High concentration methanol tolerant methanotroph and its application

Abstract

The present invention discloses a high concentration methanol tolerant methanotroph and its application, a accession number of the methanotroph in China General Microbiological Culture Collection Center being CGMCC No. 9873, deposit date being Oct. 29, 2014, category names being Methylomonas sp. ZR1. The methanotroph Methylomonas sp. ZR1 disclosed by the present invention can grow rapidly by using methane, and can tolerate with high concentration of methanol. The methanotroph Methylomonas sp. ZR1 can use C.sub.1 compounds such as methane and methanol to produce high value-added products such as carotenoids and polysaccharides, which has high application prospect in biological transformation of one-carbon chemistry.

Claims

1. A method for producing carotenoids, comprising (a) culturing a high-concentration-methanol-tolerant methanotroph named Methylomonas strain ZR1, having the accession number of CGMCC No. 9873 in the China General Microbiological Culture Collection Center in fermentation in a medium comprising at least 1% methanol or in a vessel comprising a gaseous mixture that is at least 15% methane, and (b) harvesting the carotenoids made in step (a).

2. The method of claim 1, wherein the fermentation is carried out at a temperature of 20-30 C.

3. The method of claim 2, wherein the fermentation temperature is 25 C.

4. The method of claim 3, wherein when the methanol is used as the substrate, the mass percent concentration of the methanol in the fermentation culture medium is less than or equal to 3.5%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a growth curve of the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 disclosed by the present invention with methane as substrate.

(2) FIG. 2 is a growth comparison chart of the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 disclosed by the present invention with different concentration of methanol as substrate.

(3) FIG. 3 is an absorption spectrum by full wave scanning of pigment extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce carotenoids with methane as substrate in embodiment 4 of the present invention.

(4) FIG. 4 is an absorption spectrum by full wave scanning of pigment extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce carotenoids with methane as substrate in embodiment 5 of the present invention.

(5) FIG. 5 is an absorption spectrum by full wave scanning of pigment extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce carotenoids with methane as substrate in embodiment 6 of the present invention.

(6) FIG. 6 is a HPLC spectrum of hydrolysate extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce polysaccharide with methane as substrate in embodiment 7 of the present invention.

(7) FIG. 7 is a HPLC spectrum of hydrolysate extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce polysaccharide with methane as substrate in embodiment 8 of the present invention.

(8) FIG. 8 is a HPLC spectrum of hydrolysate extracted from fermentation product, which is produced by the high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 during fermenting to produce polysaccharide with methane as substrate in embodiment 9 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention will be further described in detail with the accompanying drawings, to make those skilled in the art can implement the invention according to the text of the specification.

(10) If there is no special instruction, experimental methods used in the following embodiments are conventional methods.

(11) If there is no special instructions, materials and reagents used in the following embodiments can be obtained from commercial sources.

(12) The solvent in the NMS culture medium is water, and the solute in the NMS culture medium is KNO.sub.3 1 g/L, KH.sub.2PO.sub.4 0.717 g/L, Na.sub.2HPO.sub.4 0.272 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCl.sub.2.6H.sub.2O 0.2 g/L, sodium iron EDTA 0.005 g/L and 1 ml of trace element solution; when methane is used as carbon source or substrate, the methane accounts for 1550% of gas and air accounts for 5080% of gas; when methanol is used as carbon source or substrate, the adding content of the methanol is 1.03.5%.

(13) Solid medium is prepared by adding 15 g of agar per liter of the NMS culture medium.

(14) The trace element solution is prepared by adding EDTA 0.5 g, FeSO.sub.4.7H.sub.2O 0.2 g, H.sub.3BO.sub.3 0.03 g, ZnSO.sub.4.7H.sub.2O 0.01 g, MnCl.sub.2.4H.sub.2O 0.003 g, CoCl.sub.2.6H.sub.2O 0.02 g, CuSO.sub.4.5H.sub.2O 0.1 g, NiCl.sub.2.6H.sub.2O 0.002 g and Na.sub.2MoO.sub.4 0.003 g per liter of water.

