CN111394398A

CN111394398A - Method for preparing PHA (polyhydroxyalkanoate) by fermenting high-salt molasses serving as raw material

Info

Publication number: CN111394398A
Application number: CN202010492347.8A
Authority: CN
Inventors: 佟毅; 李义; 唐堂; 李大勇; 刘海军; 陈博; 彭超; 周卫强; 吴琼; 陈国强; 叶健文; 武丽达; 安泰
Original assignee: Tsinghua University; Cofco Nutrition and Health Research Institute Co Ltd; Jilin COFCO Bio Chemical Co Ltd; Cofco Biotechnology Co Ltd
Current assignee: Tsinghua University; Cofco Nutrition and Health Research Institute Co Ltd; Jilin COFCO Bio Chemical Co Ltd; Cofco Biotechnology Co Ltd
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2020-07-10
Anticipated expiration: 2040-06-03
Also published as: CN111394398B

Abstract

The invention relates to the field of PHA fermentation, in particular to a method for preparing PHA by fermenting high-salt molasses serving as a raw material. The method comprises the following steps: inoculating PHA fermentation strains into a fermentation medium for fermentation under the condition of fermentation to produce PHA; wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses. The method can effectively reduce the production cost of PHA, and the fermentation efficiency is also improved.

Description

Method for preparing PHA (polyhydroxyalkanoate) by fermenting high-salt molasses serving as raw material

Technical Field

The invention relates to the field of PHA fermentation, in particular to a method for preparing PHA by fermenting high-salt molasses serving as a raw material.

Background

The current society develops at a rapid pace, the environment is protected, the main melody of the contemporary chemical product is formed, and the prior fossil refining material is more and more difficult to meet the requirement of environmental protection. Under such a trend, the development and production of biodegradable materials are more urgent and necessary. PHA is a biodegradable, novel plastic material with similar material properties to traditional plastic products. PHA can be accumulated and stored in most microorganisms as an energy supply substance, and can be produced, extracted and purified on a large scale by utilizing a biological fermentation method.

In most PHA fermentation processes, a single sugar, primarily a monosaccharide, such as glucose, is often used as the primary carbon source for cultivation. PHA is accumulated mainly under unbalanced conditions of high carbon and low nitrogen, so that the use of glucose throughout the whole PHA fermentation process accounts for a large part of the overall fermentation cost. Therefore, finding an inexpensive carbon source which can replace glucose and has high fermentation performance can better save PHA fermentation cost.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a method for preparing PHA by fermenting high-salt molasses as a raw material, which can effectively reduce the production cost of PHA and improve the fermentation efficiency.

In some sugar processing plants, many industrial by-products are produced, molasses being a dark thick mass rich in sucrose, glucose, fructose. Some molasses are also enriched in salt by the processing process to form high salt molasses, which have a salt content of about 8% or more of the total composition. Although the high-salt molasses has high sugar content, the high-salt molasses has high salt concentration, high pigment content and high viscosity, so that the treatment difficulty of the high-salt molasses serving as a byproduct is increased. The inventor of the invention finds that the high-salt molasses is applied to the fermentation (halotolerant bacteria) production process of PHA to replace glucose for fermentation, on one hand, the use of the high-salt molasses reduces the addition of exogenous salt in the fermentation process, effectively improves the utilization rate of industrial byproducts, and reduces the cost; in the second aspect, the high-salt molasses is rich in various trace elements, N, P compounds, vitamins and the like which are necessary for the growth of microorganisms, can effectively promote the growth of thalli, can properly reduce the addition of corresponding raw materials in a culture medium or even directly replace conventional culture medium ingredients in the using process, reduces the cost of the culture medium, can still ensure the normal operation of PHA fermentation, and can improve the fermentation efficiency.

In order to achieve the above object, the present invention provides a method for producing PHA by fermentation, comprising inoculating a PHA fermenting strain into a fermentation medium for fermentation under conditions capable of producing PHA by fermentation;

wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses.

Preferably, the fermentation medium contains high-salt molasses, yeast powder, dipotassium phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride, and relative to 1L fermentation medium, the content of the high-salt molasses is 15-35ml, the content of the yeast powder is 15-17g, the content of the dipotassium hydrogen phosphate is 2-5g, the content of the disodium hydrogen phosphate is 5-8g, the content of the magnesium sulfate is 0.2-0.3g and the content of the sodium chloride is 45-55 g.

