U.S. patent application number 17/106052 was filed with the patent office on 2021-05-27 for method for producing bioethanol from dates.
The applicant listed for this patent is KING FAISAL UNIVERSITY. Invention is credited to SALAH MOHAMMED ALEID, Siddig Hussein Hamad.
Application Number | 20210155960 17/106052 |
Document ID | / |
Family ID | 1000005373782 |
Filed Date | 2021-05-27 |
View All Diagrams
United States Patent
Application |
20210155960 |
Kind Code |
A1 |
ALEID; SALAH MOHAMMED ; et
al. |
May 27, 2021 |
METHOD FOR PRODUCING BIOETHANOL FROM DATES
Abstract
The method for producing bioethanol from dates includes
manufacturing a suitable substrate for bioethanol from dates and
fermenting the date substrate to produce bioethanol. The date
substrate may be produced by de-pitting date fruits, heating the
flesh with water to produce a mixture, filter pressing the mixture
to produce a juice, and concentrated by vacuum drying to produce a
date substrate. The date substrate may then be fermented in either
a batch or a fed-batch culture. The fermentation may be performed
with a thermophilic yeast, such K. marxianus. In an alternative
embodiment the date substrate may be a date fruit extract.
Inventors: |
ALEID; SALAH MOHAMMED;
(Alahsa, SA) ; Hamad; Siddig Hussein; (Alahsa,
SA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KING FAISAL UNIVERSITY |
AL-AHSA |
|
SA |
|
|
Family ID: |
1000005373782 |
Appl. No.: |
17/106052 |
Filed: |
November 27, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16391497 |
Apr 23, 2019 |
10870868 |
|
|
17106052 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/36 20130101;
C12M 45/02 20130101; C12P 7/08 20130101; C12M 21/12 20130101; C12N
1/16 20130101; C12M 23/08 20130101 |
International
Class: |
C12P 7/08 20060101
C12P007/08; C12N 1/16 20060101 C12N001/16; C12M 1/00 20060101
C12M001/00; C12M 1/33 20060101 C12M001/33 |
Claims
1-19. (canceled)
20. A bioethanol produced from dates, comprising: e a fermented
date substrate, wherein the substrate is obtained by: de-pitting
date fruits to produce date fruit flesh; heating the date fruit
flesh with water to produce a mixture; filter pressing the mixture
to produce a juice; concentrating the juice to produce a date
substrate, wherein the date substrate used for the fermentation has
a starting sugar concentration ranging from 20% to 25%; and
fermenting the date substrate with yeast at between 30.degree.
C.-40.degree. C. for 32 hours, wherein the yeast is selected from
the group consisting of S. cerevisiae and S. cerevisiae NCYC 431,
to produce a 11.2-11.8% w/v bioethanol concentration.
Description
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN COMPUTER
READABLE FORM
[0001] The Applicant hereby incorporates by reference the sequence
listing contained in the ASCII text file titled
32087_09_sequence_listing_ST25.txt, created Mar. 14, 2019, and
having 17 KB of data.
BACKGROUND
1. Field
[0002] The disclosure of the present patent application relates to
biofuels, and particularly to a method for producing bioethanol
from dates.
2. Description of the Related Art
[0003] Bioethanol is ethyl alcohol produced from biological
sources. Bioethanol can be used as fuel, or as a fuel additive for
gasoline. Bioethanol is commonly made from biomass such as corn or
sugarcane. Other common sources for bioethanol include potato,
cassava, barley, wheat, and hemp.
[0004] Bioethanol is manufactured from grains by milling the grains
into meal, mixing the meal with water and alpha-amylase and
liquefying the resulting mash. This step is followed by
saccharification with a secondary enzyme to convert the liquefied
starch to fermentable sugars, fermentation with yeast, distillation
of the fermented mash to concentrate the alcohol, and dehydration
to remove any remaining water, resulting in anhydrous ethanol.
[0005] The traditional approach to manufacturing bioethanol suffers
from a number of drawbacks. The use of grains to produce bioethanol
could contribute to rising food prices. The process of producing
ethanol from grain is highly inefficient, requiring many wasteful
steps, including hydrolysis of starch by addition of enzymes and
acids.
[0006] Thus, a method for producing bioethanol from dates solving
the aforementioned problems is desired.
SUMMARY
[0007] The method for producing bioethanol from dates includes
manufacturing a suitable substrate for bioethanol from dates and
fermenting the date substrate to produce bioethanol. In one
embodiment, the date substrate can be produced by de-pitting date
fruits, heating the flesh with water to produce a mixture, filter
pressing the mixture to produce a juice, and concentrating the
juice by vacuum drying to produce a date substrate. The date
substrate may then be fermented in either a batch or a fed-batch
culture. In the batch process, the entire volume of substrate may
be added to a bioreactor along with a yeast inoculum and
fermentation may be conducted at either 30 or 40.degree. C.,
depending upon the strain of yeast used as the inoculum. In the
fed-batch process, the substrate may be fed gradually into the
bioreactor containing the yeast inoculum at a constant feeding rate
and fermentation may be conducted at either 30.degree. C. or
40.degree. C., depending upon the strain of yeast used as the
inoculum.
[0008] In an alternative embodiment, the date substrate may be date
extract.
[0009] These and other features of the present disclosure will
become readily apparent upon further review of the following
specification and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1: depicts a graph of ethanol production from date
extract in batch using a commercial strain of S. cerevisiae.
[0011] FIG. 2: depicts a graph of specific and volumetric
productivities of ethanol from date extract in batch using a
commercial strain of S. cerevisiae.
[0012] FIG. 3: depicts a graph of ethanol production from molasses
in batch using a commercial strain of S. cerevisiae.
[0013] FIG. 4: depicts a graph of specific and volumetric
productivities of ethanol from molasses in batch using a commercial
strain of S. cerevisiae.
[0014] FIG. 5: depicts a. graph of ethanol production from date
extract and molasses (1:1) in batch using a commercial strain of S.
cerevisiae.
[0015] FIG. 6: depicts a graph of specific and volumetric
productivities of ethanol from date extract and molasses (1:1) in
batch using a commercial strain of S. cerevisiae.
[0016] FIG. 7: depicts a graph of ethanol production from date
extract in batch using K. marxianus NCYC 179.
[0017] FIG. 8: depicts a graph of specific and volumetric
productivities of ethanol from date extract in batch using the
yeast K. marxianus NCYC 179.
[0018] FIG. 9: depicts a graph of ethanol production from date
extract in batch using the yeast S. cerevisiae NCYC 431.
[0019] FIG. 10: depicts a graph of the specific and volumetric
productivities of ethanol from date extract in batch using the
yeast S. cerevisiae NCYC 431.
[0020] FIG. 11: depicts a graph of ethanol production from molasses
in batch using the yeast S. cerevisiae NCYC 431.
[0021] FIG. 12: depicts a graph of specific and volumetric
productivities of ethanol from molasses in batch using the yeast S.
cerevisiae NCYC 431.
[0022] FIG. 13: depicts a graph of ethanol production from date
extract and molasses (1:1) in batch using the yeast S. cerevisiae
NCYC 431.
[0023] FIG. 14: depicts a graph of specific and volumetric
productivities of ethanol from date extract and molasses (1:1) in
batch using the yeast S. cerevisiae NCYC 431.
[0024] FIG. 15: depicts a graph of ethanol production from date
extract in fed-batch using the yeast S. cerevisiae NCYC 431.
[0025] FIG. 16: depicts a graph of specific and volumetric
productivities of ethanol from date extract in fed-batch using the
yeast S. cerevisiae NCYC 431.
[0026] FIG. 17: depicts a graph of ethanol production from molasses
in fed-batch using the yeast S. cerevisiae NCYC 431.
[0027] FIG. 18: depicts a graph of specific and volumetric
productivities of ethanol from molasses in fed-batch using the
yeast S. cerevisiae NCYC 431.
[0028] FIG. 19: depicts a graph of ethanol production from date
extract and molasses (1:1) in fed-batch using the yeast S.
cerevisiae NCYC 431.
[0029] FIG. 20: depicts a graph of specific and volumetric
productivities of ethanol from date extract and molasses (1:1) in
fed-batch using the yeast S. cerevisiae NCYC 431.
