TWO METHODS FOR PREPARING UNIVERSAL MICROBIAL MEDIUM BY EUTECTIC SYSTEM-BASED CELLULOSE LIQUEFACTION
20220396763 · 2022-12-15
Inventors
- Haitao YUE (Urumqi, CN)
- Jieyi WU (Urumqi, CN)
- Duo YANG (Urumqi, CN)
- Na WU (Urumqi, CN)
- Luyu ZHAO (Urumqi, CN)
- Xiangxiang XING (Urumqi, CN)
Cpc classification
C12N1/22
CHEMISTRY; METALLURGY
C12R2001/01
CHEMISTRY; METALLURGY
International classification
Abstract
A method for preparing a universal microbial medium by eutectic system-based cellulose liquefaction. The method includes: 1) mixing ionic liquid and cellulose, where the ionic liquid is preheated; 2) mixing a mixed solution I obtained in step 1) and distilled water; 3) filtering a mixed solution II obtained in step 2); and 4) diluting a filtrate obtained in step 3) with the distilled water to obtain the carbon source. A medium prepared from the carbon source can be used for culturing various microorganisms, with a desirable growth state of the microorganisms.
Claims
1. A method for preparing a carbon source, comprising: 1) mixing ionic liquid and cellulose, wherein the ionic liquid is preheated; 2) mixing a mixed solution I obtained in step 1) and distilled water; 3) filtering a mixed solution II obtained in step 2); and 4) diluting a filtrate I obtained in step 3) with distilled water to obtain the carbon source.
2. The method according to claim 1, wherein the ionic liquid is preheated at 70° C. to 90° C.; optionally, the ionic liquid and the cellulose are mixed in a mass ratio of 5:3 to 10:1; and optionally, the mixed solution I obtained in step 1) and the distilled water are mixed in a volume ratio of 1:2.
3. The method according to claim 1, further comprising: conducting fractionation on a diluted product I obtained in step 4) to obtain a carbon source without the ionic liquid and first recycled ionic liquid.
4. The method according to claim 1, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride.
5. The method according to claim 2, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride.
6. The method according to claim 3, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride.
7. The method for claim 1, wherein the ionic liquid and the cellulose are mixed specifically by: 1) stirring the ionic liquid and a cellulose mixed solution at a temperature between 100° C. and 140° C.; 2) stirring a resulting mixed solution at a temperature between 150° C. and 200° C.; and 3) repeating step 2) three times.
8. The method according to claim 1, wherein the diluting is conducted according to a ratio of a mass of the ionic liquid to a diluting volume at 1 g:60 mL to 1 g:30 mL; and preferably, the diluting is conducted according to the ratio of the mass of the ionic liquid to the diluting volume at 1 g:50 mL.
9. The method according to claim 3, wherein the diluting is conducted according to a ratio of a mass of the cellulose and a diluting volume at 1 g:50 mL to 1 g:30 mL; and preferably, the diluting is conducted according to the ratio of the mass of the cellulose and the diluting volume at 1 g:40 mL.
10. The method according to claim 3, further comprising the following steps: 1) mixing the first recycled ionic liquid with the cellulose at a mass ratio of 5:3 to 10:1, wherein the first recycled ionic liquid is preheated at 100° C. to 140° C.; 2) stirring a mixture I obtained in step 1) at 150° C. to 200° C.; 3) repeating step 2) two times; 4) mixing a mixture II obtained in step 3) and distilled water in a volume ratio of 1:2; 5) filtering a product A obtained in step 4); 6) diluting a filtrate A obtained in step 5) with distilled water; and 7) conducting fractionation on a diluted product A obtained in step 6) to obtain the carbon source and second recycled ionic liquid.
11. The method according to claim 1, wherein the fractionation is conducted at a vacuum pressure of −0.070 Mpa to −0.095 Mpa and 40 rpm to 70 rpm, in a water bath at 20° C. to 50° C. and then 55° C. to 80° C.
12. A method for preparing a carbon source, comprising: mixing an alkaline mixed solution I and cellulose in a volume-to-mass ratio of 50 mL:1 g to 200 mL:1 g to obtain an alkaline mixed solution II, wherein the alkaline mixed solution I comprises sodium hydroxide and urea; freezing the alkaline mixed solution II at −10° C. to −80° C. for 24 h; heating the frozen alkaline mixed solution II; subjecting the alkaline mixed solution II heated to 0° C. to −20° C. to pH adjustment to obtain an alkaline mixed solution III; and filtering the alkaline mixed solution III adjusted to a pH value of 7 to 11 to obtain a filtrate that forms the carbon source.
13. The method according to claim 12, wherein the alkaline mixed solution I comprises the sodium hydroxide with a mass percentage of 3% to 8% and the urea with a mass percentage of 6% to 14%; optionally, the alkaline mixed solution I comprises the sodium hydroxide with the mass percentage of 3% to 5% and the urea with the mass percentage of 6% to 10%; and preferably, the alkaline mixed solution I comprises the sodium hydroxide with the mass percentage of 4% and the urea with the mass percentage of 7%.
14. The method according to claim 1, wherein the cellulose is solid cellulose; optionally, the cellulose is microcrystalline cellulose; optionally, the cellulose is agricultural waste straw; and optionally, the cellulose is at least one of licorice straw, wheat straw, corn straw, sorghum straw, and cotton straw.
15. The method according to claim 12, wherein the cellulose is solid cellulose; optionally, the cellulose is microcrystalline cellulose; optionally, the cellulose is agricultural waste straw; and optionally, the cellulose is at least one of licorice straw, wheat straw, corn straw, sorghum straw, and cotton straw.
16. The method according to claim 12, wherein the pH adjustment is conducted using a dilute acid; and optionally, the dilute acid is HCl.
17. A carbon source obtained by the method according to claim 12.
18. The carbon source according to claim 17, wherein the alkaline mixed solution I comprises the sodium hydroxide with a mass percentage of 3% to 8% and the urea with a mass percentage of 6% to 14%; optionally, the alkaline mixed solution I comprises the sodium hydroxide with the mass percentage of 3% to 5% and the urea with the mass percentage of 6% to 10%; and preferably, the alkaline mixed solution I comprises the sodium hydroxide with the mass percentage of 4% and the urea with the mass percentage of 7%.
19. The carbon source according to claim 17, wherein the cellulose is solid cellulose; optionally, the cellulose is microcrystalline cellulose; optionally, the cellulose is agricultural waste straw; and optionally, the cellulose is at least one of licorice straw, wheat straw, corn straw, sorghum straw, and cotton straw.
