U.S. patent application number 14/667712 was filed with the patent office on 2016-06-30 for fungi-bacteria composite microecologics and methods for preparing and using the same.
The applicant listed for this patent is Jianmeng CHEN, Zhuowei CHENG, Jiade WANG, Jianming YU. Invention is credited to Jianmeng CHEN, Zhuowei CHENG, Jiade WANG, Jianming YU.
Application Number | 20160185638 14/667712 |
Document ID | / |
Family ID | 53309301 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160185638 |
Kind Code |
A1 |
CHEN; Jianmeng ; et
al. |
June 30, 2016 |
FUNGI-BACTERIA COMPOSITE MICROECOLOGICS AND METHODS FOR PREPARING
AND USING THE SAME
Abstract
A fungi-bacteria composite microecologies, including:
ester-degrading fungi including Trichoderma sp. LW-1 which has been
deposited in China Center for Type Culture Collection (CCTCC) with
an accession number: CCTCC NO. M2014176 and Aspergillus sp. HD-2
which has been deposited in CCTCC with an accession number: CCTCC
NO. M2014175; an alkene-degrading fungus including Ophiostoma sp.
LLC which has been deposited in CCTCC with an accession number
CCTCC NO. M2014531; a BTEX-degrading bacterium including Zoogloea
sp. HJ1 which has been deposited in CCTCC with an accession number:
CCTCC NO. M2012235; and a chlorinated hydrocarbon-degrading
bacterium including Pandoraea sp. FLX-1 which has been deposited in
CCTCC with an accession number: CCTCC NO. M2011242.
Inventors: |
CHEN; Jianmeng; (Hangzhou,
CN) ; WANG; Jiade; (Hangzhou, CN) ; CHENG;
Zhuowei; (Hangzhou, CN) ; YU; Jianming;
(Hangzhou, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHEN; Jianmeng
WANG; Jiade
CHENG; Zhuowei
YU; Jianming |
Hangzhou
Hangzhou
Hangzhou
Hangzhou |
|
CN
CN
CN
CN |
|
|
Family ID: |
53309301 |
Appl. No.: |
14/667712 |
Filed: |
March 25, 2015 |
Current U.S.
Class: |
210/601 ;
435/252.3 |
Current CPC
Class: |
B01D 2257/708 20130101;
B01D 2257/2064 20130101; B01D 2257/7022 20130101; Y02A 50/20
20180101; Y02A 50/2359 20180101; B01D 2251/95 20130101; C12N 1/14
20130101; C12N 1/20 20130101; B01D 53/85 20130101; B01D 2257/7027
20130101 |
International
Class: |
C02F 11/02 20060101
C02F011/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 2014 |
CN |
201410813739.4 |
Claims
1. A fungi-bacteria composite microecologies, comprising: a)
ester-degrading fungi comprising Trichoderma sp. LW-1 which has
been deposited in China Center for Type Culture Collection (CCTCC)
with an accession number: CCTCC NO. M2014176 and has a DNA sequence
represented by SEQ. ID. NO. 1 and Aspergillus sp. HD-2 which has
been deposited in CCTCC with an accession number: CCTCC NO.
M2014175 and has a DNA sequence represented by SEQ. ID. NO. 2; b)
an alkene-degrading fungus comprising Ophiostoma sp. LLC which has
been deposited in CCTCC with an accession number CCTCC NO. M2014531
and has a DNA sequence represented by SEQ. ID. NO. 3; c) a
BTEX-degrading bacterium comprising Zoogloea sp. HJ1 which has been
deposited in CCTCC with an accession number: CCTCC NO. M2012235 and
has a DNA sequence represented by SEQ. ID. NO. 4; and d) a
chlorinated hydrocarbon-degrading bacterium comprising Pandoraea
sp. FIX-1 which has been deposited in CCTCC with an accession
number: CCTCC NO. M2011242 and has a DNA sequence represented by
SEQ. ID. NO. 5; wherein both Zoogloea sp. HJ1 and Pandoraea sp.
FLX-1 are bacteria; and Ophiostoma sp. LLC, Trichoderma sp. LW-1,
and Aspergillus sp. HD-2 are fungi.
2. The composite microecologies of claim 1, wherein the composite
microecologies is in the form of a solid powder, and a number of
live bacteria/fungi per gram of the composite microecologies
reaches between 10.sup.8 and 10.sup.9.
3. A method for preparing the fungi-bacteria composite
microecologies of claim 1, the method comprising: 1) inoculating
liquid culture media containing inorganic salts using seed culture
tube slants of Zoogloea sp. HJ1 and Pandoraea sp. FLX-1,
respectively, for activation, in which, toluene and dichloromethane
are provided as sole carbon sources, respectively; and conducting
high-density fermentation in fermenters after activation; 2)
inoculating a potato dextrose agar (PDA) culture medium using a
seed culture plate of Ophiostoma sp. LLC for activation, conducting
high-density fermentation in a fermenter, in which, .alpha.-pinene
is provided as a sole carbon source; 3) mixings strains obtained
from the high-density fermentations of 1) and 2), sterilizing a
solid-state fermentation (SSF) culture medium, inoculating the SSF
culture medium with mixed strains for solid-state fermentation,
controlling a fermentation temperature of between 30 and 40.degree.
C. and a fermentation time of between 24 and 60 hrs; 4) vacuum
drying a product obtained from the solid-state fermentation of 3),
controlling a drying temperature at 40.degree. C. and a drying time
at between 24 and 48 hrs; and grinding a resulting product into a
powder after drying; 5) inoculating PDA culture media with seed
culture plates of Aspergillus sp. HD-2 and Trichoderma sp. LW-1 for
activation, respectively; inoculating an improved Czapek Dox
culture plate containing butyl acetate and an improved Czapek Dox
culture plate containing ethyl acetate with activated Aspergillus
sp. HD-2 and Trichoderma sp. LW-1, respectively, and acquiring a
large amounts of spores respectively from the Czapek Dox culture
plates after between 3 and 5 d cultivation; and 6) evenly mixing
the powder obtained from 4) and the spores obtained from 5) at a
weight ratio of (3-5):1, whereby obtaining a composite
microecologies.
