A method and application of stimulating microorganisms to secrete lipopeptide biosurfactants

Abstract

The present invention belongs to the technical field of microbial environmental remediation, discloses a method and application of stimulating microorganisms to secrete lipopeptide biosurfactants, and specifically discloses the application of sodium gluconate and / or vegetative Raoultella in the preparation of biosurfactants. The present invention proposes for the first time to use vegetative Raoultella and / or sodium gluconate to prepare biosurfactants, enriching the types of biosurfactant-producing bacteria and the culture materials used.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial environmental restoration, and specifically relates to a method and application of stimulating microorganisms to secrete lipopeptide biosurfactants. Background Art

[0002] With the continuous advancement of science and technology since the Industrial Revolution, people are increasingly using oil as the main energy source. Due to the characteristics of oil, it is easy to cause leakage during the process of mining, production, transportation, use and disposal. Most of the leaked oil will be released into the environment, causing harm to human health, and some will even have teratogenic, carcinogenic and mutagenic effects. Compared with physical remediation and chemical remediation, the use of microbial remediation methods for petroleum hydrocarbon pollution has gradually become the preferred method for people. It has the advantages of high efficiency, economy and no secondary pollution. However, there are still some problems in the practical application of microbial remediation methods: (1) Non-indigenous petroleum hydrocarbon degrading microorganisms are difficult to survive in petroleum hydrocarbon contaminated sites and cannot compete with indigenous microorganisms; (2) Petroleum hydrocarbon substances are generally hydrophobic, which is not conducive to microbial adsorption and uptake of hydrocarbons; (3) Petroleum hydrocarbon substances have a relatively complex structure and their degradation cycle is too long. These problems have jointly prompted researchers to find auxiliary technologies that can give full play to the effects of petroleum hydrocarbon degrading bacteria in practical applications, namely biostimulation.

[0003] Existing biostimulation mainly promotes the degradation of pollutants by increasing the activity of indigenous functional microorganisms through the addition of nutrients. These nutrients can promote the rapid growth of microorganisms, shorten the reaction time, and induce the increase of genes and enzymes related to degradation. In addition, the addition of surfactants is also a kind of biostimulation. Surfactants are mainly divided into chemical and biological types. Chemical surfactants such as Tween-80 and Triton-100 will cause secondary pollution to the environment, while biological surfactants are biodegradable, environmentally compatible, and can maintain good stability under different environments. Biosurfactants have a certain solubilizing effect on hydrocarbons; increase the contact area between microorganisms and hydrocarbons; change the hydrophobicity of the cell surface of microorganisms, which is conducive to the adsorption and uptake of hydrocarbons by microorganisms. At present, the research on microbial stimulants is still at a relatively primary level. Enzyme hydrolyzates and rice bran extracts developed by the University of Seville in Spain can significantly stimulate the degradation enzyme activity of petroleum and benzopyrene pollutants in soil; the fungal stimulant developed by the University of Düsseldorf in Germany can stimulate fungal cell growth and secondary metabolite formation through the interaction of fungal inducers with plants and microorganisms, but it is still in the laboratory research stage. Therefore, targeting microbial flora, developing small molecule peptides, polysaccharides, organic acids / esters and other substances to stimulate microbial stress responses will have great potential. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide the use of sodium gluconate and / or vegetative Raoultella in the preparation of biosurfactants.

[0005] The second aspect of the present invention aims to provide a method for preparing a biosurfactant.

[0006] The third aspect of the present invention is to provide a biosurfactant.

[0007] The purpose of the fourth aspect of the present invention is to provide the preparation method of the second aspect of the present invention or the use of the biosurfactant of the third aspect of the present invention in the degradation of petroleum hydrocarbons.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides the use of sodium gluconate and / or vegetative Raoultella in the preparation of biosurfactants.

[0010] Preferably, the biosurfactant is a lipopeptide surfactant.