20. The carbon source according to claim 17, wherein the pH adjustment is conducted using a dilute acid; and optionally, the dilute acid is HCl.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0059] The embodiments of the present disclosure are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative, which are merely intended to explain the present disclosure, rather than to limit the present disclosure.
[0060] Moreover, the terms such as “first” and “second” are used only for the purpose of description and should not be construed as indicating or implying a relative importance, or implicitly indicating a quantity of indicated technical features. Therefore, features defined by “first” and “second” may explicitly or implicitly include at least one of the features. In description of the present disclosure, “a plurality of” means at least two, for example, two or three, unless otherwise clearly and specifically limited.
[0061] Microcrystalline cellulose is a purified, partially-depolymerized cellulose, which is a white, odorless, and tasteless crystalline powder composed of porous microparticles, and uses linear polysaccharides bound by a β-1,4-glucosidic bond as a main component. The microcrystalline cellulose has a degree of polymerization of about 3,000 to 10,000 glucose molecules. In general plant fibers, microcrystalline cellulose accounts for about 73%, and the other 30% is amorphous cellulose.
[0062] A purpose of the present disclosure is to pretreat cellulose through ionic liquid and a NaOH/Urea solution system for liquefication, and to construct a novel broad-spectrum medium using liquefied cellulose as a carbon source and recover the ionic liquid. Ionic liquid for pretreating cellulose, a concentration of the ionic liquid, an addition ratio of the ionic liquid to the cellulose, and a temperature at which the ionic liquid decomposes the cellulose are screened to obtain optimum experimental conditions for the ionic liquid to dissolve the cellulose. Meanwhile, a concentration of NaOH and Urea, an addition ratio of a NaOH/Urea solution to the cellulose, and a temperature of NaOH/Urea solution pretreatment of the cellulose in a NaOH/Urea solution system are screened to obtain optimum experimental conditions for pretreatment of the cellulose with NaOH/Urea solution. The carbon source obtained under the optimal cellulose dissolving conditions is diluted in a certain number of times, the carbon source is used to prepare a broad-spectrum microbial medium, and a culture ability of the broad-spectrum medium is verified.
[0063] The method provided by the present disclosure includes the following steps:
[0064] (1) The cellulose is pretreated by the ionic liquid, and filtered and diluted; a novel medium is constructed using diluted mixed liquid as a carbon source for culturing bacteria, or the cellulose is pretreated by the ionic liquid, and subjected to filtration, dilution, and fractionation; the ionic liquid in an evaporating flask of a rotary evaporator is recovered, and a novel medium is constructed using colorless and transparent liquid in the evaporating flask of the rotary evaporator as a carbon source for culturing bacteria; the above steps are continued with the recovered ionic liquid.
[0065] (2) The cellulose is pretreated using a NaOH/Urea solution system, followed by freezing, thawing, stirring, pH adjustment, and filtration; a novel medium is constructed using a filtrate as a carbon source for culturing bacteria.
[0066] To prepare a medium with ionic liquid-pretreated cellulose as a carbon source and recycle ionic liquid, the present disclosure adopts the following technical solutions.
[0067] (1) The ionic liquid is heated to 70° C. to 90° C., mixed with the cellulose in a mass ratio of 5:3 to 10:1, heated to 100° C. to 140° C., stopped heating, and stirred. A mixture is heated to 150° C. to 200° C., stop heating, and stirred (repeating 3 times). 2 times volume of distilled water is added to a beaker, and stirred, the mixture is dissolved and filtered with a fast qualitative filter paper. The distilled water is added to dilute the filtrate, with a dilution ratio of a dilution volume and a dissolved cellulose mass of 1 mL:50 g to 1 mL:30 g, or 1 mL:60 g to 1 mL:30 g; when mixed liquid after dilution is not fractionated to be used as a carbon source to construct a medium, the obtained mixed liquid is a carbon source containing a large amount of the ionic liquid, and the ionic liquid is toxic. Therefore, to ensure the normal growth of microorganisms, it is necessary to dilute the ionic liquid in the mixed liquid. A dilution ratio is a ratio of a mass of the added ionic liquid and a dilution volume of 1 g:60 mL to 1 g:30 mL; in the mixed liquid after dilution, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by sterilization through autoclaving at 115° C. for 15 min. The strain is inoculated, changes of OD.sub.600 are observed at 37° C., 150 rpm. When the mixed liquid after dilution is fractionated, the mixed liquid obtained after fractionation is a carbon source without the ionic liquid; to ensure the growth of microorganisms, the concentration of the carbon source in the medium should be guaranteed. A dilution ratio is a ratio of a mass of the dissolved cellulose and a dilution volume of 1 g:50 mL to 1 g:30 mL.
[0068] (2) Fractionation is conducted on the mixed solution obtained in step (1) after dilution, where a vacuum pressure is −0.070 Mpa to −0.095 Mpa, a first water bath temperature is 20° C. to 50° C., a second water bath temperature is 55° C. to 80° C., that is, the boiling point changes once, and a rotation speed is 50 rpm; after the fractionation, a colorless and transparent liquid can be obtained in a collection bottle, which is a carbon source without the ionic liquid; and ionic liquid is recovered in an evaporation bottle, and the ionic liquid can be recovered multiple times.
[0069] (3) in the carbon source without ionic liquid obtained in step 2) or the carbon source with ionic liquid obtained in step 1), NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 115° C. for 15 min. The strain is inoculated, changes of OD.sub.600 are observed at 37° C., 150 rpm.
[0070] (4) The ionic liquid recovered in step (2) is heated to 100° C. to 130° C., followed by stopping heating; a resulting product was mixed with cellulose according to a mass ratio of 5:3 to 10:1, and heated to 140° C. to 180° C., followed by stopping heating; and a resulting product was stirred (repeating 3 times). 2 times volume of distilled water is added to a beaker, and stirred, the mixture is dissolved and filtered with a fast qualitative filter paper. The distilled water was added to dilute the filtrate at a ratio of constant volume and dissolved mass of 1 mL:30 g to 1 mL:60 g.
[0071] (5) Fractionation is conducted on the mixed solution obtained in step (4) after dilution, where a vacuum pressure is −0.070 Mpa to −0.095 Mpa, a first water bath temperature is 20° C. to 50° C., a second water bath temperature is 55° C. to 80° C., that is, the boiling point changes once, and a rotation speed is 50 rpm; after the fractionation, a colorless and transparent liquid can be obtained in the collection bottle; and ionic liquid is recovered in the evaporation bottle.
