CN102492605B - Bioreactor and sophorolipid continuous production method by using the same - Google Patents

Abstract

According to a bioreactor and a sophorolipid continuous production method by using the same provided by the invention, as fed-batch of a fermentation broth, a product is recovered at a liquid outlet through intravenous drip. A technology mainly comprises the following steps: centrifuging a fermentation waste liquid by a centrifuge and recovering precipitated yeast byproduct at a lower layer; mixing a centrifuged supernatant and a crude product from intravenous drip and recovering a lower layer product to a secondary purified product gathering tank 2-5 h after settlement layering; disinfecting the secondary purified product by an autoclave and storing directly, or drying at 55-75 DEG C in a drying tower to gain a constant weight and storing.

Description

Bioreactor and method for continuous production of sophorolipids

technical field

The invention relates to a bioreactor and a method for continuously producing sophorolipids.

Background technique

When a large-scale oil spill occurs, especially when the weather conditions at sea are bad, it is very difficult to recover the spilled oil using physical methods such as oil booms and oil skimmers. In this case, the oil spill dispersant can accelerate the natural dispersal process of the oil, and it is easier to break the oil into small oil droplets under the action of sea waves, accelerating the natural biodegradation of the oil spill. The use of oil spill dispersants has gradually become widely accepted and most coastline countries rely on dispersants when responding to oil spills.

Dispersants can be divided into chemical degreasers and biological degreasers. Due to the high production cost of biosurfactant, the main component of biological dispersants, the large-scale application of biological dispersants is hindered. At present, chemical dispersants are mainly used in response to marine oil spills. However, the chemical dispersant itself has certain toxicity, so governments all over the world take a cautious attitude towards the application of chemical dispersants. It is an inevitable trend for biological dispersants to gradually replace chemical dispersants. In July 2010, when dealing with crude oil pollution in Dalian, my country adopted biotechnology for the first time on a large scale to deal with oil spills, and achieved good results.

As a biodegradable biosurfactant, sophorolipid has good emulsification, dispersion, solubilization and other characteristics, and has good compatibility with the human body and the environment. Good substitute. In addition, sophorolipids have high research and development value in food, medicine, cosmetics, metallurgy, environmental protection, waste oil recovery and other fields. At present, they have been used in daily chemical, pharmaceutical, food industry, petroleum industry and other important fields. The theoretical research and application development of sophorolipids in the world have been carried out for many years, but our country is still in its infancy in this field.

The application range of sophorolipids is very wide, but its production cost remains high, which limits the large-scale commercial production process of sophorolipids. At present, there is no large-scale sophorolipid production enterprise in my country, and there is no international scope. Public sale of pure sophorolipids. Factors restricting the industrial production of sophorolipids mainly include the production capacity of strains, culture conditions, production methods, and separation and extraction methods. Among them, research on strain mutagenesis and optimization of culture conditions has been very common, and relatively good strains have also been found. , high and stable yield can be achieved under suitable culture conditions, but generally speaking, there is still a big gap between the existing technology and the low cost required for industrialized production.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for continuously producing sophorolipids with reduced production and separation costs, environmental friendliness and simple operation.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical scheme: including a tank body and a heating jacket arranged on the tank body; a sieve plate with trapezoidal holes is arranged in the tank body, and the sieve plate separates the tank body into upper It is the main fermentation area of the column and the product deposition area of the cone below; the volume ratio of the main fermentation area to the product deposition area is between 8:1-12:1, and the height-to-diameter ratio of the main fermentation area is 2: Between 1-3:1; the upper end of the main fermentation area is provided with an exhaust port, a condensed water circulation pipeline, an inoculation port, a sampling port, a feeding port, a motor and an electrode, and the main fermentation area is provided with an agitator and A main ventilation pipe; an auxiliary ventilation pipe is provided in the product deposition area, and a liquid discharge port is provided at the lower end.

Further, the agitator is an upper and lower double layer of paddles, including an upper layer of defoaming paddles and a lower layer of stirring paddles.

Further, the sieve plate is provided with isosceles trapezoidal holes, the opening rate of the upper surface of the sieve plate is 3%-6%, the opening rate of the lower surface is 75%, and the upper end aperture of the isosceles trapezoidal holes is 2mm. -4mm, the lower end aperture of the isosceles trapezoidal hole is between 6mm-20mm, the upper and lower aperture ratio of the isosceles trapezoidal hole is between 1:5-1:3, and the thickness of the sieve plate is 10mm-15mm.

