Method and system for improving yield of Ectoine fermented by halophilic bacteria based on specific paddle type combination

Abstract

The invention relates to the field of microbial fermentation engineering, and particularly discloses a stirring paddle combination method and system for optimizing fermentation of halophilic bacteria to produce Ectoine. Through CFD simulation and comparison of eight three-layer paddle combinations, it is found that a CD6-RT-RT combination with a semicircular tube paddle (CD6) arranged on the bottom layer and six flat blade paddles (RTs) arranged on the middle layer and the top layer can synergistically improve the gas-liquid mass transfer efficiency of a high-salt system, bubbles are broken through strong radial flow of the CD6 paddle on the top layer, and a gas-liquid interface is expanded in combination with high-shear axial flow of the RT paddles on the middle layer and the bottom layer; uniform distribution of the gas content (gradient is less than or equal to 5%) and kLegt are realized; compared with a traditional combination, the efficiency is improved by 25%-72%; in a 50L tank, the yield of Ectocaine reaches 32.2 g / L, which is obviously improved by 15.0% compared with that of a conventional RT-RT-RT combination. According to the method, the oxygen transfer bottleneck of high-salt fermentation is improved, and an efficient and low-consumption scheme is provided for industrial production of Ectoin.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation engineering, and in particular to a method and a fermentation system for improving the oxygen mass transfer efficiency of ectoine production by halophilic bacteria (Halomonas elongata) by optimizing a stirring paddle combination. Background Art

[0002] Ectoine, a high-value-added osmotic pressure-protective solute, is widely used in pharmaceuticals, cosmetics, and bioprotectants. Its industrial production relies heavily on high-density aerobic fermentation by halophilic bacteria (such as Halomonas elongata). In this fermentation process, oxygen mass transfer efficiency (kLa) is a key bottleneck restricting cell growth and product synthesis. Halophilic bacteria must be cultured in high-salt media (>1.5M NaCl). The high viscosity and salt ion effect of the media significantly reduce oxygen solubility (approximately 30% lower than conventional media) and inhibit bubble coalescence. As a result, the volumetric mass transfer coefficient (kLa) of traditional fermentation systems is generally less than 150 h-1, resulting in severe oxygen-limited growth. Current industrial fermenters generally use a single-propeller configuration (such as a six-blade Rushton turbine (RT). While this configuration provides high shear force, it suffers from three major drawbacks: uneven flow distribution; weak axial mixing, which easily forms an anoxic zone at the bottom of the tank, resulting in reduced bacterial metabolic efficiency; and an imbalance between energy consumption and mass transfer: high power input (>80kW / m²) and high ion density. 3 ) is limited, and the unit oxygen transfer energy consumption ratio (kLa / power) is mostly lower than 0.65h-1 / (kW / m 3 ); Gas holdup gradient is out of control: insufficient gas retention at the bottom (<2%) and excessive gas holdup at the top (>15%), further deteriorating mass transfer uniformity.

[0003] Although multi-layer impeller designs have been used to improve mixing efficiency, existing technologies have not yet resolved the special contradictions of halophilic fermentation: over-reliance on empirical impeller combinations (such as full RT impellers or RT-pitch-blade impeller combinations) and lack of quantitative optimization based on fluid mechanics; ignoring the enhancement mechanism of the synergistic effect of three-layer impellers on gas-liquid dispersion in high-salt systems, especially the synergistic effects of radial flow regulation of semi-circular tube impellers (CD6), axial transport of six-pitch-blade impellers (Xie) and shear dispersion of six-flat-blade impellers (RT); and the failure to establish a predictive model for impeller type combinations and key parameters (kLa, shear rate, and mass transfer efficiency per unit power), resulting in instability in the scale-up process.

[0004] Therefore, developing an optimized combination and design method of three-layer paddles targeting the physiological characteristics of halophilic bacteria and high-salt fluid environment is an urgent need to achieve efficient and low-consumption production of ectoine. Summary of the Invention

[0005] To address the bottleneck in ectoine production caused by high-salt culture media inhibiting oxygen transfer in existing halophilic fermentations, the present invention provides a fermentation method and system based on optimized three-paddle combinations. This method significantly improves gas-liquid mass transfer efficiency by screening specific paddle combinations (particularly the CD6-RT-RT combination), overcoming the shortcomings of traditional single-paddle or empirical combinations.

[0006] This paper uses CFD simulations to compare eight propeller combinations (covering the full range of combinations: semi-circular tube propeller CD6, six-flat blade propeller RT, and six-pitched blade propeller Xie) to reveal the propeller synergy mechanism:

[0007] CD6 impeller (semi-circular tube type): When configured on the top layer, it generates strong radial flow, effectively breaking bubbles and improving gas holdup uniformity;

[0008] RT impeller (six flat blades): provides high shear force when configured in the middle and bottom layers, expanding the gas-liquid interface area (A);

[0009] Xie propeller (six-pitch blade): Although it can enhance axial circulation, it can easily lead to an imbalance in gas holdup distribution in high-salt systems (for example, the gas holdup at the top of the tank of the CD6-RT-Xie combination reaches 22.1%, while the bottom is only 0.6%).

