# Patent

## Title
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 C1 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.

## Background
The present invention incorporates by reference the material in the ASCII text file whose name is sample sequence listing, the date of creating the ASCII text file is 29 Dec. 2015, and the size of the ASCII text file is 2552 bytes.
The methylomonas referred to as SP.ZR1 has the China General Microbiological Culture Collection Center (CGMCC) designation number CGMCC NO. 9873, having been deposited on Oct. 29, 2014.
The address of CGMCC: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing
The telephone number:0086-10-64807355
The e-mail: cgmcc@im.ac.cn
FIELD OF THE INVENTION
The present invention relates to the art of application technology about microorganisms, and particularly relates to a high concentration methanol tolerant methanotroph and its application.
DESCRIPTION OF THE RELATED ART
The world possesses a huge amount of methane, which is mainly in Siberia Marsh (about 8 ten billion tons), in the Polar Ice Sheet (about five hundred billion tons) and in the Sea Bed (about 2.5 to 10 megatons), meanwhile, there are a lot of unconventional sources of methane on earth, such as coalbed methane, landfill gas, biogas, marine methane hydrates and methane recovered from the coke oven gas and refinery gas. Methane is a greenhouse gas whose warming power of the atmosphere is 23 times stronger than carbon dioxide. Currently methane is used as fuel by human primarily. Converting methane into higher value chemicals and liquid fuels can provide significantly economic value, and environmental and strategic interests.
Currently conversion process of methane is indirect in industry, which mainly includes three steps. They are complex and multistage processes, and are operated under severe conditions such as: steam reforming at 800 ̃1000° C., and 20 ̃30 ATM; partly oxidizing at 1200 ̃1500° C. and methanol synthesizing at 200 ̃300° C. and 50 ̃100 ATM. While microorganisms can convert methane into various metabolic intermediates with different carbon chain length at room temperature and atmospheric pressure, a biotechnology of converting microorganisms into methane has good potential and prospect.
Commercial bioconversion of methane has gone through three stages: 1) producing single cell protein, 2) using methane monooxygenase to produce ethylene oxide, and 3) biodegradating chloride contaminants. Meanwhile, only using methane monooxygenase to produce ethylene oxide has achieved a certain economic benefits until now. An important factor of limiting the industrialization of converting methane by using microorganisms is low growth rate of the microorganisms.
Methanol is the main product of methane conversion process, and is the main product of coal chemical industry. Methanol is also the basic material of C1 chemical industry. Methanol has wide raw material sources, and its biggest usage is to produce other oxygen-containing organic chemicals, such as formaldehyde, acetic acid and ether. The research for biological conversion of methanol is still at the laboratory stage. Producing single cell protein by fermenting which uses methanol as substrate is the most typical application of biotechnology in the development of methanol derived product. Methanol can be directly used by methanotroph, but methanol has a relatively strong physiological toxicity to methanotroph. Methanotrophs which have been reported show poor ability to tolerate methanol. For example, Methylomonas lenta exhibits sensitiveness to culture medium added with 0.1% to 0.5% methanol. The methanotroph which has been mutagenized can tolerate 2.4% methanol, but shows poor stability. While the methanotroph disclosed by the present invention which is wild without being mutagenized can still grow when the concentration of methanol in culture medium reaches 3.5%.
At present, the patents about methanotroph in China mainly relate to cultural method of methanotroph, application of methanotroph in wastewater treatment, production of single cell protein and production of methanol. Parts of the patents have disclosed producing PHA by fermenting of methanotroph.
The methanotroph disclosed by the present invention can grow efficiently using methane, and can tolerate to high concentration of methanol. A good foundation is established for using the methanotroph to ferment methane or methanol to produce high value-added products such as carotenoids and polysaccharides.
SUMMARY OF THE INVENTION
One purpose of the present invention is to disclose a high concentration methanol tolerant methanotroph. The methanotroph disclosed by the present invention is screened from wetlands of sludge, and the methanotroph can grow rapidly by using methane, and the methanotroph can tolerate with high concentration of methanol.
