Hardware
Multiphase Flow
Bioreactor Blade Design
1. Overview
Figure 0 Flow-diagram
2. Introduction
Our final plan is to introduce different plasmids into XM01 Moraxella cell and E. coli BL21 for the degradation of polycyclic aromatic hydrocarbons (PAHs) in waste cooking oil, followed by the synthesis of α-olefins and 10-hydroxydecanoic acid using the produced MEL. This process requires multiple uses of bioreactors (fermentation tanks) to achieve the degradation of harmful substances and the production of industrial raw materials. Initially, we did not plan to design hardware and focused on experimental design and modeling. However, after conducting surveys with stakeholders in various fields (such as professors in relevant disciplines and fermentation companies), we found that due to the unique cultivation medium in our project, which involves oil-based substrates, we need special hardware equipment.
In industrial production, oil substances often need to be emulsified with other culture media to improve parameters such as dissolved oxygen levels and bacterial surface contact area, ultimately enhancing yield. Currently available emulsification equipment faces issues such as high cost and large size, and connecting multiple large-scale fermentation tanks also presents a high-cost problem.
These issues have not only posed challenges for our team, but they also limit the adoption of our production process by companies or laboratories. We hope that professionals across the country can utilize our solution as much as possible. Therefore, we will take this opportunity to further optimize our own project implementation and provide customers with suitable, feasible, and cost-effective hardware solutions.
3. Problems needs to be solved
In July, we conducted surveys to understand the needs of stakeholders regarding emulsification and separation after fermentation. We aim to continuously improve our hardware design based on the expectations and feedback from future users. We had numerous friendly exchanges with experts in the fermentation field who provided some specific requirements for our oil-based substrate project's hardware:
1.Modularity
2.Compact size
3.Cost-effectiveness
4.Commercial viability
5.User-friendliness
6.High reproducibility
Improved stirrer blades to achieve thorough mixing of multiple liquids (emulsification or semi-emulsification)
We have set these requirements as our goals and strive to meet various needs as much as possible. However, we lack expertise in using electronic equipment, so our hardware design will be based on 3D modeling and fluid simulations. We will also collaborate with other teams in the future to enhance our devices by incorporating more automated control equipment and sensors.
Following suggestions from scientists, we approached manufacturers to customize a standardized microreactor vessel (lacking components such as blades, separators, and multiple pipelines). The missing components will be filled using our 3D-printed models to test different shapes and gather more feedback from stakeholders for further improvements.
We will design, test, and optimize our hardware based on customized modular microreactors in the laboratory setting.
Figure 1 Custom modular micro fermenter
4. Iteration concept
From the beginning, we aimed to minimize reliance on electronic equipment and increase the use of replicable 3D-printed components. We hope that teams similar to ours, lacking expertise in electronic information technology, can easily construct our designed devices using the 3D-printed results. This would enable researchers in laboratories worldwide to use any commercially available 3D printer to print the components and assemble them without any knowledge of circuit connections or software applications. They would only need to follow the assembly instructions provided by us.
Additionally, we will publicly release the "stl" file formats of the models, allowing stakeholders to reference them and modify and design the dimensions according to the requirements of their specific laboratory or facility.
Figure 2 Iteration flow chart
Furthermore, our entire design process follows an iterative improvement approach:
1.Initially, we construct our 3D designs based on literature research and our design requirements.
2.We use relevant simulation software to perform fluid simulations on the constructed models. This helps explore the movement of liquids and investigate the impact of hardware structure on stirring and settling.
3.We use commercially available 3D printers, such as common home printers, to print the physical components and complete the assembly.
4.The physical prototypes are utilized and measured, and feedback and improvement suggestions are gathered from stakeholders.
5.Based on the further requirements, we refine the design concept and begin the next iteration.
This iterative process allows us to continually improve and optimize our hardware design based on real-world testing and stakeholder feedback.
5. Design
Our hardware design was not without challenges; in fact, we encountered numerous difficulties that we had to overcome one by one.
Due to the complex multiphase flow conditions (oil-water mixture) in the fermentation environment, the stirring conditions in the laboratory's bioreactors could not meet our specific requirements. We urgently needed a solution that could achieve uniform stirring without using emulsification processes and maintain relative stability in the lower solution phase (since excessive emulsification would cause subsequent separation difficulties during fermentation).
