosiruptor sp.
DIB104C or Caldicellulosiruptor sp. DIB107C, each respectively
characterized by having a 16S rDNA sequence at least 99 to 100%,
preferably 99,5 to 99,99 percent identical to SEQ ID NO. 1, SEQ ID
NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or
SEQ ID NO 7 as outlined in table 1.
[0017] In still another aspect the present invention relates to an
isolated strain comprising a Caldicellulosiruptor sp. cell
according to any of the preceding aspects.
[0018] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB004C
deposited as DSM 25177, a microorganism derived therefrom or a
Caldicellulosiruptor sp. DIB004C homolog or mutant.
[0019] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB041C
deposited as DSM 25771, a microorganism derived therefrom or a
Caldicellulosiruptor sp. DIB041C homolog or mutant.
[0020] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB087C
deposited as DSM 25772, a microorganism derived there from or a
Caldicellulosiruptor sp. DIB087C homolog or mutant.
[0021] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB101C
deposited as DSM 25178, a microorganism derived there from or a
Caldicellulosiruptor sp. DIB101C homolog or mutant.
[0022] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB103C
deposited as DSM 25773, a microorganism derived there from or a
Caldicellulosiruptor sp. DIB103C homolog or mutant.
[0023] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB104C
deposited as DSM 25774, a microorganism derived there from or a
Caldicellulosiruptor sp. DIB104C homolog or mutant.
[0024] In a further aspect, embodiments of this disclosure relate
to microorganism of the strain Caldicellulosiruptor sp. DIB107C
deposited as DSM 25775, a microorganism derived there from or a
Caldicellulosiruptor sp. DIB107C homolog or mutant.
[0025] In another aspect the present disclosure relates to a method
of producing a fermentation product comprising culturing a cell
according to the disclosure or a strain according to the disclosure
under suitable conditions.
[0026] In still another aspect, embodiments of this disclosure
relate to methods for converting lignocellulosic biomass material
to a biofuel or other carbon-based chemical, comprising the step of
contacting the lignocellulosic biomass material with a microbial
culture for a period of time at an initial temperature and an
initial pH, thereby producing an amount of a biofuel and/or other
carbon-based products; wherein the microbial culture comprises an
extremely thermophilic microorganism of the genus
Caldicellulosiruptor, in particular all microorganisms of the
strain Caldicellulosiruptor sp. as listed in table 1 with their
respective deposition numbers, microorganisms derived from either
of these strains or mutants or homologues thereof.
[0027] In still another aspect, embodiments of this disclosure
relate to methods of making ethanol from biomass material, wherein
the method comprises combining a microbial culture and the biomass
in a medium; and fermenting the biomass under conditions and for a
time sufficient to produce ethanol, in a single step process as
part of a consolidated bioprocessing (CBP) system, with a cell,
strain, microbial culture and/or a microorganism according to the
present disclosure under suitable conditions.
[0028] In still another aspect, embodiments of this disclosure
relate to methods of making lactic acid from biomass material,
wherein the method comprises combining a microbial culture and the
biomass in a medium; and fermenting the biomass under conditions
and for a time sufficient to produce lactic acid, a salt or an
ester thereof, in a single step process as part of a consolidated
bioprocessing (CBP) system, with a cell, strain, microbial culture
and/or a microorganism according to the present disclosure under
suitable conditions.
[0029] In still another aspect, embodiments of this disclosure
relate to methods of making both ethanol and lactic acid from
biomass material, wherein the method comprises combining a
microbial culture and the biomass in a medium; and fermenting the
biomass under conditions and for a time sufficient to produce
ethanol and lactic acid, a salt or an ester of the latter, in a
single step process as part of a consolidated bioprocessing (CBP)
system, with a cell, strain, microbial culture and/or a
microorganism according to the present disclosure under suitable
conditions.
[0030] In still another aspect, embodiments of this disclosure
relate to methods of making ethanol and/or lactic lactic acid from
biomass material, wherein the method comprises combining a
microbial culture and the biomass in a medium; and fermenting the
biomass under conditions and for a time sufficient to produce
ethanol and/or lactic acid, a salt or an ester of the latter, in a
single step process as part of a consolidated bioprocessing (CBP)
system, with a cell, strain, microbial culture and/or a
microorganism according to the present disclosure under suitable
conditions in combination with application of method suitable to
in-situ remove both or either fermentation product from the
fermentation broth. Suitable methods include but are not limited to
distillation, mediated distillation, extraction and
precipitation.
[0031] Further, embodiments of this disclosure relate to
compositions for converting lignocellulosic biomass or a microbial
culture comprising a cell, strain or microorganism according to the
present disclosure.
[0032] Further, embodiments of this disclosure relate to the use of
a cell, strain, microorganism and/or a microbial culture according
to the present disclosure for the production of lactic acid, a salt
or an ester thereof or for the production of ethanol.
[0033] Before the disclosure is described in detail, it is to be
understood that this disclosure is not limited to the particular
component parts of the devices described or process steps of the
methods described as such devices and methods may vary. It is also
to be understood that the terminology used herein is for purposes
of describing particular embodiments only, and is not intended to
be limiting. It must be noted that, as used in the specification
and the appended claims, the singular forms "a," "an" and "the"
include singular and/or plural referents unless the context clearly
dictates otherwise. It is moreover to be understood that, in case
parameter ranges are given which are delimited by numeric values,
the ranges are deemed to include these limitation values.
[0034] To provide a comprehensive disclosure without unduly
lengthening the specification, the applicant hereby incorporates by
reference each of the patents and patent applications cited
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 illustrates a phylogenetic tree based on 16S rDNA
genes for all Caldicellulosiruptor sp. strains comprised in the
invention as listed in table 1
[0036] FIG. 2 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB004C cell.
[0037] FIG. 3 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB041C cell.
[0038] FIG. 4 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB087C cell.
[0039] FIG. 5 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB101C cell.
[0040] FIG. 6 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB103C cell.
[0041] FIG. 7 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB104C cell.
[0042] FIG. 8 shows a 16S rDNA from Caldicellulosiruptor sp.
DIB107C cell.
[0043] FIG. 9 shows a graph indicating production of ethanol and
lactic acid by DIB004C during growth on steam-pretreated miscanthus
grass.
[0044] FIG. 10 shows a table indicating performance data from all
strains listed in table 1 and reference strain C. saccharolyticus
DSM8903 during cultivation on cellulose, cellobiose, glucose,
xylan, xylose and pretreated lignocellulosic biomass.
[0045] FIG. 11 shows a table indicating performance data from
strains DIB004C and DIB101C on various types of pretreated
lignocellulosic biomass.
DETAILED DESCRIPTION OF THIS DISCLOSURE
[0046] The present disclosure relates to methods, microorganisms,
and compositions useful for processing lignocellulosic biomass. The
disclosure relates, in certain aspects, to microorganisms which are
able to convert lignocellulosic biomass such as, for example,
miscanthus grass, to soluble products that can be used by the same
or by another microorganism to produce an economically desirable
product such as, for example, a biofuel (e.g., an alcohol and/or
hydrogen gas (H2)), polymer, or commodity carbon-based chemical
like lactic acid.
[0047] The application of this technology has the potential to
render production of carbon-based chemicals and biofuels more
economically feasible and to allow a broader range of
microorganisms to utilize recalcitrant biomass. The use of
cellulosic materials as sources of bioenergy is currently limited
by typically requiring preprocessing of the cellulosic material.
Such preprocessing methods can be expensive. Thus, methods that
reduce dependence on preprocessing of cellulosic materials may have
a dramatic impact on the economics of the use of recalcitrant
biomass for biofuels production. One challenge in converting
biomass into fermentation products is the recalcitrance and
heterogeneity of the biological material.
[0048] The present inventors have found microorganisms of the genus
Caldicellulosiruptor which have a variety of advantageous
properties for their use in the conversion of lignocellulosic
biomass material to biofuel and/or carbon-based chemicals,
preferably to lactic acid, preferably in a single step process as
part of a consolidated bioprocessing (CBP) system.