Embodiment 1

(15) Screening Method of Methanotroph Methylomonas sp. ZR1

(16) The methanotroph Methylomonas sp. ZR1 is screened from wetland of sludge, 1 g sample of wetland of sludge is inoculated directly into 250 ml anaerobic bottle containing 100 ml of the NMS culture medium, 100 ml of methane is added to the anaerobic bottle as carbon source to make methane account for 30% of gas phase in the anaerobic bottle. The methanotroph is cultured in shake at 25 C. and at 180 rpm until that OD.sub.600 of the NMS culture medium can hold steady. The methanotroph is inoculated into fresh culture medium with 1% inoculation, and is subcultured 3 times; the liquid culture is diluted and coated on coated tablet, and the coated tablet is placed in air with methane accounting for 30%. A large amount of the methanotroph can be obtained by using the screening method, wherein the methanotroph Methylomonas sp. ZR1 which generates bright orange colonies grows fastest and generates the largest colony.

(17) Genome of the methanotroph is extracted, and 16S rRNA gene sequences of the genome are amplified and sequenced; polynucleotide sequences of 16S rRNA are the same as polynucleotide sequence of SEQ ID No:1; with the 16S rRNA gene sequences compared with the Blast results in NCBI, the comparison results show that the 16S rRNA gene sequences have a 97.9% similarity with Methylomonas methanica S1.sup.T; according to the comparison results combined with physiological and biochemical characteristics of methanotroph Methylomonas sp. ZR1, the methanotroph is assigned as Methylomonas sp ZR1.

Embodiment 2

(18) Methanotroph Methylomonas sp. ZR1 Growing with Methane as Substrate

(19) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 28 C. and at 180 rpm; samples of fermentation broth are taken and cell concentration of the samples is measured every day; as shown in the FIG. 1, the cell concentration increases with the extension of fermentation time, which shows the number of the methanotroph Methylomonas sp. ZR1 gradually increases and the methanotroph Methylomonas sp. ZR1 grow well in the NMS liquid culture medium with methane as substrate.

Embodiment 3

(20) Methanotroph Methylomonas sp. ZR1 Growing with Methanol as Substrate

(21) Single colony of the methanotroph Methylomonas sp. ZR1 is obtained by the methanotroph Methylomonas sp. ZR1 growing with methanol as substrate, the single colony of the methanotroph Methylomonas sp. ZR1 is inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 28 C. and at 180 rpm. After being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, inoculated into 100 ml of fresh NMS culture medium with 5% inoculation, wherein the content of methanol in the fresh NMS culture medium is respectively 12.5 g/L, 15 g/L, 17.5 g/L, 20 g/L, 22.5 g/L, 25 g/L, 27.5 g/L, 30 g/L, 32.5 g/L and 35 g/L, and be cultured in shake for 3 days at 25 C. and at 180 rpm in a 250 ml shake flasks; cultivation results are shown in FIG. 2, which show the methanotroph Methylomonas sp. ZR1 can grow and reproduce well in the culture medium with different concentration of methanol.

Embodiment 4

(22) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids with Methane as Substrate

(23) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 25 C. and at 180 rpm, after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, and inoculated into fresh NMS culture medium with 5% inoculation, fermenting in column reactor at 28 C.; after being cultured for 2 days, fermentation broth of single colony of the methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; the methanotroph Methylomonas sp. ZR1 is collected by centrifugation for 10 mins at 8000 rpm; the methanotroph Methylomonas sp. ZR1 collected is washed twice with distilled water, vacuum freeze-dried, then dried powder of the methanotroph is obtained; the dried powder of the methanotroph Methylomonas sp. ZR1 is weighed, and is extracted 3 times with methanol solution whose volume is 10 times the volume of dried powder of the methanotroph Methylomonas sp. ZR1, then orange extraction solution of pigment is obtained; the orange extraction solution of pigment is filtered, and spin dried by a rotary evaporation apparatus, then red pigment is obtained.

(24) The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid

(25) The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 l of the pigment solution was taken, diluted with chloroform to 0.5 ml, and being added with a few drops of concentrated sulfuric acid; then the pigment solution turns from red to blue-green, which shows that the red pigment extracted is carotenoids.

(26) Full wave scanning of the red pigment is taken; 300 l of the pigment solution is taken, and is added into a quartz 96-well plate; while 300 l of dichloromethane solution is kept as control, the pigment solution is scanned from 220 nm to 700 nm, the scan results are shown in FIG. 3.

(27) The scan results show a three-finger peak which is a typical characteristic peak of carotenoid appears at around 500 nm, which also shows that the red pigment extracted is carotenoids.