[0011] Preferably, the final concentration of the sodium gluconate is 1 to 7 g / L, and further 2 to 5 g / L.

[0012] Preferably, the deposit number of the vegetative Raoultella is GDMCC No:62352.

[0013] The second aspect of the present invention provides a method for preparing a biosurfactant, comprising the following steps: inoculating a bacterial solution of Raoultella vegetabilis into a fermentation medium, culturing, and obtaining a biosurfactant.

[0014] Preferably, the fermentation medium contains sodium gluconate.

[0015] Preferably, the final concentration of the sodium gluconate is 1 to 7 g / L, further 2 to 6 g / L, and most preferably 2 to 5 g / L.

[0016] Preferably, the fermentation medium also contains n-hexadecane and an inorganic salt medium.

[0017] Preferably, the added amount of hexadecane is 0.4 v / v% to 3 v / v%, further 0.5 v / v% to 2 v / v%, and most preferably 0.5 v / v% to 1.5 v / v%.

[0018] Preferably, the inorganic salt culture medium includes K2HPO4, MgSO4, CaCl2, Na2SO4 and NH4Cl.

[0019] Preferably, the inorganic salt culture medium includes K2HPO4 1-2 g / L, MgSO4 1-3 g / L, CaCl2 0.005-0.01 g / L, Na2SO4 2-4 g / L, and NH4Cl 1-2 g / L.

[0020] Preferably, the pH value of the inorganic salt culture medium is 6.0-7.0.

[0021] Preferably, the culture conditions are 30-40° C., 100-200 rpm for 6-10 days.

[0022] More preferably, the culture conditions are 30-37° C. and 150-200 rpm for 6-10 days.

[0023] Preferably, the addition amount of the vegetative Raoultella solution is 5 v / v% to 10 v / v%; further 7 v / v% to 10 v / v%.

[0024] Preferably, the method for preparing the vegetative Raoultella bacterial liquid is: inoculating a culture medium with vegetative Raoultella and culturing.

[0025] Preferably, the culture medium is any culture medium that can be used for culturing Raoultella vegetabilis; further, it is LB culture medium.

[0026] Preferably, the culture conditions are 30-37° C. for 9-12 h.

[0027] Preferably, the preparation method further comprises solid-liquid separation to obtain bacterial sludge and crushing treatment.

[0028] Preferably, the solid-liquid separation is performed under the conditions of 4° C. and 7000-8000 rpm for centrifugation for 4-6 min.

[0029] Preferably, the crushing is carried out using an ultrasonic crusher.

[0030] Preferably, the crushing conditions are 20-30°C, 25-30KHz, and crushing for 15-25min.

[0031] Preferably, the bacterial sludge is crushed to separate the solid and liquid, and the cell crushing liquid is collected.

[0032] Preferably, the preparation method further comprises drying.

[0033] More preferably, the drying method is freeze-drying.

[0034] The third aspect of the present invention provides a biosurfactant obtained by the preparation method of the second aspect of the present invention.

[0035] Preferably, the biosurfactant contains hexadecanoic acid, octadecanoic acid, 10-octadecenoic acid, 3-hydroxydodecanoic acid methyl ester and 10-nonadecenoic acid.

[0036] Preferably, the critical micelle of the biosurfactant is about 1000 mg / L.

[0037] The fourth aspect of the present invention provides the preparation method of the second aspect of the present invention or the use of the biosurfactant of the third aspect of the present invention in the degradation of petroleum hydrocarbons.

[0038] Preferably, the usage amount of the biosurfactant is 100-2500 mg / L.

[0039] More preferably, the usage amount of the biosurfactant is 800-2500 mg / L.

[0040] More preferably, the usage amount of the biosurfactant is 1000-2500 mg / L.

[0041] The beneficial effects of the present invention are:

[0042] The present invention proposes for the first time to prepare biosurfactants using vegetative Raoultella and / or sodium gluconate, thereby enriching the types of biosurfactant-producing bacteria and the culture materials used.

