viouslydescribed (He and Sanford, 2

viously
described (He and Sanford, 2002). The medium was sterilized
by autoclaving following dispensing into sealed glass
containers. The vitamin solution was added from sterile anaerobic
stock solutions after autoclaving.2.1. Cellulosic substrates
To ensure consistency in the testing of cellulose fermentation,
only commercially available cellulosic substrates from the same
delivery were used in this study. Two powdered cellulosic substrates,
Avicel and Solka Floc, were tested in this study. Avicel
PH-101 was purchased from Sigma–Aldrich Co., St. Louis, Missouri.
Solka Floc 200 was acquired from International Fiber Corporation,
North Tonawanda, New York.
2.2. Microorganisms
Clostridium thermocellum (ATCC 35609) and Thermoanaerobacter
pseudethanolicus strain 39E (ATCC 33323) were obtained from the
American Type Culture Collection (Manassas, Virginia). Thermoanaerobacter
sp. strain X514 was originally isolated from the deep
subsurface in the Piceance Basin, Colorado (Roh et al., 2002) and is
maintained in our laboratory culture collection. Strain X514 has
been deposited at the American Type Culture Collection (ATCC
BAA-938). C. thermocellum is cellulolytic (McBee, 1954); but strains
X514 and 39E are non-cellulolytic and strictly fermentative (Roh
et al., 2002; Zeikus et al., 1980).
2.3. Medium formulation and preparation
A defined anaerobic medium was used throughout this study
according to a previously described formula (He et al., 2009). The
medium contained the following (per liter): 10.0 g NaCl, 0.5 g
MgCl26H2O, 0.2 g KH2PO4, 0.3 g NH4Cl, 0.3 g KCl, 0.015 g
CaCl22H2O, 1 mL trace element solution, 1 mL selenium-tungsten
solution, 10 mL vitamin solution, 2.52 g NaHCO3, and 0.05 mg resazurin.
The trace element solution contained the following (per liter):
1.5 g FeCl24H2O, 0.19 g CoCl26H2O, 0.1 g MnCl24H2O, 70 mg
ZnCl2, 6 mg H3BO3, 36mg Na2MoO42H2O, 24 mg NiCl26H2O, and
2 mg CuCl22H2O. The selenium-tungsten solution contained
6mg Na2SeO35H2O per liter, 8 mg Na2WO42H2O per liter, and
0.54 g of NaOH per liter. The vitamin solution contained the following
(per liter): 20 mg biotin, 20 mg folic acid, 100 mg pyridoxine
hydrochloride, 50 mg riboflavin, 50 mg thiamine, 50 mg
nicotinic acid, 50 mg pantothenic acid, 1 mg vitamin B12, 50mg
p-aminobenzoic acid, and 50 mg thioctic acid.
The pH of the medium was adjusted to 7.2 ± 0.1 by purging with
an oxygen-free nitrogen/CO2 gas mix. Anaerobic condition of the
medium was maintained by the addition of sulfide (0.048 g
Na2S9H2O) and cysteine (0.031 g L-cysteine) as reductants as previously
described (He and Sanford, 2002). The medium was sterilized
by autoclaving following dispensing into sealed glass
containers. The vitamin solution was added from sterile anaerobic
stock solutions after autoclaving.
2.4. Growth conditions
Stock cultures of all bacterial strains were stored in 15% glycerol
at 80 C. Inocula were obtained by sub-culturing aliquots of the
frozen stocks in 60-mL serum bottles with 30 ml of boiled degassed
medium closed with butyl rubber stoppers and aluminum
seals. Cultures were incubated in the dark at 60 C without constant
agitation. For routine cultivation, 1% (w/v) of cellobiose (for
the cellulolytic culture C. thermocellum) or glucose (for the noncellulolytic
39E and X514) was added as the only fermentation
substrate in defined medium. Active cultures were maintained by
transferring a 1% inoculum to fresh medium after fermentation
was completed and growth had stopped. Strict anaerobic techniques
were used throughout all experimental manipulations.
Sterile syringes and needles, used for substrate addition and
sampling, were flushed with N2 prior to use.
2.5. Cellulose fermentation
Cellulose fermentation experiments were initiated by a 1% (v/v)
inoculum of log-phase cultures (OD600  0.5) grown on cellobiose
or glucose. Mono-cultures were inoculated with C. thermocellum
only, referred to as CT mono-cultures. Co-cultures were inoculated
with strain X514 or 39E in addition to C. thermocellum, referred to
as CT-X514 or CT-39E co-cultures, respectively. In fermentation
experiments to determine the role of non-cellulolytic Thermoanaerobacter
strains in cellulose fermentation, co-cultures were
established stepwise by first initiating mono-cultures of C. thermocellum
followed by the addition of strain 39E or X514 when soluble
sugar had accumulated. When needed, yeast extract 0.6% (w/v)
was added to the defined medium during medium preparation.
In fermentation experiments where exogenous vitamin B12 was
added to the defined medium, an alternative vitamin solution
was made by omitting vitamin B12 from the formula and used for
the preparation of the defined medium. Subsequently, varying
amounts of vitamin B12 was added before inoculation. All fermentation
experiments were performed in triplicates.
2.6. Analytical procedures
To monitor the production of fermentation end products,
samples (1 mL) from the culture broth were taken periodically
via de