[0049] In particular, these microorganisms are extremely
thermophilic and show a broad substrate specificities and high
natural production of ethanol and lactic acid. Moreover, ethanol
and lactic acid fermentation at high temperatures, for example over
70.degree. C. has many advantages over mesophilic fermentation. One
advantage of thermophilic fermentation is the minimization of the
problem of contamination in batch cultures, fed-batch cultures or
continuous cultures, since only a few microorganisms are able to
grow at such high temperatures in un-detoxified lignocellulose
biomass material.
[0050] It is also an advantage that the cells, strains and
microorganisms according to the present disclosure grow on
pre-treated as well as on untreated lignocellulosic biomass
material.
[0051] The isolated cells, strains, microorganisms, compositions
and microbial cultures are capable of growing and producing
fermentation products on very high dry-matter concentrations of
lignocellulosic biomass material.
[0052] In the present context the term "lignocellulosic biomass
material" is intended to designate a untreated lignocellulosic
biomass and/or a lignocellulosic biomass which has been subjected
to a pretreatment step whereby lignocellulosic material has been at
least partially separated into cellulose, hemicellulose and lignin
thereby having increased the surface area and/or accessibility of
the material. The lignocellulosic material may typically be derived
from plant material, such as straw, hay, perennial grass, garden
refuse, comminuted wood, fruit hulls and seed hulls.
[0053] The pretreatment method most often used is steam
pretreatment, a process comprising heating of the lignocellulosic
material by steam injection to a temperature of 130-230 degrees
centigrade with or without subsequent sudden release of pressure.
Prior to or during steam pretreatment, a catalyst like a mineral or
organic acid or a caustic agent facilitating disintegration of the
biomass structure can be added optionally. Catalysts often used for
such a pretreatment include but are not limited to sulphuric acid,
sulphurous acid, hydrochloric acid, acetic acid, lactic acid,
sodium hydroxide (caustic soda), potassium hydroxide, calcium
hydroxide (lime), ammonia or the respective salts or anhydrides of
any of these agents.
[0054] Such steam pretreatment step may or may not be preceded by
another treatment step including cooking of the biomass in water or
steaming of the biomass at temperatures of 100-200.degree. C. with
or without the addition of a suitable catalyst like a mineral or
organic acid or a caustic agent facilitating disintegration of the
biomass structure. In between the cooking step and the subsequent
steam pretreatment step one or more liquid-solid-separation and
washing steps can be introduced to remove solubilized biomass
components in order to reduce or prevent formation of inhibitors
during the subsequent steam pretreatment step. Inhibitors formed
during heat or steam pretreatment include but are not limited to
furfural formed from monomeric pentose sugars,
hydroxymethylfurfural formed from monomeric hexose sugars, acetic
acid, levulinic acid, phenols and phenol derivatives.
[0055] Another type of lignocellulose hydrolysis is acid
hydrolysis, where the lignocellulosic material is subjected to an
acid such as sulfuric acid or sulfurous acid whereby the sugar
polymers cellulose and hemicellulose are partly or completely
hydrolysed to their constituent sugar monomers. A third method is
wet oxidation wherein the material is treated with oxygen at
150-185 degrees centigrade. The pretreatments can be followed by
enzymatic hydrolysis to complete the release of sugar monomers.
This pre-treatment step results in the hydrolysis of cellulose into
glucose while hemicellulose is transformed into the pentoses xylose
and arabinose and the hexoses glucose, mannose and galactose. The
pretreatment step may in certain embodiments be supplemented with
treatment resulting in further hydrolysis of the cellulose and
hemicellulose. The purpose of such an additional hydrolysis
treatment is to hydrolyze oligosaccharide and possibly
polysaccharide species produced during the acid hydrolysis, wet
oxidation, or steam pretreatment of cellulose and/or hemicellulose
origin to form fermentable sugars (e.g. glucose, xylose and
possibly other monosaccharides). Such further treatments may be
either chemical or enzymatic. Chemical hydrolysis is typically
achieved by treatment with an acid, such as treatment with aqueous
sulphuric acid or hydrochloric acid, at a temperature in the range
of about 100-150 degrees centigrade. Enzymatic hydrolysis is
typically performed by treatment with one or more appropriate
carbohydrase enzymes such as cellulases, glucosidases and
hemicellulases including xylanases.
[0056] It has been found that the microorganisms according to the
present disclosure can grow efficiently on various types of
pretreated and untreated biomass (e.g. wood incl. poplar, spruce
and cotton wood; various types of grasses and grass residues incl.
miscanthus, wheat straw, sugarcane bagasse, corn stalks, corn cobs,
whole corn plants, sweet sorghum).
[0057] As used herein "efficient" growth refers to growth in which
cells may be cultivated to a specified density within a specified
time.
[0058] The microorganisms according to the present disclosure can
grow efficiently on crystalline cellulose and steam pretreated
perennial grasses and grow efficiently on xylan. The main products
when grown on untreated biomass substrates were lactate, for
example, when the microorganisms grown on cellobiose and or xylane
the lactate yield is high.
[0059] Cellobiose is a disaccharide derived from the condensation
of two glucose molecules linked in a .beta.(1.fwdarw.4) bond. It
can be hydrolyzed to give glucose. Cellobiose has eight free
alcohol (OH) groups, one ether linkage and two hemiacetal linkages,
which give rise to strong inter- and intra-molecular hydrogen
bonds. It is a type of dietary carbohydrate also found in
mushrooms.
[0060] Xylan is a generic term used to describe a wide variety of
highly complex polysaccharides that are found in plant cell walls
and some algae. Xylans are polysaccharides made from units of
xylose.
[0061] The microorganisms according to the present disclosure also
can grow efficiently on spent biomass--insoluble material that
remains after a culture has grown to late stationary phase (e.g.,
greater than 10.sup.8 cells/mL) on untreated biomass.
[0062] The microorganisms according to the present disclosure also
grew efficiently on cellobiose, untreated switchgrass, and
untreated poplar and poplar that had been heated at 98.degree. C.
for two minutes.
[0063] Furthermore, the microorganisms according to the present
disclosure grew efficiently on both the soluble and insoluble
materials obtained after heat-treating the biomass.
[0064] It was surprisingly found that the bacterial subspecies
according to the present disclosure is capable of growing in a
medium comprising a lignocellulosic biomass material having a
dry-matter content of at least 10 percent wt/wt, such as at least
15 percent wt/wt, including at least 20 percent wt/wt, and even as
high as at least 25 percent wt/wt.
[0065] The microorganisms according to the invention are anaerobic
thermophile bacteria, and they are capable of growing at high
temperatures even at or above 70 degrees centigrade The fact that
the strains are capable of operating at this high temperature is of
high importance in the conversion of the lignocellulosic material
into fermentation products. The conversion rate of carbohydrates
into e.g. lactic acid and/or ethanol is much faster when conducted
at high temperatures. For example, the volumetric ethanol
productivity of a thermophilic Bacillus is up to ten-fold higher
than a conventional yeast fermentation process which operates at 30
degrees centigrade Consequently, a smaller production plant is
required for a given plant capacity, thereby reducing plant
construction costs. As also mentioned previously, the high
temperature reduces the risk of contamination from other
microorganisms, resulting in less downtime, increased plant
productivity and a lower energy requirement for feedstock
sterilization. The high operation temperature may also facilitate
the subsequent recovery of the resulting fermentation products.
[0066] Lignocellulosic biomass material and lignocellulose
hydrolysates contain inhibitors such as furfural, phenols and
carboxylic acids, which can potentially inhibit the fermenting
organism. Therefore, it is an advantage of the microorganisms
according to the present disclosure that they are tolerant to these
inhibitors.
[0067] The microorganisms according to the present disclosure are
novel species of the genus Caldicellulosiruptor or novel subspecies
of Caldicellulosiruptor saccharolyticus.