Preferably, the fermentation is carried out under the condition of stirring, the rotation speed of the stirring is 400-600rpm from 0h to 8-12 h;

from 8-12h to 16-20h, the stirring speed is 600-1000 rpm;

the rotation speed of the stirring is 400-600rpm from the fermentation time of 16-20h to the end of the fermentation.

Preferably, (1) the first nutrient is supplemented when the sugar content in the fermentation medium drops below 12 g/L, preferably below 10 g/L, more preferably below 5-8 g/L for the first time, the first nutrient having a carbon to nitrogen ratio of 10-20: 1, the first nutrient being supplemented in an amount of 8-12 volume percent of the fermentation medium;

(2) when the first nutrient supplement is finished, supplementing a second nutrient, wherein the carbon-nitrogen ratio of the second nutrient is 30-50: 1, the supplementation amount of the second nutrient is 5-10 volume percent of the fermentation medium;

(3) and when the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses and the supplement amount of the third nutrient is 20-30 volume percent of the fermentation medium.

Through the technical scheme, the invention can obtain the following beneficial technical effects:

1) the method can fully utilize the advantages of high sugar content and high salt concentration in the high-salt molasses, provide sufficient carbon source supply and environmental requirements for fermentation of PHA fermentation strains, and bring a new way for utilization of the industrial waste;

2) the high-salt molasses has high salt content, and the salt addition can be reduced when a culture medium is prepared, so that the production cost is reduced;

3) the high-salt molasses has low price of about 800 yuan/ton, is more economical and practical than common glucose, and can reduce the cost of carbon sources by about 700 yuan/ton;

4) the high-salt molasses has high salt content, and the higher salt content can inhibit the growth of non-salt-tolerant bacteria, so that an effective bacteriostatic action is achieved, and the additional operation and economic cost brought by fermentation tank sterilization is reduced;

5) the high-salt molasses is rich in various trace elements, N, P compounds, vitamins and the like which are necessary for the growth of microorganisms, can effectively promote the growth of thalli, can properly reduce the addition of corresponding raw materials in a culture medium or even directly replace conventional culture medium ingredients in the use process, reduces the cost of the culture medium, and improves the fermentation efficiency;

6) the high salt molasses can also be applied to fermentation and feeding as a feeding carbon source for PHA production.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention provides a method for preparing PHA through fermentation, which comprises the steps of inoculating PHA fermentation strains into a fermentation medium for fermentation under the condition of fermentation to produce PHA;

In the invention, the salt in the high-salt molasses refers to sodium chloride.

According to the invention, the high-salt molasses may be any molasses meeting the above requirements in terms of salt content and sugar content, for example, high-salt cane molasses and/or high-salt beet molasses.

According to a preferred embodiment of the invention, the high salt molasses is high salt cane molasses having a salt content of above 9 wt%, for example 9-11 wt%, and a sugar content of above 50 wt%, for example 50-70 wt%, on a dry weight basis, said sugars comprising glucose, sucrose and fructose, wherein the glucose content is 5-15 wt%, the sucrose content is 75-85 wt% and the fructose content is 5-12 wt%, based on total sugars.

According to the invention, the high-salt molasses can be crude high-salt molasses, also can be refined high-salt molasses, and also can be syrup obtained by decolorizing and diluting refined high-salt molasses.

According to the invention, the content of the high-salt molasses in the fermentation medium can be selected within a wide range, which can completely or partially replace the carbon source (glucose) in the fermentation medium, according to a preferred embodiment of the invention, which completely replaces the carbon source (glucose) in the fermentation medium, the content of the high-salt molasses is 10-60m L, for example 10m L0, 15m L1, 20m L2, 25m L3, 30m L, 35m L, 40m L, 45m L, 50m L, 55m L, 60m L, more preferably 15-35m L, relative to the 1L fermentation medium.

According to the invention, the fermentation medium may also contain other components of the PHA fermentation process, such as nitrogen sources, phosphates, magnesium salts and sodium salts.

According to the present invention, the kind of the nitrogen source is not particularly limited as long as it can provide a nitrogen source required for the PHA fermentation broth, and may be an organic nitrogen source, for example, yeast powder, corn steep liquor powder, soybean meal powder, amino acids, etc., or an inorganic nitrogen source, for example, urea, ammonium sulfate, ammonium carbonate, etc. According to a preferred embodiment of the present invention, the nitrogen source is an organic nitrogen source, more preferably yeast powder.