[0072] (6) in the solution in the collection bottle, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 110° C. for 15 min. The strain is inoculated, changes of OD.sub.600 are observed at 37° C., 150 rpm.
[0073] (7) The ionic liquid recovered in step (5) is heated to 100° C. to 130° C., followed by stopping heating; a resulting product was mixed with cellulose according to a mass ratio of 5:3 to 10:1, and heated to 140° C. to 180° C., followed by stopping heating; and a resulting product was stirred (repeating 3 times). 2 times volume of distilled water is added to a beaker, and stirred, the mixture is dissolved and filtered with a fast qualitative filter paper. The distilled water was added to dilute the filtrate at a ratio of constant volume and dissolved mass of 1 mL:30 g to 1 mL: 60 g.
[0074] (8) Fractionation is conducted on the mixed solution obtained in step (7) after dilution, where a vacuum pressure is −0.070 Mpa to −0.095 Mpa, a first water bath temperature is 20° C. to 50° C., a second water bath temperature is 55° C. to 80° C., that is, the boiling point changes once, and a rotation speed is 50 rpm; after the fractionation, a colorless and transparent liquid can be obtained in the collection bottle; and ionic liquid is recovered in the evaporation bottle.
[0075] (9) in the solution in the collection bottle, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 115° C. for 15 min. The strain is inoculated, changes of OD.sub.600 are observed at 37° C., 150 rpm.
[0076] To prepare a medium with NaOH/Urea solution system-pretreated cellulose as a carbon source, the present disclosure adopts the following technical solutions.
[0077] (1) NaOH (3% to 8%) and Urea (6% to 14%) are mixed and diluted to 1000 ml, mixed with 5 g to 20 g of cellulose, and pre-frozen at −10° C. to −80° C. for 24 h.
[0078] (2) A pre-frozen solution obtained in step (1) was placed at room temperature, heated to 0° C. to −40° C. to form an ice-water mixed state, and stirred until the solution is completely dissolved; after adjusting a pH value to 7 to 11 with HCl, the solution is filtered with gauze to collect a filtrate.
[0079] (3) in the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 115° C. for 15 min; Micrococcus luteus was inoculated, and changes of CDW are observed at 37° C., 150 rpm.
[0080] (4) After 72 h, a culture was transferred to a 50 mL centrifuge tube, centrifuged at 12,000 rpm for 10 min, and a supernatant was discarded; a resulting precipitate was pre-frozen at −70° C. for 4 h, and then lyophilized overnight in a −60° C. vacuum lyophilizer.
[0081] (5) About 20 mg of freeze-dried cell samples were accurately weighed (0.0001) and placed in an esterification tube, and 2 mL of chloroform (AR) and 2 mL of an esterification solution (including 500 ml/L anhydrous methanol+1 g/L benzoic acid+3% (v/v) concentrated sulfuric acid) were added. About 10 mg of a standard PHA sample (PHB) was treated in the same way to prepare a chloroform solution of the standard sample. The esterification tube is sealed, shaken to mix evenly, followed by conducting esterification at a constant temperature of 100° C. for 4 h. After cooling to room temperature, 1 mL of ddH.sub.2O was added, shaken until completely mixed, and allowed to stand for about 30 min for layering. After the aqueous phase and the organic phase are completely separated, a lower layer solution (chloroform phase) was subjected to GC analysis to calculate a 3HB content.
[0082] Experiments have proved that, in the present disclosure, a novel medium that can make multiple strains of bacteria of different species proliferate is constructed using ionic liquid-pretreated cellulose as a carbon source; meanwhile, the recovered ionic liquid maintains certain original properties. In addition, a novel medium that can make some bacteria proliferate while accumulating PHA is constructed using the NaOH/Urea solution system-pretreated cellulose as a carbon source.
[0083] The materials, reagents, and the like used in the following examples are all commercially available, unless otherwise specified.
[0084] The ionic liquid is purchased from Shanghai Chengjie Chemical Co., Ltd.; the rotary evaporator is purchased from Shanghai Yarong Biochemical Instrument Factory; a rapid qualitative filter paper is purchased from Hangzhou Special Paper Co., Ltd.; a gas chromatograph model is Agilent Technologies 6850, purchased from Agilent Technologies (China) Co., Ltd.; a Chinese herbal medicine grinder is purchased from Tianjin Taiste Instrument Co., Ltd.; NH.sub.4Cl, MgSO.sub.4, NaCl, Urea are purchased from Tianjin Beilian Chemcp Co., Ltd.; NaOH, hydrochloric acid, anhydrous methanol, chloroform and concentrated sulfuric acid are purchased from Tianjin Yongsheng Fine Chemical Co., Ltd.; peptone, yeast powder and agar are purchased from Beijing Aubox Biotechnology Co., Ltd.; Escherichia coli is purchased from Sangon Biotech (Shanghai) Co., Ltd.; Brevibacterium casei, Bacillus, Micrococcus luteus, Enterobacter cloacae, Halomonas, and Bacillus licheniformis are provided by the Yue Haitao experimental group of Xinjiang University; and media are prepared with distilled water unless otherwise specified.
[0085] The present disclosure is described below with reference to the specific examples. It should be noted that these examples are only illustrative and do not limit the present disclosure in any way.
Example 1 Screening of Ionic Solutions
[0086] Different ionic liquids (1-butyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium borate, 1-allyl-3-methylimidazolium chloride, and 1-butyl-3-methylimidazole acetate) pretreating cellulose were selected, to screen ionic liquid that can dissolve the cellulose efficiently, and the experimental conditions were roughly selected at the same time. The treatment methods included: 1) ionic liquid was heated in a water bath, mixed with the cellulose in proportion, followed by continuing water bath or microwave heating, where a temperature of the preheated ionic liquid was the same as that at which the two were continued to be heated after mixing; 2) the ionic liquid and the cellulose were mixed in proportion, heated, a degree of cellulose dissolution was observed, and a cellulose dissolution time was recorded. The specific experimental phenomena and experimental conclusions were shown in Table 1. After preheating to a liquid state at 110° C., the 1-butyl-3-methylimidazolium chloride was mixed with the cellulose in a mass ratio of 5:3, continued to partially dissolve; the IL turned brown and smelled like caramel, and 100 mL of water was added to let stand for stratification; it was considered that the ionic liquid could well process the cellulose under the above experimental conditions. However, other ionic liquids had a poor effect in pretreatment of cellulose, or the ionic liquid and the cellulose had a relatively large dissolving mass ratio and a long dissolution time, which were not recommended to use.