Based on simulations of fluid characteristics and the specific vessel design, we experimented with different combinations of six-flat-blade impellers and co-rotating and counter-rotating six-blade helical impellers to find a stirring system that would facilitate the desired special mixing conditions for different fluid layers.
Throughout this process, we continuously iterated and refined the design to teaddress these challenges effectively.
● First Iteration:
Regarding the impeller design, our initial concept was based on recommendations from interviews with laboratory researchers. The challenge of mixing two-phase liquids has always been a critical issue in fermenter design. Additionally, high-viscosity fluids can hinder liquid agitation, increase energy consumption, and reduce mixing efficiency, which were challenges we faced in our project. Therefore, we had some preliminary ideas for our impeller design.
Our initial design was quite rudimentary. We initially considered using two standard flat-blade impellers, one in the upper layer and one in the lower layer, as is commonly used in most laboratory bioreactors. Simultaneously, we also incorporated a unique configuration with an upper flat-blade impeller and a lower disc, hoping to prevent re-agitation of the sediment in the lower layer.
This represented the starting point of our design process, addressing the challenges associated with two-phase liquid mixing and high viscosity in the fermentation environment. Subsequent iterations would involve refining and improving this initial design.
Figure 3 straight-six-blade-turbine-blade
Figure 4 Iteration flow chart
At this stage, we used the COMSOL fluid simulation software due to its higher accuracy compared to other simulation software, as well as its capabilities for handling sliding grids and mechanical rotation. The Frozen Rotor model in COMSOL was particularly well-suited for simulating fluid behavior in bioreactors.
We simulated using a high-viscosity single-phase silicone oil as the modeling material with a rotational speed of 40 revolutions per second. The results of the simulations were measured in meters per second (m/s).
The three different impeller combinations, along with their orientations and sizes, were as follows: (The upper single flat-blade impeller configuration was used as a control.)
Figure 5 Silicone-oil-speed-distribution-of-double-straight-blade
Figure 6 Silicone-oil-velocity-equivalent-distribution-of-double-straight-blades
Figure 7 Silicone-oil-velocity-distribution-of-single-straight-blade-upper
Figure 8 Silicone-oil-velocity-equivalent-distribution-of-single-straight-blade-upper
Figure 9 Silicone-oil-velocity-distribution-of-single-straight-blade-top-single-disc-blade-bottom
Figure 10 Silicone-oil-velocity-equivalent-distribution-of-single-straight-blade-upper-single-disc-blade-lower
Based on the above, we have reached the following conclusions:
1.Comparing the three impeller configurations, we found that regardless of whether a disc or a standard flat-blade impeller was used in the lower part, there was no significant change in the flow direction. The use of the disc did not achieve the desired alteration of the lower flow direction but instead resulted in a mirrored projection in the upper part.
2.Due to the high viscosity of the silicone oil used in the simulation, even at a rotational speed of 40 revolutions per second, the flow velocities in the solution remained mostly below 1.5 m/s.
3.All of the tested impeller combinations resulted in radial flow, without achieving effective vertical mixing in the single-phase case. Therefore, it was apparent that these configurations would not be able to achieve efficient mixing in the layered two-phase fluid system.
At this point, we consulted with the laboratory researchers again for further assistance. Professor Liu Xiangming suggested using helical impellers to enable vertical flow in high-viscosity fluids. Additionally, he recommended switching to a different fluid simulation software that is more suitable for simulating multiphase flows since although COMSOL provides high accuracy, it requires more computational resources compared to other simulation software. Finally, he mentioned using SolidWorks as a modeling software to complement the simulation of multiphase flows. Professor Liu also pointed out that our initial simulation had unreasonable initial values since the viscosity of the silicone oil used was significantly different from the edible oil used in our project, and a rotational speed of 40 revolutions per second would be unattainable in the laboratory's bioreactors.
We took Professor Liu's advice and made adjustments accordingly for the next iteration of our design and simulation.
● Second iteration:
In this redesign of the impeller, we once again conducted an extensive literature search and discovered that cross-shaped impellers are widely used in stirring high-viscosity fluids, such as molten metals. Therefore, we combined the design elements of a helical impeller with cross-shaped supports as the core of our impeller. This design ensured sufficient structural strength for the helical impeller while introducing turbulent variables to enhance mixing.