[0030] FIG. 21: depicts a graph of ethanol production from date
extract in fed-batch using the yeast K. marxianus HH 5.
[0031] FIG. 22: depicts a graph of specific and volumetric
productivities of ethanol from date extract in fed-batch using the
yeast K. marxianus HH 5.
[0032] FIG. 23: depicts a graph of ethanol production from molasses
in fed-batch using the yeast K. marxianus HH 5.
[0033] FIG. 24: depicts a graph of specific and volumetric
productivities of ethanol from molasses in fed-batch using the
yeast K. marxianus HH 5.
[0034] FIG. 25: depicts a graph of ethanol production from date
extract and molasses (1:1) in fed-batch using the yeast S K.
marxianus HH 5.
[0035] FIG. 26: depicts a graph of specific and volumetric
productivities of ethanol from date extract and molasses (1:1) in
fed-batch using the yeast K. marxianus HH 5.
[0036] Similar reference characters denote corresponding features
consistently throughout the attached drawings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] As used herein, the term "about," when used to modify a
quantity, means within 10% of the modified quantity.
[0038] A method for producing bioethanol from dates includes
manufacturing a suitable substrate from dates and fermenting the
date substrate to produce bioethanol. The date substrate may be
"Dips," a date substrate produced by de-pitting date fruits,
heating the flesh with water to produce a mixture, filter pressing
the mixture to produce a juice, and concentrating the juice by
vacuum drying to produce a date substrate. The date substrate may
then be fermented in either a batch or a fed-batch culture. In the
batch process, the entire volume of substrate may be added to a
bioreactor along with a yeast inoculum and fermentation can be
conducted at about 30.degree. C. or about 40.degree. C., depending
upon the strain of yeast used as the inoculum. In the fed-batch
process, the substrate may be gradually fed into the bioreactor
containing the yeast inoculum at a constant feeding rate and
fermentation can be conducted at about 30.degree. C. or about
40.degree. C., depending upon the strain of yeast used as the
inoculum.
[0039] In an embodiment, the de-pitted date fruits may be heated
with an equal volume of water at about 80.degree. C. for about 30
minutes to produce the mixture.
[0040] The filter pressing step is particularly useful to remove
large impurities and insoluble matter. In an embodiment, the juice
may be further purified to provide a clear liquid by micro
filtration using a sheet filter system prior to the concentration
step. This embodiment is particularly suited to applications where
it is desirable to remove smaller impurities from the juice.
[0041] In an embodiment, the vacuum drying step may involve
concentrating the juice or the clear extract to about 75.degree.
Brix at a low temperature, such as about 80.degree. C.
[0042] In an alternative embodiment, the date substrate may be date
extract. The date extract may be produced by mixing date fruits 1:4
(w/w) with water, boiling the water for about 60-90 minutes, and
filtering the mixture to produce a date extract. The date extract
may then be sterilized in an autoclave at about 121.degree. C. for
about 30 minutes.
[0043] In an embodiment, the date fruits may be waste date fruits,
e.g., date fruits not fit for consumption. In a further embodiment
the waste date fruits include one or more of the Ruzeiz, Shunaizi,
and Shahal varieties of dates. In a further embodiment, the waste
date fruits may be waste date fruits harvested in Saudi Arabia. In
an embodiment of the present methods, one ton of waste date fruits
with an average of 70% sugar content can provide an extract having
about 600 kg sugars, which can provide about 300 kg to about 600
kg, e.g., 400 kg, of ethanol.
[0044] In an embodiment, the fermentation may be conducted using a
yeast such as S. cerevisiae, K. marxianus, or the like. In a
further embodiment, the fermentation may be conducted at about
30.degree. C. using a yeast strain such as S. cerevisiae, K.
marxianus, S. cerevisiae NCYC 432, K. marxianus NCYC 179, or the
like. In an alternative embodiment, the fermentation may be
conducted at about 40.degree. C. using a thermophilic yeast strain.
The thermophilic yeast strain may be a thermophilic strain of K.
marxianus, e.g., a strain isolated from local habitats in Alahsa,
Saudi Arabia. The thermophilic K. marxianus strain may include at
least one of the DNA sequences of SEQ ID NOs: 5-16. The
thermophilic yeast strain may be K. marxianus HH 5. The K.
marxianus HH 5 strain may include the DNA sequences of SEQ ID NO: 5
and/or SEQ ID NO: 6.
[0045] In an embodiment, the fermentation may be conducted using
date substrate with a starting sugar concentration ranging from
about 20% to about 25%.
[0046] The following examples illustrate the present teachings:
EXAMPLE 1
Chemical Analysis of Dips
[0047] As discussed above, Dips were manufactured by de-pitting
date fruits and heating the flesh with an equal amount of water at
80.degree. C. for 30 minutes to produce a mixture; filter pressing
the mixture to produce a juice; micro filtering the juice to a
clear liquid using a sheet filter system; and concentrating the
clear extract to 75 Brix at low temperature (80.degree. C.) by
vacuum drying. Chemical analysis of Dips manufactured according to
this process demonstrated that the Dips included the following
nutrients: about 80% sugars, mainly in form of fructose (41%) and
glucose (38%), and a small amount of sucrose (1%); about 2% crude
protein; 1.1 g/kg phosphorus; 14.9 g/kg potassium; 0.8 g/kg
magnesium; 240 ppm pantothenic acid; and 2.7 ppm biotin. (See Table
1) Therefore, Dips used as carbon and energy source in alcoholic
fermentation would be expected to yield about 400 kg ethanol (about
500 liters).
EXAMPLE 2
Chemical Analysis of Date Extracts
[0048] Waste date fruits were obtained from a date processing
factory in Al Hofuf City, Saudi Arabia. They were mainly a
collection from the varieties Ruzeiz, Shunaizi, and Shahal.
Extraction of syrup from the fruits was performed as follows: I :4
(w/w) fruit: water, boiling for about 60-90 minutes and filtration.
The resulting substrate was sterilized in 5-liter bottles by
autoclaving at 121.degree. C. for 30 minutes. The sterilized
substrate contained about 30% sugar.
[0049] The chemical composition of the date sterilized substrate
was assessed, including determining protein concentration using the
Kjeldhal method, M-Inositol concentration using and biotin and
pantothenic acid concentration according to the vitaFast vitamin
testing method, using an ELISA Reader (Multiskan EX Model No. 355,
Thermolabsystem-Finlan). M-Inositol was determined with High
Performance Liquid Chromatography (HPLC) using a Shimadzu Japan
Model 2003, equipped with a RID 10A refractive index detector, CLC
NH2 6.times.150 column, LC 10ATP pump, and CTO 10AC VP oven. The
mobile phase was 83% CH3CN:17% water (v/v), flow rate 1 ml/min.,
column pressure 200 KgF/cm.sup.2. Ethanol and sugar (glucose,
fructose, and sucrose) concentrations were determined using the
enzymatic UV method (Boehringer Mannheim). Mineral concentrations
(Mg, P, K, and S) were determined using the atomic absorption
method. The average chemical composition of the date sterilized
substrate is shown in Table 1.
TABLE-US-00001 TABLE 1 Date Extract Nutrient Analysis Chemicals
Total sugars (% w/w) Content Fruit 68-72 Thin Extract 25-30
Extracted Fruit 10.2-10.8 pH 4.3-4.5 Crude protein (% w/w) 2.0-2.5
Magnesium (g/kg) 0.5-0.9 Potassium (g/kg) 14.0-16.0 Phosphorous
(g/kg) PO.sub.4 or P 1.0-1.4 Pantothenic acid (ppm) 200-240 Biotin
(ppm) 2.0-3.0 m-Inositol (ppm) traces
[0050] The date substrate contained all nutrients needed for a
small amount of yeast growth (See Table 1) and no addition of
mineral medium was needed. The fruits contained 68-72% (w/w) sugars
on wet basis and the thin extract contained 25-30% (w/w) sugars on
wet basis, with 10.2-10.8% (w/w) sugar content remaining in the
extracted fruit. Hence, the efficiency of our extraction system was
about 85%. As a result, a ton of waste date fruits with an average
of 70% sugar content can be expected to produce a date substrate
containing about 600 kg sugars, which in turn would be expected to
produce about 300 kg bioethanol. If the efficiency of extraction is
raised, such as by using high quality equipment, the yield of
bioethanol would be predicted to increase to more than 315 kg from
a ton of waste date fruits. If the sugar content of the fruits is
increased, this would also be predicted to increase the resulting
bioethanol yield.