[0068] For example, the genus Caldicellulosiruptor includes
different species of extremely thermophilic (growth at temperature
significantly above 70.degree. C.) cellulolytic and
hemicellulolytic strictly anaerobic nonsporeforming bacteria. The
first bacterium of this genus, Caldicellulosiruptor saccharolyticus
strain Tp8T (DSM 8903) has a temperature optimum of 70.degree. C.
and was isolated from a thermal spring in New Zealand (Rainey et
al. 1994; Sissons et al. 1987). It hydrolyses a variety of
polymeric carbohydrates with the production of acetate, lactate and
trace amounts of ethanol (Donnison et al. 1988). Phylogenetic
analysis showed that it constitutes a novel lineage within the
Bacillus/Clostridium subphylum of the Gram-positive bacteria
(Rainey et al. 1994).
[0069] According to the present disclosure, the microorganisms
produce ethanol and/or lactic acid and show several features that
distinguish them from currently used microorganisms: (i) high yield
and low product inhibition, (ii) simultaneous utilization of
lignocellolytic biomass material and/or sugars, and (iii) growth at
elevated temperatures. The microorganisms according to the present
disclosure are robust thermophile organisms with a decreased risk
of contamination. They efficiently convert an extraordinarily wide
range of biomass components to carbon-based chemicals like lactic
acid or ethanol.
[0070] As mentioned above, in one aspect, the present disclosure
relates to an isolated cell comprising a 16S rDNA sequence selected
from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO
3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, and a
combination of any thereof.
[0071] In one aspect, the present disclosure pertains to an
isolated Caldicellulosiruptor sp. cell having a 16S rDNA sequence
at least 99, at least 99.3, at least 99.5, at least, 99.7, at least
99.9, at least 99.99 percent identical to either sequence listed in
table 1 or a combination thereof.
[0072] Each independently an embodiment of the invention is an
isolated cell which is Caldicellulosiruptor sp. DIB004C (DSMZ
Accession number 25177), an isolated cell which is
Caldicellulosiruptor sp. DIB041 C (DSMZ Accession number 25771), an
isolated cell which is Caldicellulosiruptor sp. DIB087C (DSMZ
Accession number 25772), an isolated cell which is
Caldicellulosiruptor sp. DIB101C (DSMZ Accession number 25178), an
isolated cell which is Caldicellulosiruptor sp. DIB103C (DSMZ
Accession number 25773), an isolated cell which is
Caldicellulosiruptor sp. DIB104C (DSMZ Accession number 25774) or
an isolated cell which is Caldicellulosiruptor sp. DIB107C (DSMZ
Accession number 25775), cells derived from either, mutants or a
homolog of either.
[0073] As used herein "mutant" or "homolog" means a microorganism
derived from the cells or strains according to the present
disclosure, which are altered due to a mutation. A mutation is a
change produced in cellular DNA, which can be spontaneous, caused
by an environmental factor or errors in DNA replication, or induced
by physical or chemical conditions. The processes of mutation
included in this and indented subclasses are processes directed to
production of essentially random changes to the DNA of the
microorganism including incorporation of exogenous DNA. All mutants
of the microorganisms comprise the advantages of being extereme
thermophile (growing and fermenting at temperatures above
70.degree. C.) and are capable of fermenting lignocellulosic
biomass to ethanol and/or lactic acid. In an advantageous
embodiment, mutants of the microorganisms according to the present
disclosure have in a DNA-DNA hybridization assay, a DNA-DNA
relatedness of at least 80%, preferably at least 90%, at least 95%,
more preferred at least 98%, most preferred at least 99%, and most
preferred at least 99,9% with one of the isolated bacterial strains
Caldicellulosiruptor sp. DIB004C, DIB041C, DIB087C, DIB101C,
DIB103C, DIB104C and DIB107C.
[0074] The invention is based on the isolated bacterial strains
Caldicellulosiruptor sp. DIB004C, DIB041C, DIB087C, DIB101C,
DIB103C, DIB104C and DIB107C that contain 16S rDNA sequences at
least 99 to 100%, preferably 99.5 to 99.99, more preferably at
least 99.99 percent identical to the respective sequences listed in
table 1.
TABLE-US-00001 TABLE 1 DSMZ accession 16SrDNA Genus Species Name
number Deposition date SEQ ID NO. Caldicellulosiruptor sp. DIB004C
DSM 25177 Sep. 15, 2011 1 Caldicellulosiruptor sp. DIB041C DSM
25771 Mar. 15, 2012 2 Caldicellulosiruptor sp. DIB087C DSM 25772
Mar. 15, 2012 3 Caldicellulosiruptor sp. DIB101C DSM 25178 Sep. 15,
2011 4 Caldicellulosiruptor sp. DIB103C DSM 25773 Mar. 15, 2012 5
Caldicellulosiruptor sp. DIB104C DSM 25774 Mar. 15, 2012 6
Caldicellulosiruptor sp. DIB107C DSM 25775 Mar. 15, 2012 7
[0075] The strains listed in table 1 have been deposited in
accordance with the terms of the Budapest Treaty on Sep. 15, 2011
with DSMZ--Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH, Inhoffenstr. 7B, 38124 Braunschweig, Germany, under the
respectively indicated DSMZ accession numbers and deposition dates,
respectively, by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE).
[0076] The microorganisms of the species Caldicellulosiruptor sp.
according to the present disclosure in particular refer to a
microorganism which belongs to the genus Caldicellulosiruptor and
which preferably has one or more of the following characteristics:
[0077] a) it is a microorganism of the genus Caldicellulosiruptor;
[0078] b) in a DNA-DNA hybridization assay, it shows a DNA-DNA
relatedness of at least 70%, preferably at least 90%, at least 95%,
more preferred at least 98%, most preferred at least 99% with
either Caldicellulosiruptor sp. strain listed in table 1 with their
respective accession numbers; and/or [0079] c) it displays a level
of 16S rDNA gene sequence similarity of at least 98%, preferably at
least 99% or at least 99.5%, more preferably 100% with either
either Caldicellulosiruptor sp. strain listed in table 1 with their
respective accession numbers; and/or [0080] d) it is capable of
surviving in high temperature conditions above 75.degree. C. [0081]
e) it is capable of surviving in high temperature conditions above
70.degree. C., and or [0082] f) it is a Gram-positive
bacterium.
[0083] Preferably, at least two or at least three, and more
preferred all of the above defined criteria a) to f) are
fulfilled.
[0084] In an advantageous embodiment, the microorganisms according
to the present disclosure in particular refer to a microorganism
which belongs to the genus Caldicellulosiruptor and which
preferably has one or more of the following characteristics:
[0085] a) It is a microorganism of the genus
Caldicellulosiruptor
[0086] b) it is a microorganism of the species Caldicellulosiruptor
saccharolyticus;
[0087] c) in a DNA-DNA hybridization assay, it shows a DNA-DNA
relatedness of at least 80%, preferably at least 90%, at least 95%,
more preferred at least 98%, most preferred at least 99%, and most
preferred at least 99.9% with one of the strains of table 1;
and/or
[0088] d) it displays a level of 16S rDNA gene sequence similarity
of at least 98%, preferably at least 99%, at least 99.5% or at
least 99.7%, more preferably 99.99% with one of the strains listed
in table 1; and/or
[0089] e) it is capable of surviving and/or growing and/or
producing a fermentation product selected from the group consisting
of acids and alcohols at temperature conditions above 70.degree.
C., in particular of above 72.degree. C.
[0090] Preferably, at least two or at least three, and more
preferred all of the above defined criteria a) to e) are
fulfilled.
[0091] The term "DNA-DNA relatedness" in particularly refers to the
percentage similarity of the genomic or entire DNA of two
microorganisms as measured by the DNA-DNA
hybridization/renaturation assay according to De Ley et al. (1970)
Eur. J. Biochem. 12, 133-142 or Hurl et al. (1983) Syst. Appl.
