Section 1: The design of our fermenter

1.1 Overview

To help our Cyanobacteria and S. oneidensis MR-1 symbiose and further achieve our goals, we built such a fermenter for cell culture. We formed this complete fermenter with partitions, making it suitable for our Cyanobacteria and S. oneidensis MR-1 cultures and easy to split and assemble. From the outside, our fermenter is like a small mushroom that is breaking out of the ground, and we hope to use it to represent new life and the future. At the same time, we have prepared sterilization equipment at the outlet of the tank to ensure that our engineered bacteria will not escape into the environment. We designed such a fermenter to help further improve and complete the experiment.

1.2 Structure

The fermenter innovatively puts forward the idea of "building block" fermenter, through the modular design, improves the operation portability and environmental adaptability of the fermentation system, and customizes the corresponding solutions for different fermentation scenarios to ensure maximum efficiency. The system provides optional modules including: stirring module, defoaming module, temperature control module, air distribution module, pH monitoring module, aerobic/anaerobic module, lightless/lighted module, and monoclonal/synergistic module. Another advantage of the modular design is the cost, which only requires the price of normal 3d printing. After obtaining the whole fermentation model family, the parts can be replaced at will, realizing the completion of industrial simulation at a cheaper price and higher fit.

1.2.1 Cyanobacteria Part

This semi-spherical fermenter is designed specifically to optimize the growth of Cyanobacteria, with a primary focusing on increasing their light receiving area but also ensure its volume. The tank boasts a total height of 15.5 cm, and we employed transparent materials in order to enhance both light efficiency and transmittance, thereby promoting better photosynthesis by the Cyanobacteria. To further support optimal culture conditions for these organisms, we included a spiral tube for temperature exchange, which allows for the inflow of thermal wastewater or thermal waste gas generated from power plants to maintain the desired temperature within the fermenter. We also prepared another four small holes on the top of the tank, one for adding culture medium，one for adding waterproof exhaust valve to balance the air pressure in the tank,one for placing temperature sensor and another for placing PH sensor. In consideration of the need to connect the filter membrane、flow switch and anode tank beneath the fermenter, we incorporated external threads at its base. These threads serve not only to facilitate this connection but also prevent potential water leakage.

1.2.2 S. oneidensis MR-1 Anode Tank

This anode pot, measuring 8.2 cm in height, is utilized for the cultivation of S. oneidensis MR-1.The upper surface of the tank features two circular openings - one serves as a connection to the Cyanobacteria culture tank above, while the smaller one is designated for the anodal elrctrode, and left the corresponding inner thread for the anode cover. The remaining openings are concentrated on one side of the tank, with the large threaded mouth serving as the point of connection for the cathode tank,the 4 cm diameter opening is more conductive to proton exchange. To facilitate the embedding of the proton membrane, a 1mm-wide gap has been left around the grid, which also acts as a safeguard against membrane damage.The small hole at the bottom of the pot is designed for waste medium discharge, while the side bend serves to input carbon dioxide,keep the anaerobic environment and maintain air pressure within the tank.

1.2.3 Cathode Tank

The counterpart of the anode tank is the cathode tank.We have also designated the position of the cathode plate on top, and left the corresponding inner thread for the cathode cover.The threaded connection on the side bears similarities to that of the anode tank, with the exception of the opposite thread direction and omitting a 1mm width.The thread in the opposite direction ensures that the cathode and anode tanks can be connected at the same time without inversion problems when installing the proton membrane connector.

1.2.4 Other Components

1) Filter Plates and Flow Switches

In order to better achieve the symbiotic relationship between Cyanobacteria and S. oneidensis MR-1, we have incorporated three components between the Cyanobacteria fermenter and the S. oneidensis MR-1 anode tank to facilitate the addition of a membrane filter while preventing leakage.

The first component is the No.1 filter plate, with one side equipped with a carbon dioxide entrance.Carbon dioxide is first passed into the S. oneidensis MR-1 anode tank to ensure its anaerobic environment, and then bypass the flow switch partially through to the cyanobacteria fermenter to participate in the reaction of cyanobacteria.This ensures that the cyanobacteria can also be given sufficient carbon dioxide when the flow switch is turned off, and the design of feeding the carbon dioxide from near the filter membrane allows the gas to blow up the cyanobacteria deposited on the filter membrane. Both sides of the No.1 filter plate are equipped with threads, with the upper thread connected to the Cyanobacteria fermenter and the lower connection to the No.2 filter plate.

The bottom-most No.2 flow switch has a similar fan-shaped structure as that of the No.1 flow switch, with its lower thread connecting to the S. oneidensis MR-1 anode tank.Its side surface also has a conduit, which can ensure that when the flow switch is closed, gas can be introduced into the Cyanobacteria fermenter to maintain an anaerobic environment.