EXAMPLE 3
Identification of Thermotolerant Yeasts
[0051] A screening program was carried out to search for
thermotolerant yeasts from the local habitat in Saudi Arabia that
can be used for ethanol production. Samples of microbially spoiled
fruits, especially date fruits, were collected from Hofuf markets
and farms. Yeasts involved in spoilage were isolated by inoculation
on PDA dishes and incubation at 35-50.degree. C. for 2-3 days.
Colonies of different forms were picked, purified by successive
streaking on PDA dishes, and the pure cultures were kept in the
refrigerator in PDA tubes for identification.
[0052] The strains were cultivated on Malt Extract Agar for 3 days
in the dark at 25.degree. C. DNA was isolated using the
MoBio-UltraClean.TM. Microbial DNA Isolation Kit according to the
manufacturer's instructions. Fragments including the 26S ribosomal
RNA gene, Large Subunit D1 and D2 region (LSU) were amplified using
the primers LROR: ACCCGCTGAACTTAAGC (SEQ ID NO: 1) and LR5:
TCCTGAGGGAAACTTCG (SEQ ID NO: 2). Fragments including the Internal
Transcribed Spacer 1 and 2 and the 5.8S gene (ITS) were amplified
using the primers LS266: GCATTCCCAAACAACTCGACTC (SEQ ID NO: 3) and
V9G TTACGTCCCTGCCCTTTGTA (SEQ ID NO: 4). The PCR fragments were
sequenced with the ABI Prism.RTM. Big Dye.TM. Terminator v. 3.0
Ready Reaction Cycle sequencing Kit. Samples were analyzed on an
ABI PRISM 3730 Genetic Analyzer and contigs were assembled using
the forward and reverse sequences with the SeqMan program from the
LaserGene package. The sequence was compared in a large yeast
database of CBS-KNAW Fungal Biodiversity Centre with sequences of
known strains.
[0053] Twenty two isolates that showed good growth at 45.degree. C.
were obtained from the screening program. Twelve of these isolates
grew at 50.degree. C., and hence were regarded as thermophilic.
Preliminary identification tests were carried out on the 12
thermophilic isolates, including morphological examination of the
yeast colonies grown on PDA, microscopic examination of the cell
form, and ability to ferment glucose. The outcome of these tests
indicated that the 12 thermophilic isolates belonged to at least to
3 different genera. Molecular identification of these 12
thermophilic isolates by DNA sequencing was carried out and the
results are shown in Table 2. The isolates belonged to three genera
and species, namely Kluyveromyces marxianus (6 isolates),
Clavispora lusitaniae (5 isolates and Wickerhamomyces anomalus (1
isolate). C. lusitaniae is an opportunistic pathogen, hence it was
not considered for ethanol production. Preliminary tests showed
that W. anomalus was not a good ethanol producer. However,
promising preliminary results for ethanol production were obtained
for the strains of K. marxianus, and the best of these isolates was
K. marxianus 05-1 or "HH5."
[0054] The isolates belonging to the same species had different
genetic sequences at the tested loci, indicating that they are
different strains of the species. The sequence of the 26S ribosomal
RNA gene, Large Subunit D1 and D2 region (LSU) of the K. marxianus
05-1 isolate includes SEQ ID. NO. 6. The sequence of the 26S
ribosomal RNA gene, Large Subunit D1 and D2 region (LSU) of the K.
marxianus 12-1 isolate includes SEQ ID. NO. 8. The sequence of the
26S ribosomal RNA gene, Large Subunit D1 and D2 region (LSU) of the
K. marxianus 14G-1 isolate includes SEQ ID. NO. 10. The sequence of
the 26S ribosomal RNA gene, Large Subunit D1 and D2 region (LSU) of
the K. marxianus 14W-1 isolate includes SEQ ID. NO. 12. The
sequence of the 26S ribosomal RNA gene, Large Subunit D1 and D2
region (LSU) of the K. marxianus 23-1 isolate includes SEQ ID. NO.
14. The sequence of the 26S ribosomal RNA gene, Large Subunit D1
and D2 region (LSU) of the K. marxianus 25-1 isolate includes SEQ
ID. NO. 16. The sequence of the Internal Transcribed Spacer 1 and 2
and the 5.8S gene (ITS) of the K. marxianus 05-1 isolate includes
SEQ ID. NO. 5. The sequence of the Internal Transcribed Spacer 1
and 2 and the 5.8S gene (ITS) of the K. marxianus 12-1 isolate
includes SEQ ID. NO. 7. The sequence of the Internal Transcribed
Spacer 1 and 2 and the 5.8S gene (ITS) of the K. marxianus 14G-1
isolate includes SEQ ID. NO. 9. The sequence of the Internal
Transcribed Spacer 1 and 2 and the 5.8S gene (ITS) of the K.
marxianus 14W-1 isolate includes SEQ ID. NO. 11. The sequence of
the Internal Transcribed Spacer 1 and 2 and the 5.8S gene (ITS) of
the K. marxianus 23-1 isolate includes SEQ ID. NO. 13. The sequence
of the Internal Transcribed Spacer 1 and 2 and the 5.8S gene (ITS)
of the K. marxianus 25-1 isolate includes SEQ ID. NO. 15.
TABLE-US-00002 TABLE 2 Identification of Yeast Isolates Using
Morphological and Molecular Characteristics Strain Name 05-1
Kluyveromyces marxianus (E. C. Hansen) Van der Walt (named K.
marxianus HH5) 12-1 Kluyveromyces marxianus (E. C. Hansen) Van der
Walt 14G-1.sub. Kluyveromyces marxianus (E. C. Hansen) Van der Walt
14W-1 Kluyveromyces marxianus (E. C. Hansen) Van der Walt 23-1
Kluyveromyces marxianus (E. C. Hansen) Van der Walt 25-1
Kluyveromyces marxianus (E. C. Hansen) Van der Walt 15-2 Clavispora
lusitaniae Rodr. Mir. 26-2 Clavispora lusitaniae Rodr. Mir. 28-2
Clavispora lusitaniae Rodr. Mir. 45-2 Clavispora lusitaniae Rodr.
Mir. 50-2 Clavispora lusitaniae Rodr. Mir. 48-2 Wickerhamomyces
anomalus (E. C. Hansen) Kurtzman, Robnett & Basehoar-Powers
EXAMPLE 4
Fermentation Experimental Design
[0055] The yeast strains were first propagated in shake flasks and
then in a bioreactor containing suitable medium, harvested by
filtration to a cake and kept in the refrigerator to be used as
inocula.
[0056] Fermentation experiments were done in batch and fed-batch
cultures using shake flasks, a BioFlo 110 bioreactor (New Brunswick
Scientific) with 10 liter working volume and an Applikon
autoclavable Bioreactor system (Applikon Biotechnology, the
Netherlands) with one liter working volume. In the batch process,
the whole substrate was added to the bioreactor at the beginning of
fermentation and the process continued to its end. In the fed-batch
process the substrate was fed to the fermenter at constant feeding
rates. Fermentations using the commercial baker's yeast, S.
cerevisiae NCYC 432, and K. marxianus NCYC 179 were at 30.degree.
C., while fermentation using the thermophilic yeast K. marxianus HH
5 (isolated by the present inventors) was at 40.degree. C. Samples
were taken from the bioreactor every 2-4 hours and the
concentrations of ethanol, sugars and yeast dry matter were
determined.
[0057] A 3 X 4 X 2 (three variables) factorial experimental design
was employed with three types of substrates (date extract,
molasses, and a mixture of 1:1 date extract/molasses), 4 production
organisms (local and imported) and two fermentation processes
(batch and fed-batch) (See Table 3). A randomized block design was
chosen to run the experiment. A collection of waste date fruits and
2 fermentation temperatures (30 and 40.degree. C.) were chosen
based on information from the literature. All of the above
parameters were used as useful means for modeling and optimizing
small-scale operations. Variables measured in this study included
yield of ethanol on substrate (g ethanol/g sugar), and the
concentrations (g/l) of ethanol, sugars, and yeast biomass.