The content of the nitrogen source can be selected from a wide range, and preferably, the content of the nitrogen source is 13 to 18g, for example, 13g, 14 g, 15g, 16g, 17g, 18g, and preferably 15 to 17g, relative to 1L fermentation medium.

According to the invention, the phosphate may be a phosphate conventionally used in PHA fermentation processes, for example, sodium salts of phosphoric acid, potassium salts of phosphoric acid, preferably, the phosphate is dipotassium hydrogen phosphate and disodium hydrogen phosphate.

The content of the phosphate can be selected from a wide range, and preferably, the content of the phosphate is 5-20g, for example, 5g, 7g, 9 g, 11 g, 13g, 15g, 17g, 19 g, 20g, and preferably 7-13g, relative to 1L fermentation medium.

According to a preferred embodiment of the invention, the phosphate is dipotassium phosphate and disodium phosphate, the content of dipotassium phosphate is 2-5g, preferably 2.5-4g, and the content of disodium phosphate is 5-8g, preferably 5.5-6.5g, relative to 1L fermentation medium.

According to the invention, the magnesium salt can be conventional soluble magnesium salts, but does not include phosphoric acid, preferably magnesium sulfate and/or magnesium chloride.

The content of the magnesium salt may be selected from a wide range, and preferably, the content of the magnesium salt is 0.1 to 0.5g, for example, 0.1 g, 0.2g, 0.3g, 0.4 g, 0.5g, and preferably 0.2 to 0.3g, based on 1L fermentation medium.

According to the invention, the sodium salt may be any of the conventional sodium salts, but excluding the sodium salt of phosphoric acid, preferably the sodium salt is sodium chloride.

The content of the sodium salt can be selected from a wide range, and preferably, the content of the sodium salt is 40 to 70g, for example, 40g, 45g, 50g, 55g, 60g, 65g, 70g, and preferably 45 to 55g, relative to 1L fermentation medium.

According to a preferred embodiment of the invention, the fermentation medium contains high-salt molasses, yeast powder, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride, wherein the content of the high-salt molasses is 15-35ml, the content of the yeast powder is 15-17g, the content of the dipotassium hydrogen phosphate is 2-5g, the content of the disodium hydrogen phosphate is 5-8g, the content of the magnesium sulfate is 0.2-0.3g and the content of the sodium chloride is 45-55g relative to 1L of the fermentation medium.

The content of each substance as described above refers to the content of each substance in the medium, and does not refer to the total amount of each substance charged.

The temperature for fermentation of PHA according to the present invention may be the conventional fermentation temperature, and preferably, the temperature is 30 to 45 ℃ and may be, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃. More preferably, the temperature is 35-39 ℃.

In accordance with the present invention, the pH of fermentation of PHA may be at its conventional fermentation pH, preferably, the pH is from 7 to 9, e.g., may be from 7, 7.5, 8, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9. more preferably, the pH is from 8.3 to 8.7. wherein the pH may be adjusted using conventional bases, e.g., from 8 to 12 moles per L mole of sodium hydroxide solution.

The amount of dissolved oxygen in PHA fermentation according to the present invention may be the amount of dissolved oxygen in conventional fermentation, preferably 1-40%, for example, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%. More preferably, the dissolved oxygen amount is 1 to 30%.

According to the invention, the conditions for fermentation of the PHA also preferably comprise a control of the aeration, preferably in a quantity comprised between 0.5 and 1.5vvm, and may be, for example, 0.5vvm, 0.6vvm, 0.7vvm, 0.8vvm, 0.9vvm, 1vvm, 1.1vvm, 1.2vvm, 1.3vvm, 1.4vvm, 1.5vvm, preferably between 1 and 1.2 vvm.

According to the present invention, the fermentation conditions of PHA also preferably include agitation, wherein the rotation speed of the agitation can be selected within a wide range, preferably, from 2 to 7L fermentation tank, the rotation speed of the agitation is 400-1000rpm, for example, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm, 750rpm, 800rpm, 850rpm, 900rpm, 950rpm, 1000 rpm.