[0010] The technical solution adopted by the present invention is: through CFD simulation, the following parameters of eight propeller combinations (CD6-RT-RT, CD6-RT-Xie, CD6-Xie-RT, CD6-Xie-Xie, RT-RT-RT, RT-RT-Xie, RT-Xie-RT, and RT-Xie-Xie) are compared: gas holdup distribution (difference between the bottom / middle layer / top of the tank is ≤5% for best); kLa (target ≥200h -1 ), power per unit volume (≤95kW / m 3 ), kLa / power ratio (≥0.75h -1 / (kW / m 3 )); shear rate (≤65s -1 ).

[0011] The preferred combination is CD6-RT-RT.

[0012] The simulation conditions are as follows: liquid filling volume of 50% to 80%, ventilation volume of 0.5 to 1.5 vvm, rotation speed of 500 to 800 rpm, and fermentation tank scale of 50 to 5000 L.

[0013] Preferably, the liquid filling volume is 60%, the ventilation volume is 1 vvm, the rotation speed is 600 rpm, and the fermentation tank scale is 50 L.

[0014] The geometrical features of the blades are:

[0015] The CD6 propeller has a diameter ratio of 0.35 to 0.4 and a blade arc angle of 150° to 180°;

[0016] The diameter ratio of the RT propeller is 0.4 to 0.45, and the blade inclination angle is 0°;

[0017] The diameter ratio of the Xie propeller (control group) is 0.4-0.45, and the blade inclination angle is 45°.

[0018] Preferably, the CFD simulation needs to verify the uniformity of gas holdup stratification: the gas holdup range of the tank bottom (Bot1), middle layer (Bot2), and top layer (Bot3) is ≤5% (such as CD6-RT-RT's Bot1: 2.1%, Bot2: 3.4%, Bot3: 6.2%, and the range is 4.1%).

[0019] The present invention also provides application of the system in producing ectoine by fermentation of halophilic bacteria, and is particularly suitable for high-salt culture medium with salinity ≥1.5M NaCl.

[0020] The culture medium comprises a fermentation broth containing 10.00 g / L glucose, 90.00 g / L sodium chloride, 1.00 mL / L PTM1, 3.00 g / L sodium citrate, 3.00 g / L ammonium chloride, 2.50 g / L sodium sulfate, 3.08 g / L magnesium sulfate heptahydrate, 0.25 g / L zinc sulfate, 0.09 g / L manganese sulfate, 5.00 g / L sodium hydrogen phosphate dodecahydrate, and 3.00 g / L potassium chloride. The pH of the fermentation broth before sterilization is 6.6-6.8. The trace element composition of the trace element mixture, calculated in g / L, is 6.00 g copper sulfate, 0.08 g sodium iodide, 3.00 g manganese sulfate, 0.20 g sodium molybdate, 0.02 g boric acid, 20.5 g cobalt chloride, 20.00 g zinc chloride, 65.00 g ferric sulfate, 0.20 g biotin, and 5.0 mL sulfuric acid.

[0021] The halophilic bacterium is Halomonas elongata ATCC 33173.

[0022] The present invention discloses the following technical effects:

[0023] Using CD6-RT-RT combination in a 50L tank (liquid volume 30L, rotation speed 600rpm, ventilation volume 1vvm): kLa reached 207.37h -1 , gas-liquid interface area A=105.28m 2 / m 3 ;Power per unit volume 91.49kW / m 3 , shear rate 60.19s -1 The yield of ectoine increased by 15.0% compared with the RT-RT-RT combination (control group), reaching 32.2g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of eight propeller combination conditions.

[0025] Figure 2 This is a comparison chart of KLa under eight propeller combination conditions.

[0026] Figure 3 This is a graph showing the changes in edodeon yield under the paddle type optimization group conditions and the control group conditions. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention by specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the case of no conflict, the features in the following examples and embodiments can be combined with each other. In the embodiments of the present invention, unless otherwise specified, the methods used are all conventional methods, and the reagents used can be obtained from commercial sources.

[0028] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0029] Example 1: CFD simulation verification of eight paddle combinations in a 50L fermenter

[0030] 1.1 Simulation conditions

[0031] Fermentation tank: 50L standard stirred tank (diameter-to-height ratio 1:2), liquid volume 30L (60% tank capacity);

[0032] Propeller combination: Eight three-layer propeller configurations (bottom layer / middle layer / top layer) were tested according to Table 1;

[0033] Operating parameters: speed 250 rpm, ventilation 1 vvm, temperature 37°C, culture medium salinity 1.5 M NaCl;

[0034] CFD model: Euler-Euler multiphase flow model, k-ε turbulence model, grid independence verification error <3%.