Microbiol. 4, 184-192. In particular, the DNA-DNA hybridization
assay preferably is performed by the DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)
Identification Service.
[0092] The term "16S rDNA gene sequence similarity" in particular
refers to the percentage of identical nucleotides between a region
of the nucleic acid sequence of the 16S ribosomal RNA (rDNA) gene
of a first microorganism and the corresponding region of the
nucleic acid sequence of the 16S rDNA gene of a second
microorganism. Preferably, the region comprises at least 100
consecutive nucleotides, more preferably at least 200 consecutive
nucleotides, at least 300 consecutive nucleotides or at least 400
consecutive nucleotides, most preferably about 480 consecutive
nucleotides.
[0093] The strains according to disclosure have the potential to be
capable of producing a number of different fermentation products,
including acids, alcohols, ketones and hydrogen. In one embodiment,
the alcohol is selected from ethanol, butanol, propanol, methanol,
propanediol and butanediol. In a further embodiment the acid is
lactic acid, propionic acid, acetic acid, succinic acid, butyric
acid or formic acid and the ketone is acetone.
[0094] The Caldicellulosiruptor sp. strains according to the
present disclosure have several highly advantageous characteristics
needed for the conversion of lignocellulosic biomass material.
Thus, these base strains possess all the genetic machinery for the
hydrolysis of cellulose and hemicelluloses and for the conversion
of both pentose and hexose sugars to various fermentation products
such as lactic acid and ethanol. As will be apparent from the below
examples, the examination of the complete 16S rDNA sequence showed
that the closely related strains may all be related to
Caldicellulosiruptor saccharolyticus although the 16S rDNA
sequences may place them in a separate subspecies or even a
different species
[0095] Furthermore, the Caldicellulosiruptor sp. strains according
to the present disclosure are cellulolytic and xylanolytic.
[0096] In a preferred embodiment, the Caldicellulosiruptor sp.
microorganism is
[0097] a) Caldicellulosiruptor sp. DIB004C, deposited on Sep. 15,
2011 under the accession number DSM 25177 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0098] b) a microorganism derived from Caldicellulosiruptor sp.
DIB004C or
[0099] c) a Caldicellulosiruptor sp. DIB004C mutant.
[0100] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0101] a) Caldicellulosiruptor sp. DIB041C, deposited on Mar. 15,
2012 under the accession number DSM 25771 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0102] b) a microorganism derived from Caldicellulosiruptor sp.
DIB041C or
[0103] c) a Caldicellulosiruptor sp. DIB041C mutant.
[0104] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0105] a) Caldicellulosiruptor sp. DIB087C, deposited on Mar. 15,
2012 under the accession number DSM 25772 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0106] b) a microorganism derived from Caldicellulosiruptor sp.
DIB087C or
[0107] c) a Caldicellulosiruptor sp. DIB087C mutant.
[0108] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0109] a) Caldicellulosiruptor sp. DIB101C, deposited on Sep. 15,
2011 under the accession number DSM 25178 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0110] b) a microorganism derived from Caldicellulosiruptor sp.
DIB101C or
[0111] c) a Caldicellulosiruptor sp. DIB101C mutant.
[0112] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0113] a) Caldicellulosiruptor sp. DIB103C, deposited on Mar. 15,
2012 under the accession number DSM 25773 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0114] b) a microorganism derived from Caldicellulosiruptor sp.
DIB103C or
[0115] c) a Caldicellulosiruptor sp. DIB103C mutant.
[0116] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0117] a) Caldicellulosiruptor sp. DIB104C, deposited on Mar. 15,
2012 under the accession number DSM 25774 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0118] b) a microorganism derived from Caldicellulosiruptor sp.
DIB104C or
[0119] c) a Caldicellulosiruptor sp. DIB104C mutant.
[0120] In another preferred embodiment, the Caldicellulosiruptor
sp. microorganism is
[0121] a) Caldicellulosiruptor sp. DIB107C, deposited on Mar. 15,
2012 under the accession number DSM 25775 according to the
requirements of the Budapest Treaty at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstra.beta.e 7B,
38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH,
Nattermannallee 1, 50829 Cologne (DE),
[0122] b) a microorganism derived from Caldicellulosiruptor sp.
DIB107C or
[0123] c) a Caldicellulosiruptor sp. DIB107C mutant.
[0124] All strains listed above and in table 1 belong to the genus
Caldicellulosiruptor and are strictly anaerobic, non-sporeforming,
non-motile, gram-positive bacteria. Cells are straight rods 0.4-0.5
.mu.m by 2.0-4.0 .mu.m, occuring both singly and in pairs. After 7
days incubation at 72.degree. C. on solid medium with agar and
cellulose as substrate both strains form circular milky colonies of
0.5-1 mm in diameter. Clearing zones around the colonies are
produced indicating cellulose degradation.
[0125] The term "a microorganism" as used herein may refer to only
one unicellular organism as well as to numerous single unicellular
organisms. For example, the term "a microorganism of the genus
Caldicellulosiruptor" may refer to one single Caldicellulosiruptor
bacterial cell of the genus Caldicellulosiruptor as well as to
multiple bacterial cells of the genus Caldicellulosiruptor.
[0126] The terms "a strain of the genus Caldicellulosiruptor" and
"a Caldicellulosiruptor cell" are used synonymously herein. In
general, the term "a microorganism" refers to numerous cells. In
particular, said term refers to at least 10.sup.3 cells, preferably
at least 10.sup.4 cells, at least 10.sup.5 or at least 10.sup.6
cells.
[0127] As mentioned above lignocellolytic biomass according to the
present disclosure can be but is not limited to grass, switch
grass, cord grass, rye grass, reed canary grass, mixed prairie
grass, miscanthus, Napier grass, sugar-methoding residues,
sugarcane bagasse, sugarcane straw, agricultural wastes, rice
straw, rice hulls, barley straw, corn cobs, cereal straw, wheat
straw, canola straw, oat straw, oat hulls, corn fiber, stover,
soybean stover, corn stover, forestry wastes, recycled wood pulp
fiber, paper sludge, sawdust, hardwood, softwood, pressmud from
sugar beet, cotton stalk, banana leaves, oil palm residues and
lignocellulosic biomass material obtained through processing of
food plants. In advantageous embodiments, the lignocellulosic
biomass material is hardwood and/or softwood, preferably poplar
wood. In advantageous embodiments, the lignocellulosic biomass
material is a grass or perennial grass, preferably miscanthus.
[0128] In advantageous embodiments, the lignocellulosic biomass
material is subjected to mechanical, thermochemical, and/or
biochemical pretreatment. The lignocellulosic biomass material
could be exposed to steam treatment. In further embodiments, the
lignocellulosic biomass material is pretreated with mechanical
comminution and a subsequent treatment with lactic acid, acetic
acid, sulfuric acid or sulfurous acid or their respective salts or
anhydrides under heat and pressure with or without a sudden release
of pressure. In another embodiment, the lignocellulosic biomass
material is pretreated with mechanical comminution and a subsequent
treatment with either sodium hydroxide, ammonium hydroxide, calcium
hydroxide or potassium hydroxide under heat and pressure with or
without a sudden release of pressure.
[0129] In advantageous embodiments, the lignocellulosic biomass
material is pretreated with mechanical comminution and subsequent
exposure to a multi-step combined pretreatment process. Such
multi-step combined pretreatment may include a treatment step
consisting of cooking in water or steaming of the lignocellulosic
biomass material at a temperature of 100-200.degree. C. for a
period of time in between 5 and 120 min. Suitable catalysts
including but not limited to lactic acid, acetic acid, sulfuric
acid, sulfurous acid, sodium hydroxide, ammonium hydroxide, calcium
hydroxide or potassium hydroxide or their respective salts or
anhydrides may or may not be added to the process. The process may
further include a step comprising a liquid-solid separation
operation, e.g. filtration, separation, centrifugation or a
combination thereof, separating the process fluid containing
partially or fully hydrolyzed and solubilized constituents of the
lignocellulosic biomass material from the remaining insoluble parts
of the lignocellulosic biomass. The process may further include a
step comprising washing of the remaining lignocellulosic biomass
material. The solid material separated from solubilized biomass
constituents may then be treated in a second step with steam under
heat and pressure with or without a sudden release of pressure at a
temperature of 150-250.degree. C. for a period of time in between 1
and 15 min. In order to increase pretreatement effectiveness, a
suitable catalyst including but not limited to lactic acid, acetic
acid, sulfuric acid, sulfurous acid, sodium hydroxide, ammonium
hydroxide, calcium hydroxide or potassium hydroxide or their
respective salts or anhydrides may be added also to the second
step.