In the course of research, the inventors of the present invention found that the final fermentation effect can be further improved by controlling different stirring speeds in different fermentation stages. Preferably, the fermentation is carried out under stirring conditions, from 0h to 8-12h (for example, 0-8h, 0-9h, 0-10h, 0-11h, 0-12 h) preferably to 9-11h, and the stirring speed is 400-600 rpm;

from fermentation for 8-12h (preferably 9-11 h) to fermentation for 16-20h (for example, 8-16h, 9-17h, 10-18h, 11-19h, 12-20h, 9-20h, 10-20h, etc., preferably 17-19h, the specific starting time depends on the ending time of the previous stage), and the rotation speed of the stirring is 600-;

from 16-20h (preferably 17-19 h) to the end of fermentation (e.g., 16h to the end of fermentation, 17h to the end of fermentation, 18h to the end of fermentation, 19h to the end of fermentation, 20h to the end of fermentation, with the start time being determined by the end time of the previous stage), the rotation speed of the stirring is 400-600 rpm.

According to the invention, the fermentation of the PHA can be carried out without feed or with feed. According to a preferred embodiment of the invention, the fermentation of PHA is a fed-batch fermentation, i.e., the fermentation is supplemented with nutrients.

The adding time of the nutrient substances can be determined according to the demand of the PHA fermentation strains on sugar, and preferably, the nutrient substances are added when the sugar content in the fermentation medium is reduced to be less than 12 g/L, preferably to be less than 10 g/L, and more preferably to be 5-8 g/L (for example, 5 g/L, 6 g/L, 7 g/L and 8 g/L).

Wherein, the addition amount of the nutrient substances is preferably controlled to be 5-20 g/L, such as 5 g/L, 6 g/L0, 7 g/L1, 8 g/L2, 9 g/L3, 10 g/L4, 11 g/L5, 12 g/L6, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L and 20 g/L.

Wherein the sugar content refers to the sugar content in the fermentation centrifugation supernatant measured by an SBA-90 biosensor analyzer.

Wherein, preferably, the sugar content in the fermentation system is monitored in real time during the fermentation process.

Wherein, when OD of the fermentation liquid is₆₀₀The fermentation can be finished when the rate of increase is less than 5/h, and according to the fermentation experience of the inventor, when the fermentation is carried out for 36 to 48 hours, preferably 39 to 42 hours, the OD is₆₀₀The speed increase of (b) can be reduced to the above level.

Wherein, OD₆₀₀Refers to the absorbance of the fermentation broth in a spectrophotometer at a wavelength of 600 nm.

Wherein, preferably, OD in the fermentation system is monitored in real time during the fermentation process₆₀₀。

According to the invention, the nutrient may be a feed in a conventional fermentation process, as long as the above requirements are met, for example, the nutrient may contain glucose. However, according to a preferred embodiment of the invention, the nutrient substance contains high salt molasses.

The inventor of the present invention further finds that the fermentation effect can be further improved by adding different nutrients at different stages of the fermentation, and preferably, the nutrient adding method comprises the following steps:

(1) when the sugar content in the fermentation medium drops below 12 g/L, preferably below 12 g/L, more preferably below 5-8 g/L for the first time, the first nutrient is supplemented with a carbon to nitrogen ratio of 10-20: 1, preferably 14-18:1, more preferably 15-17:1, in an amount of 8-12 volume percent, more preferably 9-11 volume percent of the fermentation medium.

Preferably, the first nutrient contains high salt molasses and yeast powder.

Preferably, the content of the high salt molasses is 550-830ml and the content of the yeast powder is 80-120g relative to the first nutrient of 1L.

(2) And after the first nutrient substance is supplemented, supplementing a second nutrient substance, wherein the carbon-nitrogen ratio of the second nutrient substance is 30-50: 1, preferably 34 to 42:1, further preferably 38-42: 1; the second nutrient is supplemented in an amount of 5-10 volume percent, preferably 6-8 volume percent of the fermentation medium.

Preferably, the second nutrient contains high salt molasses and yeast powder.

Preferably, the content of the high salt molasses is 830-980ml and the content of the yeast powder is 40-80g relative to the second nutrient of 1L.

Wherein the second nutrient can be supplemented immediately after the first nutrient is supplemented, or at intervals such that the sugar content of the fermentation medium is 5-20 g/L, preferably 8-15 g/L.

(3) And after the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses, and the supplement amount of the third nutrient is 20-30 volume percent, preferably 23-27 volume percent of the fermentation medium.

Wherein the third nutrient can be supplemented immediately after the second nutrient is supplemented, or at intervals such that the sugar content in the fermentation medium is 5-20 g/L, preferably 8-15 g/L.

Preferably, the fermentation is terminated after the end of the addition of the third nutrient by continuing the fermentation for 1 to 3 hours.

According to the invention, the nutrient can be supplemented in an intermittent or fed-batch manner, and the person skilled in the art can determine the nutrient according to the actual situation.

