Laser irradiation device and application method for accelerating cell growth in fermentation broth

Abstract

This invention relates to a laser irradiation device for accelerating cell growth in fermentation broth and its application method in a fermenter. The laser irradiation device includes a laser source module, a light energy transmission system, and an integrated control and synchronization system. The laser source module is located outside the fermenter and generates one or more laser beams with preset parameters. The light energy transmission system guides the laser generated by the laser source module into the fermenter. The integrated control and synchronization system enables intelligent coordinated control of the laser irradiation and fermentation process. Through a rational optical path design and intelligent control system, this device uniformly, safely, and controllably introduces laser energy with specific parameters into a sealed fermenter, thereby effectively regulating cell growth rate and metabolic activity, achieving the goal of shortening the fermentation cycle and increasing biomass or the yield of the target product.

Description

Technical Field

[0002] This invention belongs to the field of bio-fermentation technology, specifically relating to a laser irradiation device for accelerating cell growth in fermentation broth and its application method in a fermenter. Background Technology

[0003] Industrial fermentation is a core technology that utilizes the specific functions of microorganisms or animal and plant cells to cultivate them on a large scale under suitable conditions to produce a variety of high-value-added products, such as antibiotics, amino acids, organic acids, enzyme preparations, biofuels, vaccines, and single-cell fermentations. The efficiency of the fermentation process directly determines production costs and economic benefits, and its key indicators include cell growth rate, target product yield, and fermentation cycle.

[0004] Currently, methods to improve fermentation efficiency mainly focus on strain selection and modification, fermentation medium optimization, fermentation process parameter control, and the addition of chemical promoters. While these methods have improved fermentation efficiency to some extent, they still face bottlenecks. Currently, in industrial fermentation processes, cell growth regulation primarily relies on controlling parameters such as temperature, pH, and dissolved oxygen, lacking direct means to regulate cell metabolic processes.

[0005] In recent years, physical field-assisted fermentation technologies, such as ultrasound and electromagnetic fields, have provided new approaches to improving fermentation efficiency. Among these, light, as an important physical factor, has attracted considerable attention for its application in biological processes. Currently, the application of light in fermentation is mainly limited to the cultivation of photosynthetic microorganisms, such as algae and photosynthetic bacteria. For the vast majority of non-photosynthetic industrial microorganisms, such as bacteria and yeast, the traditional view is that light has no direct promoting effect on their growth, and may even inhibit it due to ultraviolet components. However, increasing fundamental research shows that low-energy lasers of specific wavelengths and doses have a significant regulatory effect on the physiological activities of non-photosynthetic cells, i.e., the photobiological regulatory effect.

[0006] However, existing technologies have the following problems and shortcomings:

[0007] 1. Lack of integrated equipment: Currently, there is no commercially available or patented equipment that effectively integrates laser technology with standard industrial fermenters and is specifically designed to accelerate the growth of non-photosynthetic microorganisms. Existing research is mostly limited to laboratory-scale petri dishes or microfluidic chips, making it difficult to directly apply to large-scale, high-density fermentation broth environments.

[0008] 2. Light energy transfer and uniformity issues: Fermentation broth is typically a high-density, high-turbidity suspension, limiting the depth to which light can penetrate. How to efficiently and uniformly transfer laser energy to the tens or even tens of thousands of liters of liquid within the fermenter, ensuring that the vast majority of cells receive an effective dose of irradiation, presents a significant technical challenge.

[0009] 3. Process control and safety issues: The introduction of lasers will generate additional heat, which may interfere with the fermenter's sophisticated temperature control system. Furthermore, the integration and operation of high-power lasers must comply with stringent safety standards to ensure the safety of operators and equipment.