[0130] In advantageous embodiments, the lignocellulosic biomass is
milled before converted into biofuels like ethanol and/or
carbon-based chemicals like lactic acid. In one embodiment, the
lignocellulosic biomass is pretreated biomass from Populus sp,
preferably pretreated with steam pretreatment or multi-step
combined pretreatment. In another embodiment, the lignocellulosic
biomass is pretreated biomass from any perennial grass, e.g.
Miscanthus sp., preferably treated with steam pretreatment or
multi-step combined pretreatment.
[0131] In advantageous embodiments the cells, strains,
microorganisms may be modified in order to obtain mutants or
derivatives with improved characteristics. Thus, in one embodiment
there is provided a bacterial strain according to the disclosure,
wherein one or more genes have been inserted, deleted or
substantially inactivated. The variant or mutant is typically
capable of growing in a medium comprising a lignocellulosic biomass
material.
[0132] In another embodiment, there is provided a process for
preparing variants or mutants of the microorganisms according to
the present disclosure, wherein one or more genes are inserted,
deleted or substantially inactivated as described herein.
[0133] In some embodiments one or more additional genes are
inserting into the strains according to the present disclosure.
Thus, in order to improve the yield of the specific fermentation
product, it may be beneficial to insert one or more genes encoding
a polysaccharase into the strain according to the invention. Hence,
in specific embodiments there is provided a strain and a process
according to the invention wherein one or more genes encoding a
polysaccharase which is selected from cellulases (such as EC
3.2.1.4); beta-glucanases, including glucan-1,3 beta-glucosidases
(exo-1,3 beta-glucanases, such as EC 3.2.1.58),
1,4-beta-cellobiohydrolases (such as EC 3.2.1.91) and
endo-1,3(4)-beta-glucanases (such as EC 3.2.1.6); xylanases,
including endo-1,4-beta-xylanases (such as EC 3.2.1.8) and xylan
1,4-beta-xylosidases (such as EC 3.2.1.37); pectinases (such as EC
3.2.1.15); alpha-glucuronidases, alpha-L-arabinofuranosidases (such
as EC 3.2.1.55), acetylesterases (such as EC 3.1.1.-),
acetylxylanesterases (such as EC 3.1.1.72), alpha-amylases (such as
EC 3.2.1.1), beta-amylases (such as EC 3.2.1.2), glucoamylases
(such as EC 3.2.1.3), pullulanases (such as EC 3.2.1.41),
beta-glucanases (such as EC 3.2.1.73), hemicellulases,
arabinosidases, mannanases including mannan
endo-1,4-beta-mannosidases (such as EC 3.2.1.78) and mannan
endo-1,6-alpha-mannosidases (such as EC 3.2.1.101), pectin
hydrolases, polygalacturonases (such as EC 3.2.1.15),
exopolygalacturonases (such as EC 3.2.1.67) and pectate lyases
(such as EC 4.2.2.10), are inserted.
[0134] In accordance with the present disclosure, a method of
producing a fermentation product comprising culturing a strain
according to the invention under suitable conditions is also
provided.
[0135] The strains according to the disclosure are strictly
anaerobic microorganisms, and hence it is preferred that the
fermentation product is produced by a fermentation process
performed under strictly anaerobic conditions. Additionally, the
strain according to invention is an extremely thermophillic
microorganism, and therefore the process may perform optimally,
when it is operated at temperature in the range of about 40-95
degrees centigrade, such as the range of about 50-90 degrees
centigrade, including the range of about 60-85 degrees centigrade,
such as the range of about 65-75 degrees centigrade
[0136] For the production of certain fermentation products, it may
be useful to select a specific fermentation process, such as batch
fermentation process, including a fed-batch process or a continuous
fermentation process. Also, it may be useful to select a
fermentation reactor such as a stirred vessel reactor, an
immobilized cell reactor, a fluidized bed reactor or a membrane
bioreactor.
[0137] In accordance with the invention, the method is useful for
the production of a wide range of fermentation products including
acids, alcohols, ketones and hydrogen. Thus fermentation products
such as ethanol, butanol, propanol, methanol, propanediol,
butanediol, lactic acid, propionic acid, acetic acid, succinic
acid, butyric acid, formic acid and acetone may be produced in
accordance with the disclosure.
[0138] The expression "comprise", as used herein, besides its
literal meaning also includes and specifically refers to the
expressions "consist essentially of" and "consist of". Thus, the
expression "comprise" refers to embodiments wherein the
subject-matter which "comprises" specifically listed elements does
not comprise further elements as well as embodiments wherein the
subject-matter which "comprises" specifically listed elements may
and/or indeed does encompass further elements. Likewise, the
expression "have" is to be understood as the expression "comprise",
also including and specifically referring to the expressions
"consist essentially of" and "consist of".
[0139] The following methods and examples are offered for
illustrative purposes only, and are not intended to limit the scope
of the present disclosure in any way.
METHODS AND EXAMPLES
[0140] In the following examples, materials and methods of the
present disclosure are provided including the determination of the
properties of the microbial strains according to the present
disclosure. It should be understood that these examples are for
illustrative purpose only and are not to be construed as limiting
this disclosure in any manner. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference in their entirety for all purposes.
Example 1
Isolation and Cultivation
[0141] All procedures for enrichment and isolation of the strains
listed in table 1 employed anaerobic technique for strictly
anaerobic bacteria (Hungate 1969). The strains were enriched from
environmental samples at temperatures higher than 70.degree. C.
with crystalline cellulose and beech wood as substrate. Isolation
was performed by picking colonies grown on solid agar medium at
72.degree. C. in Hungate roll tubes (Hungate 1969).
[0142] The cells are cultured under strictly anaerobic conditions
applying the following medium:
TABLE-US-00002 Basic medium NH4Cl 1.0 g NaCl 0.5 g MgSO4 .times.
7H2O 0.3 g CaCl2 .times. 2H2O 0.05 g NaHCO3 0.5 g K2HPO4 1.5 g
KH2PO4 3.0 g Yeast extract (bacto, BD) 0.5 g Cellobiose 5.0 g
Vitamins (see below) 1.0 ml Trace elements (see 0.5 ml below)
Resazurin 1.0 mg Na2S .times. 9H2O 0.75 g Distilled water 1000.0 ml
Trace elements stock solution NiCl.sub.2 .times. 6H.sub.2O 2 g
FeSO.sub.4 .times. 7H.sub.2O 1 g NH.sub.4Fe(III) citrate, brown, 10
g 21.5% Fe MnSO.sub.4 .times. H.sub.2O 5 g CoCl.sub.2 .times.
6H.sub.2O 1 g ZnSO.sub.4 .times. 7H.sub.2O 1 g CuSO.sub.4 .times.
5H.sub.2O 0.1 g H.sub.3BO.sub.3 0.1 g Na.sub.2MoO.sub.4 .times.