According to the present invention, the fermentation method for producing PHA may be a continuous fermentation method or a batch fermentation method.

According to the invention, the PHA fermentation strain can be various conventional halophilic fermentation strains capable of producing PHA through fermentation, and preferably, the PHA fermentation strain is halophilic monad (a)Halomonas sp.) (ii) a More preferably, the PHA fermentation strain is halomonas (M.halophila)Halomonas sp.) TD01 with preservation number of CGMCC NO.4353 (CN 201010578858.8).

According to the present invention, the inoculum size of the fermentation strain may not be particularly limited, and preferably, the inoculum size of the fermentation strain is 9 to 15 vol%, for example, 9 vol%, 10 vol%, 11 vol%, 12 vol%, 13 vol%, 14 vol%, 15 vol% with respect to 1L fermentation medium.

According to the invention, the fermentation strain inoculated into the fermentation medium is preferably an activated fermentation seed liquid, the OD of which₆₀₀The value is preferably 3 to 5.

The activation mode can adopt the conventional technical means in the field, for example, the low-temperature preserved strain is inoculated into a seed culture medium for activation culture, the seed culture medium can contain 5-10 g/L of yeast powder, 10-15 g/L of peptone and 50-60 g/L of sodium chloride, and the activation culture can be obtained by high-temperature and high-pressure sterilization.

Wherein, the conditions of the activation culture preferably comprise: culturing at 30-40 deg.C and rotation speed of 150-₆₀₀To 3-5.

Among them, the activation culture is preferably a multi-stage activation culture, for example, 2 to 3 stages, so that a sufficiently activated seed solution is obtained.

The method according to the invention also comprises the extraction of PHA from the fermentation broth, which may be conventional in the art, see for example Chung A, L iu Q, Ouyang S P, et al, Microbial production of 3-hydroxydocanoic acid by Phaoperate and Fafaca knock-out of fermentation ofPseudomonas putidaKT2442 harboring tesB gene[J].Applied Microbiology&Biotechnology, 2009, 83(3):513-519.。

The present invention will be described in detail below by way of examples. In the following examples of the present invention,

the PHA fermentation strain is halomonas (Halomonas sp.) TD01 with preservation number of CGMCC NO.4353 from CN 201010578858.8;

the high-salt cane molasses 1 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 10 weight percent, contains glucose, sucrose and fructose in a dry-solid basis, has the sugar content of about 51 weight percent, and contains 11 weight percent of glucose, 81 weight percent of sucrose and 8 weight percent of fructose in total sugar;

the high-salt cane molasses 2 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 9 weight percent, and contains glucose, sucrose and fructose in a dry-solid basis, wherein the sugar content is about 60 weight percent, and the total sugar content comprises 15 weight percent of glucose, 75 weight percent of sucrose and 10 weight percent of fructose;

the high-salt cane molasses 3 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 11 weight percent, contains about 70 weight percent of glucose, sucrose and fructose in terms of dry solids, and contains 10 weight percent of glucose, 85 weight percent of sucrose and 5 weight percent of fructose in terms of total sugar;

the high-salt beet molasses 4 is waste material produced by the sugar refining and sugar making process of sugarcane, is in a thick liquid state, has the salt content of about 8 weight percent, and contains glucose, sucrose and fructose with the sugar content of about 40 weight percent in terms of dry solids, and has the glucose content of 5 weight percent, the sucrose content of 83 weight percent and the fructose content of 12 weight percent in terms of total sugar;

the sugar content is determined according to the SBA-90 biosensing analyzer method;

the determination method of the biomass of the halomonas in the fermentation broth comprises the steps of taking 25-45 ml of the fermentation broth, centrifuging (8000 rpm, 10 min), leaving precipitate, washing for 2 times by using sterile water, drying the washed precipitate by using a vacuum freeze dryer for 48 hours, and weighing;

reference is made to Chung A, L iu Q, Ouyang S P, et al, Microbial production of 3-hydroxydecanoic acid by Phaperon and fadBA knock-out ofPseudomonas putidaKT2442 harboring tesB gene[J]. Applied Microbiology&Biotechnology,2009, 83(3): 513-;

fermenter volume 5L.

Preparation example

This preparation example is intended to illustrate the activation of the fermentation broth

Seed culture medium containing 8 g/L yeast powder, 12 g/L peptone and 55 g/L sodium chloride.