4. The method of claim 3, wherein the liquid culture medium
containing inorganic salts and culture media in the fermenters of
1) comprise: 0.376 g/L of KH.sub.2PO.sub.4, 0,456 g/L of
K.sub.2HPO.sub.4, 0.48 g/L of (NH.sub.4).sub.2SO.sub.4, 0.68 g/L of
NaNO.sub.3, 0.25 g/L of Mg(NO.sub.3).sub.2, 0.011 g/L of
CaCl.sub.2.2H.sub.2O, trace elements (0.06 g/L of
MnCl.sub.2.H.sub.2O, 0.088 g/L of ZnCL.sub.2, 0.01 g/L of KI, 0.1
g/l of NaMoO.sub.4.2H.sub.2O, and 0.05 g/I, of H.sub.3BO.sub.3, and
pH values thereof are between 7.0 and 7.2; the liquid culture
medium containing the inorganic salts and the culture media in the
fermenters are performed with moist heat sterilization at a
temperature of 121.degree. C. for between 30 and 40 min; toluene
and dichloromethane are supplied for Zoogloea sp. HJ1 and Pandoraea
sp. FLX-1 as the carbon sources in activation and high-density
fermentation, respectively; and temperatures for the activation and
fermentation cultivation of strains in 1) are controlled at between
30 and 35.degree. C. and dissolved oxygen contents are controlled
at between 2 and 3 mg/L.
5. The method of claim 3, wherein the PDA culture medium in 2)
comprises: 200 g/L of a potato, 20 g/L of glucose (or sucrose), and
20 g/L of an agar, and a pH value thereof is 6.5; a culture medium
in the fermenter in 2) comprises: 2.0 g/L of NH.sub.4Cl, 0.47 g/L
of Na.sub.2HPO.sub.4, 0.45 g/L of KH.sub.2PO.sub.4, 0.5 g/L of
MgSO.sub.4, 0.01 g/L of anhydrous CaCl.sub.2, and trace elements
(0.001 g/L of Mn.sup.2+, Fe.sup.2+, Cu.sup.2+, and Zn.sup.2+,
respectively), a pH value thereof is between 4.2 and 4.6, and a
carbon source thereof is .alpha.-pinene; both the PDA culture
medium and the culture medium of the fermenter in 2) are performed
with moist heat sterilization at a temperature of 121.degree. C.
for between 30 and 40 min; the activation and the fermentation
cultivation of Ophiostoma sp. LLC 2) are conducted at temperatures
of between 30 and 35.degree. C.; and a dissolved oxygen
concentration during the fermentation cultivation is controlled at
between 2 and 3 mg/L.
6. The method of claim 3, wherein the SSF culture medium of step 3)
comprises a solid state composite comprising between 45 and 50 wt.
of wheat bran, between 25and30wt. % of a sawdust, and between 25
and 30 wt. % of and a powdered activated carbon; an aqueous
solution having a volume of between and 2 times of that of the
solid state composite is added to yield a mixture; the aqueous
solution comprises: 20 g/L of a yeast extract, 20 g/L of a potato,
and 5 g/L, of NaCl; a pH value of the mixture is regulated to be
between 6.8 and 7.2; and the mixture is conducted with moist heat
sterilization at a temperature of 121.degree. C. for between 30 and
40 mm and then cooled to obtain the SSF culture medium.
7. The method of claim 3, wherein an inoculum of the mixed strains
in 3) is between 5 and 20%.
8. The method of claim 3, wherein in step 5), the improved Czapek
Dox culture plates comprises: 3 g/L of NaNO.sub.3, 0.5 g/L of
MgSO.sub.4, 0.5 g/L of KCl, 0.01 g/L of FeSO.sub.4, and 20 g of an
agar, and pH values thereof is between 6.0 and 6.5; butyl acetate
and ethyl acetate are supplied as carbon sources for Aspergillus
sp. HD-2 and Trichoderma sp. LW-1, respectively; and the improved
Czapek Dox culture plates are conducted with moist heat
sterilization at a temperature of 121.degree. C. for between 30 and
40 min.
9. The method of claim 3, wherein the composite microecologies
Obtained from 6) is solid powder state and is adapted to maintain
viabilities thereof after preservation on at room temperature or a
temperature of 4.degree. C. for more than 45 d.
10. A method for treating waste gas comprising chlorinated
hydrocarbons, alkenes, aromatic hydrocarbons, and esters, the
method comprising applying the fungi-bacteria composite
microeeologics of claim 1, wherein the fungi-bacteria composite
microecologies is directly added to an inoculation sludge in a
reactor; and an addition of the fungi-bacteria composite
microecologies in controlled to be between 0.5 and 2 kg per cubic
meter of a filler.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn.119 and the Paris Convention
Treaty, this application claims the benefit of Chinese Patent
Application No. 201410813739.4 tiled Dec. 24, 2014, the contents of
which are incorporated herein by reference. Inquiries from the
public to applicants or assignees concerning this document or the
related applications should be directed to: Matthias Scholl P. C.,
Attn.: Dr. Matthias Scholl Esq., 245 First Street, 18th Floor,
Cambridge, Mass. 02142.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a fun bacteria composite
microecologies and methods for preparing and using the same.
[0004] 2. Description of the Related Art
[0005] A typical process for treating organic waste gas by a
fungi-bacteria composite biological system adopts gas feeding at
high concentration and low flow rate and domestication by
circulating fluid at a low flow rate. However, as the
fungi-bacteria composite biological system is gradually formed in
the domestication process, components and contents of the
fungi-bacteria system are different subject to treating conditions,
and the formed biological system cannot be reused or
commercialized.
[0006] A typical composite microecologies includes: bacillus,
pseudomonas, alcaligenes, aspergillus, and yeast. The composite
microecologies is adapted to treat a high concentrated organic
wastewater including toxic ingredients having large molecular and
being difficult to be degraded and a high ammonia wastewater.
However, the selection of strains is blind, and the strain source
is indefinite. The conventional preparation process of the
fungi-bacteria composite microecologies neglects differences in
cultivation systems and pH values of the fungi and the bacteria.
The composite microecologies are formed by mechanically mixing the
separate solid state fermentation substances of the fungi and the
bacteria. Actually, the composite microecologies has small number
of live fungi/bacteria and activities thereof are not high.
SUMMARY OF THE INVENTION
[0007] In view of the above-described problems, it is one objective
of the invention to provide a fungi-bacteria composite
microecologies, a method for preparing the same, and a method for
applying the same. The fungi-bacteria composite microecologies has
simple preparation process, low preparation cost, convenient use,
small volume for transportation whereby realizing
industrialization, and is adapted to keep activity after long time
storage.
[0008] To achieve the above objective, in accordance with one
embodiment of the invention, there is provided a fungi-bacteria
composite microecologies. The fungi-bacteria composite
microecologies comprises: ester-degrading fungi comprising
Trichoderma sp. LW-1 which has been deposited in China Center for
Type Culture Collection (CCTCC) with an accession number: CCTCC NO.