[0010] Therefore, there is an urgent need to develop a new type of laser irradiation device that can be seamlessly integrated with industrial fermenters, achieve uniform irradiation within the fermentation liquid volume, and coordinate with the fermentation process parameters for control. This would fill the gap in existing technologies and provide a new, non-invasive, highly efficient, and controllable technical means to improve the efficiency of industrial fermentation. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a laser irradiation device for accelerating cell growth in fermentation broth and its application method in a fermenter. The device, through reasonable optical path design and intelligent control system, uniformly, safely and controllably introduces laser energy with specific parameters into a sealed fermenter, thereby effectively regulating the cell growth rate and metabolic activity, and achieving the purpose of shortening the fermentation cycle and increasing biomass or the yield of the target product.

[0012] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0013] A laser irradiation device for accelerating cell growth in fermentation broth is characterized by comprising a laser source module, a light energy transmission system, and an integrated control and synchronization system; wherein the laser source module is located outside the fermenter and is used to generate one or more laser beams with preset parameters; wherein the light energy transmission system is used to guide the laser generated by the laser source module into the fermenter; and wherein the integrated control and synchronization system is used to realize intelligent coordinated control of laser irradiation and fermentation processes.

[0014] Preferably, the laser source module includes one or more semiconductor lasers or fiber lasers, which are capable of outputting laser light with a wavelength range of 400nm to 1100nm, and whose power is continuously adjustable from 0.1W to 100W. The irradiation mode of the laser light is either continuous mode or pulsed mode.

[0015] Preferably, the aforementioned optical energy transmission system includes an optical fiber bundle, a sealed coupling interface, and an in-tank irradiation and homogenization module; wherein the optical fiber bundle consists of one or more high-power energy transmission optical fibers, used to transmit laser light from an external laser source module to the fermenter; wherein the sealed coupling interface is installed on the fermenter body and is made of high-temperature, high-pressure, and corrosion-resistant materials, used to maintain a sterile environment and airtightness inside the tank; wherein the in-tank irradiation and homogenization module is installed inside the fermenter, used to receive laser light from the optical fiber and evenly distribute it throughout the entire fermentation liquid volume.

[0016] Preferably, the above-mentioned in-tank irradiation and homogenization module includes a waterproof sealed shell, a beam collimation and shaping unit, and a beam homogenization and diffusion unit; wherein the waterproof sealed shell is made of medical-grade stainless steel or titanium alloy, and the front end is provided with a laser emission window made of high-transmittance quartz glass or sapphire; the beam collimation and shaping unit is located inside the sealed shell and is used to convert the divergent laser beam emitted from the end of the optical fiber into a parallel beam, and can shape the Gaussian distributed spot energy into a flat-top spot with uniform energy distribution through diffractive optical elements or microlens arrays; wherein the beam homogenization and diffusion unit is used to form a large-angle, uniformly distributed light field from the shaped laser beam, covering the entire fermentation liquid volume.

[0017] Preferably, the above-mentioned beam homogenization and diffusion unit adopts a rotating diffuser, which includes a miniature, high-temperature resistant, waterproof motor-driven rotating frosted glass or engineering diffuser. The rotating diffuser destroys the laser coherence through high-speed rotation, and the emitted light forms a conical light field with a large angle, no speckle, and highly uniform light intensity distribution.

[0018] Preferably, the above-mentioned beam homogenization and diffusion unit adopts an integrated light rod or light cage. The integrated light rod or light cage integrates the light-emitting ends of multiple optical fibers or multiple micro laser diodes on a hollow frame structure. Through simultaneous irradiation at multiple points and angles, combined with the macroscopic mixing of the fermentation broth, uniform irradiation in three-dimensional space is achieved.

[0019] Preferably, the aforementioned integrated control and synchronization system includes a multi-parameter sensing interface, a laser parameter control module, collaborative control logic, and a safety interlock module. The multi-parameter sensing interface is used to connect sensors on the fermenter to acquire real-time parameters such as temperature, pH, dissolved oxygen, stirring speed, and cell concentration of the fermentation broth. The laser parameter control module is used to precisely control the output wavelength, power, and operating mode of the laser source module according to a preset program or real-time feedback. The collaborative control logic has a built-in algorithm to achieve synchronous adjustment of laser irradiation and fermentation parameters, automatically switching the optimal laser irradiation scheme at different growth stages based on the real-time monitored cell growth curve. The safety interlock module can hard-wire the physical state of the laser system and the fermenter; any abnormal operation that causes laser leakage triggers the interlock to instantly cut off the laser output.