2H.sub.2O 0.1 g Na.sub.2SeO.sub.3 .times. 5H.sub.2O 0.2 g
Na.sub.2WoO.sub.4 .times. 2H.sub.2O 0.1 g Distilled water 1000.0 ml
Add 0.5 ml of the trace elements stock solution to 1 liter of the
medium Vitamine stock solution nicotinic acid 200 mg cyanocobalamin
25 mg p-aminobenzoic acid (4- 25 mg aminobenzoic acid) calcium
D-pantothenate 25 mg thiamine-HCl 25 mg riboflavin 25 mg lipoic
acid 25 mg folic acid 10 mg biotin 10 mg pyridoxin-HCl 10 mg
Distilled water 200.0 ml Add 1 ml of the vitamine stock solution to
1 liter of the medium
[0143] All ingredients except sulfide are dissolved in deionized
water and the medium is flushed with nitrogen gas (purity 99.999%)
for 20 min at room temperature. After addition of sulfide, the
pH-value is adjusted to 7.0 at room temperature with 1 M HCl. The
medium is then dispensed into Hungate tubes or serum flasks under
nitrogen atmosphere and the vessels are tightly sealed. After
autoclaving at 121.degree. C. for 20 min pH-value should be in
between 6.8 and 7.0.
[0144] Carbon sources as specified for individual experiments are
added prior to autoclaving. All applied substrate concentrations
are indicated as glucose equivalents on the basis of available mol
C (carbon).
[0145] Subsequent to autoclaving, cultures are inoculated by
injection of a seed culture through the seal septum and inoculated
in an incubator at 72.degree. C. for the time indicated.
Example 2
HPLC
[0146] Sugars and fermentation products were quantified by HPLC-RI
using a Via Hitachi LaChrom Elite (Hitachi corp.) fitted with an
Rezex ROA Organic Acid H+ (Phenomenex). The analytes were separated
isocratically with 2.5 mM H.sub.2SO.sub.4 and at 65.degree. C.
Example 3
Phylogenetic Analysis of 16S rDNA Genes
[0147] Genomic DNA was isolated from cultures grown as described
above and 16SrDNA amplified by PCR using 27F (AGAGTTTGATCMTGGCTCAG;
SEQ ID No. 8) as forward and 1492R (GGTTACCTTGTTACGACTT; SEQ ID No.
9) as reverse primer. The resulting products were sequenced and the
sequences analyzed using the Sequencher 4.10.1 software (Gene Codes
Corporation). The NCBI database was used for BLAST procedures.
[0148] Sequencing of 16S rDNA from all strains listed in table 1
revealed all these had (at least) one copy of a 16S rDNA operon
which was most closely related to Caldicellulosiruptor
saccharolyticus (Strain Tp8T=DSM8903) in the available public
databases. Alignment was carried out using ClustalW (Chenna et al.
2003) and the phylogenetic tree was constructed using software
MEGA4 (Kumar et al. 2001). The tree for all strains listed in table
1 is displayed in FIG. 1.
[0149] The 16S rDNA sequences of all strains listed in table 1 have
99% percent identity to the respective sequence of e.g.
Caldicellulosiruptor saccharolyticus (Strain Tp8T=DSM8903).
Example 4
Batch Experiments
[0150] Batch experiments with all strains were executed by
cultivation on the medium described above with the carbon source
substrates listed in FIGS. 10 and 11. Sealed Hungate tubes or serum
flaks were used for cultivation in a standard incubator at a
temperature of 72.degree. C.
[0151] The results clearly show that all strains are capable to
produce ethanol and lactic acid on soluble sugars, on soluble and
insoluble sugar polymers as well as on the pretreated
lignocellulose in the absence of free sugars.
[0152] Physiological comparison with the strain DSM8903 identified
as the most closely related to the 16S rDNA comparison indicates a
significantly higher ethanol and lactate formation in combination
with a partially decreased production of acetate on polymeric
substrates.
Example 5
Fermentation
[0153] Batch experiments with all strains, e.g. DIB004C, were
performed by cultivation on the medium described above with
addition of 20 g/L miscanthus grass pretreated with a suitable
method selected from those described above comprising heating in
the presence of dilute acid followed by sudden release of
pressure.
[0154] Temperature is controlled to 72.degree. C. and the pH-value
is controlled to 6.75.+-.0.1 throughout the fermentation. The
fermenter is purged with nitrogen to remove excess oxygen before
sodium sulphide is added as described above.
[0155] The fermentation is started by addition of a seed culture
prepared as described in example 1.
[0156] The results of the HPLC analysis as described in example 2
show parallel production of ethanol, lactic acid and acetic
acid.
[0157] The results of the product formation during a fermentation
of Caldicellulosiruptor sp. DIB004C on pretreated miscanthus grass
is shown in FIG. 9.
LIST OF ADDITIONAL REFRENCES
[0158] Rainey F A, Donnison A M, Janssen P H, Saul D, Rodrigo A,
Bergquist P L, Daniel R M, Stackebrandt E, Morgan H W. (1994)
Description of Caldicellulosiruptor saccharolyticus gen. nov., sp.
nov: an obligately anaerobic, extremely thermophilic, cellulolytic
bacterium. FEMS Microbiol Lett. 120:263-266.
[0159] Sissons C H, Sharrock K R, Daniel R M, Morgan H W. (1987)
Isolation of cellulolytic anaerobic extreme thermophiles from New
Zealand thermal sites. Appl Environ Microbiol. 53:832-838.
[0160] Donnison A M, Brockelsby C M, Morgan H W, Daniel R M. (1989)
The degradation of lignocellulosics by extremely thermophilic
microorganisms. Biotechnol Bioeng. 33:1495-1499.
[0161] Hungate R E. (1969) A roll tube method for cultivation of
strict anaerobes. In: Methods in Microbiology Eds. Norris J R and
Ribbons D W. pp 118-132. New York: Academic Press.
[0162] Chenna R, Sugawara H, Koike T, Lopez R, Gibson T J, Higgins
D G, Thompson J D. (2003) Multiple sequence alignment with the
Clustal series of programs. Nucleic Acids Res. 13:3497-3500.
[0163] Kumar S, Tamura K, Jakobsen I B, Nei M. (2001) MEGA2:
molecular evolutionary genetics analysis software. Bioinformatics.
17:1244-1245.