Further, one purpose of the present invention is to provide an application of a high concentration methanol tolerant methanotroph. The methanotroph disclosed by the present invention can produce carotenoids in liquid fermentation culture medium with methanol or methane as substrate.
Further, one purpose of the present invention is to provide an application of a high concentration methanol tolerant methanotroph. The methanotroph disclosed by the present invention can produce polysaccharide in liquid fermentation culture medium with methanol or methane.
To achieve the above objects and other objects, the present invention adopts the technical scheme, the present invention provides:
A high concentration methanol tolerant methanotroph named Methylomonas sp. ZR1, an accession number of the methanotroph in China General Microbiological Culture Collection Center of the methanotroph is CGMCC No. 9873, and deposit date of the methanotroph is Oct. 29, 2014.
An application of the high concentration methanol tolerant methanotroph is for producing carotenoids.
Preferably, the carotenoids is produced by fermentation product which is obtained from inoculating and fermenting the methanotroph with methane or methanol as substrate in a fermentation temperature of 20 ̃30° C.
Preferably, the fermentation temperature is 25° C.
Preferably, when the methanol is used as substrate, the mass percent concentration of the methanol in the fermentation culture medium is less than or equal to 3.5%.
An application of the high concentration methanol tolerant methanotroph is for producing polysaccharide.
Preferably, polysaccharides are produced by the fermentation product, which is obtained from inoculating and fermenting of the methanotroph with methane or methanol as substrate in a fermentation temperature of 20 ̃30° C.
Preferably, the fermentation temperature is 25° C.
Preferably, when the methanol is used as substrate, the mass percent concentration of the methanol in the fermentation culture medium is less than or equal to 3.5%.
Preferably, the polysaccharide is heteropolysaccharide which mainly includes glucosamine, glucose and mannose.
Morphological characteristics of colonies of the methanotroph which is disclosed by the present invention are as follows:
circular smooth colonies with central uplift and smooth edges are generated on NMS culture medium, the colonies of the methanotroph show brightly orange and thick, Gram staining of the cells of the methanotroph is negative.
The beneficial effects of the present invention are as follows:
The advantages the methanotroph disclosed by the present invention are as follows:
{circle around (1)} being easy to cultivate: the methanotroph can grow well in NMS culture medium with methane as carbon source, wherein recipe of the NMS culture medium is simple, wherein there is no need to add organic nitrogen into the NMS culture medium;
{circle around (2)} growing rapidly with methane as carbon source: single colony of the methanotroph is picked and inoculated into the NMS culture medium with methane as substrate and with inorganic as nitrogen source; after being cultured for 48 h, the cell concentration of the methanotroph can reach 108 cfu/ml;
{circle around (3)} tolerating with high concentration of methanol: the methanotroph can grow well in the NMS culture medium with methanol as substrate, wherein concentration of methanol is 35 g/L; after being cultured for 3 days, OD600 of the NMS culture medium can reach 2.5;
{circle around (4)} producing carotenoids and polysaccharides with methane and methanol as substrates:
After being cultured for 10 days with methane as substrate, fermentation broth of the methanotroph appears to be bright orange viscous liquid because of carotenoids and polysaccharides produced by the methanotroph;
After being cultured for 3 days with methanol as substrate, fermentation broth of the methanotroph appears to be bright orange viscous liquid because of carotenoids and polysaccharides produced by the methanotroph;
As described above, a high concentration methanol tolerant methanotroph Methylomonas sp. ZR1 is obtained by screening in the present invention, and the methanotroph Methylomonas sp. ZR1 can use methane to grow rapidly, and can use C1 compounds such as methane and methanol to produce high value-added products such as carotenoids and polysaccharides. The methanotroph Methylomonas sp. ZR1 has good potential and prospect in biological transformation of C1 compounds. The methanotroph Methylomonas sp. ZR1 disclosed by the present invention is applied to produce polysaccharide and carotenoids, wherein the producing process is simple and the operation of the producing is easy. The methanotroph Methylomonas sp. ZR1 can grow rapidly by using methane or methanol, and can produce carotenoids and polysaccharides with high added value. Meanwhile the methanotroph Methylomonas sp. ZR1 can tolerate with high concentration of methanol and has a high industrial application value.