TABLE-US-00003 TABLE 3 Experimental Design Independent Variable
Factor Substrate 100% Dips 100% 1:1 Dips/ Molasses Molasses
Production Organism Local isolate Imported strains (one strain)
(three strains) Production process Batch Fed-Batch
[0058] Ethanol and sugar (glucose, fructose, and sucrose)
concentrations were determined using the enzymatic UV method
(Boehringer Mannheim). Yeast biomass concentration was determined
by centrifugation of 5 ml broth at 2000 rpm for 5 minutes in dry
centrifugal tube, and overnight drying at 105.degree. C.
[0059] The yeasts used were S. cerevisiae NCYC 431, Kluyveromyces
marxianus NCYC 179, a commercial baker's yeast strain of the
species S. cerevisiae purchased from the local market, and K.
marxianus HH 5 (isolated by the present inventors). Batch and
fed-batch fermentation processes were used at different final sugar
concentrations. For comparison, substrates of pure molasses and a
1:1 mixture of date extract and molasses were also used for
production. The fermentation experiments were done using shake
flasks, a Biollo 110 bioreactor (New Brunswick Scientific) with 10
liter working volume and an Applikon autoclavable Bioreactor system
(Applikon Biotechnology, the Netherlands) with one liter working
volume.
[0060] Date substrate was extracted as described in Example 1. The
fruits used were waste fruits collected from a date processing
factory in Hofuf City, mainly made of the varieties Ruzeiz,
Shunaizi, and Shahal. Because yeast growth during ethanol
production is normally minimal, sugars in the production substrate
are mainly converted to ethanol and no significant amount of yeast
biomass is formed.
[0061] Four yeast strains were used for production including a
commercial baker's yeast strain of the species S. cerevisiae, K.
marxianus NCYC 179, S. cerevisiae NCYC 431 and the strain K.
marxianus HH 5 (isolated by the present inventors). Production was
in batch and fed-batch cultures at fermentation temperatures of 30
and 40.degree. C. Three substrates were used, namely pure date
extract, pure molasses and a mixture of date extract and molasses
at 1:1 ratio, with sugar concentrations of 20 to 25%. The results,
all of which were averages of three runs, are presented in FIGS. 1
to 26.
EXAMPLE 5
Fermentation Testing with Commercial S. Cerevisiae
[0062] The strain of the baker's yeast from the species S.
cerevisiae was used for ethanol production in batch processes at
fermentation temperatures of 30.degree. C. on substrates of pure
date extract, pure molasses and a 1:1 mixture of date extract and
molasses. Samples were taken every 4 hours and the concentrations
of ethanol, sugar and yeast dry matter were measured. The results
are shown in FIGS. 1-6.
[0063] Ethanol production from date extract continued at a
relatively high rate during the first 12 hours of fermentation, the
concentration increased steadily during this period to reach 85.2
g/l (FIG. 1). After that, the production rate decreased towards the
end of the fermentation period of 32 hours and the final ethanol
concentration reached was 112.1 g/l (11.2% w/v). The course of
ethanol productivity is illustrated in FIG. 2. The specific and
volumetric productivities were relatively high during the first 12
hours of fermentation, after which they decreased steadily to reach
very low values towards the end of the fermentation period. During
the first 4 hours of fermentation the specific productivity was
0.92 g ethanol per g yeast per hour (g/g. h) and the volumetric
productivity was 9.7 g ethanol per liter reactor volume per hour
(g/l. h). At the end of fermentation after 32 hours, the specific
productivity decreased to 0.05 g/g. h and the volumetric
productivity decreased to 0.5 g/l. h. It is apparent that increased
ethanol concentration in the fermentation medium was inhibitory to
production, and the maximum amount produced and tolerated by the
yeast was 11.2% (w/v). The specific productivity is mainly a
measure of the efficiency of the yeast in ethanol production and
the volumetric productivity is mainly a measure of the efficiency
of the bioreactor system. The overall yield of ethanol on sugar was
91.5% of the theoretical. There was no significant yeast growth
during fermentation, as yeast biomass concentration remained almost
constant during the whole period of fermentation (FIG. 1). The
sugar was almost completely consumed, with only traces remaining at
the end of the fermentation period (FIG. 1). Generally, the values
obtained for the yield of ethanol on sugar and for the specific and
volumetric productivities are comparable to values reported for
different organisms and substrates.
[0064] Substrates of pure molasses and a 1:1 mixture of date
extract and molasses were used for ethanol production under
conditions similar to those of the pure date extract. The results
of molasses fermentation are presented in FIGS. 3 and 4. These
results were quite similar to those of pure date extract (FIG. 3).
Most production occurred in the first 12 hours of fermentation,
after which production rate started to slow down and the final
ethanol concentration reached was 108.4 g/l (10.8% w/v). The
specific and volumetric productivities were also comparable to
those of the pure date extract (FIG. 4). The specific productivity
after 4 hours of fermentation was 1.05 g/g. h and the volumetric
was 10.0 g/l. h, and both decreased to 0.06 g/g. h and 0.6 g/l. h
at the end of fermentation, respectively. The yield of ethanol on
sugar was 90.3% of the theoretical, which was a little lower than
that of pure date extract, but statistical analysis showed that the
difference was not significant. No significant yeast growth
occurred and the sugar provided was almost completely utilized with
only traces remaining at the end of the fermentation period (FIG.
3). A similar result was obtained using the 1:1 date extract and
molasses mixture (FIGS. 5 and 6). The final ethanol concentration
was 114.0 g/l (11.4% w/v), the specific and volumetric
productivities 0.94 g/g. h and 9.2 g/l. h after 4 hours and 0.05
g/g. h and 0.5 g/l. h at the end of fermentation, respectively. The
yield of ethanol on sugar was 90.1% of the theoretical and was not
significantly different from the yields obtained for the other two
substrates as statistical analysis showed. Again, no significant
yeast growth occurred and the sugar was largely consumed with very
small amount remaining at the end of fermentation (FIG. 5). It can
therefore be said that this commercial yeast strain of the species
S. cerevisiae can produce ethanol from date extract in efficiency
comparable to that of the reference substrate molasses. That is to
say that date extract substrate is quite suitable for ethanol
production.
EXAMPLE 6
Fermentation Testing with K. Marxianus NCYC 179
[0065] The strain Kluyveromyces marxianus NCYC 179 was used for
ethanol production in batch processes at fermentation temperatures
of 30.degree. C. on a substrate of pure date extract. Samples were
taken every 4 hours and the concentrations of ethanol, sugar and
yeast dry matter were measured. The results are shown in FIGS. 7
and 8.
[0066] The performance of this yeast was generally weak. The
concentration of ethanol after the first 4 hours of fermentation
was only 25.6 g/l, which was much lower than the concentration
reached from the commercial baker's yeast reported above (FIG. 7).
The maximum ethanol concentration was 71 g/l (7.1% w/v); it was
reached after 28 hours of fermentation. The specific and volumetric
productivities were also much lower than the values reported above
for the commercial baker's yeast. They were 0.62 g/g. h for the
specific productivity and 6.3 for the volumetric productivity after
4 hours of fermentation and 0.05 g/g. h and zero after 28 hours,
respectively (FIG. 8). No increase in ethanol concentration
occurred when fermentation was continued to 36 hours. The yield was
only 54.9% of the theoretical, which was much lower than that
obtained from the commercial yeast. The sugar was not fully
utilized, and 95.6 g/l remained unconsumed in the fermentation
broth. This suggests that this yeast cannot produce and tolerate
more than 7% w/v ethanol. For this reason, further experiments were
not performed with this yeast strain.
EXAMPLE 7
Fermentation Testing with S. Cerevisiae NCYC 431
[0067] The yeast strain S. cerevisiae NCYC 431 was used for ethanol
production in hatch processes at fermentation temperatures of
30.degree. C. on substrates of pure date extract, pure molasses and
a 1:1 mixture of date extract and molasses. Samples were taken
every 4 hours and the concentrations of ethanol, sugar and yeast
dry matter were determined. The results are shown in FIGS.