M2014176 and has a DNA sequence represented by SEQ. ID. NO. 1 and
Aspergillus sp. HD-2 which has been deposited in CCTCC with an
accession number: CCTCC NO. M2014175 and has a DNA sequence
represented by SEQ. ID. NO. 2; an Acme-degrading fungus comprising
Ophiostoma sp. LLC which has been deposited in CCTCC with an
accession number CCTCC NO. M2014531 and has a DNA sequence
represented by SEQ. ID. NO. 3; a BTEX-degrading bacterium
comprising Zoogloea sp. HP which has been deposited in CCTCC with
an accession number: CCTCC NO. M2012235 and has a DNA sequence
represented by SEQ. ID. NO. 4; and a chlorinated
hydrocarbon-degrading bacterium comprising Pandaraea sp. FLX-1
which has been deposited in CCTCC with an accession number: CCTCC
NO. M2011242 and has a DNA sequence represented by SEQ. ID. NO. 5.
Both Zoogloea sp. HJ1 and Pandoraea sp. FLX-1 are bacteria.
Ophiostoma sp. LLC, Trichoderma sp. LW-1, and Aspergillus sp.
11.D-2 are fungi. The above fungi/bacteria are adapted to decompose
different kinds of pollutants, the strains have a broad substrate
range, and the growth of microbes do not affect one another, so
that the composite microecologies is capable of degrading waste gas
containing a plurality of volatile organic compounds (VOCs).
[0009] In a class of this embodiment, the composite microecologies
is in the form of a solid powder, and a number of live
bacteria/fungi per gram of the composite microecologies reaches
between 10.sup.8 and 10.sup.9. The solid powder is convenient for
transportation, a large number of the microbes exist in per gram of
the solid power, thereby decreasing the dosage in practical
use.
[0010] In accordance with one embodiment of the invention, there is
provided a method for preparing the fungi-bacteria composite
microecologies, and the method comprises: [0011] 1) inoculating
liquid culture media containing inorganic salts using seed culture
tube slants of Zoogloea sp. HJ1 and Pandoraea sp. FLX-1,
respectively, for activation, in which, toluene and dichloromethane
are provided as sole carbon sources, respectively; and conducting
high-density fermentation in fermenters after activation; [0012] 2)
inoculating a potato dextrose agar (PDA) culture medium using a
seed culture plate of Ophiostoma sp. for activation, conducting
high-density fermentation in a fermenter, in which, .alpha.-pinene
is provided as a sole carbon source; [0013] 3) mixing strains
obtained from the high-density fermentations of 1) and 2),
sterilizing a solid-state fermentation (SSF) culture medium,
inoculating the SSP culture medium with mixed strains for
solid-state fermentation, controlling a fermentation temperature of
between 30 and 40.degree. C. and a fermentation time of between 24
and 60 hrs; [0014] 4) vacuum drying a product obtained from the
solid-state fermentation of 3), controlling a drying temperature at
40.degree. C. and a drying time at between 24 and 48 hrs; and
grinding a resulting product into a powder after drying; [0015] 5)
inoculating PDA culture media with seed culture plates of
Aspergillus sp. HD-2 and Trichoderma sp. LW-1 for activation,
respectively; inoculating an improved Czapek Dox culture plate
containing butyl acetate and an improved Czapek Dox culture plate
containing ethyl acetate with activated Aspergillus sp. HD-2 and
Trichoderma sp., respectively, and acquiring a large amounts of
spores respectively from the Czapek Dox culture plates after
between 3 and 5 d cultivation; and [0016] 6) evenly mixing the
powder obtained from 4) and the spores obtained from 5) at a weight
ratio of (3-5):1, whereby obtaining a composite microecologies. In
the above process, different strains are cultivated in particular
cultivation systems, and the drying time and drying temperature are
reasonably controlled, whereby acquisition of a large amount of
strains and the activities thereof are ensured. The fungal spores
and the solid state fermentation powder are mixed, so that the
separate degradation activities of the fungi and the bacteria are
kept to the maximum extent.
[0017] In a class of this embodiment, the liquid culture medium
containing inorganic salts and culture media in the fermenters of
1) comprise: 0.376 g/L of KH.sub.2PO.sub.4, 0.456 g/L of
K.sub.2HPO.sub.4, 0.48 g/L, of (NH.sub.4).sub.2SO.sub.4, 0.68 g/L
of NaNO.sub.3, 0.25 g/L of Mg(NO.sub.3).sub.2, 0.011 g/L of
CaCl.sub.2.2H.sub.2O, trace elements (0.06 g/L of
MnCl.sub.2.H.sub.2O, 0.088 g/L of ZnCl.sub.2, 0.01 g/L of KI, 0.1
g/L of NaMoO.sub.2.2H.sub.2O, and 0.05 g/L of H.sub.3BO.sub.3, and
pH values thereof are between 7.0 and 7.2. The liquid culture
medium containing the inorganic salts and the culture media in the
fermenters are performed with moist heat sterilization at a
temperature of 121.degree. C. for between 30 and 40 min. Toluene
and dichloromethane are supplied for Zoogloea sp. HJ1 and Pandoraea
sp. FLX-1 as the carbon sources in activation and high-density
fermentation, respectively. Temperatures for the activation and
fermentation cultivation of strains in 1) are controlled at between
30 and 35.degree. C. and dissolved oxygen contents are controlled
at between 2 and 3 mg/L. The above technical parameters enable the
cultivation systems and cultivation environments to be suitable for
growth of the bacteria, so that a large amount of bacteria are
acquired in a relatively short period.
[0018] In a class of this embodiment, the PDA culture medium in 2)
comprises: 200 g/L of a potato, 20 g/L of glucose (or sucrose), and
20 g/L of an agar, and a pH value thereof is 6.5. A culture medium
in the fermenter in 2) comprises: 2.0 g/L of NH.sub.4Cl, 0.47 g/L
of Na.sub.2HPO.sub.4, 0.45 g/L of KH.sub.2PO.sub.4, 0.5 g/L of
MgSO.sub.4, 0.01 g/L of anhydrous CaCl.sub.2, and trace elements
(0.001 g/L of Mn.sup.2+, Fe.sup.2+, Cu.sup.2+, and Zn.sup.2+,
respectively), a pH value thereof is between 4.2 and 4.6, and a
carbon source thereof is u-pinene. Both the PDA culture medium and
the culture medium of the fermenter in 2) are performed with moist
heat sterilization at a temperature of 121.degree. C. for between
30 and 40 min. The activation and the fermentation cultivation of
Ophiostoma sp. LLC 2) are conducted at temperatures of between 30
and 35.degree. C. A dissolved oxygen concentration during the
fermentation cultivation is controlled at between 2 and 3 m/L. The
above technical parameters enable the cultivation systems and
cultivation environments to be suitable for growth of the fungi, so
that a large amount of the fungi are acquired in a relatively short
period.