Figure 11 Cross-propeller-blade
Video 2-1 Cross helix - trajectory animation
Video 2-2 Cross helix - velocity shear diagram
Video 2-3 Cross helix immiscible soybean oil - velocity isosurface
In terms of parameter settings, we based our simulation on a microreactor module, taking into account the suggestions of stakeholders. We changed the fluid simulation software to SolidWorks and reduced the rotational speed to 20 rad/s. The three phases, from top to bottom, were set as air, soybean oil, and water, providing density and dynamic viscosity variations. The surrounding walls were set as ideal walls, while the top plate was set to atmospheric static pressure.
In the simulation, this impeller design demonstrated a significant improvement in promoting the mixing of the two-phase fluid, resulting in more pronounced and unstable turbulence compared to the flat-blade turbine impeller. The overall effect was good. However, the decrease in rotational speed resulted in lower flow velocities, indicating some limitations of this approach.
After sharing the simulation results with Professor Liu Guanglei and Professor Li Jing, both provided similar suggestions:
1.Reduce the overall size of the impeller.
2.Segment the helical blades to prevent filling the entire fermentation vessel.
3.Hollow out the internal helix and retain only the flat supports to facilitate the upward movement of the liquid in the center of the helical blades.
These suggestions aim to further optimize the impeller design and address the limitations identified in the simulation.
● Third iteration:
In this latest impeller design iteration, we reduced the size of the helical blades and hollowed out the interior while keeping the other parameters unchanged. We then initiated another round of fluid simulation.
Figure 12 Cross-propeller-blade-alter
Video 3-1 Cross helix modification (change)- flow trajectories
Video 3-2 Cross helicity(change) - velocity shear diagram
Video 3-2 Cross helix(change) - immiscible soybean oil - velocity isosurface
We discovered that after reducing the size of the impeller blades and hollowing out the interior, the fluid velocity actually increased, and there was more significant particle collision and mixing at the interface between the two phases.
After submitting the engineering drawings to the printing service provider and explaining our experimental situation and objectives, they also gave us some advice. Considering that we need to securely fix the impeller blades deep into the shaft, if the contact area at the connection point with the rod is too large, it may result in excessive friction and difficulty in insertion. Additionally, a longer overall impeller blade may not find a suitable angle for insertion due to limitations with the ventilation shaft in our bioreactor. Finally, considering the structural strength, the cross-shaped supports may still be slightly insufficient. It is recommended to thicken the blades to some degree and increase their quantity. Metal printing is preferred to ensure the strength of the impeller blades.
● Fourth iteration:
We divided the aforementioned impeller into upper and lower sections and increased the number of internal flat blades to six. Through simulation, we observed changes in the distribution of mixing velocities and found that the impeller's performance was similar to that in the fourth iteration.
Figure 12/13 Six-blade-propeller-blade-alignment-left/Six-blade-propeller-blade-inversion-right
Video 4-1 Twin contra-rotating propeller blade helix trajectory simulation
Video 4-2 Twin contra-rotating propeller blade helix immiscible soybean oil - velocity isosurface
Video 4-3 Twin contra-rotating propeller blade helix trajectory simulation
Following that, we printed two sets of clockwise and counterclockwise helical impeller blades for actual fluid physical modeling observations. (Clockwise from bottom to top is referred to as "clockwise"; otherwise, it is "counterclockwise"). In total, nine combinations were tested using the clockwise helical, counterclockwise helical, and six-blade flat turbine impeller blades in the physical modeling experiments. We observed and recorded the actual time required for mixing in different combinations and the time needed for the final settling and layering. Utilizing the Analytic Hierarchy Process (AHP), we determined the optimal combination for our project.
Because sample 1 was found to have uneven composition and serious oxidation problems after comparison test, it was no longer included in the scope of consideration in the subsequent evaluation, but the mixed test video was retained.