9-14.
[0068] Ethanol production from date extract substrate started at a
relatively high rate compared to the other yeast strains discussed
above. The concentration reached 45 g/l in the first 4 hours and
continued to increase at a decreasing rate till it reached 117.6
g/l (11.8% w/v) at the end of fermentation of 32 hours (FIG. 9).
The specific productivity started at 1.05 g/g. h in the first 4
hours and ended at 0.03 g/g. h at the end of fermentation, while
the volumetric productivity was 11.3 and 0.4 g/l. h after 4 and 32
hours of fermentation, respectively (FIG. 10). The yield of ethanol
on sugar was 94.6% of the theoretical, indicating that sugar was
mainly converted into ethanol and no significant amounts of other
metabolic products were formed. Sugar provided was consumed to
traces remaining and no significant yeast growth occurred.
[0069] Ethanol production from molasses substrate using the yeast
strain S. cerevisiae NCYC 431 is presented in FIGS. 11 and 12.
Ethanol concentration after 4 hours was 50.5 g/l and reached 118.2
g/l (11.8% w/v) at the end of the fermentation period of 32 hours
(FIG. 11). The specific and volumetric productivities were high,
starting at 1.15 g/g. h and 12.6 g/l. h in the first 4 hours,
respectively and ending at 0.05 g/g. h and 0.6 WE h after 32 hours,
respectively (FIG. 12). The yield of ethanol on sugar was also
high, reaching a value of 96.6% of the theoretical, but statistical
analysis showed that this result was not significantly different
from the 94.6% obtained using the date extract substrate. As
expected, sugar provided was almost completely consumed and no
significant yeast growth occurred.
[0070] Ethanol production from the mixed substrate using strain S.
cerevisiae NCYC 431 (1:1 date extract and molasses) followed a
quite similar trend to the other substrates presented above (FIGS.
13 and 14). Ethanol concentration in the first 4 hours was 47.9 g/l
and reached 113.8 g/l (11.4% w/v) after 32 hours of fermentation
(FIG. 13). The specific and volumetric productivities were 1.13
g/g. h and 12 g/l. h after 4 hours, respectively and ended at 0.01
g/g. h and 0.2 g/l. h after 32 hours, respectively (FIG. 14). The
yield of ethanol on sugar was 95.4% of the theoretical, it was the
second highest, but was not significantly different from the yields
of pure date extract and pure molasses.
[0071] Statistical analysis comparing the yields of ethanol on
sugar obtained from the baker's yeast strain S. cerevisiae and from
the yeast strain S. cerevisiae NCYC 431 showed that the yields of
the latter were significantly higher. It can therefore be concluded
that the yeast strain S. cerevisiae NCYC 431 was the best producer
of ethanol from date extract substrate. Production by this yeast
stain was therefore further tested in fed-batch experiments.
[0072] The strain S. cerevisiae NCYC 431 was used for ethanol
production in a fed-batch process of fermentation to attempt to
further improve its productivity. Production was at a temperature
of 30.degree. C. on substrates of pure date extract, pure molasses
and a 1:1 mixture of date extract and molasses. The substrates were
fed at constant rates of 200 ml/h and feeding time was 15 hours to
deliver 3 liters of substrate containing about 50% sugars. Samples
were taken every 2 hours and the concentrations of ethanol, sugar
and yeast dry matter were measured. The results are shown in FIGS.
15-20.
[0073] The results of the experiments using date extract as
substrate are shown in FIGS. 15 and 16. Ethanol concentration
reached 18.8 g/l in the first 2 hours of fermentation (FIG. 15).
The concentration increased steadily and reached its maximum of
121.5 g/l (12.2% w/v) in 24 hours. The total amount of ethanol
produced was not much higher than that obtained in the batch
culture discussed above. The main difference was in the
fermentation time, which was 24 hours in the fed-batch process
compared to 32 hours in the batch process. The specific
productivity decreased much slower compared to that of the batch
process (FIG. 16). In the batch process the yeast was affected by
the combined inhibitory effect of high concentrations of sugar and
ethanol, whereas in the fed-batch process there was no inhibitory
effect of sugar because its concentration remained relatively low
throughout the fermentation time. Sugar concentration in the
fermentation medium increased at a low rate till the end of
substrate feeding after 15 hours, then decreased quickly to reach
traces at the end of fermentation. No significant yeast growth
occurred. The volumetric productivity in turn remained also
relatively high during the whole time of fermentation indicating
the absence of the combined inhibitory effect of sugar and ethanol
(FIG. 16). Ethanol yield on sugar was 97.2% of the theoretical,
indicating a high efficiency in converting sugar into ethanol
without formation of significant amounts of other metabolic
products.
[0074] Molasses substrate gave quite similar results to date
extract substrate. As can be seen in FIGS. 17 and 18, production
proceeded in the same way as those of the date extract substrate.
The concentration of ethanol reached 20.3 g/l in the first two
hours of fermentation and continued to increase steadily to reach
its Maximum of 119.9 g/l (12% w/v) after 24 hours of fermentation
(FIG. 17). The specific productivity remained fairly high over the
whole period of fermentation (FIG. 18). Sugar concentration
increased slowly to reach 46.3 g/l towards the end of substrate
feeding, and decreased quickly after that to reach very low levels
(FIG. 18). No significant amount of yeast growth was noticed. The
yield of ethanol on sugar was 96.4% of the theoretical, which was a
little lower than that obtained from the date extract but the
difference was not significant as statistical analysis
revealed.
[0075] The substrate obtained from mixing date extract with
molasses at a 1:1 ratio was utilized by the yeast in a similar
efficiency as the former substrates discussed above (FIGS. 19 and
20). Ethanol concentration was 17.6 g/l after 2 hours of
fermentation and reached 116.3 g/l (11.6% w/v) at the end of
fermentation after 24 hours (FIG. 19). The specific productivity
remained relatively high during the whole fermentation period at
values of 0.45 and 0.2 g/g. h (FIG. 20). Sugar concentration
increased slowly during substrate feeding, and then decreased
quickly to reach traces at the end of fermentation (FIG. 19). No
significant yeast growth occurred. The yield of ethanol on sugar
was 95.1% of theoretical and was not significantly different from
the yields of the other two substrates discussed above.
[0076] Date extract gave ethanol yields comparable to those
obtained from the reference substrate molasses.
EXAMPLE 8
Fermentation Testing with K. Marxianus HH5
[0077] The strain Kluyveromyces marxianus HH5, isolated by the
research team from local habitats in Saudi Arabia, was used for
ethanol production in batch processes at fermentation temperatures
of 40.degree. C. on substrates of pure date extract, pure molasses
and a 1:1 mixture of date extract and molasses. The results are
shown in FIGS. 21 to 26.
[0078] Production in a substrate of date extract containing about
20% sugar started at a relatively high rate. Ethanol concentration
reached 40.5 g/l in the first 4 hours, which is comparable to
values obtained from the other yeasts described above (FIG. 21).
Production continued at a reduced rate, and the maximum ethanol
concentration was 92.7 g/l, reached in 24 hours of fermentation,
which then remained almost constant till the end of fermentation
after 28 hours. About 13 g/l sugar remained unconsumed in the
fermentation broth and no significant increase in yeast
concentration was observed. The yield of ethanol on sugar was 92.7%
of the theoretical, which was quite good for a wild yeast strain.
The yeast seemed to have converted most consumed sugars to ethanol
without significant amounts of other metabolic products.
[0079] When a substrate of date extract containing about 25% sugar
concentration was used, the rate of production decreased
drastically. Only 24.5 g/l ethanol concentration was reached in the
first 4 hours of fermentation compared to 40.5 g/l when 20% sugar
concentration was used (FIG. 22). Production continued after that
at a low rate and the highest ethanol concentration was 81.2 g/l,
reached after 28 hours of fermentation, and no further increase in
ethanol concentration was observed when fermentation time was
extended to 32 hours. This suggests that this yeast is sensitive to
high sugar concentration. The yield of ethanol on sugar was 64.7%
of the theoretical, which was very low compared to the yield of
92.7% obtained when a 20% sugar concentration was used. Therefore,
it can be concluded that this yeast can produce and tolerate up to
about 9% w/v or 11% v/v ethanol concentration. This high
productivity is particularly of interest given that it can be
achieved at a fermentation temperature of 40.degree. C. The
combination of high productivity and higher fermentation
temperature suggests that this method could reduce the costs of
cooling during fermentation and heating during distillation.