[0019] In a class of this embodiment, the SSF culture medium of
step 3) comprises a solid state composite comprising between 45 and
50 wt. % of a wheat bran, between 25 and 30 wt. % of a sawdust, and
between 25 and 30 wt. % of and a powdered activated carbon. An
aqueous solution having a volume of between 1 and 2 times of that
of the solid state composite is added to yield a mixture. The
aqueous solution comprises: 20 g/L of a yeast extract, 20 g/L of a
potato, and 5 g/L of NaCl. A pH value of the mixture is regulated
to be between 6.8 and 7.2. The mixture is conducted with moist heat
sterilization at a temperature of 121.degree. C. for between 30 and
40 min and then cooled to obtain the SSF culture medium. The SSF
culture medium is capable of acquiring a relative large quantity of
biomass within a relatively short period, besides, a large quantity
of the biomass is absorbed per unit volume.
[0020] In a class of this embodiment, an inoculum of the mixed
strains in 3) is between 5 and 20%. Thus, the inoculation microbes
grow well in the cultivation system.
[0021] In a class of this embodiment, in step 5), the improved
Czapek Dox culture plates comprises: 3g/L of NaNO.sub.3, 0.5 g/L,
of MgSO.sub.4, 0.5 g/L, of KCl, 0.01 g/L of FeSO.sub.4, and 20 g of
an agar, and pH values thereof is between 6.0 and 6.5. Butyl
acetate and ethyl acetate are supplied as carbon sources for
Aspergillus sp. HD-2 and Trichoderma sp. LW-1, respectively. The
improved Czapek Dox culture plates are conducted with moist heat
sterilization at a temperature of 121.degree. C. for between 30 and
40 min. Thus, the cultivated microbes have relatively good
degradation capability on butyl acetate and ethyl acetate.
[0022] In a class of this embodiment, the composite microecologies
obtained from 6) is in a solid powder state and is adapted to
maintain viabilities thereof after preservation at room temperature
or a temperature of 4.degree. C. for more than 45 d. Thus, the
degradation activity of the composite microecologies is well
kept.
[0023] In accordance with one embodiment of the invention, there is
provided a method for treating waste gas comprising chlorinated
hydrocarbons, alkenes, aromatic hydrocarbons, and esters comprising
applying the fungi-bacteria composite microecologies. The
fungi-bacteria composite mieroecologics is directly added to an
inoculation sludge in a reactor. An addition of the fungi-bacteria
composite microecologies in controlled to be between 0.5 and 2 kg
per cubic meter of a filler. When the above composite
microecologies is added, the time for initiating the reactor is
obviously shortened, and the fungi and bacteria symbiotics is
formed, thereby accelerating the formation of the biofilm.
[0024] Advantages according to embodiments of the invention are
summarized as follows:
[0025] The composite microecologies of the invention adopts
bacteria and fungi possessing particular VOCs degradation
activities and is adapted to effectively overcome shortages of the
conventional reactor inoculated with activated sludge. Not only is
the number of the fungi bacteria possessing high degradation
activities per unit volume significantly improved and the
initiating time of the reactor shortened, but also advantages of
the fungi and bacteria are presented, and the composite
microecologies has improved adaptability on the environment and
great development potential and application prospect in engineering
practice of the purification of the waste gas.
[0026] The method for preparing the fungi-bacteria composite
microecologies of the invention simple process, low price of the
raw materials. Each volume unit of the prepared composite
microecologies contains a large number of live microbes and has
high degradation activity. The composite microecologies is capable
of recovering the degradation activity in a short period after low
temperature storage and is applicable for biological purification
of the industrial waste gas. A fungi-bacteria symbiotic system is
formed during the application process, and separate characters of
the two types of microbes are ensured.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The invention is described hereinbelow with reference to the
accompanying drawings, in which:
[0028] FIG. 1 is a flow chart of a method for preparing a
fungi-bacteria composite microecologies according to one embodiment
of the invention;
[0029] FIG. 2A shows a test result of degradation activity
stability of a fungi-bacteria composite microecologies after 48 hrs
cultivation according to one embodiment of the invention;
[0030] FIG. 2B shows a test result of degradation activity
stability of a fungi-bacteria composite microecologies after 72 hrs
cultivation according to one embodiment of the invention;
[0031] FIG. 3 shows removal efficiencies of different waste gases
by waste purification devices initiated by a domesticated activated
sludge mixed with a fungi-bacteria composite microecologies;
and
[0032] FIG. 4 shows removal efficiencies of different waste gases
by waste purification devices initiated only by a domesticated
activated sludge.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0033] For further illustrating the invention, experiments
detailing a fungi-bacteria composite microecologies and methods for
preparing and using the same are described below. It should be
noted that the following examples are intended to describe and not
to limit the invention.
EXAMPLE 1
Preparation of Fungi-Bacteria Composite Microecologies
[0034] Microbes related in the invention are all deposited in China
Center fur Type Culture Collection (CCTCC), Wuhan University,
Wuhan, 430072, China. The microbes are as follows: [0035] ethyl
acetate-degrading fungus Trichoderma sp. LW-1, accession number for
the deposit: CCTCC NO. M2014176, date of the deposit: May 9, 2014;
[0036] butyl acetate-degrading fungus Aspergillus sp. HD-2,
accession number for the deposit: CCTCC NO. M2014175, date of the
deposit: May 9, 2014; [0037] .alpha.-pinene-degrading fungus
Ophiostoma sp. LLC, accession number for the deposit: CCTCC NO.
M2014531, date of the deposit: Nov. 5, 2014; [0038]
toluene-degrading bacterium Zoogloea sp. HJ1, accession number for
he deposit: CCTCC NO. M2012235, date of the deposit: Jun. 21, 2012,
disclosed in Chinese patent application number 201310281412.2; and
[0039] dichloromethane-degrading bacterium Pandoraea sp. FLX-1,
accession number for the deposit: CCTCC NO. M2011242, date of the
deposit: Jul. 14, 2011, disclosed in Chinese patent application
number 201110370070.2.