[0043] The present invention provides a method for preparing a lipopeptide biosurfactant, wherein the addition of sodium gluconate can effectively stimulate the secretion of biosurfactants by bacteria and increase the yield of biosurfactants. The preparation method does not require complex equipment, has a short fermentation cycle, and the fermentation process is simple and easy, which is conducive to the development, application and promotion of lipopeptide biosurfactants.

[0044] The present invention provides a lipopeptide biosurfactant. The addition of the lipopeptide biosurfactant can effectively improve the degradation of n-hexadecane by vegetative Raoultella, and the degradation effect is improved by 341.2% compared with that without adding the lipopeptide biosurfactant. The characteristics of low degradation efficiency and long degradation cycle of petroleum hydrocarbon pollution by microorganisms are overcome, and the needs of bioremediation of petroleum hydrocarbon polluted environments can be met. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Diagram of the extraction system with and without sodium gluconate added during the preparation of biosurfactants (left picture) and without sodium gluconate added (right picture).

[0046] Figure 2 This is the emulsification index result diagram of the cell lysis solution of Example 1 after being placed for 180 hours.

[0047] Figure 3It is a graph showing the results of an acid bromophenol blue experiment, wherein 1 to 4 represent distilled water, the biosurfactant prepared in Example 1, hexadecyltrimethylammonium bromide and sodium dodecyl sulfate, respectively.

[0048] Figure 4 It is a graph showing the results of a methylene blue-chloroform color development experiment, wherein 1 to 4 represent distilled water, the biosurfactant prepared in Example 1, hexadecyltrimethylammonium bromide and sodium dodecyl sulfate, respectively.

[0049] Figure 5 It is a graph of the experimental results of agarose immunoblotting, wherein 1 to 4 represent the biosurfactant prepared in Example 1, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate and the biosurfactant prepared in Example 1, respectively.

[0050] Figure 6 It is a graph showing the results of a thin layer chromatography experiment, wherein X1 and X2 both represent the biosurfactants prepared in Example 1, wherein rat represents a rhamnolipid standard product, gly represents a glycine standard product, and egg represents a phosphatidylcholine standard product.

[0051] Figure 7 This is the Fourier infrared spectrum of the biosurfactant prepared in Example 1.

[0052] Figure 8 This is a gas chromatography-mass spectrometry result diagram of the biosurfactant prepared in Example 1.

[0053] Fig. 9 This is a graph showing the relationship between the biosurfactant concentration and surface tension obtained in Example 1.

[0054] Fig.10 The graphs show the degradation results of n-hexadecane by Raoultella vegetabilis without adding the biosurfactant prepared in Example 1 and with adding different concentrations of the biosurfactant prepared in Example 1. DETAILED DESCRIPTION

[0055] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0056] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0057] The taxonomic name of Raoultella vegetabilis used hereinafter is Raoultella planticola The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 6, 2022, with the deposit number GDMCC No: 62352.

[0058] Example 1 Preparation of biosurfactant

[0059] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0060] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and use a pH meter to adjust the pH of the inorganic salt culture medium to 6.5±0.2;

[0061] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0062] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0063] (4) adding 1 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0064] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0065] Example 2 Preparation of biosurfactant

[0066] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0067] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0068] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0069] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0070] (4) adding 0.5 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0071] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0072] Example 3 Preparation of biosurfactant

[0073] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0074] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0075] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0076] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0077] (4) adding 1.5 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0078] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0079] Example 4 Preparation of biosurfactant

[0080] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0081] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0082] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0083] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0084] (4) adding 2 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0085] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0086] Example 5 Preparation of biosurfactant

[0087] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0088] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0089] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0090] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0091] (4) adding 2.5 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0092] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0093] Example 6 Preparation of biosurfactant