ผลลัพธ์ (ไทย) 1:

viously described and tea 2002) medium was sterilized by autoclaving to pay into a sealed glass container solutions vitamins were added from sterile anaerobic stock solution behind. autoclaving.2.1 surface time to test the cellulose enzyme only in the surface of a ship that is used to study the surface of the powder, two outs and Avicel Solka Floc was tested in this study Avicel PH. -101 -Aldrich purchased from Sigma, St. Louis, Missouri Solka Floc 200 from International fiber Corporation , North Tonawanda, NY 2.2. microbial infection clostridium thermocellum (ATCC 35609) and Thermoanaerobacter Pseudethanolicus species 39E (ATCC 33323) was coming from. a series of American culture type (Manassas, Virginia) Thermoanaerobacter species BSP X 514 was originally extracted from deep beneath the surface in the Piceance Basin, Colorado (Roh et al. 2002) and maintained in collections. News culture laboratory strains X 514 was deposited at the American Type culture collection. (ATCC BAA-938) C. Thermocellum a cellulolytic (McBee, 1954); but the strain X 514 and 39E are fermented and strictly non-cellulolytic Twitter et al. Two thousand and two Zeikus et al. The 1,980th) 2.3. Central determination and is prepared to use intermediaries. the determined throughout the study , according to the formula described previously (he et al. 2009) to the following address (per liter): 10.0 g NaCl, 0.5 g MgCl2 6H2O, KH2PO4 0.2 g, 0.3 g NH4Cl,. 0.3 g KCl, 0.015 g CaCl2 2H2O solution micronutrients 1 mL, 1 mL selenium tungsten solution. Troubleshooting Vitamins 10 ml. 2.52 g NaHCO3 and resazurin 0.05 mg modified elements Following (l) the following: 1.5 G FeCl2 4H2O, 6H2O COCl 2 00:19 G, 0.1 G MnCl2 4H2O, 70 mg ZnCl2, H3BO3 6 mg 2H2O Na2MoO4. 36 mg, 24 mg NiCl2 6H2O and 2 mg CuCl2 2H2O solution tungsten selenium contained 6 mg Na2SeO3 5H2O per liter 8 mg Na2WO4 2H2O per liter and 0.54 g of NaOH per liter solution of vitamins are the following (per liter): Boca Rio. Martin, 20 mg folic acid, 20 mg pyridoxine 100 mg hydrochloride at 50 mg riboflavin thiamine 50 mg, 50 mg acid, nicotinic, 1 mg of vitamin B12 acid pantothenate Nick. 50 mg, 50 mg acid, p-aminobenzoic acid and thioctic 50 mg pH of updates to 7.2 ± 0.1 by washing with an oxygen-free, nitrogen / CO2 gas mix is a condition of the central part of the sulfide (0.048 g Na2S. 9H2O) and rectum (0.031 g L-cysteine) is reductants as previously described and tea. 2002) medium was sterilized by autoclaving to pay into a sealed glass container solutions vitamins were added from sterile anaerobic stock solution behind. autoclaving 2.4 The conditions for the growth of each strain of bacteria culture products were stored in glycerol 15% at 80 C. Inocula by sub-culturing aliquots of the frozen shares in serum 60 ml bottle containing 30 ml of boiling. degassed closed with a rubber butyl and aluminum middle C was incubated culture in the dark at 60 C is not constant agitation for planting a 1% (w / v) of cellobiose (for thermocellum culture cellulolytic C. .) or glucose (for Noncellulolytic 39E and X 514) only added to the fermentation substrate in defining the management culture by transferring inoculum 1% fresh medium after fermentation is complete. And stop growing techniques, rigorous control experiments all syringes and needles. For added texture And the sample was rinsed with N2 before use 2.5. Cellulose fermentation, the fermentation of cellulose are starting 1% (v / v) inoculum. The recording process of the culture (OD600 0.5) planted cellobiose or glucose mono-cultures inoculated with C. thermocellum otherwise known as mono culture CT- cultural experience. inoculated strains of C. thermocellum X 514 or 39E also referred to as CT - X 514 or CT-39E, respectively, co-culture fermentation experiments to determine the role. Thermoanaerobacter cellulolytic not breed in fermenting cellulose. Culture was founded The first initial mono culture of C. thermocellum stepwise by adding or 514 X 39E of the species in water has accumulated sugar when necessary, yeast extract 0.6% (w / v) were given a moderate increase moderately during the preparation of the trial. fermented vitamins B12, which is outside the designated media added. Alternative solutions vitamins do Without the vitamin B12 and the formulas used for the preparation of media defined differently in later times the amount of vitamin B 12 were added before inoculation , fermentation, all experiments performed in. Triplicates 2.6 Diagnostic procedures to monitor the production of fermentation end products samples (1 mL) of broth cultures taken periodically to the desktop.