Inoculating Halomonas into seed culture medium, performing primary activation culture at 37 deg.C and 200rpm, and culturing to OD₆₀₀Reaching about 4 to obtain first-grade seed liquid;

inoculating the primary seed liquid into a seed culture medium at an inoculation amount of 10 volume percent, performing secondary activation culture at 37 ℃ and 200rpm, and culturing to OD₆₀₀And obtaining a secondary seed liquid when the yield reaches about 4, and obtaining a fermented seed liquid.

Example 1

This example illustrates the method of fermentative preparation of PHA provided by the present invention

The fermentation medium comprises 25m L of high-salt molasses 1, 17g of yeast powder, 3.5g of dipotassium hydrogen phosphate, 6.2g of disodium hydrogen phosphate, 0.25g of magnesium sulfate and 55g of sodium chloride relative to 1L of the fermentation medium, and the pH value is adjusted to 8.5.

And a first feeding step, wherein the first feeding step uses high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source, the carbon-nitrogen ratio is 16:1, the first feeding step uses high-salt molasses 1640 m L and yeast powder 108g relative to a first nutrient substance of 1L, and the volume ratio of the first feeding step to the fermentation medium is 9: 100.

And a second feeding material, wherein the high-salt molasses 1 is used as a carbon source, the yeast powder is used as a nitrogen source, the carbon-nitrogen ratio is 40:1, the molasses is 900m L, the yeast powder is 69.4g relative to the second nutrient substance of 1L, and the volume ratio of the feeding material to the fermentation medium is 6: 100.

Feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 23: 100.

Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 10 volume percent, and performing fermentation culture at 37 ℃ and a ventilation amount of 1vvm, wherein the pH value in the fermentation process is controlled to be about 8.5, and the dissolved oxygen amount is controlled to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-10h, and the stirring speed is 400 rpm; fermenting for 10-18h, wherein the rotating speed of stirring is 800 rpm; fermenting for 18h until the fermentation is finished, wherein the rotating speed of the stirring is 400 rpm.

And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.

When the sugar content in the fermentation system drops below 6 g/L for the first time, feeding the first material, feeding the second material when the first material is finished, and feeding the third material when the second material is finished.

The feeding flow acceleration keeps the sugar content in the fermentation liquor at about 10 g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.

The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.

Example 2

The fermentation medium contained 35ml of high-salt molasses 1, 15g of yeast powder, 2.5g of dipotassium hydrogen phosphate, 6.5g of disodium hydrogen phosphate, 0.3g of magnesium sulfate and 45g of sodium chloride, and the pH value was adjusted to 8.3, relative to 1L.

And feeding I, namely taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source, wherein the carbon-nitrogen ratio is 15:1, wherein the high-salt molasses 1783 m L and the yeast powder are 120g relative to the first nutrient substance of 1L, and the volume ratio of the feeding I to the fermentation medium is 10: 100.

And a second feeding material, wherein the high-salt molasses 1 is used as a carbon source, the yeast powder is used as a nitrogen source, the carbon-nitrogen ratio is 38:1, the molasses is 830m L, the yeast powder is 80g relative to the second nutrient substance of 1L, and the volume ratio of the feeding material to the fermentation medium is 7: 100.

Feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 25: 100.

Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 12 volume percent, performing fermentation culture at 35 ℃ under the condition of 1.1vvm ventilation rate, and simultaneously controlling the pH value to be about 8.3 and the dissolved oxygen amount to be 10-30 percent in the fermentation process. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-9h, and the stirring speed is 500 rpm; fermenting for 9-17h, wherein the rotating speed of stirring is 900 rpm; fermenting for 17h until the fermentation is finished, wherein the rotating speed of the stirring is 500 rpm.

When the sugar content in the fermentation system drops below 5 g/L for the first time, feeding the first material, feeding the second material when the first material is finished, and feeding the third material when the second material is finished.

The feeding flow acceleration keeps the sugar content in the fermentation liquor at about 8 g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.

Example 3

The fermentation medium comprises 15ml of high-salt molasses 1, 16g of yeast powder, 4g of dipotassium hydrogen phosphate, 5.5g of disodium hydrogen phosphate, 0.2g of magnesium sulfate and 50g of sodium chloride, relative to 1L fermentation medium, and the pH value is adjusted to 8.7.

The first material supplement comprises high-salt molasses 1 serving as a carbon source and yeast powder serving as a nitrogen source, wherein the carbon-nitrogen ratio is 17:1, the high-salt molasses 1560 m L and the yeast powder 91g are relative to the first nutrient substance 1L, and the volume ratio of the first material supplement to the fermentation medium is 11: 100.