Another technical problem to be solved by the present invention is to provide a bioreactor with reduced production and separation costs, environmental friendliness, and simple operation

In order to solve the above-mentioned technical problems, the present invention adopts the following technical scheme: add the sterilized fermentation medium into the bioreactor, add the cultivated seed liquid through the inoculation port, and replenish the bioreactor in batches at a rate of 50mL/d-80mL/d. Add rapeseed oil, 55h-96h before fermentation, the upper and lower tanks are ventilated with oxygen at the same time, the speed is controlled to keep the dissolved oxygen above 50%, and the ventilation at the lower end of the fermentation is stopped at 96h. After 3h-5h, the valve is opened, and the settled crude product is discharged statically. Supplement the fermentation broth in batches at a rate of 40mL/d-100mL/d, and finally can ferment continuously for 306h-322h, with a cumulative recovery of 4.75kg-6.75kg of crude product, and the production efficiency per unit time and unit fermenter volume is higher than that of single batch fermentation More than 3 times, the effective fermentation time has been extended by more than 4 times; after the fermentation waste liquid is centrifuged, the supernatant is mixed with the recovered crude product, and after mixing and sedimentation for 2 hours, the lower layer product is recovered to the secondary purification product collection tank; after secondary purification, the product is sterilized in a sterilizing tank, and then dried at 70°C to constant weight to obtain 2.92kg-3.96kg of pure sophorolipids.

Further, the fermentation is aerobic fermentation under the conditions of 25-30° C. and pH 3.0-4.0.

Further, the medium components are glucose 130g/L-150g/L, yeast powder 3g/L-10g/L and urea 1g/L.

Further, the medium components are glucose 150g/L, yeast powder 3g/L, peptone 0.7g/L, sodium citrate 5g/L, MgSO4 7H2O4g/L, (NH4)2SO4 2g/L, KH2PO4 1g/L , NaCl0.1g/L and CaCl2 2H2O0.1g/L.

Further, the seed liquid is a bacterial cell enrichment liquid obtained by culturing the cells in a seed medium at 30° C. for 48 hours, and the culture condition: the pH value is 6; the components of the seed medium are: glucose 100 g/L, yeast powder 10g/L and urea 1g/L.

The bioreactor of the present invention and the method for continuously producing sophorolipids thereof aim to improve the yield of sophorolipids by converting the existing batch fermentation production into continuous production; reduce separation costs, such as compared to traditional separation processes , The present invention does not use organic solvent extraction at all, is environmentally friendly, and has no product loss; while the separation operation is performed, yeast by-products can be recovered incidentally, without additional operations and costs; the entire process is simple to operate and easy to control equipment. The present invention realizes the continuous production of sophorolipids by designing a new reactor, and at the same time takes the new separation method as the main line, designs a complete production process of efficient and energy-saving continuous production, separation and recovery of by-products of sophorolipids, The production cost can be greatly saved, and it is possible to realize the large-scale continuous production of the sophorolipid.

Description of drawings

Fig. 1 is the schematic diagram of bioreactor of the present invention;

Fig. 2 is a block diagram of the production process of the method for continuously producing sophorolipids of the present invention.

Detailed ways

In the present embodiment, with reference to Fig. 1, the bioreactor includes a tank body and a heating jacket 14 arranged on the tank body; a sieve plate 17 with trapezoidal holes is arranged in the tank body, and the sieve plate 17 will The tank body is divided into the main fermentation area with the cylinder above and the product deposition area with the cone below; the volume ratio of the main fermentation area to the product deposition area is between 8:1-12:1, and the height of the main fermentation area is The diameter ratio is between 2:1-3:1; the upper end of the main fermentation area is provided with an exhaust port 10, a condensed water circulation pipeline 11, an inoculation port 12, a sampling port 20, a feeding port 21, a motor 22 and an electrode ( Not shown), the main fermentation area is provided with a stirrer and the main ventilation pipe 19; the product deposition area is provided with an auxiliary ventilation pipe 15, and the lower end is provided with a liquid outlet 16.

