阅读说明：本技术 一种槐糖脂生物表面活性剂的多级连续流加双相发酵工艺 (Multi-stage continuous fed-batch dual-phase fermentation process of sophorolipid biosurfactant ) 是由 梁生康 李俊峰 王哲 罗成坤 于 2020-04-05 设计创作，主要内容包括：本发明公开了一种槐糖脂生物表面活性剂的多级连续流加双相发酵工艺,其步骤包括：将多个体积相同的发酵罐依次连通,前一个发酵罐部分发酵液打入其紧连的发酵罐中,该发酵液中不仅其中含有高密度的菌细胞,可以作为接种液快速利用水溶性底物导致菌体细胞快速增殖,而且含有一定量槐糖脂,可以有效乳化增溶新补加的油溶性底物,缩短发酵周期；多个发酵罐相互连通,依次接种,连续补料,形成多级连续流加发酵工艺。本发明的优点在于可以大大缩短槐糖脂的发酵周期,提高发酵效率。同时,各主体发酵罐相互独立操作,单独补料和排放反应物料,可以大大避免相互感染杂菌的机会。(The invention discloses a multi-stage continuous fed-batch dual-phase fermentation process of sophorolipid biosurfactant, which comprises the following steps: a plurality of fermentation tanks with the same volume are communicated in sequence, partial fermentation liquor of the previous fermentation tank is injected into the fermentation tank which is connected with the previous fermentation tank, the fermentation liquor not only contains high-density bacterial cells which can be used as an inoculation liquid to quickly utilize a water-soluble substrate to cause the bacterial cells to quickly proliferate, but also contains a certain amount of sophorolipid, can effectively emulsify and solubilize a newly-supplemented oil-soluble substrate, and shortens the fermentation period; the plurality of fermentation tanks are communicated with each other, and are sequentially inoculated and continuously fed to form a multi-stage continuous fed-batch fermentation process. The invention has the advantages of greatly shortening the fermentation period of sophorolipid and improving the fermentation efficiency. Meanwhile, the main fermentation tanks are operated independently, and the reaction materials are fed and discharged independently, so that the chance of mutual infection of mixed bacteria can be greatly avoided.)

1. A multi-stage continuous fed-batch two-phase fermentation process of sophorolipid biosurfactant is characterized by comprising the following steps:

(1) filling a seed culture medium into a seed tank, sterilizing, inoculating candida, keeping the temperature at 25-35 ℃, the aeration quantity at 0.4-0.8 vvm, the stirring speed at 800 rpm and the tank pressure at 0.105-0.15 MPa, fermenting for a period of time, and then completely inoculating the seed liquid into a 1 st fermentation tank filled with a sterilized fresh fermentation culture medium in advance;

(2) after the fermentation liquid in the 1 st fermentation tank is inoculated, the temperature is kept at 25-35 ℃, the aeration rate is 0.4-0.8 vvm, the stirring speed is 300-; then, periodically adding 60-180g/L glucose solution and vegetable oil to the 1 st fermentation tank in batches, wherein the addition amounts are 2-10% and 1-4% of the volume of the fermentation tank respectively, continuously ventilating, stirring and fermenting until the fermentation is finished, and discharging the fermentation liquor into a storage tank;

(3) repeating the step (2) in the 2 nd fermentation tank until reaching the nth fermentation tank;

(4) adding a certain volume of fresh sterilized culture medium into the 1 st fermentation tank through a material supplementing tank, taking the fermentation liquor added from the nth fermentation tank as inoculation liquid, and continuing to perform the step (2);

(5) by analogy, a multi-stage series continuous flow-adding two-phase fermentation process is formed among n fermentation tanks;

(6) after the fermentation liquor in the storage tank is kept stand for 0.5-10h, the sophorolipid automatically settles on the bottom layer of the fermentation liquor, the middle layer is a yeast aggregate, and the upper layer is fermentation supernatant; standing for separation to obtain sophorolipid of lower layer, and directly preserving.

2. The multi-stage continuous fed-batch biphasic fermentation process of claim 1, wherein the number of fermentors n = 3-8.

3. The multi-stage continuous fed-batch biphasic fermentation process of claim 1, wherein said seed medium is: glucose 20g, peptone 20g, and yeast powder 10g were dissolved in 1L of tap water, and sterilized at 115 ℃ for 30 min.

4. The multi-stage continuous fed-batch biphasic fermentation process of claim 1, wherein said fermentation medium is: adding 2.5g of yeast powder, 5g of sodium citrate, 2g of ammonium sulfate, 1g of monopotassium phosphate and 100g of glucose into 1L of tap water, and sterilizing at 115 ℃ for 30 min; 100g of vegetable oil, sterilized separately at 115 ℃ for 30 min.