[0020] The method for applying the laser irradiation device of the present invention in a fermenter is characterized by comprising the following steps:

[0021] S1 Installation and Sterilization: The in-tank irradiation and homogenization module is installed in a predetermined position inside the fermenter and connected to an external fiber optic bundle through a sealed coupling interface to perform online sterilization of the entire fermenter;

[0022] S2 Inoculation and Initial Culture: The target microorganism is inoculated into the sterilized culture medium for initial culture;

[0023] S3 Start-up and Parameter Setting: When the cells enter the logarithmic growth phase or at a preset time point, laser irradiation is started through an integrated control and synchronization system. The wavelength, power, irradiation mode and collaborative control strategy of the laser are set according to the target strain and fermentation purpose.

[0024] S4 Real-time Monitoring and Automatic Adjustment: Throughout the fermentation process, the integrated control and synchronization system continuously monitors various parameters and automatically adjusts the laser parameters and the temperature control system of the fermenter according to the preset collaborative control logic to maintain optimal fermentation conditions.

[0025] S5 End and Harvest: After fermentation is complete, turn off the laser and harvest the product and clean the equipment according to the standard procedure.

[0026] Preferably, when used to promote Clostridium growth, a laser with a wavelength of 808 nm or 810 nm is selected, and irradiation is performed in continuous mode, with the power density controlled between 0.1 and 1.5 W / cm². 2 Within a certain range, until the cell density reaches a stable phase.

[0027] Preferably, when used to regulate yeast metabolism, the power density is controlled at 0.1-1.5 W / cm² during the stationary phase of the later stage of fermentation. 2 Pulsed irradiation with a 630 nm laser within the range for 1 minute followed by a 5-minute pause induces the oversynthesis of specific secondary metabolites by applying moderate light stress.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. Significantly improve fermentation efficiency: Through the precise control of laser photobiological regulation effect, it directly acts on cell growth and metabolism, which can effectively shorten the lag phase of cell growth and increase the specific growth rate in the logarithmic phase, thereby shortening the entire fermentation cycle by 10-30%.

[0030] 2. Increase the yield of target products: By increasing the final cell biomass (cell density) or by inducing the synthesis of target metabolites through light stress at a specific stage, the total yield and productivity of target products can be significantly improved.

[0031] 3. Non-invasive and high cleanliness: As a physical stimulation method, laser does not require the addition of any chemical substances to the fermentation broth, avoiding the introduction of impurities and the increase in downstream separation costs, and fully meets the high cleanliness requirements of biopharmaceutical and other industries.

[0032] 4. Universality and flexibility: By replacing the laser source module with different wavelengths and adjusting the control program, this device can be applied to a variety of different industrial microorganisms, and the irradiation scheme can be flexibly adjusted according to different fermentation goals (promoting growth or inducing synthesis).

[0033] 5. High integration and intelligence: The laser irradiation system is deeply integrated with the temperature control, sensing and safety systems of the fermenter, realizing fully automatic collaborative control and safety assurance, reducing the difficulty of operation and improving the stability and repeatability of the process.

[0034] 6. Solved the problem of light energy transmission: The innovative design of the in-tank irradiation and homogenization module, combined with the mechanical stirring of the fermentation broth, effectively overcomes the light shielding effect of high-density culture medium and achieves a quasi-uniform light field distribution within a large-scale fermentation broth volume. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the overall structure of the laser irradiation device of the present invention;

[0036] Figure 2 This is a schematic diagram of the structure of the in-can irradiation and homogenization module of the present invention (implementation using a rotating diffuser). Detailed Implementation

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0038] The present invention provides a laser irradiation device for accelerating cell growth in fermentation broth, comprising:

[0039] 1. Laser Source Module 1: Located outside the fermenter, this module generates one or more laser beams with specific parameters. Optionally, this module includes one or more semiconductor lasers or fiber lasers capable of outputting lasers with preset wavelengths (e.g., preferably between 400nm and 1100nm, specifically 450nm, 630nm, 808nm, etc., depending on the target microorganism), adjustable power (e.g., continuously adjustable within the range of 0.1W to 100W), and selectable irradiation modes (continuous mode or pulsed mode).