2) Connecting Pipe

We also have a separate connecting pipe designed between the cathode tank and anode tank. It has completely symmetrical positive-reverse thread structure. And since the screw starting point of the cathode and anode tanks is the same but with opposite rotation direction, we can separate them without rotating the tank body to replace the proton membrane.

3) Threaded cover

The cathode, like the anode, is equipped with a threaded electrode cover to ensure that the culture medium does not flow out of the electrode port and can easily replace the electrode. In addition, each cover has 2mm holes, leaving room for the insertion of the electrode in advance.

Considering that the initial addition of media requires a faster rate of adding samples, but the subsequent replenishment of media requires the use of catheters for continuous adding samples, and requires a closed internal environment, we set a large feeding port on the Cyanobacteria fermenter, and equipped it with a corresponding connecting port for the use of catheter sampling. This is also in the form of threads to ensure better tightness.

1.2.5 Procedures of Use

1) Assemble the proton membrane and connect the cathode and anode tanks.

2) Add the electrolyte and S. oneidensis MR-1,cover the cathode and anode cover as well.

3)Install the filter and flow part,connect the tube between No.1filter plate and No.2 flow switch.Turn off the flow switch,connect the Cyanobacteria fermenter and add Synechocystis sp. PCC 6803.

4)Plug in the warm water, turn on the flow switch properly and done!

1.3 Electric Part

Our hardware also needs to realize the detection of the growth condition of the medium, such as temperature, ph value, etc., and also according to the temperature to control the in and out of the hot air to ensure the appropriate temperature of the medium, as well as according to the ph value to control the replenishment and flow of the medium.
Therefore, we built a set of simple control board based on STM32F103C8T6 master to realize these functions.
The wiring diagram on the breadboard is as follows:

1.3.1 Temperature Acquisition Section

In the temperature acquisition section, we have used DS18B20 module.The DS18B20 module provides 12-bit temperature readout with a temperature range of -55° to 125°. The temperature information is sent to and from the DS18B20 via a single wire interface. Therefore, only one wire (and ground) needs to be connected from the CPU to the DS18B20. The power required to read, write, and complete temperature conversions can be supplied from the data line itself, and no external power supply is required.
The DS18B20 stores the temperature value in 12 bits, with the remaining high bits being sign bits. All 1s are negative and all 0s are positive. Thus the DS18B20 can achieve an accuracy of 0.0625°.

For the hardware connection, the DS18B20 needs to be connected to a temperature probe for data acquisition, and itself needs to be connected to a 3-5V supply, ground, and a data cable.

1.3.2 PH Detection Section

For the PH detection part we chose a PH sensor module.
The module is connected to the PH composite electrode through a BNC connector and expanded with a DS18B20 temperature sensor interface, which is convenient for the software to carry out temperature compensation design. Adjusting the 10K blue potentiometer can be adjusted for amplification.

1.3.3 The module pins are defined as follows：

Before using the PH sensor, we need to use the two-point calibration method. Since our medium environment is acidic, the PO port output voltage can be measured in a standard buffer with a pH of 6.86 and a pH of 4.00, and then a V-PH curve can be fitted in EXCEL based on these two points, from which the pH value of the medium can be calculated.

1.3.4 Solenoid Valve Control Section

Our solenoid valve is a normally closed type, with an operating voltage of DC12V.
Since the maximum output voltage of STM32 is limited, in order to drive this 12V solenoid valve, we need to borrow an external 12V battery (of course, in the future, after the industrialization of maturity, we can realize the self-sufficiency of the power supply), an NMOS FET, and a 10K pull-up resistor to form a driver circuit.

PA6 is the microcontroller output pin. In order to utilize the microcontroller to output 12V, we configure the output mode of PA6 pin as open-drain output mode to realize that when the output is low, the output is 0, the NMOS tube is cut off, and the solenoid valve is closed; and when the output is high, the output is high resistive so that the NMOS tube is turned on, and the solenoid valve is opened.
The value of PA6 determines the opening and closing of the solenoid valve, and the microcontroller judges the value of PA6 according to the acquired information such as temperature and PH value to realize the entry and exit of hot air and the replenishment of culture medium.

1.3.5 Main Function Code

The main function code is as follows:

Ph calibration curves for calculations in the main function

1.3.6 Other:

The appendix should have Keil project files;

To verify that both the temperature sensing and ph sensing modules in the circuit work correctly and accurately, we tested them and express the results in the video below. As it shows, they all work well.

1.4 Mathematical modeling part

Synechocystis sp. PCC 6803 was the first phototrophic organism to be fully sequenced. As a result of the genome sequencing and the establishment of various databases, Synechocystis sp. PCC 6803 provides an extremely versatile and easy model to study the genetic systems of photosynthetic organisms. Light energy is the most important energy substrate for the growth of algal cells, so how to make Synechocystis sp. PCC 6803 use light energy reasonably and efficiently is one of the major problems that our hardware needs to solve. By means of literature search and experimental verification, we fit the cell growth curve and the light attenuation curve in the bacterial solution.