TABLE-US-00001 TABLE 1 Types of ionic Ionic Microcrystalline Experimental Dissolution No. solutions solution/g cellulose/g temperature Treatment time Dissolved state Conclusion 1 1-butyl-3- 10 1 90° C. IL and 40 min Microcrystalline Microcrystalline methylimidazolium microcrystalline cellulose cellulose chloride cellulose dissolves, but has a are newly-added desirable co-heated, microcrystalline dissolution stirred for cellulose has effect, but 20 sec increased the ionic every 5 viscosity and solution and min the cellulose microcrystalline does not cellulose dissolve has a large mass ratio and a long dissolution time 2 1-butyl-3- 5 3 110° C. IL is 1 min Partially Microcrystalline methylimidazolium preheated dissolved, part cellulose chloride to liquid of the has a state and microcrystalline desirable mixed with cellulose dissolution microcrystalline became lumps, effect and a cellulose the solution is short by stirring brown and dissolution smells like time caramel, 100 mL of water is added to allow stand for stratification 3 1-butyl-3- 5 3 90° C. IL and 4 h Partially Microcrystalline methylimidazolium microcrystalline dissolved, part cellulose chloride cellulose of the has a are microcrystalline common co-heated, cellulose dissolution stirred for became lumps effect and a 20 sec long every 5 dissolution min time 4 1-butyl-3- 10 1 90° C. IL and 40 min Microcrystalline Microcrystalline methylimidazolium microcrystalline cellulose cellulose borate cellulose mixed with has a poor are ionic liquid to dissolution co-heated, form lumps effect stirred for 20 sec every 5 min 5 1-butyl-3- 2 1.5 100° C. IL is 4 h Microcrystalline Microcrystalline methylimidazolium preheated cellulose cellulose borate to liquid mixed with has a poor state, ionic liquid to dissolution microcrystalline form lumps effect cellulose is added, and stirred for 20 sec every 5 min 6 1-butyl-3- 2 1.5 100° C. IL is 4 h Microcrystalline Microcrystalline methylimidazolium preheated cellulose cellulose borate to liquid mixed with has a poor state, ionic liquid to dissolution microcrystalline form lumps effect cellulose is added, and stirred for 20 sec every 5 min 7 1-butyl-3- 10 6 90° C. IL and 4 h Microcrystalline Microcrystalline methylimidazolium microcrystalline cellulose cellulose borate cellulose mixed with has a poor are ionic liquid to dissolution co-heated, form lumps effect stirred for 20 sec every 5 min 8 1-allyl-3- 10 1 90° C. IL and 40 min Microcrystalline Microcrystalline methylimidazolium microcrystalline cellulose cellulose chloride cellulose dissolves, but has a are newly-added desirable co-heated, microcrystalline dissolution stirred for cellulose has effect and a 20 sec increased long every 5 viscosity and dissolution min microcrystalline time cellulose does not dissolve 9 1-allyl-3- 5 1 100° C. IL is 20 min Dissolved, but Microcrystalline methylimidazolium heated to the solution has cellulose chloride 100° C., a high viscosity has a microcrystalline and is in a form common cellulose is of a hard dissolution added, and lamella after effect and a stirred for adding water long 20 sec dissolution every 5 time min 10 1-allyl-3- 5 2 100° C. The 10 min Dissolved, but Microcrystalline methylimidazolium microcrystalline the solution has cellulose chloride cellulose is an extremely has a soaked in viscosity, common IL for 20 h, almost being dissolution co-heated solid effect and a in a water long bath, dissolution stirred for time 20 sec every 5 min 11 1-allyl-3- 5 3 90° C. IL and 4 h Microcrystalline Microcrystalline methylimidazolium microcrystalline cellulose cellulose chloride cellulose mixed with has a poor are ionic liquid to dissolution co-heated, form lumps effect stirred for 20 sec every 5 min 12 1-butyl-3- 10 1 90° C. IL and 40 min Microcrystalline Microcrystalline methylimidazole microcrystalline cellulose cellulose acetate cellulose dissolves, but has a are newly-added desirable co-heated, microcrystalline dissolution stirred for cellulose has effect and a 20 sec increased long every 5 viscosity and dissolution min microcrystalline time cellulose does not dissolve 13 1-butyl-3- 10 6 90° C. IL and 4 h Microcrystalline Microcrystalline methylimidazole microcrystalline cellulose cellulose acetate cellulose mixed with has a poor are ionic liquid to dissolution co-heated, form lumps effect stirred for 20 sec every 5 min
Example 2 Screening of Addition Ratio of Ionic Liquid and Cellulose and Experimental Temperatures
[0087] According to the results of Example 1, this example aimed to further screen a mixing ratio of the ionic liquid (1-butyl-3-methylimidazolium chloride) and the cellulose and suitable experimental conditions The treatment method included: 1) after microwave heating, the 1-butyl-3-methylimidazolium chloride was mixed with the cellulose in proportion, and the microwave heating was continued; 2) the 1-butyl-3-methylimidazolium chloride was mixed with the cellulose in proportion, followed by microwave heating; during the heating, the stirring treatment was conducted, a degree of dissolution was observed, and a dissolution time was recorded. The specific operation and experimental results were shown in Table 2. The cellulose in treatment 6 to 10 is fully dissolved, and the ionic solution and the microcrystalline cellulose has a mass ratio of 5:2 to 10:1; the experimental conditions are as follows: the IL is heated to 90° C., added with cellulose, stirred for 20 sec, and heated to 120° C., stirred for 20 sec, and repeatedly heated to over 120° C.; when heated to 120° C., the system turns black and has a caramel smell, and the cellulose is dissolved after repeated heating to 180° C.