[0040] 2. Optical Energy Transmission System 2: Used to efficiently and safely introduce the laser generated by the laser source module into the fermenter. This system includes: Fiber Bundle 2-1: One or more high-power energy transmission fibers that transmit the laser from the external laser source module to the fermenter. Sealed Coupling Interface 2-2: One or more interfaces installed on the fermenter body (such as the top cover or side wall). This interface is made of high-temperature and high-pressure resistant, corrosion-resistant materials (such as 316L stainless steel and sapphire glass windows) to ensure the maintenance of a sterile environment and airtightness inside the tank during sterilization (such as 121°C moist heat sterilization) and fermentation. In-Tank Irradiation and Homogenization Module 2-3: This is the core component of the invention, installed inside the fermenter, used to receive the laser from the optical fiber and evenly distribute it throughout the entire fermentation liquid volume.

[0041] The in-can irradiation and homogenization module includes:

[0042] Waterproof sealed housing 2-3-1: Made of medical-grade stainless steel or titanium alloy, hollow inside, with a highly polished surface for easy cleaning and sterilization. It features a laser emission window at the front, made of high-transmittance quartz glass or sapphire.

[0043] Beam collimation and shaping unit 2-3-2: Located inside the sealed housing, it receives the diverging laser beam emitted from the end of the optical fiber and converts it into a parallel beam through a collimating lens. Beam shaping elements, such as diffractive optical elements (DOEs) or microlens arrays, can be selectively configured to shape the Gaussian-distributed beam energy into a flat-top beam with a more uniform energy distribution.

[0044] Beam homogenization and diffusion unit 2-3-3: This unit is crucial for achieving uniform illumination over a large volume. This invention proposes two preferred embodiments:

[0045] Method 1 uses a rotating diffuser, which employs a miniature, high-temperature resistant, waterproof motor-driven rotating frosted glass or engineering diffuser. When the shaped laser beam passes through the high-speed rotating diffuser, the coherence is destroyed, and the emitted light forms a cone-shaped light field with a large angle, no speckle, and highly uniform light intensity distribution, which covers the entire fermentation liquid volume as the stirring paddle moves.

[0046] Method 2: Integrated light rod or cage. This light rod or cage integrates the light-emitting ends of multiple optical fibers or multiple miniature laser diodes onto a specially designed hollow frame structure (light rod or cage). This frame rotates with the main agitator or is fixedly installed at a specific position inside the tank, achieving uniform irradiation in three-dimensional space through simultaneous irradiation at multiple points and angles, combined with the macroscopic mixing of the fermentation broth.

[0047] 3. Integrated Control and Synchronization System 3: A central control unit based on a programmable logic controller (PLC) or industrial computer, used to achieve intelligent coordinated control of the laser irradiation and fermentation processes. This system includes:

[0048] Multi-parameter sensing interface 3-1: Connects to existing sensors on the fermenter to acquire real-time data on the fermentation broth's temperature, pH, dissolved oxygen, stirring speed, and cell concentration (OD value) measured by an online turbidimeter or densitometer.

[0049] Laser parameter control module 3-2: Precisely controls the output wavelength, power and working mode of the laser source module according to a preset program or real-time feedback.