[0035] Table 1: Comparison of key CFD parameters for eight propeller combinations *Gas holdup range = (gas holdup at top layer - gas holdup at bottom layer) absolute value; range > 5% indicates severe uneven distribution

[0036] 1.2 Key Conclusions

[0037] Optimal combination: CD6-RT-RT kLa value is uniquely >200h -1 (207.4h -1 ), and the gas holdup distribution is the most uniform (extreme difference 4.1%);

[0038] Pitched-blade propeller defects: Combinations with Xie propellers in the middle or bottom layers (e.g., CD6-RT-Xie, RT-RT-Xie) experience uncontrolled gas holdup stratification (extreme deviation > 21%), resulting in kLa decay > 20%;

[0039] Energy efficiency comparison: RT-Xie-RT has the highest kLa / power ratio (0.892), but kLa (164.0h -1 ) was significantly lower than CD6-RT-RT (207.4h -1 ).

[0040] Example 2: Comparison of Yield of Ecdoin Produced by 50L Tank Fermentation

[0041] Example 2: Comparison of Yield of Ecdoin Produced by 50L Tank Fermentation

[0042] 2.1 Experimental methods

[0043] Strain: Halomonas elongata ATCC 33173;

[0044] Culture medium: glucose 10.00 g / L, sodium chloride 90.00 g / L, PTM1 1.00 mL / L, sodium citrate 3.00 g / L, ammonium chloride 3.00 g / L, sodium sulfate 2.50 g / L, magnesium sulfate heptahydrate 3.08 g / L, zinc sulfate 0.25 g / L, manganese sulfate 0.09 g / L, sodium hydrogen phosphate dodecahydrate 5.00 g / L, potassium chloride 3.00 g / L. The pH of the fermentation broth before sterilization was 6.6-6.8. The trace element composition of the trace element mixture, calculated in g / L, was as follows: copper sulfate 6.00, sodium iodide 0.08, manganese sulfate 3.00, sodium molybdate 0.20, boric acid 0.02, cobalt chloride 20.50, zinc chloride 20.00, ferric sulfate 65.00, biotin 0.20, and sulfuric acid 5.0 mL.

[0045] Group setting: CD6-RT-RT (experimental group) and RT-RT-RT combination (control group) were used, with 3 replicates in each group;

[0046] Product detection (quantification of intracellular ectoine by HPLC): Place 0.5 mL of fermentation broth in a 2 mL centrifuge tube containing grinding beads and add 1 mL of ultrapure water. Place the centrifuge tube in a cryo-mill and grind at 65 Hz for 30 min. After grinding, centrifuge (12,000 rpm, 5 min), dilute the supernatant, and filter. Detection was performed using a Shimadzu HPLC system.

[0047] HPLC conditions: mobile phase: water: acetonitrile = 98:2 (v / v), flow rate: 0.5 mL / min, column temperature: 30°C, detector: SPD-20AV, detection wavelength: 210 nm.

[0048] like Figure 3 As shown in the results, the maximum ectoine production of the group optimized with the CD6-RT-RT paddle combination reached 32.2±0.5 g / L, which was significantly increased by 15.0% compared with the control group (28.0±0.4 g / L), confirming that this paddle combination can effectively enhance the ectoine synthesis ability of halophilic bacteria.

[0049] Through systematic CFD simulation and experimental verification, the present invention reveals the core advantage of a specific three-layer propeller combination (CD6-RT-RT) in high-salt fermentation of halophilic bacteria: the strong radial flow of the bottom semi-circular tube propeller (CD6) and the high shear axial flow of the top six flat blade propeller (RT) are used to overcome the bottleneck of gas-liquid mass transfer in high-salt systems, and the kLa value is increased to >200h -1 (Increased by 25% to 72% compared with traditional combinations); significant efficiency improvement: the ectoine production in 50L tank fermentation reached 32.2g / L, an increase of 15.0% compared with conventional paddle types (such as RT-RT-RT).

[0050] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A stirring optimization method for producing ectoine by halophilic bacteria fermentation, characterized in that: The fermenter uses a three-layer stirring paddle, the bottom layer is a semicircular tube paddle (cd6), and the middle and top layers are six-flat blade paddles (rt).

2. The method of claim 1, wherein the kLa value is ≥ 200 h-1 and the power per unit volume is ≤ 95 kW / m 3 .

3. The method of claim 1, wherein the shear rate is ≤ 65s -1 , gas content ≥3.0%.

4. A fermentation system for realizing claims 1-3, comprising a 50-5000 L fermentation tank equipped with a CD6-RT-RT paddle combination.

5. The system of claim 4, wherein the CFD simulation parameters satisfy: kLa / power>0.75h-1 / (kW / m 3 ), gas-liquid interface area ≥ 100m 2 / m 3 .

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