BRIEF DESCRIPTION OF THE DRAWINGS
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.
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.
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.
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.
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.
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.
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.
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
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.
If there is no special instruction, experimental methods used in the following embodiments are conventional methods.
If there is no special instructions, materials and reagents used in the following embodiments can be obtained from commercial sources.
The solvent in the NMS culture medium is water, and the solute in the NMS culture medium is KNO3 1 g/L, KH2PO4 0.717 g/L, Na2HPO4 0.272 g/L, MgSO4.7H2O 1 g/L, CaCl2.6H2O 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 15 ̃50% of gas and air accounts for 50 ̃80% of gas; when methanol is used as carbon source or substrate, the adding content of the methanol is 1.0 ̃3.5%.
Solid medium is prepared by adding 15 g of agar per liter of the NMS culture medium.
The trace element solution is prepared by adding EDTA 0.5 g, FeSO4.7H2O 0.2 g, H3BO3 0.03 g, ZnSO4.7H2O 0.01 g, MnCl2.4H2O 0.003 g, CoCl2.6H2O 0.02 g, CuSO4.5H2O 0.1 g, NiCl2.6H2O 0.002 g and Na2MoO4 0.003 g per liter of water.
Embodiment 1
Screening Method of Methanotroph Methylomonas sp. ZR1
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 OD600 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.
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 S1T; 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
Methanotroph Methylomonas sp. ZR1 Growing with Methane as Substrate
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
Methanotroph Methylomonas sp. ZR1 Growing with Methanol as Substrate
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, OD600 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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids with Methane as Substrate
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, OD600 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.
The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid
The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 μl 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.
Full wave scanning of the red pigment is taken; 300 μl of the pigment solution is taken, and is added into a quartz 96-well plate; while 300 μl 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.
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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids by Fermentation with Methane as Substrate
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, OD600 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.
The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid
The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 μl 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.
Full wave scanning of the red pigment is taken, 300 μl of the pigment solution is taken, and is added into quartz 96-well plate; while 300 μl 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,
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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Carotenoids with Methane as Substrate
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, OD600 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.
The Color Reaction of the Red Pigment in Concentrated Sulfuric Acid
The extracted red pigment is re-dissolved in 1 ml of dichloromethane, then pigment solution is obtained; 50 μl 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.
Full wave scanning of the red pigment is taken, 300 μl of the pigment solution is taken, and added to a quartz 96-well plate, while 300 μl 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.
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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate
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, OD600 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 6 ̃8 times repeatedly. The deproteinized polysaccharide solution is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.
Qualitative Reaction of Polysaccharide Solution
The Free Polysaccharide Analysis of Polysaccharide Solution
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.
MoLish Reaction
5 mg of dry polysaccharide samples are dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 μL of the polysaccharide solution is added into a clean glass test tube, and 100 μl 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.
HPLC Analysis of Hydrolysate of Polysaccharide
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.
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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate
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, OD600 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 6 ̃8 times repeatedly. The polysaccharide solution removed protein is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.
Qualitative Reaction of Polysaccharide Solution
The Free Polysaccharide Analysis of Polysaccharide Solution
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.
MoLish Reaction
5 mg of dry polysaccharide samples is dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 μL of the polysaccharide solution is added into a clean glass test tube, then 100 μl 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.
HPLC Analysis of Hydrolysate of Polysaccharide
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.
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
Fermenting Methanotroph Methylomonas sp. ZR1 to Produce Polysaccharide with Methane as Substrate
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, OD600 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 6 ̃8 times repeatedly. The deproteinized polysaccharide solution is concentrated under reduced pressure, freeze-dried, then dry polysaccharide samples are obtained.
Qualitative Reaction of Polysaccharide Solution
The Free Polysaccharide Analysis of Polysaccharide Solution
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.
MoLish Reaction
5 mg of dry polysaccharide samples is dissolved in 1 ml of distilled water, then polysaccharide solution is obtained; 200 μL of the polysaccharide solution is added to a clean glass test tube, and 100 μl 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.
HPLC Analysis of Hydrolysate of Polysaccharide
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.
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.
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.

## 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%.