[0050] Collaborative Control Logic 3-3: Built-in algorithms enable synchronized adjustment of laser irradiation and fermentation parameters. For example, the controller monitors the fermentation broth temperature in real time. When the temperature exceeds a set threshold (e.g., ±0.1°C within the 30°C-40°C range) due to the laser heating effect, the system automatically reduces the laser power or switches to pulse mode to reduce heat input, and simultaneously instructs the fermenter's cooling jacket to increase the cooling water flow. Furthermore, the system can adjust the cooling water flow based on the real-time monitored cell growth curve (OD). 600 The laser irradiation scheme automatically switches to the optimal mode at different growth stages (such as logarithmic phase and plateau phase) to maximize the promotion of growth or induce product synthesis.

[0051] Safety interlock module 3-4: Adhering to international laser safety standards such as IEC 60825-1 and ANSI Z136.1, this module hard-wires the physical state of the laser system and the fermenter (e.g., whether the lid is locked and the observation port is closed). Any abnormal operation that could lead to laser leakage will trigger the interlock, instantly cutting off the laser output and ensuring that the fermenter remains in a safe Class 1 laser product environment during operation, protecting the safety of operators.

[0052] The method for applying the laser irradiation device of the present invention in a fermenter includes the following steps:

[0053] S1 Installation and Sterilization: Install the in-tank irradiation and homogenization module in a predetermined position within the fermenter (e.g., suspended in the middle of the fermentation broth via the top cover flange) and connect it to an external fiber optic bundle via a sealed coupling interface. Then, perform routine in-line sterilization (SIP) on the entire fermenter system, including this module.

[0054] S2 Inoculation and Initial Culture: Following standard procedures, the target microorganism is inoculated into the sterilized culture medium, and initial culture begins.

[0055] S3 Start-up and Parameter Setting: Laser irradiation is initiated via an integrated control system at the cell's entry into the logarithmic growth phase or a preset time point. Based on the target bacterial species and fermentation objective, the laser wavelength, power, irradiation mode, and synergistic control strategy are set (as in Example 1, promoting Clostridium growth, a laser with a wavelength of 808 nm or 810 nm is selected, irradiated in continuous mode, and the power density is controlled within a range that produces a significant promoting effect but with minimal thermal effect (0.1-1.5 W / cm²). 2 (This process continues until the cell density reaches a stable state; for example, in Example 2, yeast metabolism is regulated by pulsed irradiation with a low-power 630 nm laser during the later stages of fermentation (stable state). This induces the oversynthesis of specific secondary metabolites by applying moderate light stress.)

[0056] S4 Real-time Monitoring and Automatic Adjustment: Throughout the fermentation process, the integrated control system continuously monitors various parameters and automatically adjusts laser parameters and fermenter temperature control systems according to preset collaborative control logic to maintain optimal fermentation conditions.

[0057] S5 End and Harvest: After fermentation is complete, turn off the laser system and harvest the product and clean the equipment according to the standard procedure.

[0058] Example 1

[0059] This embodiment discloses a laser irradiation application for accelerating the growth of Clostridium butyricum (accession number CGMCC No. 6317, disclosed in Chinese patent CN201210568673.8) in a 1 L fermenter.

[0060] 1. Preparation: Add the fermentation culture to a 1 L fully automatic fermenter, transfer it to a fully automatic sterilizer for overall sterilization, and after the laser system is installed, inoculate with Clostridium butyricum.

[0061] The fermentation medium used consisted of: 5 g / L yeast extract, 2 g / L ammonium sulfate, 0.2 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, and 1 mL / L each of calcium chloride solution, Fe solution, and trace element solution. The components of the Fe solution and trace element solution are shown below. After preparation, the medium was stored at 4°C.

[0062] The Fe solution composition is: FeSO4•7H2O 5g / L, HCl (37%) 4 mL / L.

[0063] The trace element solution composition is as follows: CoCl2•6H2O 0.2g / L, ZnCl2 0.07g / L, CuCl2•2H2O 0.02g / L, MnCl2•4H2O 0.1g / L, Na2MoO4•2H2O 0.035g / L, H3BO3 0.06g / L, NiCl2•6H2O 0.025g / L, and HCl (37%) 0.9 mL / L.