Wherein, the agitator is an upper and lower double-layer paddle, including an upper defoaming paddle 13 and a lower paddle 18 . The sieve plate 17 is provided with an isosceles trapezoidal hole, the opening rate of the upper surface of the sieve plate 17 is 3%-6%, the opening rate of the lower surface is 75%, and the upper end aperture of the isosceles trapezoidal hole is 2mm-6%. 4mm, the diameter of the lower end of the isosceles trapezoidal hole is between 6mm-20mm, the ratio of the upper and lower apertures of the isosceles trapezoidal hole is between 1:5-1:3, and the thickness of the sieve plate is 10mm-15mm. This design is because the small pore size on the upper surface can effectively prevent the bacteria from settling to the lower end during the production process, and the large pore size on the lower surface is conducive to returning the deposited bacteria and materials to the main fermentation area on the upper part under aeration conditions; product deposition The auxiliary ventilation pipeline 15 in the area can provide oxygen for the product deposition area in the early stage of production, and at the same time play a stirring role in accelerating mass transfer. At the same time, the existence of the sieve plate 17 can effectively slow down the vortex when the product is discharged, so that the fluid in the tank is in a laminar flow state, and when the deposited product is recovered by static dripping, unnecessary back-mixing of the product and the fermentation liquid can be avoided.

As shown in Figure 2, the method for continuously producing sophorolipids includes the following equipment: bioreactor 1, centrifuge 2, crude product collection tank 3, yeast collection tank 4, fermentation broth supernatant liquid collection tank 5, secondary purification product Collection tank 6, sterilization tank 7, drying equipment 8 and pure product storage tank 9.

The method for continuous production of sophorolipids is as follows: the total height of the bioreactor is 0.4cm, the height ratio of the cylinder and the cone is 3:1, the diameter of the cylinder tank is 0.2m, the thickness of the sieve plate is 10mm, and the ratio of the upper and lower apertures of the trapezoidal holes is The ratio is 1:3, the upper surface aperture is 3mm, and the reactor with an effective volume of 10L is a specific example, wherein the inner diameter of the main ventilation pipe 19 is 1cm, and the inner diameter of the auxiliary ventilation pipe 15 is 0.8cm, and the upper and lower sides can be stopped at any time respectively. or start ventilation. Production examples are as follows:

Example 1

Taking the strain 1 whose sophorolipid yield was 90 g/L during batch fermentation as the production strain as an example.

Add 6L of sterilized fermentation medium into the device, and the components of the medium are glucose 130g/L, yeast powder 10g/L, and urea 1g/L. Add the cultured seed liquid through the inoculation port, the seed liquid is the bacterial cell enrichment liquid obtained by culturing the thalline through the seed medium at 30°C for 48 hours, the culture condition: the pH value is 6; the composition of the seed medium is : Glucose 100g/L, yeast powder 10g/L and urea 1g/L; then add rapeseed oil in batches at a rate of 50mL/d. Add rapeseed oil in batches at a high speed. 96 hours before the fermentation, the upper and lower tanks were ventilated with oxygen at the same time, and the rotating speed was controlled to keep the dissolved oxygen above 50%. Ferment until the aeration at the lower end stops for 96 hours, open the valve after 5 hours, and discharge the sedimented crude product statically, while replenishing the fermentation broth in batches at a rate of 40mL/d. Finally, continuous fermentation can be carried out for 306 hours, and 4.75 kg of crude product can be recovered accumulatively. The production efficiency per unit time and unit fermentation tank volume is more than 3 times higher than that of single batch fermentation, and the effective fermentation time is more than 4 times longer. Wherein, the fermentation is aerobic fermentation under the conditions of 25-30° C. and pH 3.0-4.0.

After centrifuging the fermentation waste liquid, take the supernatant and mix it with the recovered crude product. After mixing and settling for 2 hours, the lower layer product is recovered to the secondary purification product collection tank; the secondary purification product is sterilized After sterilization, it was dried at 70°C to constant weight to obtain 2.92 kg of pure sophorolipid. Compared with the traditional organic solvent extraction separation process, this separation process saves 6 liters of ethyl acetate, and greatly reduces the relevant distillation energy consumption and condensation water, and the separation cost is only about 1/4 of the traditional process. At the same time, 0.26 kg of yeast by-products were obtained.