And a second feeding step, wherein high-salt molasses 1 is used as a carbon source, yeast powder is used as a nitrogen source, the carbon-nitrogen ratio is 42:1, 875 m of molasses L and 58g of yeast powder are used relative to a second nutrient substance of 1L, and the volume ratio of the second feeding step to the fermentation medium is 8: 100.

Feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 27: 100.

Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 15 volume percent, performing fermentation culture at 39 ℃ and under the condition of 1.2vvm ventilation rate, and simultaneously controlling the pH value in the fermentation process to be about 8.7 and the dissolved oxygen amount to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-11h, and the stirring speed is 600 rpm; fermenting for 11-19h, wherein the rotating speed of stirring is 1000 rpm; fermenting for 19h until the fermentation is finished, wherein the rotating speed of the stirring is 600 rpm.

When the sugar content in the fermentation system drops below 8 g/L for the first time, feeding the first material, feeding the second material when the first material is finished, and feeding the third material when the second material is finished.

The feeding flow acceleration keeps the sugar content in the fermentation liquor at about 15 g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.

Example 4

The fermentation medium contained 40ml of high-salt molasses 1, 10g of yeast powder, 2.0g of dipotassium hydrogen phosphate, 5.0g of disodium hydrogen phosphate, 0.15g of magnesium sulfate and 40g of sodium chloride, relative to 1L fermentation medium, and the pH was adjusted to 8.0.

And feeding I, namely taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source, wherein the carbon-nitrogen ratio is 18:1, wherein the high-salt molasses is 1750 m L and the yeast powder is 116 g relative to the first nutrient substance of 1L, and the volume ratio of the feeding I to the fermentation medium is 8: 100.

And a second feeding material, wherein the high-salt molasses 1 is used as a carbon source, the yeast powder is used as a nitrogen source, the carbon-nitrogen ratio is 34:1, the molasses is 900m L, the yeast powder is 74 g relative to the second nutrient substance of 1L, and the volume ratio of the feeding material to the fermentation medium is 5: 100.

Feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 20: 100.

The seed solution prepared in the preparation example was inoculated into a fermentation medium at an inoculum size of 10 volume percent, and the fermentation was carried out at 30 ℃ and a ventilation of 1.5vvm while controlling the pH during the fermentation to about 8. In addition, the rotation speed was controlled to be about 800rpm during the fermentation.

When the sugar content in the fermentation system drops below 10 g/L for the first time, feeding the first material, feeding the second material when the first material is finished, and feeding the third material when the second material is finished.

The feeding flow acceleration keeps the sugar content in the fermentation liquor at about 5 g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.

Example 5

The fermentation medium comprises 15ml of high-salt molasses 1, 20g of yeast powder, 5g of dipotassium hydrogen phosphate, 8g of disodium hydrogen phosphate, 0.5g of magnesium sulfate and 70g of sodium chloride, relative to 1L fermentation medium.

Feeding: example 1 was used to feed one. The volume ratio of the fed material to the fermentation medium was 42: 100.

Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 10 volume percent, performing fermentation culture at 45 ℃ and under the condition of 0.5vvm ventilation rate, and simultaneously controlling the pH value in the fermentation process to be about 9 and the dissolved oxygen amount to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-12h, and the stirring speed is 400 rpm; fermenting for 12-20h, wherein the rotating speed of stirring is 800 rpm; fermenting for 20h until the fermentation is finished, wherein the rotating speed of the stirring is 400 rpm.

When the sugar content in the fermentation system first drops below 12 g/L, only one feed is fed.

The feeding speed of the feeding material is kept to keep the sugar content in the fermentation liquor at about 20 g/L, and after the feeding material feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.

Example 6

PHA production was carried out according to the procedure of example 1, except that the high-salt molasses 1 was replaced with an equal amount of high-salt cane molasses 2.

Example 7

PHA production was carried out according to the method of example 1, except that the high-salt molasses 1 was replaced with an equal amount of high-salt cane molasses 3.

Example 8

PHA production was performed as in example 1, except that the high salt molasses 1 was replaced with an equal amount of high salt beet molasses 4.

Comparative example 1

This comparative example serves to illustrate a reference fermentative PHA production process

PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with an equal amount of low-salt molasses, and the sodium chloride content in the fermentation medium was adjusted so as to be the same as that in the fermentation medium of example 4.