[0040] Separation, purification, and identification of Ophiostoma
sp. LLC are as follows:
[0041] A biofilm is collected from an a-pinene-treating biofilter
and placed in an culture medium containing inorganic salts fix
cultivation. A concentration of .alpha.-pinene is gradually
increased within a range of between 50 and 200 mg/L. When the
concentration of a-pinene decreases, 200 mL of a mixed solution is
smeared on a solid state culture medium containing inorganic salts
containing a-pinene as a sole carbon source and is continuously
streaked for separation, so that a purified strain is finally
obtained. The purified strain is inoculated on a slant culture
medium and the slant culture medium is then stored in a
refrigerator at a temperature of 4.degree. C.
[0042] PCR amplification of a genomic DNA of the above purified
strain is conducted using universal primers of internal transcribed
spacer (ITS) of fungi to obtain a target DNA fragment. The sequence
of the target DNA fragment is compared with genomic sequence from
NCBI database, which indicates that the purified strain and the
strain Ophiostoma stenoceras have 100% sequence homology. Clustal.
X2. 0 and MEGA 4.0 (1000 times sampling analyses) are adopted to
construct a phylogenetic tree. From genetic distance and ITS
sequence comparison, the purified strain is identified as
Ophiostoma sp. and is denominated as LLC.
[0043] Separation, purification, and identification of Aspergillus
sp. HD-2 are as follows:
[0044] A sludge from a wastewater treatment plant is air-aerated
for three days. 50 mL of a supernatant is then collected and
centrifuged, and a deposited sludge is added to a brine bottle
containing 50 mL of a liquid culture medium containing inorganic
salts which is previously sterilized at a temperature of
110.degree. C. for 40 min, and antibiotics (0.001 g of streptomycin
and gentainicin) is added. After a bottle plug is inserted, 5 .mu.L
of butyl acetate (a concentration of which is approximately 88
mg/L) is added. The brine bottle is placed on a shaking table and
is cultivated at a temperature of 30.degree. C. and at a rotational
speed of 160 rpm. The concentration of butyl acetate is then
gradually increased. When butyl acetate is obviously degraded, 2 mL
of a mixed strain solution is collected and smeared on a Czapek Dox
culture medium. In the absence of the carbon source. A filter paper
having a diameter of 1 cm is placed on a central position of a
cover of a petri dish, and 5 .mu.L of butyl acetate is dropped on
the filter paper. The mixed strain solution is continuously
streaked for separation, and a purified strain is obtained. A PDA
slant medium is inoculated with the purified strain and then stored
in a refrigerator at a temperature of 4.degree. C.
[0045] PCR amplification of a genomic DNA of the purified strain is
conducted by universal primers of ITS of fungi to obtain a target
DNA fragment. The sequence of the target DNA fragment is compared
with the genomic sequence from NCBI database, which indicates that
the ITS sequence of the purified strain and the ITS sequence of
strain Aspergillus fumigatus have 100% sequence homology.
Thereafter, Clustal X2. 0 and MEGA 4.0 (1000 times sampling
analyses) are adopted to construct the phylogenetic tree. From
genetic distance and ITS sequence comparison, the purified strain
is identified as Aspergillus fumigatus, and is denominated as
HD-2.
[0046] Separation, purification and identification of Trichoderma
sp. LW-1 are as follows:
[0047] An activated sludge is collected from an aeration tank of a
wastewater treatment plant. The activated sludge is washed by a tap
water for five times and air-aerated for 48 hrs for the purpose of
removing organic compound residue as much as possible. After that,
an inorganic culture solution is prepared, and ethyl acetate is
used as the sole carbon source for domestication of the activated
sludge. The inorganic culture solution is replaced with fresh one
every 3 d, and after 40 d of domestication, separation can be
performed. 50 mL of a supernatant is collected from a domestication
bottle and is centrifuged, a deposited sludge is then collected and
added to a brine bottle containing 50 mL of a sterilized culture
medium containing inorganic salts, and antibiotics are added. After
that, a bottle plug is inserted, and ethyl acetate is added (a
concentration of which is 50 mg/L). The brine bottle is placed on
the shaking table at the temperature of 30.degree. C. and the
rotational speed of 160 rpm. The concentration of ethyl acetate is
gradually increased, and when obvious degradation of ethyl acetate
occurs, 2 mL of a mixed strain solution is smeared on a solid state
culture medium containing inorganic salts containing ethyl acetate
and continuously streaked for separation, whereby a purified strain
is finally obtained. A PDA slant culture medium is inoculated with
the purified strain and is stored at the refrigerator at the
temperature of 4.degree. C.
[0048] Based on the homology of ITS sequence, Clustal X2. 0 and
MEGA 4.0 (1000 times sampling analyses) are adopted to construct
the phylogenetic tree. From genetic distance and ITS sequence
comparison, the purified strain is identified as Trichoderma
viride. It is indicated from Biolog FF rnicroplate that the strain
has a relative good conformity degree with Trichoderma viride SIM
index within the system, which indicates that the separated strain
LW-1 belongs to Trichoderma viride.
[0049] As shown in FIG. 1, preparation of a fungi-bacteria
composite microecologies is specifically as follows:
[0050] 1) Activation of Strains
[0051] Inorganic liquid media are inoculated from seed culture tube
slants of Zoogloea sp. HJ1 and Pandoraea sp. FLX-I, respectively,
for activation. The liquid culture media containing inorganic salts
comprise: 0.376 g/L of KH.sub.2PO.sub.4, 0.456 g/L of
K.sub.2HPO.sub.4, 0.48 g/L of (NH.sub.4).sub.2SO.sub.4, 0.68 of
NaNO.sub.3, 0.25 g/L of Mg(NO.sub.3).sub.2, 0.011 g/L of
CaCl.sub.2.2H.sub.2O, trace elements (0.06 g/L of
MnCl.sub.2.H.sub.2O, 0.088 g/L of ZnCl.sub.2, 0.01 g/L of KI, 0.1
g/L of NaMoO.sub.4.2H.sub.2O, and 0.05 g/L of H.sub.3BO.sub.3). pH
values of the liquid culture media are regulated to be 7.0. Both
the culture media are conducted with moister heat sterilization at
a temperature of 121.degree. C. for between 30 and 40 min. The
culture media are then cooled and inoculated with the strains.
Zoogloea sp. HJ1 and Pandoraea sp. FLX-1 are supplied with toluene
and dichloromethane as sole carbon sources, respectively, and
cultivated on a shaking table at a temperature of 32.degree. C.
After 5 d cultivation, inoculation solutions are obtained.