TABLE-US-00002 TABLE 2 Ionic Micro- solution crystalline No. Gram cellulose (g) Treatment Dissolved state Conclusion Treat- 10 1 IL and microcrystalline cellulose are Microcrystalline cellulose Poor ment 1 co-heated to 90° C., stirred for 20 sec, does not dissolve, and the dissolution heated to 90° C., stirred for 20 sec, and microcrystalline cellulose effect the operations are repeated is mixed with ionic liquid to forms lumps, with a poor dissolution effect Treat- 10 1 IL is heated to 90° C. and added with After repeated heating Common ment 2 microcrystalline cellulose, stirred for 20 several times, a small part dissolution sec, heated to 90° C., stirred for 20 sec, of microcrystalline effect and operations are repeated cellulose is dissolved in the ionic liquid, and most of fibers are mixed with part of the ionic liquid to form lumps Treat- 10 1 IL is heated to 90° C. and added with After repeated heating Common ment 3 microcrystalline cellulose, stirred for 20 several times, part of dissolution sec, heated to 100° C., stirred for 20 sec, microcrystalline cellulose effect and heating to 100° C. is repeated is dissolved, and part of fibers are mixed with part of the ionic liquid to form lumps Treat- 10 1 IL is heated to 90° C. and added with After repeated heating Common ment 4 microcrystalline cellulose, stirred for 20 several times, the system dissolution sec, heated to 110° C., stirred for 20 sec, turns yellow, part of effect and heating to 110° C. is repeated microcrystalline cellulose is dissolved, and part of fibers are mixed with part of the ionic liquid to form lumps Treat- 10 1 IL is heated to 90° C. and added with When heated to 120° C., the Common ment 5 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect and heating to 120° C. is repeated repeated heating several times, the microcrystalline cellulose is partially dissolved, and part of fibers are mixed with part of ionic liquid to form bumps Treat- 10 1 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 6 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect and heating to 180° C. is repeated repeated heating to 180° C., the microcrystalline cellulose dissolves Treat- 8 1 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 7 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect and heating to 180° C. is repeated repeated heating to 180° C., the microcrystalline cellulose dissolves Treat- 5 1 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 8 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 180° C., stirred for 20 sec, a caramel smell; after effect and heating to 180° C. is repeated repeated heating to 180° C., the microcrystalline cellulose dissolves Treat- 5 1.5 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 9 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect and heating to 180° C. is repeated repeated heating to 180° C., the microcrystalline cellulose dissolves Treat- 5 2 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 10 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect and heating to 180° C. is repeated repeated heating to 180° C., the microcrystalline cellulose dissolves Treat- 5 2.5 IL is heated to 90° C. and added with When heated to 120° C., the Desirable ment 11 microcrystalline cellulose, stirred for 20 system turns black and has dissolution sec, heated to 120° C., stirred for 20 sec, a caramel smell; after effect, an and heating to 180° C. is repeated repeated heating to 120° C., added the microcrystalline amount of cellulose is mostly micro- dissolved, and flaky crystal insolubles are formed line cellulose has exceeded a maximum dissolving amount of cellulose under this condition
Example 3 Screening of Ionic Solution Concentrations in Microbial Medium
[0088] Due to the toxicity of ionic liquid, if the carbon source contains a large amount of ionic liquid, the prepared medium is not conducive to the growth of strains. Therefore, a purpose of this example is to screen a concentration range of the ionic liquid contained in the medium. In an LB solid medium (including yeast powder: 10 g/L, peptone: 5 g/L, NaCl: 10 g/L, and agar: 18 g/L), different proportions of ionic liquid (1-butyl-3-methylimidazolium chloride) were added, followed by coating with Escherichia coli, and a growth state for 48 h was observed. The specific results are shown in Table 3. The Escherichia coli grows normally when the ionic solution concentration is 2%; and the growth of Escherichia coli is inhibited when the ionic liquid concentration reaches not less than 3%. Therefore, the concentration of the ionic liquid in the medium during microbial culture should not exceed 3%.
TABLE-US-00003 TABLE 3 Ionic solution Growth concentration state of strains 0% Normal 1% Normal 2% Normal 3% Slightly inhibited growth 4% Inhibited growth 5% Inhibited growth 6% No growth 7% No growth
Example 4 Verification of Strain Culture Ability of Novel Microbial Medium with Ionic Liquid-Pretreated Cellulose as Carbon Source
[0089] In this example, a carbon source obtained by optimizing experimental conditions is applied; functional verification is conducted on a medium containing a liquid cellulose-based carbon source and a medium prepared from a fractionated product obtained after recovery of ionic liquid; and an ability of a novel medium is determined to culture multiple strains of different species.
Experiment 1
[0090] 1) Licorice straw was pulverized using a Chinese herbal medicine grinder, and sieved through a 30-mesh sieve to obtain a licorice straw powder. Ionic liquid (1-butyl-3-methylimidazolium chloride) was mixed with the licorice straw powder in a mass ratio of 5:2, the ionic liquid was added to a beaker and heated to 90° C., and the ionic liquid was completely melted into transparent, colorless and viscous liquid. The licorice straw powder was added to the completely melted ionic liquid, stirred with a glass rod, and heated to 120° C.; when a mixture of the licorice straw powder and the ionic liquid began to turn black, heating was terminated, followed by stirring for 20 sec. The mixture was heated to 180° C., followed by stopping heating, and stirring for 20 sec (repeating 3 times). 2 times volume of distilled water was added to a beaker, and stirred with the glass rod, the mixture was dissolved and filtered with a rapid qualitative filter paper. Distilled water was added to a filtrate to control a concentration of the ionic liquid in the carbon source, to further control a concentration of the ionic liquid in the new medium. A mass of the ionic liquid and a dilution volume had a ratio of 1 g:50 mL.
[0091] In the solution after dilution, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Brevibacterium casei, Bacillus, Micrococcus luteus and Escherichia coli with a 3% inoculum (OD.sub.600=0.5 to 0.6) were inoculated into the mixture above. Shaker culture was conducted at 37° C., 150 rpm. From a growth curve (
Experiment 2
[0092] 1) The ionic liquid and the microcrystalline cellulose were mixed in a mass ratio of 5:2. The ionic liquid was added to the beaker and heated to 90° C., and the ionic liquid was completely melted into transparent, colorless and viscous liquid. The microcrystalline cellulose was added to the completely melted ionic liquid, and stirred with the glass rod for 20 sec. Heating was continued to 120° C., a mixture of the microcrystalline cellulose and the ionic liquid began to turn black, and heating was terminated, followed by stirring with the glass rod for 20 sec. The mixture was heated to 180° C., followed by stopping heating, and stirring for 20 sec (repeating 3 times). 2 times volume of distilled water was added to a beaker, and stirred with the glass rod, the mixture was dissolved and filtered with a rapid qualitative filter paper. The distilled water was added to dilute a filtrate, where a mass of dissolved microcrystalline cellulose and a dilution volume had a ratio of 1 g:40 mL.