[0094] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0095] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0096] (2) Add sodium gluconate to the inorganic salt medium to a final concentration of 3 g / L, sterilize at 121° C. for 20 min, and set aside;

[0097] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0098] (4) adding 1 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0099] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0100] Example 7 Preparation of biosurfactant

[0101] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0102] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0103] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 4 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0104] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0105] (4) adding 1 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0106] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0107] Example 8 Preparation of biosurfactant

[0108] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0109] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0110] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 5 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0111] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0112] (4) adding 1 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0113] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0114] Example 9 Preparation of biosurfactant

[0115] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0116] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0117] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0118] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0119] (4) adding 1 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 37° C. and 150 rpm for 9 days;

[0120] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0121] Example 10 Preparation of biosurfactant

[0122] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0123] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, adjust pH to 6.5±0.2, sterilize at 121℃ for 20 min, and set aside;

[0124] (2) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0125] (3) adding n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (1) in an amount of 1 v / v%, and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 30° C. and 150 rpm for 9 days;

[0126] (4) The fermentation liquid after culture was poured out, and centrifuged at 4°C, 8000 rpm for 5 min to collect the bacteria, which were then disrupted by an ultrasonic cell disruptor at a time of 20 min, a temperature of 25°C, and a frequency of 25 kHz. The cell disrupted liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, and the secretion of biosurfactant was detected by referring to the emulsification index determination method described below.

[0127] When Raoultella vegetabilis used n-hexadecane as the sole carbon source, it was unable to secrete biosurfactants ( Figure 1 ).

[0128] Example 11 Preparation of biosurfactant

[0129] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0130] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0131] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 2 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0132] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0133] (4) adding 0.2 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 37° C. and 150 rpm for 9 days;

[0134] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0135] Example 12 Preparation of biosurfactant

[0136] A method for stimulating microorganisms to secrete biosurfactants, comprising the following steps:

[0137] (1) Prepare inorganic salt culture medium: K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, and adjust the pH to 6.5±0.2;

[0138] (2) adding sodium gluconate to the inorganic salt medium to a final concentration of 4 g / L, sterilizing at 121° C. for 20 min, and setting aside;

[0139] (3) The vegetative Raoultella was cultured in LB medium (10 g / L peptone, 5 g / L yeast powder and 10 g / L sodium chloride, sterilized at 121°C for 20 min) and cultured at 37°C for 10 h to obtain the vegetative Raoultella;

[0140] (4) adding 0.2 v / v% of n-hexadecane filtered through a 0.22 μm filter membrane to the inorganic salt medium of step (2), and adding 10 v / v% of Raoultella vegetabilis bacterial solution, and culturing in a shaker at 37° C. and 150 rpm for 9 days;

[0141] (5) The fermentation liquid after culture was poured out, centrifuged at 4°C, 8000 rpm for 5 min, the bacteria were collected, and the bacteria were disrupted by an ultrasonic cell disruptor with a disruption time of 20 min, a disruption temperature of 25°C, and a disruption frequency of 25 kHz; the cell disruption liquid was freeze-dried by a vacuum freeze dryer to obtain a freeze-dried crude extract, that is, a lipopeptide biosurfactant.

[0142] Effect Example

[0143] 1. Determination of emulsification index

[0144] The emulsification index of the cell lysis solution that has not been freeze-dried by a vacuum freeze dryer in Example 1 was determined. The specific steps are as follows: take six graduated test tubes at room temperature, mark three of each, AB, and add 5 mL of liquid paraffin to each test tube; then add 5 mL of the test solution (A is the cell lysis solution, B is deionized water, as a blank control group) to each test tube; then oscillate with a vortex instrument for 2 minutes, and measure the emulsion layer height (H1, cm) and the total liquid height (H, cm) in the test tube after standing for different 24 hours, 72 hours, and 180 hours, and calculate the emulsification index. The calculation formula of the emulsification index is: emulsification index (%) = H1 / H×100%.