[0052] PDA culture media are inoculated with seed culture plates of
Ophiostoma sp. LLC, Aspergillus sp. HD-2, and Trichoderma sp. LW-1
respectively, for activation. The PDA culture media comprise: 200
g/L of a potato, 20 g/L of glucose (or sucrose), and 20 g/L of an
agar. pH values of the PDA culture media are regulated to be 6.5.
The PDA culture media are performed with moist heat sterilization
at the temperature of 121.degree. C. for between 30 and 40 min.
After the PDA culture media are cooled, the strains are
respectively smeared on the solid culture medium plates and
cultivated at the temperature of 32.degree. C. Inoculums are
obtained after 5 d cultivation.
[0053] 2) Acquisition of Strains
[0054] The inoculation solutions of strains Zoogloea sp. HJ1 and
Pandoraea sp. FLX-1 after activation are inoculated in fermenters
containing liquid culture media containing inorganic salts for
conducting high-density cultivation. Toluene and dichloromethane
are continuously fed as the sole carbon sources, respectively. The
temperature, the pH value, and the dissolved oxygen concentration
are monitored on line and are controlled at 32.degree. C., 7.0, and
between 2 and 3 mg/L, respectively. A large amount of strains are
acquired after 3 d cultivation and then centrifuged for
accumulation.
[0055] The activated strain Ophiostoma sp. LLC is inoculated in a
fermenter containing a liquid culture medium containing inorganic
salts for high-density fermentation. .alpha.-pinene is continuously
fed into the fermenter as the carbon source. The liquid culture
medium containing inorganic salts comprises: 2.0 g/L of NH.sub.4Cl,
0.47 g/L of Na.sub.2HPO.sub.4, 0.45 g/L of KH.sub.2PO.sub.4, 0.5
g/L of MgSO.sub.4, 0.01 g/L of anhydrous CaCl.sub.2, and trace
elements (0.001 g/L of Mn.sup.2+, Fe.sup.2+, Cu.sup.2+, and
Zn.sup.2+, respectively). The temperature, the pH value, and the
dissolved oxygen concentration are monitored on line and are
controlled at 32.degree. C., 4.4, and between 2 and 3 mg/L
respectively. A large amount of mycelia are acquired after 3 d
cultivation and then centrifuged for accumulation.
[0056] The inoculation solutions of Aspergillus sp. HID-2 and
Trichoderma sp. LW-1 after activation are inoculated on improved
Czapek Dox culture plates for high-density cultivation. Butyl
acetate and ethyl acetate are supplied as the carbon sources,
respectively. The improved Czapek Dox culture plates comprise: 3
g/L of NaNO.sub.3, 0.5 g/L of MgSO.sub.4, 0.5 g/L of KCl, 0.01 g/L
of FeSO.sub.4, and 20 g of the agar. pH values of the improved
Czapek Dox culture plates are 6.0. The improved Czapek Dox culture
plates are placed in an incubator for cultivation at the
temperature of between 30 and 35.degree. C. After 5 d cultivation,
a large amount of spores are obtained from the plates,
respectively.
[0057] 3) Preparation of Composite Microecologies
[0058] Strains of Zoogloea sp. HJ1, Pandoraea sp. FLX-1, and
Ophiostoma sp. LLC obtained from high-density fermentation are
evenly mixed and then inoculated on a SSF culture medium for solid
state fermentation. The fermentation temperature is 35.degree. C.,
and the fermentation time is 40 hrs. The SSF culture medium in 3)
comprises a solid state composite comprising between 45 and 50 wt.
% of a wheat bran, between 25 and 30 wt. % of a sawdust, and
between 2.5 and 30 wt. % of and a powdered activated carbon. An
aqueous solution having a volume of between 1 and 2 times of that
of the solid state composite is added to the solid state composite
to yield a mixture. The aqueous solution comprises: 20 g/L of a
yeast extract, 20 g/L of a potato, and 5 g/L of NaCl. A pH value of
the mixture is regulated to be between 6.8 and 7.2. The mixture is
conducted with moist heat sterilization at the temperature of
121.degree. C. for between 30 and 40 min and then cooled to obtain
the SSF culture medium, which is then inoculated with a strain
suspension for cultivation.
[0059] A product obtained from the solid state fermentation is
vacuum dried at a drying temperature of 40.degree. C. for 24 hrs,
and is further ground to yield a powder. The powder is then mixed
with spores of Aspergillus sp. HD-2 and Trichoderma sp. LW-1 at a
weight ratio of 4:1,
EXAMPLE 2
Performance Measurement of the Fungi-Bacteria Composite
Microecologies
[0060] The constructed fungi-bacteria composite microecologies are
performed with biomass and degradation activity stability tests.
The method for testing the biomass is specifically conducted as
follows: 1 g of composite microecologies is inoculated on a
sterilized LB solid culture medium and cultivated at the
temperature of 30.degree. C. for 24 hrs, and the cell number of the
bacteria is then measured; and 1 g of composite microecologies is
inoculated on a sterilized PDA solid culture medium and cultivated
at the temperature of 35.degree. C. for 48 hrs, and the weight of
the fungi is then measured. The measurement of the cell number of
the bacteria adopts dilution smear, that is, conducting gradient
dilution before counting the number of colonies. The measurement of
the fungi biomass adopts dry weight method, that is, mycelia are
scrapped from the PDA culture medium and dried at the temperature
of 80.degree. C. before weighing. The LB solid culture medium
comprises: 10 g/L of a tryptone, 5 g/L of a yeast extract, 10 g/L
of NaCl, and 15 g/L of the agar, and a pH value of the LB solid
culture medium is 7.2. The PDA culture medium comprises: 200 g/L,
of a potato, 20 of glucose (or sucrose), and 20 of the agar, and a
pH value thereof is 6.5.
[0061] It is indicated from the results that the number of
effective live colonies on the LB plate (diluted to 10.sup.-6)
after 24 hrs cultivation is 298; and the number of the effect by
live colonies on the LB plate (diluted to 10.sup.-7) after 48 hrs
is 31. After conversion, the composite microecologies contains
6.08.times.10.sup.8 live bacteria per gram of a dried composite
microecologies. An effective weight increase of the PDA plate is
0.057 g after 24 hrs cultivation and 0.248 g after 48 hrs
cultivation. After conversion, the composite microecologies
contains 0.4296 g of fungi per gram of the dried composite
microecologies.
[0062] The degradation activity stability test is conducted as
follows: 2 g of the fungi-bacteria composite microecologies after 0
h, 1 d, 5 d, 15 d, 30 d, and 45 d storage in the 4.degree. C.
refrigerator are inoculated to sterilized liquid culture media
containing inorganic salts, respectively. The liquid culture media
are added with a-pinene, ethyl acetate, butyl acetate, toluene, and
dichloromethane to enable concentrations thereof reach 50 mg/L
respectively. After that, the culture media are sealed and shaking
cultivated, and removal efficiencies of each pollutant are measured
after 48 hrs and 72 hrs, respectively.