[0093] 2) A solution obtained in the previous step was fractionated using a rotary evaporator at a pressure of −0.080 Mpa, at 25° C. and then 80° C. in a water bath, respectively (a boiling point changed once), and a rotation speed of 50 rpm; after the fractionation, colorless and transparent liquid was obtained in a collection bottle, and ionic liquid was obtained in an evaporation bottle.
[0094] (3) In a recovery bottle solution (a carbon source without ionic liquid), NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Brevibacterium casei, Bacillus, Micrococcus luteus, Enterobacter cloacae, Halomonas, and Escherichia coli with a 3% inoculum (OD.sub.600=0.5 to 0.6) were inoculated into the mixture above. Shaker culture was conducted at 37° C., 150 rpm. From a growth curve (
Experiment 3
[0095] The ionic liquid recovered in Experiment 2 of the example was mixed with microcrystalline cellulose in a mass ratio of 5:2. The ionic liquid was added to the beaker, heated to 130° C., microcrystalline cellulose was added to the ionic liquid, and stirred with a glass rod for 20 sec. The mixture was heated to 180° C., followed by stirring for 20 sec (repeating 3 times). 2 times volume of distilled water was added to a beaker, and stirred with the glass rod, the mixture was dissolved and filtered with a rapid qualitative filter paper. The distilled water was added to dilute a filtrate, where a mass of dissolved microcrystalline cellulose and a dilution volume had a ratio of 1 g:40 mL.
[0096] 2) A solution obtained in the previous step was fractionated using a rotary evaporator at a pressure of −0.075 Mpa, at 35° C. and then 75° C. in a water bath, respectively (a boiling point changed once), and a rotation speed of 50 rpm; after the fractionation, colorless and transparent liquid was obtained in a collection bottle, and ionic liquid was obtained in an evaporation bottle.
[0097] (3) in the solution in the recovery bottle, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Brevibacterium casei and Escherichia coli with a 3% inoculum (OD.sub.600=0.5 to 0.6) were inoculated into the mixture above. Shaker culture was conducted at 37° C., 150 rpm. The changes of OD.sub.600 with time are shown in Table 4. From the OD.sub.600, it can be seen that the Brevibacterium casei and Escherichia coli can proliferate in this medium, indicating that the medium obtained by this method has certain microbial culture ability.
TABLE-US-00004 TABLE 4 OD.sub.600 OD.sub.600 OD.sub.600 OD.sub.600 Item 0 h 24 h 48 h 72 h Escherichia coli 0.0730 0.1465 0.1978 0.1998 Brevibacterium casei 0.0632 0.1525 0.1834 0.1820
Experiment 4
[0098] The ionic liquid recovered in Experiment 3 of the example was mixed with microcrystalline cellulose in a mass ratio of 5:2. The ionic liquid was added to the beaker, heated to 130° C., microcrystalline cellulose was added to the ionic liquid, and stirred with a glass rod for 20 sec; heating was continued to 180° C., stirred with the glass rod for 20 sec, repeating 3 times. 2 times volume of distilled water was added to a beaker, and stirred with the glass rod, the mixture was dissolved and filtered with a rapid qualitative filter paper. The distilled water was added to dilute a filtrate, where a mass of dissolved microcrystalline cellulose and a dilution volume had a ratio of 1 g:40 mL.
[0099] 2) A solution obtained in the previous step was fractionated using a rotary evaporator at a pressure of −0.075 Mpa, at 35° C. and then 75° C. in a water bath, respectively (a boiling point changed once), and a rotation speed of 50 rpm; after the fractionation, colorless and transparent liquid was obtained in a collection bottle, and ionic liquid was obtained in an evaporation bottle.
[0100] (3) in the solution in the recovery bottle, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) are added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Brevibacterium casei and Escherichia coli with a 3% inoculum (OD.sub.600=0.5 to 0.6) were inoculated into the mixture above. Shaker culture was conducted at 37° C., 150 rpm. The changes of OD.sub.600 with time are shown in Table 5. From the OD.sub.600, it can be seen that the Brevibacterium casei and Escherichia coli can proliferate in this medium, indicating that the medium obtained by this method has certain microbial culture ability.
TABLE-US-00005 TABLE 5 OD.sub.600 OD.sub.600 OD.sub.600 OD.sub.600 Item 0 h 24 h 48 h 72 h Escherichia coli 0.0716 0.1321 0.1821 0.1878 Brevibacterium casei 0.0655 0.1644 0.1964 0.1920
Example 5 Screening of Experimental Temperatures of NaOH/Urea and Cellulose
[0101] In this example, experimental conditions for treating cellulose with a NaOH/Urea solution system were screened. The specific experimental conditions, operations and experimental results were as follows:
[0102] 1) A solution of 4% NaOH/7% Urea was prepared, and 1 g/L microcrystalline cellulose was added to the solution; a resulting NaOH/Urea-microcrystalline cellulose solution was turbid at room temperature, and the microcrystalline cellulose precipitated to a bottom of the solution after standing. This indicates that microcrystalline cellulose is insoluble in this solution system at room temperature.
[0103] 2) A solution of 4% NaOH/7% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −10° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and a NaOH/Urea-microcrystalline cellulose solution was gradually clarified, indicating that the cellulose was completely dissolved. Therefore, this experimental condition could be used to pretreat microcrystalline cellulose.
[0104] 3) A solution of 4% NaOH/7% Urea was prepared, 15 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and flakes of cellulose were precipitated at the bottom of the NaOH/Urea-cellulose solution, indicating that the solution system had reached the highest cellulose dissolving capacity.
Example 6 Screening of Concentrations of NaOH and Urea
[0105] In this example, concentrations of NaOH and Urea were screened. The specific experimental conditions, operations and experimental results were as follows:
[0106] 1) A 3% NaOH solution was prepared, Urea at concentrations of 7, 8, 10, 12, and 14(%) were added to the solution, respectively, 5 g of microcrystalline cellulose was added to the NaOH/Urea solution systems of different concentrations, frozen at −20° C., and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the microcrystalline cellulose was insoluble.