Embodiment 5

(28) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids by Fermentation with Methane as Substrate

(29) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 30 C. and at 180 rpm; after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, and inoculated into fresh NMS culture medium with 5% inoculation, fermenting in column reactor at 28 C., and after being cultured for 5 days, fermentation broth of single colony of methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; the methanotroph Methylomonas sp. ZR1 is collected by centrifuging for 10 mins at 8000 rpm; the methanotroph Methylomonas sp. ZR1 collected is washed twice with distilled water, vacuum freeze-dried, then dried powder of the methanotroph Methylomonas sp. ZR1 is obtained; the dried powder of the methanotroph Methylomonas sp. ZR1 is weighed, extracted 3 times with methanol solution whose volume is 10 times of dried powder of the methanotroph Methylomonas sp. ZR1, then orange extraction solution of pigment is obtained; the orange extraction solution of pigment is filtered, and is dried by the rotary evaporation apparatus, then red pigment is obtained.

(30) The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid

(31) The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 l of the pigment solution was taken, diluted with chloroform to 0.5 ml, and being added with a few drops of concentrated sulfuric acid, then the pigment solution turns from red to blue-green, which shows that the red pigment extracted is carotenoids.

(32) Full wave scanning of the red pigment is taken, 300 l of the pigment solution is taken, and is added into quartz 96-well plate; while 300 l of dichloromethane solution is kept as control, the pigment solution is scanned from 220 nm to 700 nm, the scan results are shown in FIG. 4,

(33) The scan results show a three-finger peak which is a typical characteristic peak of carotenoid appears at around 500 nm, which also shows that the red pigment extracted is carotenoids.

Embodiment 6

(34) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids with Methane as Substrate

(35) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 28 C. and at 180 rpm, after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, and inoculated into fresh NMS culture medium with 5% inoculation, fermenting in column reactor is conducted at 28 C., and after being cultured for 10 days, fermentation broth of single colony of methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; the methanotroph is collected by centrifuging for 10 mins at 8000 rpm; the methanotroph collected is washed twice with distilled water, vacuum freeze-dried. then is dried powder of the methanotroph is obtained; dried powder of the methanotroph is weighed, extracted 3 times with methanol solution whose volume is 10 times the volume of dried powder of the methanotroph, then orange extraction solution of pigment is obtained; the orange extraction solution of pigment is filtered, and is dried by the rotary evaporation apparatus, then red pigment is obtained.

(36) The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid

(37) The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 l of the pigment solution was taken, diluted with chloroform to 0.5 ml, and being added with a few drops of concentrated sulfuric acid, then the solution turns from red to blue-green, which shows that the red pigment extracted is carotenoids.

(38) Full wave scanning of the red pigment is taken, 300 l of the pigment solution is taken, and added to a quartz 96-well plate, while 300 l of dichloromethane solution is kept as control, the pigment solution is scanned from 220 nm to 700 nm, the scan results are shown in FIG. 5.

(39) The scan results show a three-finger peak which is a typical characteristic peak of carotenoid appears at around 500 nm, which also shows that the red pigment extracted is carotenoids.

Embodiment 7

(40) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate

(41) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 20 C. and at 180 rpm, after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, and inoculated into fresh NMS culture medium with 5% inoculation, fermenting in column reactor at 20 C., and after being cultured for 3 days, fermentation broth of single colony of methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; supernatant of the orange viscous liquid is collected by centrifuging for 10 mins at 8000 rpm; volume of the supernatant collected is condensed 10 times through using a rotary evaporator; the supernatant condensed is added with 95% ethanol to make the concentration of ethanol reach 70%, then the supernatant is remained at 4 C. overnight; after being centrifuged for 10 mins at 5000 rpm, supernatant is collected and is washed by ethanol, acetone and petroleum ether in turn, and being dried at 50 C. in an oven, then EPS raw material (polysaccharide) is obtained. The EPS raw material is re-dissolved in distilled water, and then is added into a dialysis bag whose cutoff molecular weight is 3.5 KDa. After being dialyzed for 3 days, and being desalted, polysaccharide solution desalted is obtained. The polysaccharide solution desalted is added with a Sevage solution of the same volume (the volume ratio of chloroform to n-butanol is 4 to 1), and swinging violently for 5 mins, centrifugating for 10 mins at 8000 rpm, collecting the upper polysaccharide solution, then removing protein 68 times repeatedly. The deproteinized polysaccharide solution is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.

(42) Qualitative Reaction of Polysaccharide Solution

(43) The Free Polysaccharide Analysis of Polysaccharide Solution

(44) 5 mg of dry polysaccharide samples are dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; reducing sugars in the polysaccharide solution are determined by using 3,5-dinitrosalicylic acid, then measurement results show that the polysaccharide solution extracted do not contain free reducing polysaccharides.