[0145] The results are shown in Table 1. After the cell lysate obtained in Example 1 was mixed with liquid paraffin and allowed to stand for 24 hours and 72 hours, the emulsification index was 60.51±1.62% and 61.51±1.98% respectively. After standing for 180 hours, the emulsification index was still 58.33±5.89% ( Figure 2 ), the measurement results of the biosurfactants prepared in Examples 2 to 10 were not significantly different from those in Example 1.

[0146] Table 1 Determination results of emulsification index

[0147]

[0148] 2. Determination of biosurfactant type

[0149] (1) Acid bromophenol blue test

[0150] A certain amount of the biosurfactant prepared in Example 1 was taken and dissolved in deionized water to make the final concentration of the biosurfactant 1%; the dissolved biosurfactant was added to a test tube of bromophenol blue solution (75 mL 0.2 mol / L sodium acetate solution, 925 mL 0.2 mol / L acetic acid solution and 20 mL 0.1% bromophenol blue solution (0.1 g bromophenol blue dissolved in 100 mL 95% ethanol), pH adjusted to 3.6-3.9, prepared and used immediately), anionic surfactant sodium dodecyl sulfate, cationic surfactant hexadecyltrimethylammonium bromide and deionized water were used as control groups, and their color changes were observed.

[0151] Bromophenol blue is an anionic dye that can form a stable complex with a cationic surfactant, and the color is sky blue or blue-purple or somewhere in between. Figure 3 As shown, the color of the No. 2 scale test tube (biosurfactant prepared in Example 1) is green, which is the same as the color of the No. 1 (deionized water) and No. 4 scale test tubes (anionic surfactant sodium dodecyl sulfate), while the No. 3 scale test tube (cationic surfactant hexadecyltrimethylammonium bromide) is blue, forming a stable complex with the bromophenol blue solution. Therefore, it is preliminarily determined that the substance may be an anionic surfactant or a non-ionic surfactant.

[0152] (2) Methylene blue-chloroform color development experiment

[0153] In order to further determine whether the biosurfactant belongs to anionic surfactant or nonionic surfactant. The methylene blue-chloroform method is used to determine the type of active agent, and the specific steps are as follows: the biosurfactant prepared in Example 1 is dissolved in deionized water (to a final concentration of 0.2%), added to a methylene blue-chloroform solution (a mixed solution of 8 mL of methylene blue solution and 5 mL of chloroform, wherein the preparation method of the methylene blue solution is: weigh 0.015 g of methylene blue, mix with distilled water, add 6 g of concentrated sulfuric acid and 25 g of anhydrous sodium sulfate, dissolve with distilled water and make up to 495 mL), shake vigorously to separate the layers, and use anionic surfactant sodium dodecyl sulfate, cationic surfactant hexadecyltrimethylammonium bromide and deionized water as control groups, respectively, and observe the color of the water layer and the chloroform layer.

[0154] Methylene blue is a cationic colored substance. When it encounters anionic surfactants, it will generate products that are insoluble in water but soluble in chloroform, making the chloroform layer darker in color. If the test solution contains cationic surfactants, due to the combination of anionic and cationic surfactants, methylene blue will be separated from the anionic surfactant and return to the water from the chloroform, making the chloroform layer lighter in color. Figure 4 As shown, the chloroform layer of the No. 4 scale test tube (sodium dodecyl sulfate) has a dark color while the water layer is almost colorless. The water layer of the No. 3 scale test tube (hexadecyl trimethyl ammonium bromide) has a darker color while the chloroform layer has a lighter or colorless color. The No. 1 (deionized water) and No. 2 scale test tubes (biosurfactant prepared in Example 1) have roughly the same color, and the water layer is an emulsion, indicating that the biosurfactant prepared in Example 1 is a non-ionic surfactant.