Video T-1 Six-blade spiral (Sample 2) (2023)
Video T-2 Upper reverse six-blade spiral lower straight plate turbine (Sample 2)
Video T-3 Double reversed six-blade spiral (Sample 2)
Video T-4 Inverted six-blade spiral (Sample 2)
Video T-5 Upper six-blade spiral lower straight plate turbine (Sample 2)
Video T-6 Double straight six-leaf spiral (Sample 2)
Video T-7 Upper straight turbine lower reverse six-blade spiral (Sample 2)
Video T-8 Upper straight turbine down six-blade spiral (Sample 2)
Video T-9 Double straight turbine blades (Sample 2)
Video T-10 Double straight turbine blades (Sample 1)
Figure 15 Hierarchical-analysis-score-chart
Figure 16 Fermentation-efficiency-score
Figure 17 Materiality-evaluation-matrix
Figure 18 Score(first sample is not include)
Video Actual fermentation process
The final conclusion is that the combination of clockwise helical for the upper impeller and counterclockwise helical for the lower impeller showed higher initial and complete mixing efficiency. Additionally, it demonstrated excellent layering efficiency after agitation cessation. This combination is deemed the most suitable impeller configuration for our fermentation project. We applied this blade combination into the fermentation process of MEL, and successfully produced a certain amount of MEL, which proved the reliability and practicability of this hardware.
Design of Accelerated Settling
and Separation Tank
1.Introduction
After the fermentation process is complete, we need to separate the desired product – MEL. However, there is a challenge in achieving clear stratification and further separation at this stage.
The conventional method for separating MEL involves allowing it to settle in a 1-liter measuring cylinder, but this method is highly inefficient. Hence, we took this opportunity to develop a unique device tailored for the separation of similar organic macromolecules in our project.
2.Problem needs to be solved
Following discussions with personnel from various laboratories during our field research, we discovered that many researchers were facing the same challenges. When it comes to using centrifugation for separation, there are several issues. First, there is often a lack of a proper pretreatment process, resulting in low separation efficiency. Second, many laboratories do not have the necessary conditions for using a centrifuge, or they encounter situations where the centrifuge's capacity is not suitable for achieving effective separation of their products. As a result, stakeholders have once again raised the following demands:
1.The addition of specialized equipment to achieve efficient separation.
2.Low cost
3.Easily reproduced and manufactured
4.Compatible with other similar separation requirements implementations while addressing the pain points of the project
3.Design
Figure 1 Schematic diagram of 3D model of accelerated settling box
According to the principles of settling in shallow pools, increasing the pool area can accelerate settling under constant flow conditions, and reducing the height will decrease the volume of the settling pool. The principle of accelerated settling in inclined plate settling tanks lies in the effective increase of the contact surface area between the liquid in the tank and the walls. Simultaneously, it reduces the hydraulic radius, lowering the Reynolds number of the liquid, resulting in a highly stable laminar flow, greatly improving hydraulic conditions. The initial fluid simulation of our model design confirmed this point.
The design of the accelerated settling tank in this experiment is based on the inclined plate settling tank commonly used in water treatment processes. Following its principles, we designed our own settling device for separating MEL (or for accelerated purification and separation) generated in the bioreactor. By optimizing the device's structure to improve hydraulic conditions, we accelerate settling and reduce the need for a centrifuge.
From left to right, and from top to bottom, the four sections of pipes are: the inlet pipe, upper clear liquid outlet pipes 1 and 2, and MEL concentrate outlet pipe. The upper clear liquid will be recirculated to the bioreactor through a peristaltic pump to continue participating in synthesis.
Table 1 Component cost
* The above parameters are based on the standard mode of Bambu Lab P1P 0.4mm nozzle to create three-dimensional PLA consumables calculation, including necessary support consumption
And all of this is achieved through the assembly of 3D-printed parts, without utilizing any electrical circuitry!
We initially designed a box with one inlet and two outlets. Inside, at the starting point, there is a baffle to facilitate the first round of collision for colloids and suspended particles. The subsequent design involving reflux and inclined plate grates aims to achieve static self-blockage and self-locking of the liquid. The upper part of the last two outlets is used for the separation of the upper clear liquid, while the lower part is for the liquid containing a high concentration of MEL. Then we carried out the fluid simulation experiment, The inlet flow rate of the upper left is 10m/s, and the flow rate of the two outlet pipes of the lower level is set to 2.5m/s, reflecting the situation in the actual use scenario where the fluid enters quickly and for a short time due to high pressure and the peristaltic pump pumps out the target liquid steadily in the outlet pipe.