[0107] 2) A 3.5% NaOH solution was prepared, Urea at of 7, 8, 10, 12, and 14(%) were added to the solution, respectively, microcrystalline cellulose was added to the NaOH/Urea solution systems of different concentrations, frozen at −20° C., and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and dissolved amounts of the microcrystalline cellulose were 10 g/L, 10 g/L, 9 g/L, 9 g/L, and 8 g/L in sequence. The results are shown in
[0108] 3) A 4% NaOH solution was prepared, Urea at of 7, 8, 10, 12, and 14(%) were added to the solution, respectively, microcrystalline cellulose was added to the NaOH/Urea solution systems of different concentrations, frozen at −20° C., and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and dissolved amounts of the microcrystalline cellulose were 14 g/L, 14 g/L, 14 g/L, 13 g/L, and 12 g/L in sequence. The results are shown in
[0109] 4) A 5% NaOH solution was prepared, Urea at of 7, 8, 10, 12, and 14(%) were added to the solution, respectively, microcrystalline cellulose was added to the NaOH/Urea solution systems of different concentrations, frozen at −20° C., and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and dissolved amounts of the microcrystalline cellulose were 18 g/L, 18 g/L, 16 g/L, 15 g/L, and 15 g/L in sequence. The results are shown in
[0110] 5) A 7% NaOH solution was prepared, Urea at of 7, 8, 10, 12, and 14(%) were added to the solution, respectively, microcrystalline cellulose was added to the NaOH/Urea solution systems of different concentrations, frozen at −20° C., and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and dissolved amounts of the microcrystalline cellulose were 12 g/L, 12 g/L, 10 g/L, 10 g/L, and 9 g/L in sequence. The results are shown in
[0111] 6) By using a dissolved amount of microcrystalline cellulose as a response value, a response analysis table was constructed for a test value and a predicted value of the cellulose, as shown in Table 6. A two-dimensional contour map and a three-dimensional response surface map were drawn, respectively (as shown in
TABLE-US-00006 TABLE 6 Studentized r internal Studentized Fit standard Actual Predicted residual external value Cook's Running order value Value Residual Influence error residual error effect distance sequence 1 14 9.04 4.96 0.09 1.29 1.31 0.40 0.03 8 2 0 9.79 −9.79 0.22 −2.76 −3.27 −1.73 0.35 13 3 8 4.77 3.23 0.24 0.92 0.92 0.51 0.04 6 4 9 8.94 0.06 0.10 0.02 0.02 0.01 0.00 5 5 0 5.86 −5.86 0.17 −1.60 −1.65 −0.74 0.08 7 6 18 15.48 2.52 0.13 0.67 0.67 0.26 0.01 15 7 16 17.95 −1.95 0.17 −0.53 −0.52 −0.24 0.01 16 8 12 5.26 6.74 0.09 1.76 1.84 0.58 0.05 2 9 18 13.04 4.96 0.14 1.33 1.35 0.54 0.05 14 10 11 8.78 2.22 0.21 0.62 0.61 0.32 0.02 12 11 0 2.12 −2.12 0.18 −0.58 −0.57 −0.27 0.01 1 12 13 13.75 −0.75 0.12 −0.20 −0.20 −0.07 0.00 10 13 10 9.88 0.12 0.11 0.03 0.03 0.01 0.00 4 14 11 7.61 3.39 0.08 0.88 0.88 0.26 0.01 3 15 14 11.41 2.59 0.08 0.67 0.66 0.20 0.01 9 16 14 17.20 −3.20 0.15 −0.87 −0.86 −0.37 0.02 17 17 14 13.24 0.76 0.26 0.22 0.22 0.13 0.00 18 18 12 12.87 −0.87 0.10 −0.23 −0.22 −0.07 0.00 11 19 0 3.49 −3.49 .sup. 0.43 # −1.15 −1.16 −1.01 0.17 19 20 12 6.87 5.13 0.27 1.50 1.54 0.95 0.14 20 21 12 9.44 2.56 0.22 0.72 0.71 0.38 0.02 21 22 11 12.17 −1.17 0.23 −0.33 −0.33 −0.18 0.01 22 23 10 11.69 −1.69 0.29 −0.50 −0.49 −0.31 0.02 23 24 8 7.99 0.01 .sup. 0.57 # 0.01 0.01 0.01 0.00 24 25 0 −2.81 2.81 0.28 0.82 0.82 0.51 0.04 25 26 0 0.31 −0.31 0.18 −0.08 −0.08 −0.04 0.00 26 27 0 2.62 −2.62 0.16 −0.71 −0.70 −0.30 0.02 27 28 0 4.83 −4.83 0.18 −1.33 −1.35 −0.64 0.07 28 29 0 3.82 −3.82 0.19 −1.06 −1.06 −0.51 0.04 29 30 0 −0.41 0.41 0.36 0.13 0.13 0.09 0.00 30
TABLE-US-00007 TABLE 7 NaOH Urea Microcrystalline NaOH Urea Microcrystalline No. (g/L) (g/L) cellulose (g/L) No. (g/L) (g/L) cellulose (g/L) 8 40 70 14 17 50 120 14 13 50 60 0 18 50 140 14 6 35 140 8 11 40 120 12 5 35 120 9 19 70 60 0 7 40 60 0 20 70 70 12 15 50 80 18 21 70 80 12 16 50 100 16 22 70 100 11 2 35 70 12 23 70 120 10 14 50 70 18 24 70 140 8 12 40 140 11 25 30 60 0 1 35 60 0 26 30 70 0 10 40 100 13 27 30 80 0 4 35 100 10 28 30 100 0 3 35 80 11 29 30 120 0 9 40 80 14 30 30 140 0
Example 7 Screening of pH of NaOH/Urea Solution System-Pretreated Cellulose System
[0112] In this example, the experiment was continued on the basis of Example 6. By observing the pretreatment of cellulose with the NaOH/Urea solution system at different pH values, and the treatment of microcrystalline cellulose with the NaOH/Urea solution system, changes of cellulose in this solution system due to different experimental operations were observed. The specific experimental conditions, operations and experimental results were as follows:
[0113] 1) A solution of 5% NaOH/10% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was gradually clear (pH >14), 1 mL of the solution was used as a spare sample. The pH was adjusted to 10 with HCl, the solution turned turbid, and 1 mL of the solution was used as a spare sample. The solution was filtered through gauze and 1 mL of a filtrate was used as a spare sample.
[0114] 2) 1 μL of each of the three spare samples was applied to a glass slide, the slide glass was fixed with a liquid conductive adhesive on a pie-shaped battery, followed by spraying gold, and the battery was placed on a scanning electron microscope stage to collect images after spraying gold.