5. The multi-stage continuous fed-batch biphasic fermentation process of claim 1, wherein said vegetable oil is one or more of soybean oil, peanut oil, rapeseed oil, corn oil, olive oil, cottonseed oil, and oleic acid.

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a multistage continuous fed-batch dual-phase fermentation process of a sophorolipid biosurfactant.

Background

As an important chemical product, the surfactant is known as industrial monosodium glutamateAnd has wide application in various industries such as chemistry and the like. Sophorolipids are the most extensively studied and widely used class of biosurfactants. A number of Candida strains have been found (Candidasp.) can utilize a water-soluble and oil-soluble biphasic carbon source to produce sophorolipid in high yield. Compared with the traditional chemical synthesis surfactant, the sophorolipid biosurfactant has the following remarkable characteristics and advantages: (1) high surface/interfacial activity; (2) low toxicity or no toxicity, and is environment-friendly; (3) is easy to be biologically degraded; (4) good temperature resistance, salt resistance and acid and alkali resistance. Based on the characteristics, the sophorolipid biosurfactant can partially replace a chemically synthesized surfactant and is widely applied to the industries of daily chemical industry, petroleum exploitation, textile printing and dyeing, food fermentation and the like. Microbial fermentation is an important way and method for producing sophorolipid biosurfactant. At present, the bottleneck limiting the wide application of sophorolipid biosurfactant in various industrial industries is the overhigh production cost. It is urgently needed to improve the production efficiency of sophorolipid and reduce the production cost by optimizing the fermentation process of sophorolipid.

The fermentation kinetics research of sophorolipid biosurfactant shows that the sophorolipid is applied to candida (C)Candidasp.) in the process of synthesizing the sophorolipid biosurfactant by using a water-soluble and oil-soluble two-phase carbon source as a substrate, firstly, using a water-soluble carbon source such as glucose and the like to ensure that a large amount of thallus cells grow and reproduce, when reaching a growth stationary phase, the thallus cells begin to use the oil-soluble carbon source and secrete and metabolize sophorolipid, the strain enters the stationary phase from a lag phase and usually lasts for more than 4 hours, and the whole fermentation period is mostly more than 196 hours. The reason is that the oil-soluble substrate has poor affinity with the surface of hydrophilic bacteria cells, so that the bacteria cells can utilize the oil-soluble substrate in a large amount after long-time induction to generate sophorolipid and emulsification and dispersion of oil drops. Therefore, in order to shorten the fermentation period, the fermentation process should be improved, and the time for the strain to induce and produce sophorolipid to increase the oil-soluble substrate should be reduced.

At present, in terms of sophorolipid fermentation process, three fermentation modes of single-pot batch fermentation (U.S. Pat. No. 3205150; U.S. Pat. No. 3321648), single-pot fed-batch fermentation (Chinese patent: CN 101845469A) and single-pot continuous fed-batch fermentation (Chinese patent: CN 102492605A) have been developed on a pilot plant and pilot plant scale. But still has the outstanding problems of long fermentation period, low fermentation efficiency and the like. At present, the commonly used single-tank batch fermentation operation mode is to add the substrate once and take out all reaction materials after the reaction is finished, so that the high-concentration oil-soluble substrate can block the oxygen mass transfer rate, and the product concentration or other metabolites can inhibit the growth of strains, thereby causing longer fermentation period, low substrate utilization rate and low sophorolipid yield. The single-tank fed-batch fermentation can improve the utilization rate of the substrate and keep a higher dissolved oxygen level of a fermentation system by feeding the oil-soluble substrate in batches; however, the reaction materials are taken out once after the fermentation is finished, the growth of the strains is limited due to high-concentration products, and in the next culture period, the strains have longer time for generating the sophorolipid under the induction action of the oil-soluble substrate, the fermentation period is prolonged, and the fermentation efficiency is reduced. And single-tank continuous feeding fermentation is easy to be infected with bacteria, and the process control requirement is higher, so that the requirement of industrial large-scale production is difficult to meet.

Disclosure of Invention

Aiming at the outstanding problem that the fermentation period is long due to the fact that sophorolipid biosurfactant is required to be induced and generated when oil-soluble substrates are ingested and utilized by candida, the invention aims to provide a sophorolipid biosurfactant multi-stage continuous fed-batch dual-phase fermentation process with short fermentation period and high fermentation efficiency according to the growth of sophorolipid fermentation strains and the generation characteristics and the kinetic characteristics of products.