Sequence CWU 1
1
911396DNACaldicellulosiruptorsource1..1396/organism="Caldicellulosiruptor"
/note="16SrDNA" /mol_type="unassigned DNA" 1ttacgacttc accccaatca
tcagccccac cttcaacaca gcttaacctg tgtcttcagg 60tgttgctgac tctcatggtg
tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg 120cggcatgctg
atccgcgatt actagcgatt ccgacttcat gcaggcgagt tgcagcctgc
180aatccgaact gggggtgctt ttttgggatt cgctccggct cgcgccttcg
cacgccctct 240gtagcaccca ttgtagcacg tgtgtagccc agggcataag
gggcatgatg atttgacgtc 300atccccacct tcctccgcct catcgacggc
agtcccctta gagtgcccac cattacgcgc 360tggcaactaa gggcaggggt
tgcgctcgtt gcgggactta acccaacatc tcacgacacg 420agctgacgac
aaccatgcac cacctgtgtc cgggctcctg ctctcatcga acaggcaccc
480caccctttcg ggcaggtccc cggcatgtca agccctggta aggttcttcg
cgttgcttcg 540aattaaacca catgctccac cgcttgtgcg ggcccccgtc
aattcctttg agtttcaacc 600ttgcggccgt actccccagg cgggatgctt
attgtgttaa ctacggcacg gaggagtcct 660tctcccccac acctagcatc
catcgtttac agcgtggact accagggtat ctaatcctgt 720tcgctcccca
cgctttcgtg cctcagcgtc agttacggtc cagacggccg ccttcgccac
780tggtgttcct cccgatatct acgcatttca ccgctacacc gggaattccg
ccgtcctctc 840ccgcactcaa gctatgcagt attaagcgca atccttaggt
tgagcctaag gctttcacgc 900ttaactcgca tagccgccta cgcacccttt
acgcccagta attccggaca acgctcgcca 960cctacgtatt accgcggctg
ctggcacgta gttagccgtg gctttttaaa cgggtactat 1020ctcctacttc
tccccgtcca aagaggttta caccccgaag ggcttcttcc ctcacgcggc
1080gtcgctgcgt caggcttccg cccattgcgc aagattcccc gctgctgcct
cccgtaggag 1140tgtgggccgt gtctcagtcc cactgtggcc gtacaccctc
tcaggccggc tacccgtcgt 1200cgccttggta ggccgttacc ccaccaacta
gctgatgggc cgcgagccca tccccagcca 1260gtatagcctc cccggctacc
ctttcaccac atcaccatgc gatgacgtgg tcccatcggg 1320tattagcagc
cctttcgagc tgttatcccc gtgctggggg taggttgctc acgtgttact
1380cacccgtccg ccgcta
139621480DNACaldicellulosiruptorsource1..1480/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 2ctcaggacga
acgctggcgg cgtgcctaac gcatgcaagt cgagcggagg tagccatgaa 60ggtgaagagc
tggagtggct atcttagcgg cggacgggtg agtaacacgt gagcaaccta
120ccctcagcac ggggataaca gctcgaaagg gctgctaata cccgatggga
ccacggcatc 180gcatgatgtt gtggtgaaag ggtagccgtg gaggctatac
cggctgggga tgggctcgcg 240gcccatcagc tagttggtgg ggtaacggcc
taccaaggct acgacgggta gccggcctga 300gagggtggtc ggccacagtg
ggactgagac acggcccaca ctcctacggg aggcagcagc 360ggggaatctt
gcgcaatggg cgaaagcctg acgcagcgac gccgcgtgag ggaggaagcc
420cttcggggtg taaacctctt tggacgggga gaaggaggag atagtacccg
tttaaaaagc 480cacggctaac tacgtgccag cagccgcggt aatacgtagg
tggcgagcgt tgtccggaat 540tactgggcgt aaagggtgcg taggcggcta
tgcaagttaa gcgtgaaatc ttggggctca 600accccaaggc tgcgcttaat
actgcatagc ttgagtgcgg gagaggacgg cggaattccc 660ggtgtagcgg
tgaaatgcgt agatatcggg aggaacacca gtggcgaagg cggccgtctg
720gaccgtaact gacgctgagg cacgaaagcg tggggagcga acaggattag
ataccctggt 780agtccacgct gtaaacgatg gatgctaggt gtgggggaga
aggactcctc cgtgccgtag 840ttaacacaat aagcatcccg cctggggagt
acggccgcaa ggttgaaact caaaggaatt 900gacgggggcc cgcacaagcg
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 960taccagggct
tgacatgccg ggaacctgcc cgaaagggtg gggtgcctgc gcgatgagtg
1020caggagcccg gacacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga
gatgttgggt 1080taagtcccgc aacgagcgca acccctgccc ttagttgcca
gcacgtaatg gtgggcactc 1140taaggggact gccgccgatg aggcggagga
aggtggggat gacgtcaaat catcatgccc 1200cttatgccct gggctacaca
cgtgctacaa tgggtgctac agagggttgc gaaggcgcga 1260gccggagcta
atcccaaaaa agcaccccca gttcggattg caggctgcaa ctcgcctgca
1320tgaagtcgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg
ttcccgggcc 1380ttgtacacac cgcccgtcac accatgagag tcagcaacac
ctgaagacac agggcagctg 1440tgttgaaggt ggggctgatg attggggtga
agtcgtaaca
148031481DNACaldicellulosiruptorsource1..1481/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 3tcaggacgaa
cgctggcggc gtgcctaacg catgcaagtc gagcggagat ggtggttgaa 60ggtgatgagc
tggaggctgc catcttagcg gcggacgggt gagtaacacg tgagcaacct
120acccccagca cggggataac agctcgaaag ggctgctaat acccgatggg
accacgtcat 180cgcatggtga tgtggtgaaa gggtagccgg ggaggctata
ctggctgggg atgggctcgc 240ggcccatcag ctagttggtg gggtaacggc
tcaccaaggc gacgacgggt agccggcctg 300agagggtgta cggccacagt
gggactgaga cacggcccac actcctacgg gaggcagcag 360cggggaatct
tgcgcaatgg gcggaagcct gacgcagcga cgccgcgtga gggaagaagc
420ccttcggggt gtaaacctct ttggacgggg agaagtagga gatagtaccc
gtttaaaaag 480ccacggctaa ctacgtgcca gcagccgcgg taatacgtag
gtggcgagcg ttgtccggaa 540ttactgggcg taaagggtgc gtaggcggct
atgcgagtta agcgtgaaag ccttaggctc 600aacctaagga ttgcgcttaa
tactgcatag cttgagtgcg ggagaggacg gcggaattcc 660cggtgtagcg
gtgaaatgcg tagatatcgg gaggaacacc agtggcgaag gcggccgtct
720ggaccgtaac tgacgctgag gcacgaaagc gtggggagcg aacaggatta
gataccctgg 780tagtccacgc tgtaaacgat ggatgctagg tgtgggggag
aaggactctt ccgtgccgta 840gttaacacaa taagcatccc gcctggggag
tacggccgca aggttgaaac tcaaaggaat 900tgacgggggc ccgcacaagc
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 960ttaccagggc
ttgacatgcc ggggacctgc ccgaaagggt ggggtgcctg ttcgatgaga
1020gcaggaaccc ggacacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg
agatgttggg 1080ttaagtcccg caacgagcgc aacccctgcc cttagttgcc
agcgggtaat ggtgggcact 1140ctaaggggac tgccgtcgat gaggcggagg
aaggtgggga tgacgtcaaa tcatcatgcc 1200ccttatgccc tgggctacac
acgtgctaca atgggtgcta cagagggcgt gcgaaggcgc 1260gagccggagc
gaatcccaaa aaagcacccc cagttcggat tgcaggctgc aactcgcctg
1320catgaagtcg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata
cgttcccggg 1380ccttgtacac accgcccgtc acaccatgag agtcagcaac
acctgaagac acaggttaag 1440ctgtgttgaa ggtggggctg atgattgggg
tgaagtcgta a
148141212DNACaldicellulosiruptorsource1..1212/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 4cctgtgtctt
caggtgttgc tgactctcat ggtgtgacgg gcggtgtgta caaggcccgg 60gaacgtattc
accgcggcat gctgatccgc gattactagc gattccgact tcatgcaggc
120gagttgcagc ctgcaatccg aactgggggt gcttttttgg gattcgctcc
ggctcgcgcc 180ttcgcacgcc ctctgtagca cccattgtag cacgtgtgta
gcccagggca taaggggcat 240gatgatttga cgtcatcccc accttcctcc
gcctcatcga cggcagtccc cttagagtgc 300ccaccattac gcgctggcaa
ctaagggcag gggttgcgct cgttgcggga cttaacccaa 360catctcacga
cacgagctga cgacaaccat gcaccacctg tgtccgggct cctgctctca
420tcgaacaggc accccaccct ttcgggcagg tccccggcat gtcaagccct