Video B-1 Accelerated sinker box trace animation
Video B-2 Inclined plate settling particle track side
Video B-3 Inclined plate settler particle track front
Video B-4 Inclined plate settler particle track rotation
Video B-5 Inclined plate settler velocity contour view
Video B-6 Inclined plate settler velocity contour side
One of the key factors to accelerate the settling of colloids and suspended particles and facilitate subsequent separation is to reduce their flow velocity and adjust the liquid pressure within the water. From the animation, it is evident that the flow velocity at the entrance is quite high, but after impacting the first intercepting plate in our design, the flow velocity rapidly decreases. When it reaches the main body of the inclined plate, the flow velocity becomes nearly zero due to the extremely slow laminar flow speed around the plates.
Within the pipes designed around the main body of the inclined plate, the fluid exchanges occur at a slow pace, allowing the viscous fluid from the plates to effectively brake the circulating fluid, resulting in a significant drop in the flow velocity throughout the box. This successfully accomplishes our goal of using the principle of inclined plate settling to decelerate and accelerate settling.
Similarly, Professor Liu also provided us with some recommendations for our accelerated settling tank, suggesting that we add another higher clear liquid outlet to handle the separation of different proportions of the liquid.
4.Parts construction
Considering the worldwide distribution of 3D printing and its consumables, as well as the performance of various materials, we initially ruled out 3D printing technologies based on photopolymerization (SLA). While SLA offers high precision, it employs toxic and harmful materials, making it unsuitable for our application. Secondly, we abandoned less common techniques such as Selective Laser Sintering (SLS), Electron Beam Melting (EBM), and Three-Dimensional Powder Binding (3DP). Ultimately, we opted for a versatile printing method led by Fused Deposition Modeling (FDM) and chose environmentally friendly PLA resin as the consumable material for our accelerated settling tank[1,2,3].
PLA 3D printing process
Table 2 Print parameters
* Note: Please reduce the first layer printing speed to 1/3 of the normal printing speed
To address the substantial contact with microorganisms in our project, we recommend that laboratories and companies intending to use our accelerated settling tank solution for an extended period of time switch to ABS (a consumable material with high heat resistance, good toughness, and resistance to biological corrosion, widely used in the production of LEGO bricks)[4].
If there is a high industrial demand for equipment accuracy, we suggest replacing all printing technologies with photopolymerization or metal powder bed fusion techniques and entrusting the task to relevant specialized companies.
5.Parts assembly
6.Conclusion
From the results of simulation, it can be seen that the macromolecular organic particles in the solution will settle at the bottom of the accelerated settling box due to hydraulic action, which can achieve the basic requirements set by us. At the same time, the design of the individual pipes can be used to achieve our final solution: After the box is sealed, it is directly connected to the fermentation circulation system. After the fermentation is completed, part of the liquid containing MEl is pumped into the box with peristaltic pump, and then the supernatant is pumped back into the fermenter at the appropriate position of the pipe mouth to continue the follow-up reaction and improve the yield. The liquid containing more MEL in the lower layer will be pumped out by another pipeline to the collection vessel for subsequent extraction, separation and purification.
Attach
ment
1.Patent
2.Three-dimensional model
Below are the model files for the blades used in each iteration of our project:
six-leaf-helix-anticlockwisesix-leaf-helix-clockwise
The following is the model file of the accelerated settling box in our project:
Refer
ences
[1] TANG Tongming, Zhang Zheng, Deng Jiawen et al. Research status and development trend of 3D printing technology based on FDM [J]. New Chemical Materials, 2015,43 (06) : 228-230+234.
[2] LI Xiaoli, Ma Jianxiong, Li Ping et al.3D printing technology and application trend [J]. Automation Instrument,2014,35(01):1-5.
[3] ZHU Yanqing, Shi Jifu, WANG Leilei et al. Development Status of 3D printing technology [J]. Manufacturing Technology & Machine Tool,2015,No.642(12):50-57.
[4] FANG L H. Research on ABS 3D printing materials based on FDM [D]. South China University of Technology,2016.