[0155] (3) Agarose immunodiffusion co-precipitation experiment

[0156] In order to further determine that the biosurfactant prepared in Example 1 belongs to the type of non-ionic surfactant, an agarose immunodiffusion coprecipitation experiment was performed using the biosurfactant. The specific steps are as follows: a 1% agarose solution was prepared, heated in a microwave oven to dissolve, and then poured into a culture dish with a diameter of 9 cm. After it solidified, four small holes were punched on the solid surface along the same straight line using a gun tip. The biosurfactant prepared in Example 1 (dissolved in deionized water to a final concentration of 1%) was added to the small holes at both ends (20 μL / hole), and 20 μL of 1% hexadecyltrimethylammonium bromide and 1% sodium dodecyl sulfate were added to the two middle holes respectively. The solution was allowed to stand at room temperature to observe whether immunocoprecipitation occurred.

[0157] The ions of the surfactant solution can diffuse on the agarose gel solid. When the ion types of the two solutions are opposite, the positive and negative charged ions will combine to form a co-precipitation. Figure 5 It can be seen that the anionic and cationic surfactants have opposite ionic types and form a precipitate after combining, while the biosurfactant prepared in Example 1 does not form a precipitate with hexadecyltrimethylammonium bromide or sodium dodecyl sulfate, which further confirms that the biosurfactant prepared in Example 1 is a non-ionic surfactant.

[0158] (4) Thin layer chromatography

[0159] In order to further explore whether the biosurfactant prepared in Example 1 belongs to glycolipid surfactant, lipopeptide surfactant or phospholipid surfactant, thin layer chromatography was used for determination, and the specific steps were as follows: the biosurfactant prepared in Example 1 was dissolved in methanol to prepare a solution with a concentration of 0.1 g / mL, and a capillary was used to spot 4 to 5 times at a distance of 1 cm from the lower end of the thin layer chromatography plate, and the sample point diameter was less than 2 mm; after drying, the silica gel plate was placed obliquely in a developing tank for development; when the developing agent (chloroform: methanol: acetic acid = 65:15:1) ran to 1 cm from the upper end of the silica gel plate, the silica gel plate was taken out, marked, and dried in an oven; 0.1 g / mL rhamnolipid, 0.1 g / mL glycine, and 0.1 g / mL lecithin were used as standard samples of glycolipid, lipopeptide and phospholipid surfactants as control groups respectively; phenol-sulfuric acid reagent, phosphomolybdic acid-ethanol or ninhydrin-acetone reagent was used to spray the dried silica gel plate for color development, and then it was allowed to stand at room temperature to observe the staining.

[0160] Phenol-sulfuric acid reagent, phosphomolybdic acid-ethanol and ninhydrin-acetone reagent can make glycolipid surfactants appear brown spots, phospholipid surfactants appear light blue spots, and lipopeptide surfactants appear purple spots. Figure 6 It can be seen that the biosurfactant prepared in Example 1 can only be dyed purple-red by the ninhydrin-acetone reagent, so it is determined that the biosurfactant prepared in Example 1 is a lipopeptide biosurfactant.

[0161] The measurement results of the biosurfactants prepared in Examples 2 to 9 are consistent with those in Example 1.

[0162] 3. Structural Characterization of Biosurfactants

[0163] In order to explore the structure of the lipopeptide biosurfactant prepared in Example 1, Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry were used to characterize the structure.

[0164] (1) Fourier transform infrared spectroscopy

[0165] The specific experimental steps of the experiment are as follows: the lipopeptide biosurfactant prepared in Example 1 and dried potassium bromide are mixed in a ratio of 1:100 and ground into powder, and then tableted using a tablet press, and then subjected to Fourier infrared spectroscopy scanning with a scanning range of 4000 cm -1 ~400cm -1 , resolution 4cm -1 .