[0080] The yeast used molasses as substrate for ethanol production
in a quite similar way as its use of date extract, confirming
suitability of date extract as substrate for ethanol production. As
can be seen in FIG. 23, ethanol concentration was 38.4 g/l after 4
hours of fermentation and reached 94.8 g/l in 24 hours then
remained almost constant till 28 hours of fermentation. This result
confirms that the yeast K. marxianus HH 5 can produce and tolerate
ethanol concentrations in range of about 9-10% w/v (11-12% v/v).
About 6 g/l sugar remained unconsumed and no significant yeast
growth occurred. The yield of ethanol on sugar was 95.8% of the
theoretical, which was higher than that obtained from date extract
but the difference was not significant as statistical analysis
showed. It is apparent that most of the sugar consumed was used for
ethanol production and no significant amounts of other metabolic
products were formed. When sugar concentration in the molasses
substrate was raised to 25%, the performance of the yeast became
very weak. The highest ethanol concentration reached was about 7.1%
w/v (about 9% v/v) in 28 hours of fermentation, which was the
lowest concentration obtained (FIG. 24). The yield of ethanol on
sugar was only 56.9% of the theoretical, and about 98 g/l sugar
remained unconsumed. This is a very low yield compared to the 95.8%
obtained from molasses substrate containing 20% sugar, and confirms
the result achieved when date extract substrate containing 25%
sugar was used. This indicates again that the yeast K. marxianus HH
5 is sensitive to high sugar and ethanol concentrations.
[0081] Substrates of mixed date extract and molasses that contained
20% sugar gave a little lower substrate containing 20% sugar, and
confirmed the result achieved when date extract substrate
containing 25% sugar was used. This indicates again that the yeast
K. marxianus HH 5 is sensitive to high sugar and ethanol
concentrations.
[0082] Substrates of mixed date extract and molasses that contained
20% sugar gave a little lower ethanol yields compared to substrates
of pure date extract and pure molasses containing the same sugar
concentrations. The maximum ethanol concentration reached was 8.7%
w/v (11% v/v) in 28 hours of fermentation and the yield of ethanol
on sugar was 85.7% of the theoretical (FIG. 25). No considerable
yeast growth occurred and about 17 g/l sugar remained in the
fermentation broth unconsumed. In substrates containing sugar
concentrations of about 25%, the yield of ethanol on sugar dropped
to 63.3% of the theoretical compared to the 85.7% obtained from the
substrates containing 20% sugar. The maximum ethanol concentration
reached was 7.9 w/v (10% v/v) in 28 hours of fermentation (FIG. 26)
with no significant yeast growth and about 85 g/l sugar remaining
unconsumed.
[0083] It is to be understood that the method for producing
bioethanol from dates is not limited to the specific embodiments
described above, but encompasses any and all embodiments within the
scope of the generic language of the following claims enabled by
the embodiments described herein, or otherwise shown in the
drawings or described above in terms sufficient to enable one of
ordinary skill in the art to make and use the claimed subject
matter.
Sequence CWU 1
1
16117DNAArtificial Sequenceprimer 1acccgctgaa cttaagc
17217DNAArtificial SequencePrimer 2tcctgaggga aacttcg
17322DNAArtificial Sequenceprimer 3gcattcccaa acaactcgac tc
22420DNAArtificial Sequenceprimer 4ttacgtccct gccctttgta
2051000DNAKluyveromyces marxianus 5ttagtgaggc ctcaggattt gcttagagaa
gggggcaact ccatctcaga gcgaaaaatc 60tggtcaaact tggtcattta gaggaactaa
aagtcgtaac aaggtttccg taggtgaacc 120tgcggaagga tcattaaaga
ttatgaatga atagattact gggggaatcg tctgaacaag 180gcctgcgctt
aattgcgcgg ccagttcttg attctctgct atcagttttc tatttctcat
240cctaaacaca atggagtttt ttctctatga actacttccc tggagagctc
gtctctccag 300tggacataaa cacaaacaat attttgtatt atgaaaaact
attatactat aaaatttaat 360attcaaaact ttcaacaacg gatctcttgg
ttctcgcatc gatgaagaac gcagcgaatt 420gcgatatgta ttgtgaattg
cagattttcg tgaatcatca aatctttgaa cgcacattgc 480gccctctggt
attccagggg gcatgcctgt ttgagcgtca tttctctctc aaacctttgg
540gtttggtagt gagtgatact cgtctcgggt taacttgaaa gtggctagcc
gttgccatct 600gcgtgagcag ggctgcgtgt caagtctatg gactcgactc
ttgcacatct acgtcttagg 660tttgcgccaa ttcgtggtaa gcttgggtca
tagagactca taggtgttat aaagactcgc 720tggtgtttgt ctccttgagg
catacggctt taaccaaaac tctcaaagtt tgacctcaaa 780tcaggtagga
gtacccgctg aacttaagca tatcaataag cggaggaaaa gaaaccaacc
840gggattgcct tagtaacggc gagtgaagcg gcaaaagctc aaatttgaaa
tctggcgtct 900tcgacgtccg agttgtaatt tgaagaaggc gactttgtag
ctggtccttg tctatgttcc 960ttggaacagg acgtcataga gggtgagaat
cccgtgtggc 10006800DNAKluyveromyces marxianus 6ttagtacggc
gaagtgaagc ggcaaaagct caaatttgaa atctggcgtc ttcgacgtcc 60gagttgtaat
ttgaagaagg cgactttgta gctggtcctt gtctatgttc cttggaacag
120gacgtcatag agggtgagaa tcccgtgtgg cgaggatccc agttatttgt
aaagtgcttt 180cgacgagtcg agttgtttgg gaatgcagct ctaagtgggt
ggtaaattcc atctaaagct 240aaatattggc gagagaccga tagcgaacaa
gtacagtgat ggaaagatga aaagaacttt 300gaaaagagag tgaaaaagta
cgtgaaattg ttgaaaggga agggcatttg atcagacatg 360gcgtttgctt
cggctttcgc tgggccagca tcagttttag cggttggata aatcctcggg
420aatgtggctc tgcttcggta gagtgttata gcccgtggga atacagccag
ctgggactga 480ggattgcgac ttttgtcaag gatgctggcg taatggttaa
atgccgcccg tcttgaaaca 540cggaccaagg agtctaacgt ctatgcgagt
gtttgggtgt aaaacccgta cgcgtaatga 600aagtgaacgt aggtgagggc