ggtaaggttc 480ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg
tgcgggcccc cgtcaattcc 540tttgagtttc aaccttgcgg ccgtactccc
caggcgggat gcttattgtg ttaactacgg 600cacggaggag tccttctccc
ccacacctag catccatcgt ttacagcgtg gactaccagg 660gtatctaatc
ctgttcgctc cccacgcttt cgtgcctcag cgtcagttac ggtccagacg
720gccgccttcg ccactggtgt tcctcccgat atctacgcat ttcaccgcta
caccgggaat 780tccgccgtcc tctcccgcac tcaagctatg cagtattaag
cgcaatcctt aggttgagcc 840taaggctttc acgcttaact cgcatagccg
cctacgcacc ctttacgccc agtaattccg 900gacaacgctc gccacctacg
tattaccgcg gctgctggca cgtagttagc cgtggctttt 960taaacgggta
ctatctccta cttctccccg tccaaagagg tttacacccc gaagggcttc
1020ttccctcacg cggcgtcgct gcgtcaggct tccgcccatt gcgcaagatt
ccccgctgct 1080gcctcccgta ggagtgtggg ccgtgtctca gtcccactgt
ggccgtacac cctctcaggc 1140cggctacccg tcgtcgcctt ggtaggccgt
taccccacca actagctgat gggccgcgag 1200cccatcccca gc
121251253DNACaldicellulosiruptorsource1..1253/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 5cgacttcacc
ccaatcatca gccccacctt caacacagct taacctgtgt cttcaggtgt 60tgctgactct
catggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg
120catgctgatc cgcgattact agcgattccg acttcatgca ggcgagttgc
agcctgcaat 180ccgaactggg ggtgcttttt tgggattcgc tccggctcgc
gccttcgcac gccctctgta 240gcacccattg tagcacgtgt gtagcccagg
gcataagggg catgatgatt tgacgtcatc 300cccaccttcc tccgcctcat
cgacggcagt ccccttagag tgcccaccat tacgcgctgg 360caactaaggg
caggggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc
420tgacgacaac catgcaccac ctgtgtccgg gctcctgctc tcatcgaaca
ggcaccccac 480cctttcgggc aggtccccgg catgtcaagc cctggtaagg
ttcttcgcgt tgcttcgaat 540taaaccacat gctccaccgc ttgtgcgggc
ccccgtcaat tcctttgagt ttcaaccttg 600cggccgtact ccccaggcgg
gatgcttatt gtgttaacta cggcacggag gagtccttct 660cccccacacc
tagcatccat cgtttacagc gtggactacc agggtatcta atcctgttcg
720ctccccacgc tttcgtgcct cagcgtcagt tacggtccag acggccgcct
tcgccactgg 780tgttcctccc gatatctacg catttcaccg ctacaccggg
aattccgccg tcctctcccg 840cactcaagct atgcagtatt aagcgcaatc
cttaggttga gcctaaggct ttcacgctta 900actcgcatag ccgcctacgc
accctttacg cccagtaatt ccggacaacg ctcgccacct 960acgtattacc
gcggctgctg gcacgtagtt agccgtggct ttttaaacgg gtactatctc
1020ctacttctcc ccgtccaaag aggtttacac cccgaagggc ttcttccctc
acgcggcgtc 1080gctgcgtcag gcttccgccc attgcgcaag attccccgct
gctgcctccc gtaggagtgt 1140gggccgtgtc tcagtcccac tgtggccgta
caccctctca ggccggctac ccgtcgtcgc 1200cttggtaagc cgttacccca
ccaactagct gatgggccgc gagcccatcc cca
125361255DNACaldicellulosiruptorsource1..1255/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 6gacttcaccc
caatcatcag ccccaccttc aacacagctt aacctgtgtc ttcaggtgtt 60gctgactctc
atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc
120atgctgatcc gcgattacta gcgattccga cttcatgcag gcgagttgca
gcctgcaatc 180cgaactgggg gtgctttttt gggattcgct ccggctcgcg
ccttcgcacg ccctctgtag 240cacccattgt agcacgtgtg tagcccaggg
cataaggggc atgatgattt gacgtcatcc 300ccaccttcct ccgcctcatc
gacggcagtc cccttagagt gcccaccatt acgcgctggc 360aactaagggc
aggggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct
420gacgacaacc atgcaccacc tgtgtccggg ctcctgctct catcgaacag
gcaccccacc 480ctttcgggca ggtccccggc atgtcaagcc ctggtaaggt
tcttcgcgtt gcttcgaatt 540aaaccacatg ctccaccgct tgtgcgggcc
cccgtcaatt cctttgagtt tcaaccttgc 600ggccgtactc cccaggcggg
atgcttattg tgttaactac ggcacggaag agtccttctc 660ccccacacct
agcatccatc gtttacagcg tggactacca gggtatctaa tcctgttcgc
720tccccacgct ttcgtgcctc agcgtcagtt acggtccaga cggccgcctt
cgccactggt 780gttcctcccg atatctacgc atttcaccgc tacaccggga
attccgccgt cctctcccgc 840actcaagcta tgcagtatta agcgcaatcc
ttaggttgag cctaaggctt tcacgcttaa 900ctcgcatagc cgcctacgca
ccctttacgc ccagtaattc cggacaacgc tcgccaccta 960cgtattaccg
cggctgctgg cacgtagtta gccgtggctt tttaaacggg tactatctcc
1020tacttctccc cgtccaaaga ggtttacacc ccgaagggct tcttccctca
cgcggcgtcg 1080ctgcgtcagg cttccgccca ttgcgcaaga ttccccgctg
ctgcctcccg taggagtgtg 1140ggccgtgtct cagtcccact gtggccgtac
accctctcag gccggctacc cgtcgtcgcc 1200ttggtgagcc gttaccccac
caactagctg atgggccgcg agcccatccc cagcc
125571466DNACaldicellulosiruptorsource1..1466/organism="Caldicellulosirup-
tor" /note="16SrDNA" /mol_type="unassigned DNA" 7gacttcaccc
ccaatcatca gccccacctt caacacagct taacctgtgt cttcaggtgt 60tgctgactct
catggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg
120catgctgatc cgcgattact agcgattccg acttcatgca ggcgagttgc
agcctgcaat 180ccgaactggg ggtgcttttt tgggattcgc tccggctcgc
gccttcgcac gccctctgta 240gcacccattg tagcacgtgt gtagcccagg
gcataagggg catgatgatt tgacgtcatc 300cccaccttcc tccgcctcat
cgacggcagt ccccttagag tgcccaccat tacgcgctgg 360caactaaggg
caggggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc
420tgacgacaac catgcaccac ctgtgtccgg gctcctgctc tcatcgaaca
ggcaccccac 480cctttcgggc aggtccccgg catgtcaagc cctggtaagg
ttcttcgcgt tgcttcgaat 540taaaccacat gctccaccgc ttgtgcgggc
ccccgtcaat tcctttgagt ttcaaccttg 600cggccgtact ccccaggcgg
gatgcttatt gtgttaacta cggcacggag gagtccttct 660cccccacacc
tagcatccat cgtttacagc gtggactacc agggtatcta atcctgttcg
720ctccccacgc tttcgtgcct cagcgtcagt tacggtccag acggccgcct
tcgccactgg 780tgttcctccc gatatctacg catttcaccg ctacaccggg
aattccgccg tcctctcccg 840cactcaagct atgcagtatt aagcgcaatc
cttaggttga gcctaaggct ttcacgctta 900actcgcatag ccgcctacgc
accctttacg cccagtaatt ccggacaacg ctcgccacct 960acgtattacc
gcggctgctg gcacgtagtt agccgtggct ttttaaacgg gtactatctc
1020ctacttctcc ccgtccaaag aggtttacac cccgaagggc ttcttccctc
acgcggcgtc 1080gctgcgtcag gcttccgccc attgcgcaag attccccgct
gctgcctccc gtaggagtgt 1140gggccgtgtc tcagtcccac tgtggccgta
caccctctca ggccggctac ccgtcgtcgc 1200cttggtgagc cgttacctca
ccaactagct gatgggccgc gagcccatcc ccagccggat 1260tactcctttc
accacatcac catgcgatga cgtggtccca tcgggtatta gcagcccttt
1320cgagctgtta tccccgtgct gggggtaggt tgctcacgtg ttactcaccc
gtccgccgct 1380aagatggcag cctccagctc atcaccttca accaccatct
ccgctcgact tgcatgcgtt 1440aggcacgccg ccagcgttcg tcctga
1466820DNAArtificial Sequencesource1..20/organism="Artificial
Sequence" /note="Primer for 16SrDNA amplification"
/mol_type="unassigned DNA" 8agagtttgat cmtggctcag
20919DNAArtificial Sequencesource1..19/organism="Artificial
Sequence" /note="16SrDNA reverse Primer" /mol_type="unassigned DNA"
9ggttaccttg ttacgactt 19
* * * * *