[0063] It is known from results shown in FIGS. 2A-2B, after 48 hrs
cultivation, a large amount of fungi/bacteria grow in the
cultivation solutions that have been stored in the refrigerator for
0 h, 1 d, and 5 d. It is indicated by the degradation activity test
that the composite microecologies has certain removal effects on
the five pollutants. Removal effects (85% in average) on
.alpha.-pinene, ethyl acetate, and butyl acetate are relatively
good, while the removal effects (approximately 60%) on toluene and
dichloromethane are relatively low. The composite rnicroecologics
that are stored for a relatively long period do not present too
strong of the degradation performance after 48 hrs cultivation, and
the removal efficiency of the five pollutants are only between 10%
and 20% After 72 hrs cultivation, the composite microecologics that
have been stored for 15 d, 30 d, and 45 d grow in good conditions
in the cultivation solution, and the removal efficiency of the five
pollutants obviously increases, removal efficiencies of
.alpha.-pinene, ethyl acetate, and ethyl propionate are higher than
80%, and removal efficiencies of toluene and dichloromethane are
higher than 50%. Thus, the constructed fungi-bacteria composite
microecologics possesses relatively good degradation activity at
low temperature, and the longer period the preservation time is,
the longer period it quires to recover the degradation activity
thereof.
Example 3
Fungi-Bacteria Composite Microecologies
[0064] Fungi-bacteria composite microecologies is added with 1.5 kg
of the fungi-bacteria composite microecologies per cubic meter of a
filler, and a certain amount of domesticated activated sludge is
added to inoculate and initiate a reactor. Meanwhile, the
domesticated activated sludge is used as a control group.
.alpha.-pinene, butyl acetate, ethyl acetate, toluene, and
dichloromethane are supplied as waste gas sources, concentrations
of the waste gases are controlled at 50 mg/m.sup.3, and a retention
time thereof is 45 s.
[0065] As illustrated in FIGS. 3-4, it is know that for the reactor
initiated by the sludge mixed with the composite microecologies,
removal efficiencies of ethyl acetate and butyl acetate after 3 d
reach 85% above and remain stable, the removal efficiency of
u-pinene after 4 d reaches 85% above and remains stable, the
removal efficiency of toluene after 6 d reaches 75% above, and the
removal efficiency of dichloromethane reaches 70% above after 8 d
and remains stable. Besides, a biofilm is apparently formed on a
surface of the finer, which indicates that the biofilm formation is
basically successful. For the reactor inoculated only by the
activated sludge, the removal efficiencies of pollutants are very
slow, and the removal efficiencies of a-pinene, ethyl acetate,
butyl acetate, toluene, and dichloromethane after 20 d are
maintained at 80%, 85%, 85%, 65%, and 50%, which indicates the
biofilm is basically formed. Thus, the bioreactor inoculated with
the domesticated activated sludge mixed with the composite
icroecologics is adapted to obviously shorten the time for biofilm
formation, and the removal efficiencies of different pollutants
thereof are higher than the reactor inoculated only by activated
sludge.
[0066] While particular embodiments of the invention have been
shown and described, it will be obvious to those skilled in the art
that changes and modifications may be made without departing from
the invention in its broader aspects, and therefore, the aim in the
appended claims is to cover all such changes and modifications as
fall within the true spirit and scope of the invention.
Sequence CWU 1
1
51599DNATrichoderma viride 1taggtgaacc tgcggaggga tcattaccga
gtttacaact cccaaaccca atgtgaacca 60taccaaactg ttgcctcggc ggggtcacgc
cccgggtgcg tcgcagcccc ggaaccaggc 120gcccgccgga gggaccaacc
aaactctttt ctgtagtccc ctcgcggacg ttatttctta 180cagctctgag
caaaaattca aaatgaatca aaactttcaa caacggatct cttggttctg
240gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa
ttcagtgaat 300catcgaatct ttgaacgcac attgcgcccg ccagtattct
ggcgggcatg cctgtccgag 360cgtcatttca accctcgaac ccctccgggg
ggtcggcgtt ggggacctcg ggagccccta 420agacgggatc ccggccccga
aatacagtgg cggtctcgcc gcagcctctc ctgcgcagta 480gtttgcacaa
ctcgcaccgg gagcgcggcg cgtccacgtc cgtaaaacac ccaacttctg
540aaatgttgac ctcggatcag gtaggaatac ccgctgaact taagcatatc aataagcgg
5992516DNAAspergillus fumigatus 2cctgcggaag gatcattacc gagtgagggc
cctctgggtc caacctccca cccgtgtcta 60tcgtaccttg ttgcttcggc gggcccgccg
tttcgacggc cgccggggag gccttgcgcc 120cccgggcccg cgcccgccga
agaccccaac atgaacgctg ttctgaaagt atgcagtctg 180agttgattat
cgtaatcagt taaaactttc aacaacggat ctcttggttc cggcatcgat
240gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga
atcatcgagt 300ctttgaacgc acattgcgcc ccctggtatt ccggggggca
tgcctgtccg agcgtcattg 360ctgccctcaa gcacggcttg tgtgttgggc
ccccgtcccc ctctcccggg ggacgggccc 420gaaaggcagc ggcggcaccg
cgtccggtcc tcgagcgtat ggggctttgt cacctgctct 480gtaggcccgg
ccggcgccag ccgacaccca acttta 51631342DNAOphiostoma stenoceras