ccgcaagggt gcatcatcga ccgatcctga tgtcttcgga 660tggatttgag
taagagcata gctgttggga cccgaaagat ggtgaactat gcctgaatag
720ggtgaagcca gaggaaactc tggtggaggc tcgtagcggt tctgacgtgc
aaatcgatcg 780tcgaatttgg gtatagggcg 80071000DNAKluyveromyces
marxianus 7ttagtgaggc ctcaggattt gcttagagaa gggggcaact ccatctcaga
gcgaaaaatc 60tggtcaaact tggtcattta gaggaactaa aagtcgtaac aaggtttccg
taggtgaacc 120tgcggaagga tcattaaaga ttatgaatga atagattact
gggggaatcg tctgaacaag 180gcctgcgctt aattgcgcgg ccagttcttg
attctctgct atcagttttc tatttctcat 240cctaaacaca atggagtttt
ttctctatga actacttccc tggagagctc gtctctccag 300tggacataaa
cacaaacaat attttgtatt atgaaaaact attatactat aaaatttaat
360attcaaaact ttcaacaacg gatctcttgg ttctcgcatc gatgaagaac
gcagcgaatt 420gcgatatgta ttgtgaattg cagattttcg tgaatcatca
aatctttgaa cgcacattgc 480gccctctggt attccagggg gcatgcctgt
ttgagcgtca tttctctctc aaacctttgg 540gtttggtagt gagtgatact
cgtctcgggt taacttgaaa gtggctagcc gttgccatct 600gcgtgagcag
ggctgcgtgt caagtctatg gactcgactc ttgcacatct acgtcttagg
660tttgcgccaa ttcgtggtaa gcttgggtca tagagactca taggtgttat
aaagactcgc 720tggtgtttgt ctccttgagg catacggctt taaccaaaac
tctcaaagtt tgacctcaaa 780tcaggtagga gtacccgctg aacttaagca
tatcaataag cggaggaaaa gaaaccaacc 840gggattgcct tagtaacggc
gagtgaagcg gcaaaagctc aaatttgaaa tctggcgtct 900tcgacgtccg
agttgtaatt tgaagaaggc gactttgtag ctggtccttg tctatgttcc
960ttggaacagg acgtcataga gggtgagaat cccgtgtggc
10008800DNAKluyveromyces marxianus 8cttagtacgg cgagtgaagc
ggcaaaagct caaatttgaa atctggcgtc ttcgacgtcc 60gagttgtaat ttgaagaagg
cgactttgta gctggtcctt gtctatgttc cttggaacag 120gacgtcatag
agggtgagaa tcccgtgtgg cgaggatccc agttatttgt aaagtgcttt
180cgacgagtcg agttgtttgg gaatgcagct ctaagtgggt ggtaaattcc
atctaaagct 240aaatattggc gagagaccga tagcgaacaa gtacagtgat
ggaaagatga aaagaacttt 300gaaaagagag tgaaaaagta cgtgaaattg
ttgaaaggga agggcatttg atcagacatg 360gcgtttgctt cggctttcgc
tgggccagca tcagttttag cggttggata aatcctcggg 420aatgtggctc
tgcttcggta gagtgttata gcccgtggga atacagccag ctgggactga
480ggattgcgac ttttgtcaag gatgctggcg taatggttaa atgccgcccg
tcttgaaaca 540cggaccaagg agtctaacgt ctatgcgagt gtttgggtgt
aaaacccgta cgcgtaatga 600aagtgaacgt aggtgagggc ccgcaagggt
gcatcatcga ccgatcctga tgtcttcgga 660tggatttgag taagagcata
gctgttggga cccgaaagat ggtgaactat gcctgaatag 720ggtgaagcca
gaggaaactc tggtggaggc tcgtagcggt tctgacgtgc aaatcgatcg
780tcgaatttgg gtatagggcg 80091000DNAKluyveromyces marxianus
9tagtgaggcc tcaggatttg ctttagagaa gggggcaact tccatctcag tgcgaaaaat
60ctggtcaaac ttggtcattt agaggaacta aaagtcgtaa caaggtttcc gtaggtgaac
120ctgcggaagg atcattaaag attatgaatg aatagattac tgggggaatc
gtctgaacaa 180ggcctgcgct taattgcgcg gccagttctt gattctctgc
tatcagtttt ctatttctca 240tcctaaacac aatggagttt tttctctatg
aactacttcc ctggagagct cgtctctcca 300gtggacataa acacaaacaa
tattttgtat tatgaaaaac tattatacta taaaatttaa 360tattcaaaac
tttcaacaac ggatctcttg gttctcgcat cgatgaagaa cgcagcgaat
420tgcgatatgt attgtgaatt gcagattttc gtgaatcatc aaatctttga
acgcacattg 480cgccctctgg tattccaggg ggcatgcctg tttgagcgtc
atttctctct caaacctttg 540ggtttggtag tgagtgatac tcgtctcggg
ttaacttgaa agtggctagc cgttgccatc 600tgcgtgagca gggctgcgtg
tcaagtctat ggactcgact cttgcacatc tacgtcttag 660gtttgcgcca
attcgtggta agcttgggtc atagagactc ataggtgtta taaagactcg
720ctggtgtttg tctccttgag gcatacggct ttaaccaaaa ctctcaaagt
ttgacctcaa 780atcaggtagg agtacccgct gaacttaagc atatcaataa
gcggaggaaa agaaaccaac 840cgggattgcc ttagtaacgg cgagtgaagc
ggcaaaagct caaatttgaa atctggcgtc 900ttcgacgtcc gagttgtaat
ttgaagaagg cgactttgta gctggtcctt gtctatgttc 960cttggaacag
gacgtcatag agggtgagaa tcccgtgtgg 100010800DNAKluyveromyces
marxianus 10ttagtacggc gtagtgaagc ggcaaaagct caaatttgaa atctggcgtc
ttcgacgtcc 60gagttgtaat ttgaagaagg cgactttgta gctggtcctt gtctatgttc
cttggaacag 120gacgtcatag agggtgagaa tcccgtgtgg cgaggatccc
agttatttgt aaagtgcttt 180cgacgagtcg agttgtttgg gaatgcagct
ctaagtgggt ggtaaattcc atctaaagct 240aaatattggc gagagaccga
tagcgaacaa gtacagtgat ggaaagatga aaagaacttt 300gaaaagagag
tgaaaaagta cgtgaaattg ttgaaaggga agggcatttg atcagacatg
360gcgtttgctt cggctttcgc tgggccagca tcagttttag cggttggata
aatcctcggg 420aatgtggctc tgcttcggta gagtgttata gcccgtggga
atacagccag ctgggactga 480ggattgcgac ttttgtcaag gatgctggcg
taatggttaa atgccgcccg tcttgaaaca 540cggaccaagg agtctaacgt
ctatgcgagt gtttgggtgt aaaacccgta cgcgtaatga 600aagtgaacgt
aggtgagggc ccgcaagggt gcatcatcga ccgatcctga tgtcttcgga
660tggatttgag taagagcata gctgttggga cccgaaagat ggtgaactat
gcctgaatag 720ggtgaagcca gaggaaactc tggtggaggc tcgtagcggt
tactgacgtg caaatcgatc 780gtcgaatttg ggtatagggc
800111000DNAKluyveromyces marxianus 11gtgaggcctc aggatttgct
tagagaaggg ggcaactcca tctcagagcg aaaaatctgg 60tcaaacttgg tcatttagag
gaactaaaag tcgtaacaag gtttccgtag gtgaacctgc 120ggaaggatca
ttaaagatta tgaatgaata gattactggg ggaatcgtct gaacaaggcc
180tgcgcttaat tgcgcggcca gttcttgatt ctctgctatc agttttctat
ttctcatcct 240aaacacaatg gagttttttc tctatgaact acttccctgg
agagctcgtc tctccagtgg 300acataaacac aaacaatatt ttgtattatg
aaaaactatt atactataaa atttaatatt 360caaaactttc aacaacggat
ctcttggttc tcgcatcgat gaagaacgca gcgaattgcg 420atatgtattg
tgaattgcag attttcgtga atcatcaaat ctttgaacgc acattgcgcc
480ctctggtatt ccagggggca tgcctgtttg agcgtcattt ctctctcaaa
cctttgggtt 540tggtagtgag tgatactcgt ctcgggttaa cttgaaagtg
gctagccgtt gccatctgcg 600tgagcagggc tgcgtgtcaa gtctatggac
tcgactcttg cacatctacg tcttaggttt 660gcgccaattc gtggtaagct
tgggtcatag agactcatag gtgttataaa gactcgctgg 720tgtttgtctc
cttgaggcat acggctttaa ccaaaactct caaagtttga cctcaaatca
780ggtaggagta cccgctgaac ttaagcatat caataagcgg aggaaaagaa