1.4.1 Fitting of light attenuation curve

In the mixed homogeneous microalgae culture system, the light attenuation caused by algal fluid in the concentration range is generally considered to conform to such Lambert-Beer law:
$I=I_0\cdot\exp{\left(-aXL\right)}$
$A\left(X\right)=\alpha X$
or
$A\left(X\right)=\alpha_mX+\left(\alpha_0-\alpha_m\right)X\exp{\left(-\varepsilon x\right)}$

I₀ - the incident light intensity, I - the light intensity at the optical path L(cm), α - the specific extinction coefficient(m²/kg), α₀ - the specific extinction coefficient at X=0, α_m - the minimum specific extinction coefficient, A(X) - the optical attenuation coefficient, ε - proportional coefficient.^[1]-[2]
With the help of the two groups of data, we have carried out the fitting respectively to get two relations:

With reference to the literature, we know that the optimal cultivation light intensity of PCC is 300-1000μE/m2·s^[3], and Synechocystis sp. PCC 6803 has the highest biomass productivity and efficiency in converting light energy to biomass at a culture density of 760mg/L^[4]. Thus, we substitute the second type of optical attenuation coefficient expression to get:
$L=\frac{\ln{\frac{I_0}{I}}}{A(X)}$
Then, we plugged in the transmittance of our materialcan (0.93) and further introduced the most suitable amount of liquid in our fermentation tank is 4.719-11.730 cm. Under these conditions, we chose 8 cm as our filling volume.

1.4.2 Construction of cell growth dynamics model

We cultured the cells in a 250ml shaker bottle with a liquid volume of 150ml, at a culture temperature of 30℃ and incident light intensity of 100μE/(m2·s). Since the algal cell biomass curve is similar to the cell growth curve during microbial fermentation, we can describe the algal cell growth with the help of microbial fermentation growth kinetics. Using light energy as a substrate, the growth rate of cells in the growth phase can be expressed as follows by Logistic equation description:
$\frac{dx}{dt}=\mu_{max}\left(1-\frac{x}{x_{max}}\right)x$
μmax is the maximum specific cell growth rate (1/h), and xmax is the maximum cell concentration (g/L). When t=t0 and x = x0, the integral transformation of the equation yields:
$x=\frac{x_0e^{\mu_mt}}{1-\frac{x_0}{x_m}\left(1-e^{\mu_mt}\right)}$
After fitting the test data, the cell growth kinetics equation and the cell growth curve over time was obtained:

The fitting equation is:
$x=\frac{x_0e^{0.68t}}{1-\frac{x_0}{0.616}\left(1-e^{0.68t}\right)},R^2=0.962083$
In fact, we also know that Synechocystis sp. PCC 6803 PCC6803 has the highest biomass productivity and efficiency in converting light energy to biomass at a culture density of 760mg/L.[4] Therefore, with this growth model, we can more clearly define the cycle of media replacement and keep our system operating efficiently.

Section 2:"Family" of modular fermentation units

We are trying to build a fermentation facility for our co-culture system to test it in the laboratory and to prepare for the practical application of the project in a thermal power plant in the future. In the process of design and research, we found that in the current synthetic biology related projects, synthetic biology practitioners sometimes do not have the perfect fermentation engineering reserves to connect the results of the laboratory level with the industrial applications, and in the amplification process before putting into production, we often encountered that the fermentation results are inconsistent with the simple shake flask culture, and the lack of a miniature fermentation device to predict the effects of the project, which led to some cost wastage.
Therefore, we constructed a set of miniature modular fermentation equipment system, through the research of the current pilot and even the production of fermentation equipment and reasonably reduced and modularized to build a set of miniature fermentation system, so as to be able to simulate the effect of the pilot and even production in the laboratory. The fermentation equipment system consists of several modules, through the replacement of different parts within the module, and then realize the culture of different strains and under different conditions, and accordingly build a set of fermentation system for our co-culture system.

2.1 Mixing Module

In the fermentation process, due to the different viscosity of different liquids and other reasons, it may lead to different reaction degrees in different positions in the tank, so there is a need to increase the contact area by stirring and accelerate the reaction. Moreover, in order to better simulate the industrial application scenario, we set up two models of six-flat-blade turbine and six-curved blade turbine, both of which can withstand different stress levels. Users can choose the right mixing paddle according to the liquid viscosity in the tank. If the speed needs to be increased, the electric control parameters can be adjusted to achieve the purpose.