Comparative example 2

PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with a mixture of equal amounts of glucose, sucrose and fructose, and the content of sodium chloride in the fermentation medium was adjusted so as to be the same as that in the fermentation medium of example 4.

Comparative example 3

PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with an equal amount of glucose, and the sodium chloride content in the fermentation medium was adjusted to be the same as that in the fermentation medium of example 4.

TABLE 1

As can be seen from the results in Table 1, the embodiment 4 is compared with the comparative examples 1-2, the technical scheme of the invention can effectively reduce the production cost of PHA, and the embodiment 4 is compared with the comparative example 3, the technical scheme of the invention not only can effectively reduce the production cost of PHA, but also can improve the biomass of thalli in fermentation liquor and the yield of PHA. Comparing examples 1-3 with examples 4-5, it can be seen that controlling the fermentation process in the preferred mode of the present invention can further reduce the cost and increase the biomass of the cells and the yield of PHA in the fermentation broth. Comparing examples 1, 6 and 7 with example 8, it can be seen that selecting a specific high salt molasses and controlling the salt content and sugar content of the high salt molasses within the range of the present invention can further improve the fermentation effect.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A method for preparing PHA through fermentation is characterized in that PHA fermentation strains are inoculated into a fermentation medium for fermentation under the condition of fermentation to produce PHA;

2. The process according to claim 1, wherein the high salt molasses content is 10-60m L relative to 1L fermentation medium.

3. The process of claim 1, wherein the fermentation medium further comprises a nitrogen source, phosphate, magnesium salt, and sodium salt;

relative to 1L fermentation medium, the content of nitrogen source is 13-18g, the content of phosphate is 5-20g, the content of magnesium salt is 0.1-0.5g, and the content of sodium salt is 40-70 g.

4. The method according to claim 3, wherein the fermentation medium contains high-salt molasses, yeast powder, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride, and the content of the high-salt molasses is 15-35ml, the content of the yeast powder is 15-17g, the content of the dipotassium hydrogen phosphate is 2-5g, the content of the disodium hydrogen phosphate is 5-8g, the content of the magnesium sulfate is 0.2-0.3g and the content of the sodium chloride is 45-55g relative to 1L of the fermentation medium.

5. The method of any one of claims 1-4, wherein the conditions of the fermentation comprise: the temperature is 30-45 deg.C, pH is 7-9, and dissolved oxygen is 1-40%.

6. The method according to any one of claims 1 to 4, wherein the fermentation is carried out under stirring at a speed of 400-1000 rpm.

7. The method as claimed in claim 6, wherein the stirring speed is 400-600rpm from 0h to 8-12 h;

from 8-12h to 16-20h, the stirring speed is 600-1000 rpm;

8. The process according to any one of claims 1 to 4 and 7, wherein the fermentation is carried out under aeration with an aeration rate of 0.5 to 1.5 vvm.

9. The method according to any one of claims 1 to 4 and 7, wherein the method further comprises feeding nutrients during the fermentation, wherein the amount of the nutrients fed is such that the sugar content in the fermentation medium is controlled to be 5-20 g/L.

10. The method of claim 9, wherein the nutrient feed is initiated when the sugar content of the fermentation medium drops below 12 g/L during the fermentation.

11. The method of claim 10, wherein the method of nutrient supplementation comprises:

(1) supplementing a first nutrient when the sugar content in the fermentation medium is reduced to below 12 g/L for the first time, wherein the carbon-nitrogen ratio of the first nutrient is 10-20: 1, and the supplemented amount of the first nutrient is 8-12 volume percent of the fermentation medium;

(2) and after the first nutrient substance is supplemented, supplementing a second nutrient substance, wherein the carbon-nitrogen ratio of the second nutrient substance is 30-50: 1, the supplementation amount of the second nutrient is 5-10 volume percent of the fermentation medium;

(3) and after the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses, and the supplement amount of the third nutrient is 20-30 volume percent of the fermentation medium.

12. The method of claim 11, wherein the first nutrient comprises high salt molasses and yeast powder; and/or

The second nutrient substance contains high-salt molasses and yeast powder; and/or

The third nutrient is high salt molasses.

13. The method as claimed in claim 1, wherein the fermentation strain PHA is Halomonas salina (Zymomonas salina)Halomonas sp.)。

14. The method of claim 1, wherein the fermentation is a continuous fermentation or a batch fermentation.