accaaccggg 840attgccttag taacggcgag tgaagcggca aaagctcaaa
tttgaaatct ggcgtcttcg 900acgtccgagt tgtaatttga agaaggcgac
tttgtagctg gtcctttgtc tatgttcctt 960ggaacaagga cgtcataaga
agggtgagaa tcccgtgtgg 100012800DNAKluyveromyces marxianus
12cttagtacgg cgtagtgaag cggcaaaagc tcaaatttga aatctggcgt cttcgacgtc
60cgagttgtaa tttgaagaag gcgactttgt agctggtcct tgtctatgtt ccttggaaca
120ggacgtcata gagggtgaga atcccgtgtg gcgaggatcc cagttatttg
taaagtgctt 180tcgacgagtc gagttgtttg ggaatgcagc tctaagtggg
tggtaaattc catctaaagc 240taaatattgg cgagagaccg atagcgaaca
agtacagtga tggaaagatg aaaagaactt 300tgaaaagaga gtgaaaaagt
acgtgaaatt gttgaaaggg aagggcattt gatcagacat 360ggcgtttgct
tcggctttcg ctgggccagc atcagtttta gcggttggat aaatcctcgg
420gaatgtggct ctgcttcggt agagtgttat agcccgtggg aatacagcca
gctgggactg 480aggattgcga cttttgtcaa ggatgctggc gtaatggtta
aatgccgccc gtcttgaaac 540acggaccaag gagtctaacg tctatgcgag
tgtttgggtg taaaacccgt acgcgtaatg 600aaagtgaacg taggtgaggg
cccgcaaggg tgcatcatcg accgatcctg atgtcttcgg 660atggatttga
gtaagagcat agctgttggg acccgaaaga tggtgaacta tgcctgaata
720gggtgaagcc agaggaaact ctggtggagg ctcgtagcgg ttctgacgtg
caaatcgatc 780gtcgaatttg ggtatagggc 800131000DNAKluyveromyces
marxianus 13ttagtgaggc ctcaggattt gcttagagaa gggggcaact ccatctcaga
gcgaaaaatc 60tggtcaaact tggtcattta gaggaactaa aagtcgtaac aaggtttccg
taggtgaacc 120tgcggaagga tcattaaaga ttatgaatga atagattact
gggggaatcg tctgaacaag 180gcctgcgctt aattgcgcgg ccagttcttg
attctctgct atcagttttc tatttctcat 240cctaaacaca atggagtttt
ttctctatga actacttccc tggagagctc gtctctccag 300tggacataaa
cacaaacaat attttgtatt atgaaaaact attatactat aaaatttaat
360attcaaaact ttcaacaacg gatctcttgg ttctcgcatc gatgaagaac
gcagcgaatt 420gcgatatgta ttgtgaattg cagattttcg tgaatcatca
aatctttgaa cgcacattgc 480gccctctggt attccagggg gcatgcctgt
ttgagcgtca tttctctctc aaacctttgg 540gtttggtagt gagtgatact
cgtctcgggt taacttgaaa gtggctagcc gttgccatct 600gcgtgagcag
ggctgcgtgt caagtctatg gactcgactc ttgcacatct acgtcttagg
660tttgcgccaa ttcgtggtaa gcttgggtca tagagactca taggtgttat
aaagactcgc 720tggtgtttgt ctccttgagg catacggctt taaccaaaac
tctcaaagtt tgacctcaaa 780tcaggtagga gtacccgctg aacttaagca
tatcaataag cggaggaaaa gaaaccaacc 840gggattgcct tagtaacggc
gagtgaagcg gcaaaagctc aaatttgaaa tctggcgtct 900tcgacgtccg
agttgtaatt tgaagaaggc gactttgtag ctggtccttg tctatgttcc
960ttggaacagg acgtcataga gggtgagaat cccgtgtggc
100014820DNAKluyveromyces marxianus 14gacaccggga tgccttagta
acggcgaagt gaagcggcaa aagctcaaat ttgaaatctg 60gcgtcttcga cgtccgagtt
gtaatttgaa gaaggcgact ttgtagctgg tccttgtcta 120tgttccttgg
aacaggacgt catagagggt gagaatcccg tgtggcgagg atcccagtta
180tttgtaaagt gctttcgacg agtcgagttg tttgggaatg cagctctaag
tgggtggtaa 240attccatcta aagctaaata ttggcgagag accgatagcg
aacaagtaca gtgatggaaa 300gatgaaaaga actttgaaaa gagagtgaaa
aagtacgtga aattgttgaa agggaagggc 360atttgatcag acatggcgtt
tgcttcggct ttcgctgggc cagcatcagt tttagcggtt 420ggataaatcc
tcgggaatgt ggctctgctt cggtagagtg ttatagcccg tgggaataca
480gccagctggg actgaggatt gcgacttttg tcaaggatgc tggcgtaatg
gttaaatgcc 540gcccgtcttg aaacacggac caaggagtct aacgtctatg
cgagtgtttg ggtgtaaaac 600ccgtacgcgt aatgaaagtg aacgtaggtg
agggcccgca agggtgcatc atcgaccgat 660cctgatgtct tcggatggat
ttgagtaaga gcatagctgt tgggacccga aagatggtga 720actatgcctg
aatagggtga agccagagga aactctggtg gaggctcgta gcggttctga
780cgtgcaaatc gatcgtcgaa tttgggtata gggcgaaaga
820151000DNAKluyveromyces marxianus 15tagtgaggcc tcaggatttg
cttagagaag ggggcaactc catctcagag cgaaaaatct 60ggtcaaactt ggtcatttag
aggaactaaa agtcgtaaca aggtttccgt aggtgaacct 120gcggaaggat
cattaaagat tatgaatgaa tagattactg ggggaatcgt ctgaacaagg
180cctgcgctta attgcgcggc cagttcttga ttctctgcta tcagttttct
atttctcatc 240ctaaacacaa tggagttttt tctctatgaa ctacttccct
ggagagctcg tctctccagt 300ggacataaac acaaacaata ttttgtatta
tgaaaaacta ttatactata aaatttaata 360ttcaaaactt tcaacaacgg
atctcttggt tctcgcatcg atgaagaacg cagcgaattg 420cgatatgtat
tgtgaattgc agattttcgt gaatcatcaa atctttgaac gcacattgcg
480ccctctggta ttccaggggg catgcctgtt tgagcgtcat ttctctctca
aacctttggg 540tttggtagtg agtgatactc gtctcgggtt aacttgaaag
tggctagccg ttgccatctg 600cgtgagcagg gctgcgtgtc aagtctatgg
actcgactct tgcacatcta cgtcttaggt 660ttgcgccaat tcgtggtaag
cttgggtcat agagactcat aggtgttata aagactcgct 720ggtgtttgtc
tccttgaggc atacggcttt aaccaaaact ctcaaagttt gacctcaaat
780caggtaggag tacccgctga acttaagcat atcaataagc ggaggaaaag
aaaccaaccg 840ggattgcctt agtaacggcg agtgaagcgg caaaagctca
aatttgaaat ctggcgtctt 900cgacgtccga gttgtaattt gaagaaggcg
actttgtagc tggtccttgt ctatgttcct 960tggaacagga cgtcatagag
ggtgagaatc ccgtgtggcg 100016800DNAKluyveromyces marxianus
16ttagtacggc gagtgaagcg gcaaaagctc aaatttgaaa tctggcgtct tcgacgtccg
60agttgtaatt tgaagaaggc gactttgtag ctggtccttg tctatgttcc ttggaacagg
120acgtcataga gggtgagaat cccgtgtggc gaggatccca gttatttgta
aagtgctttc 180gacgagtcga gttgtttggg aatgcagctc taagtgggtg
gtaaattcca tctaaagcta 240aatattggcg agagaccgat agcgaacaag
tacagtgatg gaaagatgaa aagaactttg 300aaaagagagt gaaaaagtac
gtgaaattgt tgaaagggaa gggcatttga tcagacatgg 360cgtttgcttc
ggctttcgct gggccagcat cagttttagc ggttggataa atcctcggga
420atgtggctct gcttcggtag agtgttatag cccgtgggaa tacagccagc
tgggactgag 480gattgcgact tttgtcaagg atgctggcgt aatggttaaa
tgccgcccgt cttgaaacac 540ggaccaagga gtctaacgtc tatgcgagtg
tttgggtgta aaacccgtac gcgtaatgaa 600agtgaacgta ggtgagggcc
cgcaagggtg catcatcgac cgatcctgat gtcttcggat 660ggatttgagt
aagagcatag ctgttgggac ccgaaagatg gtgaactatg cctgaatagg
720gtgaagccag aggaaactct ggtggaggct cgtagcggtt actgacgtgc
aaatcgatcg 780tcgaatttgg gtatagggcg 800
* * * * *