Example 8 Novel Medium Obtained from NaOH/Urea Solution System-Pretreated Cellulose
[0115] In this example, functional verification of a NaOH/Urea-cellulose-based medium was conducted to determine an ability of the novel medium to culture multiple strains of different species.
Experiment 1
[0116] 1) A solution of 4% NaOH/7% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was gradually clarified. After adjusting the pH value to 10 with HCl, the solution was filtered with gauze to collect a filtrate.
[0117] 2) in the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. The filtrate was inoculated with Halomonas, Bacillus, Micrococcus luteus, Brevibacterium casei, Bacillus licheniformis (OD.sub.600=0.5 to 0.6), followed by conducting shake culture at 37° C., 150 rpm; all strains grew normally. After 72 h, a culture was transferred to a 50 mL centrifuge tube, centrifuged at 12,000 rpm for 10 min, and a supernatant was discarded; a resulting precipitate was pre-frozen at −70° C. for 4 h, and then lyophilized overnight in a −60° C. vacuum lyophilizer. A dry weight was weighed using a ten-thousandth balance, and dry cell weights (CDWs) were 0.381 g/L, 0.403 g/L, 0.522 g/L, 0.343 g/L, and 0.362 g/L, respectively. The Halomonas, Bacillus, Micrococcus luteus, Brevibacterium casei and Bacillus licheniformis can proliferate in this medium, indicating that the medium obtained by this method has certain microbial culture ability.
Experiment 2
[0118] 1) A solution of 4% NaOH/7% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was gradually clarified. After adjusting the pH value to 10 with HCl, the solution was filtered with gauze to collect a filtrate.
[0119] 2) in the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Micrococcus luteus with a 3% inoculum (OD.sub.600=0.5 to 0.6) was inoculated into the mixture above. Shaker culture was conducted at 37° C., 150 rpm. After 72 h, 1 mL of a bacterial solution was placed in a 1.5 mL EP tube, centrifuged at 4,000 rpm for 5 min, and 1 μL of a lower layer precipitate was smeared on a glass slide, the slide glass was fixed with a liquid conductive adhesive on a pie-shaped battery, followed by spraying gold, and the battery was placed on a electron microscope stage to collect images after spraying gold (
Experiment 3
[0120] 1) A solution of 4% NaOH/7% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was gradually clarified. After adjusting the pH value to 10 with HCl, the solution was filtered with gauze to collect a filtrate.
[0121] 2) in the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Micrococcus luteus with a 3% inoculum (OD.sub.600=0.5 to 0.6) was inoculated into the mixture above. Shake fermentation culture was conducted at 37° C., 150 rpm for 72 h. A pH value, CDW and a cellulose content in the fermentation system (indirectly determined by a Fehling's reagent method) were measured every 8 h. It is found that with the increase of culture time, the pH value and glucose content are in a decreasing trend, while the CDW is in a continuous increasing trend, and an average CDW is about 0.52 g/L, as shown in
Experiment 4
[0122] 1) A solution of 4% NaOH/7% Urea was prepared, 14 g/L microcrystalline cellulose was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was gradually clarified. After adjusting the pH value to 10 with HCl, the solution was filtered with gauze to collect a filtrate.
[0123] 2) in the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), and NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min. Micrococcus luteus with a 3% inoculum (OD.sub.600=0.5 to 0.6) was inoculated into the mixture above. Shake fermentation culture was conducted at 37° C., 150 rpm for 72 h. After 72 h, a culture was transferred to a 50 mL centrifuge tube, centrifuged at 12,000 rpm for 10 min, and a supernatant was discarded; a resulting precipitate was pre-frozen at −70° C. for 4 h, and then lyophilized overnight in a −60° C. vacuum lyophilizer.
[0124] 3) Preparation of an esterification solution: 500 mL of anhydrous methanol (AR) was added with about 1 g/L benzoic acid and 3% (v/v) concentrated sulfuric acid (mass fraction 98%) carefully and slowly.
[0125] 4) About 20 mg of freeze-dried cell samples were accurately weighed (0.0001 g) and placed in an esterification tube, and 2 mL of the chloroform (AR) and 2 mL of the esterification solution were added. About 10 mg of a standard polyhydroxy alkanoate (PHA) sample (PHB) was treated in the same way to prepare a chloroform solution of the standard sample. The esterification tube is sealed, shaken to mix evenly, followed by conducting esterification at a constant temperature of 100° C. for 4 h. After cooling to room temperature, 1 mL of ddH.sub.2O was added, shaken until completely mixed, and allowed to stand for about 30 min for layering. After the aqueous phase and the organic phase were completely separated, a lower layer solution (chloroform phase) was subjected to GC analysis to obtain results as shown in
Experiment 5
[0126] 1) Licorice straw was pulverized using a Chinese herbal medicine grinder, and sieved through a 30-mesh sieve to obtain a licorice straw powder. A solution of 4% NaOH/7% Urea was prepared, 10 g/L licorice straw powder was added to the solution, frozen at −20° C. for 24 h, and thawed at room temperature; when being heated to −14° C., the solution was in a state of an ice-water mixture. The stirring was conducted, and the solution was partially precipitated. After being adjusted to a pH value of 10 with HCl, the solution was filtered with gauze to collect a filtrate.
[0127] 2) In the filtrate, NH.sub.4Cl (2 g/L), MgSO.sub.4 (0.2 g/L), NaCl (10 g/L) were added in proportion, followed by conducting autoclaving at 115° C. for 15 min; Micrococcus luteus (OD.sub.600=0.5 to 0.6) was inoculated with a 3% inoculum. It is found that the OD.sub.600 is 0.31 at 0 h, and the OD.sub.600 increases to 0.75 after 48 h. This proves that Micrococcus luteus can proliferate in this medium, indicating that the medium obtained by this method has a certain ability to culture microorganisms.
[0128] In this specification, descriptions of reference terms such as “one embodiment”, “some embodiments”, “an example”, “a specific example” and “some examples” indicate that specific features, structures, materials or characteristics described in combination with the embodiment(s) or example(s) are included in at least one embodiment or example of the present disclosure. In this specification, schematic representation of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, a person skilled in the art may combine different embodiments or examples described in this specification and characteristics of the different embodiments or examples without mutual contradiction.
[0129] Although the embodiments of the present disclosure have been illustrated and described above, it will be appreciated that the above embodiments are illustrative and should not be construed as limiting the scope of the present disclosure. Changes, modifications, substitutions and variations can be made to the above embodiments by a person of ordinary skill in the art within the scope of the present disclosure.