[0166] The results of Fourier transform infrared spectroscopy are as follows Figure 7 As shown. At 3000~3800cm -1In the range of , there is a broad and strong absorption peak, and the maximum absorption value appears at 3235.00cm -1 , mainly produced by -OH and -NH stretching vibration; 2923.56cm -1 The absorption peak at 1648.36cm indicates the presence of CH2 and CH3; -1 The absorption peak at 1545.67cm is speculated to be the stretching vibration of CO-N bond; -1 The absorption peaks at 1432.85cm are NH bending and CN stretching; -1 The absorption peak at 992.68cm may be the bending vibration of CH, and it is speculated that there is a carbon chain structure; -1 There is an absorption peak at , which represents the stretching vibration of the CO bond; according to the results of Fourier infrared spectroscopy, it is shown that the lipopeptide biosurfactant prepared in Example 1 has the functional group characteristics of lipopeptide compounds, namely, aliphatic hydrocarbon group and peptide group.

[0167] (2) Gas chromatography-mass spectrometry

[0168] The fatty acid components in the lipopeptide biosurfactant prepared in Example 1 were further analyzed by gas chromatography-mass spectrometry. The specific experimental steps were as follows: 0.02 g of the lipopeptide biosurfactant prepared in Example 1 was weighed, and fully oscillated and dissolved by 2 mL of n-hexane, then mixed with 2 mL of 0.5 M sulfuric acid-methanol solution, and heated in a 70 ° C water bath for 60 min; after cooling, 2 mL of saturated sodium chloride solution was added to separate the solution, and then 1 g of anhydrous sodium sulfate was used for dehydration, and the solution was filtered through a 0.2 μm membrane for on-machine detection. The gas phase conditions were an injection port temperature of 280 ° C, a carrier gas of helium, a carrier gas flow rate of 1 mL / min, a split ratio of 10:1, and an injection volume of 1 μL; the heating program was an initial temperature of 60 ° C, maintained for 2 min, and increased to 280 ° C at 10 ° C / min, and maintained for 10 min; mass spectrometry conditions: the ion source was an EI source, the ionization voltage was 70 eV, the ion source temperature was 230 ° C, and the quadrupole temperature was 150 ° C.

[0169] Test results such as Figure 8 As shown, the fatty acid components in the lipopeptide biosurfactant prepared in Example 1 contain hexadecanoic acid, octadecanoic acid, 10-octadecenoic acid, 3-hydroxydodecanoic acid methyl ester and 10-nonadecenoic acid, and the measurement results of the biosurfactants prepared in Examples 2 to 9 are consistent with those in Example 1.

[0170] 4. Determination of critical micelle concentration of biosurfactants

[0171] The lipopeptide biosurfactant prepared in Example 1 was diluted to different concentrations (0 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L, 250 mg / L, 500 mg / L, 1000 mg / L, 2500 mg / L, 5000 mg / L), and the surface tension at different concentrations was measured by the platinum plate method. The instrument for testing the surface tension was a fully automatic surface tension meter. The surface tension-concentration logarithmic curve (such as Fig. 9 As shown), the surfactant concentration corresponding to the inflection point of the curve is the critical micelle concentration of the lipopeptide surface activity, and its critical micelle concentration is 1000 mg / L. The measurement results of the biosurfactants prepared in Examples 2 to 9 are not significantly different from those in Example 1.

[0172] 5. Effect of biosurfactants on the degradation of n-hexadecane by Raoultella vegetata