ผลลัพธ์ (ไทย) 2:

viously
described (He and Ford, 2002), the media has been sterilized
by steam sterilization to pay into a sealed glass
container solution added vitamins from anaerobic barren
solution after stock. autoclaving.2.1 cellulose surface
to meet the test of the fermentation of cellulose
only commercially available surface of the cellulose
shipments were used in this study, two surface cellulose powder,
Avicel? and Solka Floc?, has. tested in this study Avicel
PH-101, purchased from Sigma-Aldrich Co., St. Louis.
Solka Floc obtained from International fiber Corporation, 200
North David, New York.
2.2 microorganism
Clostridium thermocellum (ATCC 35609) and Thermoanaerobacter.
strain pseudethanolicus 39E (ATCC 33323) was obtained from
the American Type Culture Collection. (Manassas, VA) Thermoanaerobacter
SP strain X514 was separated from the deep
subsurface of the watershed Piceance Colorado (TA et al., 2002) and has been
preserved in a collection Culture Our ​​lab strain X514 has.
the American Type culture collection deposited. (ATCC
BAA-938) C. thermocellum cellulose (McBee, 1954); however, strains
X514 and will not break down cellulose and ferment 39E strictly (TA
, et al., 2 002 ;. Zeikus, et al, 1 980).
2.3 in. define medium and arrangements
defined medium anaerobic was used in this study, all
the formulas as previously described (he et al., 2009)
medium is as follows (per liter). : 10.0 g of sodium chloride, 0.5 G
? MgCl2 6H2O 0.2 g KH2PO4 0.3 g NH4Cl 0.3 g of potassium chloride, 0.015 g
? CaCl2 2H2O, 1 mL, 1 mL of the solution to elemental selenium, tungsten
solution. 10 ml vitamin solution 2.52 grams NaHCO3 and 0.05 mg resazurin
solution elements are as follows. (L):
1.5 g FeCl2 4H2O 0.19 g COCl2 6H2O, 0.1 g MnCl2 4H2O 70 mg?
ZnCl2, 6 mg H3BO3, 36mg Na2MoO4? 2H2O 24 mg NiCl2? 6H2O and
2 mg CuCl2? 2H2O solution selenium tungsten
6mg Na2SeO3? 5H2O per liter, 8 mg Na2WO4? 2H2O per liter and
0.54 grams per liter NaOH solution vitamins are as follows
(per liter): 20 mg biotin 20. mg folic acid, 100 mg of pyridoxine
hydrochloride, 50 mg vitamin riboflavin 50 mg 50 mg
acid, nicotinic 50 mg acid pantothenic 1 mg vitamin B 12, 50mg
and 50 P -aminobenzoic acid. acid mg thioctic.
the pH of the medium was adjusted to 7.2 ± 0.1 by washing with
mixed gas / CO2, nitrogen, oxygen-free anaerobic conditions of the
medium being preserved by the addition of sulphide (0.048 g
Na2S? 9H2O) and cysteine ​​(0.031 g L-cysteine) is reductants previously
described. (He and Ford, 2002), the media has been sterilized
by steam sterilization to pay into a sealed glass
container solution added vitamins from anaerobic barren
solution after stock. autoclaving.
2.4 growth conditions
Stock. Bacterial cultures were stored in 15% glycerol
at? 80? C Inocula from small aliquots feast of
shares frozen bottles of serum 60 ml to 30 ml degassed boiled
Central closed with a rubber butyl and aluminum
seals cultures were incubated in the dark at 60. C without constant
agitation for regular cultivation, 1% (w / v) cellobiose (for
cell culture Lucie. thermocellum) or glucose (for Noncellulolytic
39E and X514) is recorded as a fermentation
substrate in the media, the culture, the use of care by
transferring the infection of 1% to the Live After fermentation
is complete and thrive. stop technique using oxygen rigorous
been used throughout the routine experiment.
sterile syringes and needles used for adding texture and
sampling were washed with N2 before use.
2.5 cellulose fermentation
of cellulose fermentation was initiated. by 1% (v / v)
inoculum of culture log phase (OD600? 0.5) grown on cellobiose
or glucose mono-cultures were infected with C. thermocellum
only called CT mono-cultural co-cultures were inoculated
with the strain X514 or 39E in addition to C. thermocellum called
a CT-X514 or CT-39E, respectively, co-culture fermentation
experiments to determine the role of non-cellulose. Thermoanaerobacter
strains to ferment cellulose co-culture was
established as the first step by starting a series of mono-culture. thermocellum
along with the addition of stress or 39E X514 when soluble
sugar accumulation when needed, yeast extract 0.6% (w / v)
was added to the medium, as defined in the process of preparing the media.
In the fermentation of vitamin B12. the exterior has been
enhanced medium that is defined as a solution vitamins choice
was made ​​by omitting vitamin B12 from the formula used for the
preparation of the medium defined later, different
doses of the vitamin. B12 was added prior to the vaccination of all birds
in the experiment was conducted. Triplicates.
2.6 analysis
to monitor the production of end products of fermentation
samples (1 ml) of broth cultures were periodically
through.