(45) MoLish Reaction

(46) 5 mg of dry polysaccharide samples are dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 L of the polysaccharide solution is added into a clean glass test tube, and 100 l of 6% phenol is added into the clean glass test tube, and 1 ml of concentrated sulfuric acid is added into the clean glass test tube, then color reacting solution of the dry polysaccharide samples is obtained; water is used as a negative control, and glucose is used as a positive control; the solution of the dry polysaccharide samples and the solution of the glucose all show yellow, color of the water do not change.

(47) HPLC Analysis of Hydrolysate of Polysaccharide

(48) 5 mg of dry polysaccharide samples are dissolved in 1 ml of TFA aqueous solution whose concentration is 4 M; after being hydrolyzed for 8 h at 115 C., hydrolysate of polysaccharide is obtained; HPLC analysis of the hydrolysate of polysaccharide is taken; the HPLC analysis is taken by using BioRad42A column, with ultrapure water as mobile phase, at a flow rate of 0.6 ml/min and at 55 C.; the HPLC analysis is taken by Refractive Index Detector, then HPLC analysis results are shown in FIG. 6.

(49) The HPLC analysis results show that one peak of the hydrolysate of polysaccharide appears at 6.2 min, which is consistent with the time that peak of glucosamine standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 16.2 min, which is consistent with the time that peak of glucose standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 17.8 min, which is consistent with the time that peak of mannose standard sample appears. The HPLC analysis results show the polysaccharides which are produced by the methanotroph Methylomonas sp. ZR1 are mainly heteropolysaccharide which is consisted of glucosamine, glucose and mannose.

Embodiment 8

(50) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate

(51) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source). to culture at 25 C. and at 180 rpm; after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reaches 0.7, and inoculated into fresh NMS culture medium with 5% inoculation; fermenting in column reactor at 28 C., and after being cultured for 6 days, fermentation broth of single colony of methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; supernatant of the orange viscous liquid is collected by centrifuging for 10 mins at 8000 rpm; volume of the supernatant collected is condensed 10 times through using a rotary evaporator; the supernatant condensed is added with 95% ethanol to make the concentration of ethanol reach 70%, then the supernatant is remained at 4 C. overnight; after being centrifuged for 10 mins at 5000 rpm, supernatant is collected, and is washed by ethanol, acetone and petroleum ether in turn, and being dried at 50 C. in an oven, then EPS raw material (polysaccharide) is obtained. The EPS raw material is re-dissolved in distilled water, and then is added into a dialysis bag whose cutoff molecular weight is 3.5 KDa, and after being dialyzed for 3 days, and being desalted, then polysaccharide solution desalted is obtained. The polysaccharide solution desalted is added with a Sevage solution of the same volume (the volume ratio of chloroform to n-butanol is 4 to 1), and swinging violently for 5 mins, centrifugating for 5 mins at 8000 rpm, collecting the upper polysaccharide solution, then removing protein 68 times repeatedly. The polysaccharide solution removed protein is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.

(52) Qualitative Reaction of Polysaccharide Solution

(53) The Free Polysaccharide Analysis of Polysaccharide Solution

(54) 5 mg of dry polysaccharide samples are dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; reducing sugars in the polysaccharide solution are determined by using 3,5-dinitrosalicylic acid, then the measurement results show that the polysaccharide solution extracted do not contain free reducing polysaccharides.

(55) MoLish Reaction

(56) 5 mg of dry polysaccharide samples is dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 L of the polysaccharide solution is added into a clean glass test tube, then 100 l of 6% phenol is added into the clean glass test tube, and 1 ml of concentrated sulfuric acid is added into the clean glass test tube, then color reacting solution of the dry polysaccharide samples is obtained; water is used as a negative control, and glucose is used as a positive control; the solution of the dry polysaccharide samples and the solution of the glucose all show yellow, color of the water do not change.

(57) HPLC Analysis of Hydrolysate of Polysaccharide

(58) 5 mg of dry polysaccharide samples are dissolved in 1 ml of TFA aqueous solution whose concentration is 4 M; after being hydrolyzed for 8 h at 115 C., hydrolysate of polysaccharide is obtained; HPLC analysis of the hydrolysate of polysaccharide is taken; the HPLC analysis is taken by using BioRad42A column, with ultrapure water as mobile phase, at a flow rate of 0.6 ml/min and at 55 C.; the HPLC analysis is taken by Refractive Index Detector then HPLC analysis results are shown in FIG. 7.