The invention is based on candidaCandidasp.) the dynamic characteristics of the fermentation of sophorolipid biosurfactant synthesized by using water-soluble and oil-soluble biphase carbon sources as substrates, on the basis of single-tank fed-batch fermentation and single-tank continuous fermentation, the multi-stage continuous flow-feeding biphase fermentation process of sophorolipid biosurfactant is designed and developed, a plurality of fermentation tanks with the same volume are sequentially communicated, the part of fermentation liquor of the former fermentation tank is injected into the fermentation tank which is connected immediately, the fermentation liquor not only contains high-density bacterial cells, but also can be used as inoculum for quickly utilizing water-soluble substrates to lead the bacterial cells to be quickly culturedThe sophorolipid is proliferated quickly, contains a certain amount of sophorolipid, can effectively emulsify and solubilize newly-supplemented oil-soluble substrates, and shortens the fermentation period; the plurality of fermentation tanks are communicated with each other, and are sequentially inoculated and continuously fed to form a multi-stage continuous fed-batch fermentation process.

The specific technical scheme of the invention is as follows:

the invention is completed by a set of multi-stage series fermentation equipment. The main body of the fermentation equipment is composed of n (n = 3-8) fermentation tanks which are connected in series, wherein the 1 st fermentation tank is connected with the 2 nd fermentation tank, the 2 nd fermentation tank is connected with the 3 rd fermentation tank, and so on, the n-1 st fermentation tank is connected with the n-th fermentation tank, and the n-th fermentation tank is connected with the 1 st fermentation tank. Each fermentation tank is communicated with a seed tank and a feed supplement tank, an independent connecting pipeline is arranged between each fermentation tank and the storage tank, and meanwhile, real-time monitoring probes for temperature, pH, dissolved oxygen and the like are installed, and the fermentation tanks are sequentially communicated.

The specific process flow of the invention is as follows:

a multi-stage continuous fed-batch dual-phase fermentation process of sophorolipid biosurfactant comprises the following steps:

(1) the seed culture medium is filled in a seeding tank, and after sterilization, the strain candida (Candidasp.) Keeping the temperature at 25-35 ℃, the aeration rate at 0.4-0.8 vvm, the stirring speed at 800 rpm and the tank pressure at 0.105-0.15 MPa, and after fermenting for a period of time, completely inoculating the seed liquid into the 1 st fermentation tank which is filled with a sterilized fresh fermentation culture medium in advance;

(2) after the fermentation liquid in the 1 st fermentation tank is inoculated, the temperature is kept at 25-35 ℃, the aeration rate is 0.4-0.8 vvm, the stirring speed is 300-; then, periodically adding 60-180g/L glucose solution and vegetable oil to the 1 st fermentation tank in batches, wherein the addition amounts are 2-10% and 1-4% of the volume of the fermentation tank respectively, continuously ventilating, stirring and fermenting until the fermentation is finished, and discharging the fermentation liquor into a storage tank;

(3) repeating the step (2) in the 2 nd fermentation tank until reaching the nth fermentation tank;

(4) adding a certain volume of fresh sterilized culture medium into the 1 st fermentation tank through a material supplementing tank, taking the fermentation liquor added from the nth fermentation tank as inoculation liquid, and continuing to perform the step (2);

(5) by analogy, a multi-stage series continuous flow-adding two-phase fermentation process is formed among n fermentation tanks;

(6) after the fermentation liquor in the storage tank is kept stand for 0.5-10h, the sophorolipid automatically settles on the bottom layer of the fermentation liquor, the middle layer is a yeast aggregate, and the upper layer is fermentation supernatant; standing for separation to obtain sophorolipid of lower layer, and directly preserving.

The number of fermentors n = 3-8.

The seed culture medium is as follows: glucose 20g, peptone 20g, and yeast powder 10g were dissolved in 1L of tap water, and sterilized at 115 ℃ for 30 min.

The fermentation medium is as follows: adding 2.5g of yeast powder, 5g of sodium citrate, 2g of ammonium sulfate, 1g of monopotassium phosphate and 100g of glucose into 1L of tap water, and sterilizing at 115 ℃ for 30 min; 100g of vegetable oil, sterilized separately at 115 ℃ for 30 min.

The vegetable oil is one or more of soybean oil, peanut oil, rapeseed oil, corn oil, olive oil, cottonseed oil and oleic acid.

The invention has the advantages that the continuous fermentation process of the sophorolipid biosurfactant consists of a plurality of fermentation tanks with the same volume which are communicated in sequence, so that the high substrate utilization rate and the high dissolved oxygen level can be maintained by batch feeding; meanwhile, part of fermentation liquor of the previous fermentation tank is used as seed liquor, not only contains high-density thallus cells, but also contains sophorolipid generated in the previous fermentation period, so that newly supplemented oil-soluble substrate can be effectively emulsified and solubilized, the absorption and conversion rate of the thallus cells to the oil-soluble substrate is improved, and the fermentation period is greatly shortened. The two components supplement each other, so that the fermentation period of the sophorolipid can be greatly shortened, and the fermentation efficiency is improved. Meanwhile, the main fermentation tanks are operated independently, and the reaction materials are fed and discharged independently, so that the chance of mutual infection of mixed bacteria can be greatly avoided.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

The invention is explained in further detail below by means of specific embodiments with reference to the drawings.