[0064] 2. Fermentation and Irradiation: The fermentation temperature was set at 37°C, and the pH was maintained at 7.0. After fermentation began, when the cell density sensor detected OD... 600 When the value reaches 0.8, the central controller automatically starts the laser system and outputs an 808 nm continuous laser with a power density of 0.5 W / cm².

[0065] 3. Collaborative Control: Temperature sensors monitor the liquid temperature in real time. If the temperature rises above 37.2°C, the central controller will first reduce the laser power to 0.4 W / cm². If the temperature continues to rise, it will instruct the control valve of the cooling jacket to increase the opening and increase the cooling water flow until the temperature drops back to 37°C, with the power density controlled within the range of 0.1-5 W / cm². Synchronized Growth Stages: The controller plots a real-time growth curve based on data from the cell density sensor. During the logarithmic growth phase, irradiation is maintained at 0.5 W / cm². When the growth rate begins to slow down and enters the plateau phase, the system automatically reduces the laser power to 0.2 W / cm² to save energy.

[0066] Through this embodiment, compared with the control group that did not undergo laser irradiation but was fermented under the same conditions, the cell density (OD) of Clostridium butyricum was significantly higher. 600 This represents a 19.6% increase, resulting in a significant improvement in production efficiency.

[0067] Example 2

[0068] This embodiment describes the application of the device of the present invention in the fermentation of Pichia pastoris (GS115, preserved in the laboratory) to improve the expression level of yeast proteins. The device structure is basically the same as in Embodiment 1, but the laser source module is replaced with a wavelength switchable laser (630 nm / 830 nm). The application method is as follows:

[0069] 1. Primary and secondary seed culture

[0070] 1.1 Seed culture medium preparation: 2% tryptone, 2% glucose, 1% yeast extract, bring to a final volume of 1 L, and adjust the pH to 6.0. Dispense the prepared culture medium into Erlenmeyer flasks, seal the flasks with gauze, and then wrap the gauze with kraft paper. Transfer to an autoclave and sterilize at 121 °C for 20 min.

[0071] 1.2 Seed culture: Inoculate the strain at a rate of 1% into an Erlenmeyer flask containing 100 mL of YPD medium. Incubate at 30 ℃ and 250 rpm for approximately 18 h on a constant temperature shaker. Determine whether to continue culturing based on the condition of the seed culture. Transfer the qualified seed culture to a 1.5 L fermenter.

[0072] 2. Preparation and fermentation of BSM medium

[0073] BSM Culture Medium Ingredient List

[0074] Raw material name quantity unit Remark <![CDATA[CaSO4]]> 0.465 g ultrasonic dissolution <![CDATA[MgSO4•7H2O]]> 7.45 g glycerin 20.0 g Yeast paste 2.50 g Dissolve in hot water at approximately 50°C trypsin 2.50 g Dissolve in hot water at approximately 50°C KOH 2.00 g <![CDATA[K2SO4]]> 9.10 g Add the second to last <![CDATA[85 %H3PO4]]> 13.4 mL Finally add Defoaming oil 0.50 mL ammonia Adjust pH to 5 Final adjustment

[0075] 2.1 Growth Stage of the Strains: During the early fermentation stage (0-48 hours), 0.1 L of seed culture was transferred to a 1.5 L fermenter. Fermentation was carried out at dissolved oxygen (DO) > 40%, pH 5.5, temperature 30℃, and stirring speed 450 rpm. To rapidly accumulate yeast cells, a low-power (0.3 W / cm²) laser with an 830 nm wavelength was used. 2 Continuous irradiation was used to promote cell proliferation. The irradiation continued until glycerol in the culture medium was depleted (manifested as a rapid rise in DO from 0), at which point samples were taken to measure OD. 600 If the required concentration of 150 or higher is not reached, add sterile glycerin (glycerin:water = 1:1) in several batches until the OD reaches the target level. 600 Once you reach 150, you will move on to the next stage.