(59) The HPLC analysis results show one peak of the hydrolysate of polysaccharide appears at 6.2 min, which is consistent with the time that peak of glucosamine standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 16.2 min, which is consistent with the time that peak of glucose standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 17.8 min, which is consistent with the time that peak of mannose standard sample appears. The HPLC analysis results show the polysaccharides which are produced by the methanotroph Methylomonas sp. ZR1 are mainly heteropolysaccharide, which includes glucosamine glucose and mannose.

Embodiment 9

(60) Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate

(61) Single colony of the methanotroph Methylomonas sp. ZR1 formed on the plate is picked, inoculated into 30 ml of NMS liquid culture medium (using methane as carbon source) to culture at 28 C. and at 180 rpm, after being cultured for 2 days, OD.sub.600 of the NMS liquid culture medium reached 0.7, and inoculated into fresh NMS culture medium with 5% inoculation, fermenting in column reactor at 28 C., and after being cultured for 10 days, fermentation broth of single colony of methanotroph Methylomonas sp. ZR1 appears to be orange viscous liquid; supernatant of the orange viscous liquid is collected by centrifuging for 10 mins at 8000 rpm; volume of the supernatant collected is condensed 10 times through using a rotary evaporator; the supernatant condensed is added with 95% ethanol to make the concentration of ethanol reach 70%, then the supernatant is remained at 4 C. overnight; after being centrifuged for about 10 mins at 5000 rpm, supernatant is collected, and is washed by ethanol, acetone and petroleum ether in turn, and being dried at 50 C. in an oven, then EPS raw material (polysaccharide) is obtained. The EPS raw material is re-dissolved in distilled water, and then is added into a dialysis bag whose cutoff molecular weight is 3.5 KDa. After being dialyzed for 3 days, and being desalted, then polysaccharide solution desalted is obtained. The polysaccharide solution desalted is added with a Sevage solution of the same volume (the volume ratio of chloroform to n-butanol is 4 to 1), and swinging violently for 5 mins, centrifugating for 10 mins at 8000 rpm, collecting the upper polysaccharide solution, then removing protein 68 times repeatedly. The deproteinized polysaccharide solution is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.

(62) Qualitative Reaction of Polysaccharide Solution

(63) The Free Polysaccharide Analysis of Polysaccharide Solution

(64) 5 mg of dry polysaccharide samples are dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; reducing sugars in the polysaccharide solution are determined by using 3,5-dinitrosalicylic acid, then measurement results show that the polysaccharide solution extracted does not contain free of reducing polysaccharides.

(65) MoLish Reaction

(66) 5 mg of dry polysaccharide samples is dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 L of the polysaccharide solution is added to a clean glass test tube, and 100 l of 6% phenol is added into the clean glass test tube, and 1 ml of concentrated sulfuric acid is added into the clean glass test tube, then color reacting solution of the dry polysaccharide samples is obtained; water is used as a negative control, and glucose is used as a positive control; the solution of the dry polysaccharide samples and the solution of the glucose all show yellow, color of the water do not change.

(67) HPLC Analysis of Hydrolysate of Polysaccharide

(68) 5 mg of dry polysaccharide samples dissolved in 1 ml of TFA aqueous solution whose concentration is 4 M, after being hydrolyzed for 8 h at 115 C., hydrolysate of polysaccharide is obtained; HPLC analysis of the hydrolysate of polysaccharide is taken. the HPLC analysis is taken by using BioRad42A column, with ultrapure water as mobile phase, at a flow rate of 0.6 ml/min, at 55 C.; the HPLC analysis is taken by Refractive Index Detector, then HPLC analysis results are shown in FIG. 8.

(69) The HPLC analysis results show that one peak of the hydrolysate of polysaccharide appears at 6.2 min, which is consistent with the time that peak of glucosamine standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 16.2 min, which is consistent with the time peak of glucose standard sample appears, and another peak of the hydrolysate of polysaccharide appears at 17.8 min, which is consistent with the time peak of mannose standard sample appears. The HPLC analysis results show the polysaccharides which are produced by the methanotroph Methylomonas sp. ZR1 are mainly heteropolysaccharide, which includes glucosamine, glucose and mannose.

(70) Although the embodiments of the present invention have been disclosed above, but it is not limited to the use of the specification and embodiments listed. It can be applied to various fields suitable for the present invention. Those skilled in the art can easily modify. Therefore, without departing from the general concept of the scope defined by the claims and the equivalents, the present invention is not limited to the specific details and illustrations herein illustrated and described herein.