[0173] The lipopeptide biosurfactant prepared in Example 1 was added to a Raoultella vegetative n-hexadecane degradation medium (K2HPO4·3H2O 1.3 g / L, MgSO4·7H2O 1.0 g / L, CaCl2 0.005 g / L, Na2SO4 2.0 g / L, NH4Cl 1.0 g / L, pH 6.5±0.2, with 0.2 v / v% n-hexadecane added; the medium used n-hexadecane as the sole carbon source) (the final concentrations of the lipopeptide biosurfactant were 0 mg / L, 1000 mg / L and 2500 mg / L, respectively). A blank control group was cultured only with the Raoultella vegetative n-hexadecane degradation medium at 30°C for 6 days. Raoultella vegetative was inoculated into the above medium, and the inoculation amount of Raoultella vegetative was 10 v / v% (bacterial concentration 10 9 cfu / mL~10 11 cfu / mL), cultured at 30°C for 6 days, the content of n-hexadecane in the culture medium was detected by gas chromatography, and the residual rate of n-hexadecane was calculated. The residual rate calculation formula is as follows: residual rate (%) = (the content of n-hexadecane in the culture medium before culture - the content of n-hexadecane in the culture medium after culture) / the content of n-hexadecane in the culture medium before culture × 100%. The improvement effect calculation formula is as follows: ((the residual rate of the blank control group - the residual rate without adding lipopeptide biosurfactant) - (the residual rate of the blank control group - the residual rate without adding lipopeptide biosurfactant)) / (the residual rate of the blank control group - the residual rate without adding lipopeptide biosurfactant) × 100%.

[0174] By detecting the content of n-hexadecane in the culture medium, the residual rate of n-hexadecane in the culture medium of the blank control group was 97.32%, when the lipopeptide biosurfactant prepared in Example 1 was not added, the residual rate of n-hexadecane was 85.39%, when 1000 mg / L of the lipopeptide biosurfactant prepared in Example 1 was added, the residual rate of n-hexadecane was 44.69%, relative to the case where the lipopeptide biosurfactant prepared in Example 1 was not added, the degradation effect of Raoultella vegetabilis on n-hexadecane was improved by 341.2% ( Fig.10 ). When the lipopeptide biosurfactant prepared in Example 1 was added to 2500 mg / L, the degradation rate of n-hexadecane by Raoultella vegetative was reduced, but the effect was still better than that without adding the lipopeptide biosurfactant prepared in Example 1. The reason for this is that high concentrations of lipopeptide biosurfactants may have a certain antibacterial effect on Raoultella vegetative.

[0175] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of sodium gluconate in promoting the growth of Raoultella vegetabilis to prepare a biosurfactant, wherein the biosurfactant is a lipopeptide biosurfactant containing hexadecanoic acid, octadecanoic acid, 10-octadecenoic acid, 3-hydroxydodecanoic acid methyl ester and 10-nonadecenoic acid.

2. A method for preparing a biosurfactant, comprising the following steps: inoculating the bacterial liquid of Raoultella vegetabilis into a fermentation medium, culturing, and obtaining a biosurfactant; The fermentation medium contains sodium gluconate; The fermentation medium also contains n-hexadecane and an inorganic salt medium; The biosurfactant is a lipopeptide biosurfactant, which contains hexadecanoic acid, octadecanoic acid, 10-octadecenoic acid, 3-hydroxydodecanoic acid methyl ester and 10-nonadecenoic acid.

3. The preparation method according to claim 2, characterized in that: The final concentration of the sodium gluconate is 1-7 g / L.

4. The preparation method according to claim 3, characterized in that: The added amount of hexadecane is 0.4 v / v% to 3 v / v%.

5. The preparation method according to any one of claims 2 to 4, characterized in that The culture conditions are 30-40° C. and 150-200 rpm for 6-10 days.

6. The preparation method according to any one of claims 2 to 4, characterized in that The preparation method also includes solid-liquid separation to obtain bacterial sludge and crushing treatment.

7. A biosurfactant obtained by the preparation method according to any one of claims 2 to 6; the biosurfactant is a lipopeptide biosurfactant containing hexadecanoic acid, octadecanoic acid, 10-octadecenoic acid, 3-hydroxydodecanoic acid methyl ester and 10-nonadecenoic acid.

8. Use of the biosurfactant prepared by the preparation method according to any one of claims 2 to 6 or the biosurfactant according to claim 7 in the degradation of petroleum hydrocarbons.

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