2.2 Antifoam Module

During the reaction process, especially when stirred, because some fermentation media themselves contain many surface active substances, and the strain reaction will also produce surface active substances, all tanks are prone to foam. Foam can reduce production capacity, cause waste of raw materials, affect bacterial respiration, and even lead to bacterial contamination. So we built two defoaming devices to ensure fermentation efficiency, uniformity and safety. The rake type is simple and easy to use, but the defoaming effect is general; The rotary disk defoamer is more effective but requires electricity, and the user can freely choose according to the number of bubbles.

2.3 Temperature control module

During fermentation, some strains have higher requirements for temperature, so it is necessary to control the temperature in the tank to ensure the fermentation environment. We built two kinds of temperature control tube and temperature control coil to better fit the fermenter.

2.4 Air Distribution Module

In our fermenter, a single orifice tube type gas intake device has been used. If a more effective ventilation device is required, the annular air distribution tube below can also be used. The device can reduce the diameter of bubbles entering the culture medium, increase the dissolution amount, increase the contact area, and better ensure the gas supply and reaction.

2.5 Other tank modules

During co-bacterial culture, the upper fermenter can select the printing material according to whether the light is needed. Below is an example of a fermenter that does not require light.

When the lower strain of co-culture does not produce electricity and there is no need for electron enrichment, the lower fermenter can be replaced with the one shown in the following figure.

When it is just a single culture, the fermenter below can be used in combination with the various parts mentioned earlier.

This fermenter focuses on solving the difficult problem of laboratory-to-industrial crossing in synthetic biology.it is able to meet the process optimization and characterization requirements of the biotechnology and biopharmaceutical industries, and provides excellent functionality and outstanding choices for microbial processes, making it an ideal scaled-down model for large-scale processes. The tanks are designed with similar geometrical structure, with continuous and consistent mixing and aerating strategies, overcoming the problems that need to be considered for industrial applications such as oxygen uptake efficiency and shear resistance in laboratory shaking operations, and autonomously realizing operations such as replenishment, temperature control, and oxygen control, which will be well suited to provide data samples for testing and feasibility analysis for fermentation-based projects.
The team has already conducted various types of user tests, and the SUS scale shows high user satisfaction. Scenario tests with fermentation engineering laboratories and relevant enterprises cooperating with the school have confirmed its high fermentation level.
In addition, all the design and operation methods of this fermentation system are publicly available for reference and replication by other teams.

Section 3: Industrialization thoughts

In considering the transformation of the laboratory model into an industrial scenario, the general idea is as follows: To begin with, multiple laboratory models are presented in a form similar to side by side. Keep the small mushroom shape of the cyanobacteria tank, and combine the anode and cathode tanks of S. oneidensis MR-1, roughly as shown in Figure 31.

After taking into account that such industrialization will consume too much material, it is necessary to add samples several times when adding samples, and the volume of cyanobacteria is significantly reduced, so on the premise of taking into account the lighting requirements of cyanobacteria, the cyanobacteria tank is combined into a large capsule shape tank, roughly shaped as shown in Figure 32.

Then, considering that in real industrialization, the filter membrane can be directly installed at the mouth of the pipe, and the flow switch can be realized through the water valve, the real industrialization idea is finally formed, that is Figure 33.

Considering that it is the hot gas that provides heat to the cyanobacteria tank in the real industrialization, the gas enters from the lower left corner of the tank and is discharged from the upper right corner, which can ensure that the gas is in full contact with the medium and the heat transfer is carried out as far as possible. In addition, since the culture of S. oneidensis MR-1 requires an anaerobic environment, it is also necessary to inject CO₂ into S. oneidensis MR-1, so it is also adopted to pass into the upper right corner of the left corner. In addition, in order to prevent CO₂ from entering the cyanobacteria fermenter retrograde from the culture medium flow pipe, the flow pipe is curved so as to form a water seal to prevent CO₂ from entering the cyanobacteria fermenter, thereby reducing the temperature of the cyanobacteria fermenter medium. In the real industrial implementation, a part of the hot CO₂ produced by the power plant is first cooled to become CO₂ at room temperature, and the CO₂ at room temperature can be directly passed into the S. oneidensis MR-1 tank to ensure an anaerobic environment. Then take part of the normal temperature CO₂ and hot CO₂ quantitative ratio, into the cyanobacteria fermenter, in order to play the role of heating and heat preservation. When regulating the temperature of the cyanobacteria tank, it is only necessary to adjust the ratio of cold and hot gases to regulate the temperature. In addition, we took inspiration from the design of the solar panels to add a certain Angle to the support of the cyanobacteria fermenter. Different regions can adjust the tilt Angle according to their latitude, so as to ensure that the cyanobacteria fermenter can better receive light.

In the future, the cyanobacteria fermenter may also be replaced by special plastic bags, so as to better reduce costs. In summary, we consider the problems that may be encountered in the process of real industrialization, can better simulate industrial problems from the perspective of the laboratory, and put forward reasonable industrialization ideas. Looking at the big world from a small model can help promote the development of synthetic biology.