Example 2

Take the strain 2 whose sophorolipid yield is 120g/L during batch fermentation as the production strain as an example.

Add 6L sterilized fermentation medium into the device, the medium components are glucose 150g/L, yeast powder 3g/L, peptone 0.7g/L, sodium citrate 5g/L, MgSO4 7H2O4g/L, (NH4 )2SO4 2g/L, KH2PO4 1g/L, NaCl0.1g/L, CaCl2 2H2O0.1g/L. Add the cultured seed liquid through the inoculation port, the seed liquid is the bacterial cell enrichment liquid obtained by culturing the thalline through the seed medium at 30°C for 48 hours, the culture condition: the pH value is 6; the composition of the seed medium is : Glucose 100g/L, yeast powder 10g/L and urea 1g/L; then add rapeseed oil in batches at a rate of 80mL/d. 55 hours before the fermentation, the upper and lower tanks were ventilated with oxygen at the same time, and the rotating speed was controlled to keep the dissolved oxygen at a relatively high level. Ferment until the aeration at the lower end stops for 96 hours, open the valve after 3 hours, and discharge the sedimented crude product statically, and replenish the fermentation broth in batches at a rate of 100mL/d. Finally, continuous fermentation can be carried out for 322 hours, and 6.75kg of crude product can be recovered accumulatively. The production efficiency per unit time and unit fermentation tank volume is 3.5 times higher than that of single batch fermentation, and the effective fermentation time is extended by more than 4 times.

After centrifuging the fermentation waste liquid, take the supernatant and mix it with the recovered crude product. After mixing and settling for 2 hours, the lower layer product is recovered to the secondary purification product collection tank; the secondary purification product is sterilized After sterilization, it was dried at 70°C to constant weight to obtain 3.96 kg of pure sophorolipid. Using this separation process saves 8 liters of ethyl acetate compared with the traditional organic solvent extraction separation process, and greatly reduces the relevant distillation energy consumption and condensation water, and the separation cost is only about 1/4 of the traditional process. At the same time, 0.35 kg of yeast by-product was obtained.

The bioreactor of the present invention and the method for continuously producing sophorolipids thereof have the advantages that: by designing a novel bioreactor, the existing batch fermentation production mode is changed into a continuous production mode, which greatly improves the production capacity of the culture medium. Utilization efficiency, equipment efficiency; by using the fermentation supernatant to re-mix with the crude product, further purify the sophorolipids, since the fermentation supernatant is a saturated solution of the sophorolipids, the loss of the product is reduced, while compared to Using the traditional method of organic solvent extraction, since no organic solvent is used at all, the present invention saves energy, is environmentally friendly, and has low cost; while the separation operation is performed, yeast by-products can be recovered incidentally without any additional operations and costs; the entire process The production process is complete, the operation steps are simple, and the equipment is easy to control.

The present invention has been described in detail above. The foregoing description is only a preferred embodiment of the present invention, and should not limit the implementation scope of the present invention. within the scope of the invention.

Claims (1)

1. a bio-reactor, is characterized in that: comprise tank body, and be arranged on the heating jacket on tank body; In described tank body, be provided with the sieve plate with trapezoidal hole, described sieve plate is the main fermentation region of cylinder above tank body is separated into and is the product deposition region of cone below; The volume ratio of described main fermentation region and product deposition region is 8: 1-12: between 1, the aspect ratio in the region of mainly fermenting is 2: 1-3: between 1; The upper end in described main fermentation region arranges venting port, condensate water circulatory pipeline, inoculation mouth, thief hole, material-feeding port, motor and electrode, is provided with agitator and main breather line in described main fermentation region; Described product is provided with assisted ventilation pipeline in deposition region, lower end is provided with leakage fluid dram, described agitator is Dual-layer blade, comprise the froth breaking oar on upper strata and the stirring rake of lower floor, on described sieve plate, offer isosceles trapezoid hole, the percentage of open area of sieve plate upper surface is 3%-6%, the percentage of open area of lower surface is 75%, the aperture, upper end in isosceles trapezoid hole is between 2mm-4mm, the aperture, lower end in isosceles trapezoid hole is between 6mm-20mm, top and bottom, isosceles trapezoid hole aperture ratio is 1: 5-1: between 3, plate thickness is 10mm-15mm.