[0076] 2.2 Starvation Phase: Once the bacterial strain reaches the required biomass, stop adding glycerol and wait for dissolved oxygen (DO) to rise rapidly. At this time, do not add any carbon source and maintain the bacterial strain in a starved state for 30 minutes to avoid affecting the bacterial strain's utilization of methanol. During this phase, the temperature is lowered to 28°C.

[0077] 2.3 Methanol Induction Phase: After fermentation enters the stationary phase (48 hours later), the primary target is protein expression. The central controller automatically switches the laser wavelength to 630 nm and adopts a pulsed mode (1 minute irradiation, 5 minutes off), reducing the total average power density to 0.1 W / cm². 2 The aim of this study was to regulate key metabolic pathways in yeast and increase protein expression levels through light stress of a specific wavelength. The pH was maintained at 5.0, and the temperature at 28°C, and methanol induction was initiated. For the first 12 hours of induction, methanol (containing 1.2% PTM, with 600 mL of trace elements added per 50 L of methanol) was added at a rate of 1.5 mL / L / h; after 12 hours of induction, methanol was added at a rate of 3 mL / L / h. OD was measured online and offline. 600 OD appears 600 Fermentation is terminated when the temperature drops. During induction, DO... 600The dissolved oxygen level should be controlled between 10% and 30%. If dissolved oxygen rises, the methanol addition rate can be increased appropriately. If dissolved oxygen falls below the lower limit, the methanol addition rate should be slowed down or even stopped (for no more than 10 seconds) to maintain the dissolved oxygen control range.

[0078] The results show that the fermenter employing this staged, dual-wavelength irradiation strategy has a lower final OD value. 600 The yield was 15.8% higher than that of the control group under the same fermentation conditions but without laser irradiation, demonstrating that the device of the present invention can be used to promote protein expression in Pichia pastoris.

[0079] The above description is only a preferred embodiment of the present invention. For those skilled in the art, designing different forms of structures based on the teachings of the present invention does not require creative labor. All equivalent changes, modifications, substitutions and variations made in accordance with the scope of the patent application of the present invention without departing from the principles and spirit of the present invention shall be covered by the present invention.

Claims

1. A laser irradiation device for accelerating cell growth in fermentation broth, characterized in that, It includes a laser source module (1), a light energy transmission system (2), and an integrated control and synchronization system (3); wherein the laser source module (1) is located outside the fermenter and is used to generate one or more laser beams with preset parameters; wherein the light energy transmission system (2) is used to guide the laser generated by the laser source module into the fermenter; wherein the integrated control and synchronization system (3) is used to realize intelligent collaborative control of laser irradiation and fermentation process.

2. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 1, characterized in that, The laser source module (1) includes one or more semiconductor lasers or fiber lasers, which are capable of outputting lasers with a wavelength range of 400nm to 1100nm, and whose power is continuously adjustable in the range of 0.1W to 100W. The irradiation mode of the laser is either continuous mode or pulsed mode.

3. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 1, characterized in that, The optical energy transmission system (2) includes an optical fiber bundle (2-1), a sealed coupling interface (2-2), and an in-tank irradiation and homogenization module (2-3); wherein the optical fiber bundle consists of one or more high-power energy transmission optical fibers, used to transmit laser from an external laser source module to the fermenter; wherein the sealed coupling interface is installed on the fermenter body and is made of high-temperature, high-pressure, and corrosion-resistant materials, used to maintain a sterile environment and airtightness inside the tank; wherein the in-tank irradiation and homogenization module is installed inside the fermenter, used to receive laser from the optical fiber and evenly distribute it throughout the entire fermentation liquid volume.

4. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 3, characterized in that, The in-canister irradiation and homogenization module (2-3) includes a waterproof sealed shell (2-3-1), a beam collimation and shaping unit (2-3-2), and a beam homogenization and diffusion unit (2-3-3); wherein the waterproof sealed shell is made of medical-grade stainless steel or titanium alloy, and the front end is provided with a laser emission window made of high-transmittance quartz glass or sapphire. The beam collimation and shaping unit is located inside the sealed shell and is used to convert the divergent laser beam emitted from the end of the optical fiber into a parallel beam. It can also shape the Gaussian distributed spot energy into a flat-top spot with uniform energy distribution through diffractive optical elements or microlens arrays. The beam homogenization and diffusion unit is used to form a large-angle, uniformly distributed light field from the shaped laser beam, covering the entire fermentation liquid volume.

5. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 4, characterized in that, The beam homogenization and diffusion unit employs a rotating diffuser, which includes a miniature, high-temperature resistant, waterproof motor-driven rotating frosted glass or engineered diffuser. This rotating diffuser disrupts laser coherence through high-speed rotation, resulting in a conical light field with a large angle, no speckle, and highly uniform intensity distribution.

6. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 4, characterized in that, The beam homogenization and diffusion unit adopts an integrated light rod or light cage. The integrated light rod or light cage integrates the light-emitting ends of multiple optical fibers or multiple micro laser diodes on a hollow frame structure. Through simultaneous irradiation at multiple points and angles, combined with the macroscopic mixing of the fermentation broth, uniform irradiation in three-dimensional space is achieved.

7. The laser irradiation device for accelerating cell growth in fermentation broth according to claim 1, characterized in that, The integrated control and synchronization system (3) includes a multi-parameter sensing interface (3-1), a laser parameter control module (3-2), a collaborative control logic (3-3), and a safety interlock module (3-4). The multi-parameter sensing interface is used to connect sensors on the fermenter to obtain parameters such as temperature, pH, dissolved oxygen, stirring speed, and cell concentration of the fermentation broth in real time. The laser parameter control module is used to precisely control the output wavelength, power, and working mode of the laser source module according to a preset program or real-time feedback. The collaborative control logic has a built-in algorithm to achieve synchronous adjustment of laser irradiation and fermentation parameters, and automatically switches the optimal laser irradiation scheme at different growth stages according to the real-time monitored cell growth curve. The safety interlock module can hard-wire the physical state of the laser system and the fermenter. Any abnormal operation that causes laser leakage will trigger the interlock to instantly cut off the laser output.

8. A method for applying the laser irradiation device as described in any one of claims 1-7 in a fermenter, characterized in that, Includes the following steps: S1 Installation and Sterilization: The in-tank irradiation and homogenization module is installed in a predetermined position inside the fermenter and connected to an external fiber optic bundle through a sealed coupling interface to perform online sterilization of the entire fermenter; S2 Inoculation and Initial Culture: The target microorganism is inoculated into the sterilized culture medium for initial culture; S3 Start-up and Parameter Setting: When the cells enter the logarithmic growth phase or at a preset time point, laser irradiation is started through an integrated control and synchronization system. The wavelength, power, irradiation mode and collaborative control strategy of the laser are set according to the target strain and fermentation purpose. S4 Real-time Monitoring and Automatic Adjustment: Throughout the fermentation process, the integrated control and synchronization system continuously monitors various parameters and automatically adjusts the laser parameters and the temperature control system of the fermenter according to the preset collaborative control logic to maintain optimal fermentation conditions. S5 End and Harvest: After fermentation is complete, turn off the laser and harvest the product and clean the equipment according to the standard procedure.

9. The application method according to claim 8, characterized in that, When used to promote Clostridium growth, a laser with a wavelength of 808 nm or 810 nm is selected, and irradiation is performed in continuous mode with a power density controlled at 0.1-5 W / cm². 2 Within a certain range, until the cell density reaches a stable phase.

10. The application method according to claim 8, characterized in that, When used to regulate yeast metabolism, during the stationary phase of late fermentation, the power density is controlled at 0.1-1.5 W / cm². 2 Pulsed irradiation with a 630 nm laser within the range for 1 minute followed by a 5-minute pause induces the oversynthesis of specific secondary metabolites by applying moderate light stress.

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