3aggtataagc aattataccg cgaaactgcg aatggctcat taaatcagtt atcgtttatt
60tgatagtacc ttactacttg gataaccgtg gtaattctag agctaataca tgctgaaaac
120cccgacttcg gaagggatgt atttattaga ttaaaaacca atgcccttcg
gggctccctg 180gtgattcata ataacttctc gaatcgcacg gccttgcgcc
ggcgatggtt cattcaaatt 240tctgccctat caactttcga cggctgggtc
ttggccagcc atggtgacaa cgggtaacgg 300agggttaggg ctcgaccccg
gagaaggagc ctgagaaacg gctactacat ccaaggaagg 360cagcaggcgc
gcaaattacc caatcccgac acggggaggt agtgacaata aatactgata
420cagggctctt ttgggtcttg taattggaat gagtacaatt taattccctt
aacgaggaac 480aattggaggg caagtctggt gccagcagcc gcggtaattc
cagctccaat agcgtatatt 540aaagttgttg cagttaaaaa gctcgtagtt
gaaccttggg cctggctggc cggtccgcct 600caccgcgtgc actggtccgg
ccgggtcttt ccctctgggg agccgcatgc ccttcactgg 660gtgtgtcggg
gaaccaggac ttttactttg aaaaaattag agtgttcaaa gcaggcttat
720gctcggatac attagcatgg aataatagaa taggacgtgc ggttctattt
tgttggtttc 780taggaccgcc gtaatgatta atagggacag tcgggggcat
cagtattcaa ttgtcagagg 840tgaaattctt ggatttattg aagactaact
actgcgaaag catttgccaa ggatgttttc 900attaatcagg aacgaaagtt
aggggatcga agacgatcag ataccgtcgt agtcttaacc 960ataaactatg
ccgactaggg atcggacgat gttatttttt gactcgttcg gcaccttaca
1020cgaaagtaca agtttctggg ttctgggggg agtatggtcg caaggctgaa
acttaaagaa 1080aattgacgga agggcaccac caggggtgga atctgcggct
taatttgact caacacgggg 1140aaactcacca ggtccagaca cgatgaggat
tgacagattg agagctcttt cttgatttcg 1200tgggtggtgg tgcatggccg
ttcttagttg gtggagtgat ttgtctgcct aatcgcgata 1260acgaacgaga
ccttaagctg ctaaatagcc cgcgttgctt tggcagcgcg ctggcttctt
1320agagggacta tccgctcaag cc 134241440DNAZoogloea resiniphila
4ttggggcggc agctttccat gcaagtcgaa cggcagcacg ggcttcggcc tggtggcgag
60tggcgaacgg gtgagtaatg catcggaacg tacccagtcg tgggggataa cgtagcgaaa
120gttacgctaa taccgcatac gtcctgaggg agaaagcggg ggaccgtaag
gcctcgcgcg 180attggagcgg ccgatgtcgg attagctagt tggtggggta
aaggcctacc aaggcgacga 240tccgtagcgg gtctgagagg atgatccgcc
acactgggac tgagacacgg cccagactcc 300tacgggaggc agcagtgggg
aattttggac aatgggcgaa agcctgatcc agccatgccg 360cgtgagtgaa
gaaggccttc gggttgtaaa gctctttcag acggaaagaa atcttctggg
420ctaataccct gggaggatga cggtaccgta agaagaagca ccggctaact
acgtgccagc 480agccgcggta atacgtaggg tgcgagcgtt aatcggaatt
actgggcgta aagcgtgcgc 540aggcggtgat gtaagacaga tgtgaaatcc
ccgggctcaa cctgggaact gcgtttgtga 600ctgcatcact cgagtacggc
agagggaggt ggaattccgc gtgtagcagt gaaatgcgta 660gagatgcgga
ggaacaccga tggcgaaggc agcctcctgg gccagtactg acgctcatgc
720acgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccc
taaacgatgt 780caactagttg ttcggtgagg agactcattg agtaacgcag
ctaacgcgtg aagttgaccg 840cctggggagt acggccgcaa ggttaaaact
caaaggaatt gacggggacc cgcacaagcg 900gtggatgatg tggattaatt
cgatgcaacg cgaaaaacct tacctaccct tgacatgcca 960ggaacttgcc
agagatggct tggtgctcga aagagagcct ggacacaggt gctgcatggc
1020tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc
aacccttgtc 1080attagttgcc atcattaagt tgggcactct aatgagactg
ccggtgacaa accggaggaa 1140ggtggggatg acgtcaagtc ctcatggccc
ttatgggtag ggcttcacac gtcatacaat 1200ggtcggtaca gagggttgcc
aagccgcgag gtggagccaa tcccagaaag ccgatcgtag 1260tccggattgg
agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgcagatca
1320gcatgctgcg gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca
ccatgggagt 1380ggggtttacc agaagtaggt agcttaaccg caaggagggc
gctaccacgt agctcgtccc 144051471DNAPandoraea pnomenusa 5cgccgtggcg
gctgccatta acatgcagtc gaacggcagc acgggtgctt gcacctggtg 60gcgagtggcg
aacgggtgag taatacatcg gaacgtacct tgtagtgggg gatagctcgg
120cgaaagccgg attaataccg catacgctct gaggaggaaa gcgggggacc
ttcgggcctc 180gcgctacaag agcggccgat gtcagattag ctagttggtg
aggtaaaagc tcaccaaggc 240gacgatctgt agctggtctg agaggacgac
cagccacact gggactgaga cacggcccag 300actcctacgg gaggcagcag
tggggaattt tggacaatgg gcgaaagcct gatccagcaa 360tgccgcgtgt
gtgaagaagg ccttcgggtt gtaaagcact tttgtccgga aagaaatcct
420ctgggttaat acctcggggg gatgacggta ccggaagaat aagcaccggc
taactacgtg 480ccagcagccg cggtaatacg tagggtgcaa gcgttaatcg
gaattactgg gcgtaaagcg 540tgcgcaggcg gttttgtaag acggatgtga
aatccccggg cttaacctgg gaactgcatt 600cgtgactgca aggctagagt
atggcagagg ggggtagaat tccacgtgta gcagtgaaat 660gcgtagagat
gtggaggaat accgatggcg aaggcagccc cctgggccaa tactgacgct
720catgcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca
cgccctaaac 780gatgtcaact agttgttggg gattcatttc cttagtaacg
tagctaacgc gtgaagttga 840ccgcctgggg agtacggtcg caagattaaa
actcaaagga attgacgggg acccgcacaa 900gcggtggatg atgtggatta
attcgatgca acgcgaaaaa ccttacctac ccttgacatg 960tacggaatcc
tgctgagagg tgggagtgct cgaaagagaa ccgtaacaca ggtgctgcat
1020ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag
cgcaaccctt 1080gtccttagtt gctacgcaag agcactctaa ggagactgcc
ggtgacaaac cggaggaagg 1140tggggatgac gtcaagtcct catggccctt
atgggtaggg cttcacacgt catacaatgg 1200tcggtacaga gggctgccaa
accgcgaggt ggagctaacc ccagaaaacc gatcgtagtc 1260cggatcgcag
tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc
1320atgtcgcggt gaatacgttc ccgggtcttg tacacaccgc ccgtcacacc
atgggagtgg 1380gttttgccag aagtaggtag cctaaccgca aggagggtgc
ttaccacggc aggattcatg 1440actgggggaa gtcgaatcaa gtgtctgcca c
1471
* * * * *