Our project is about efficient cracking of alginate oligosaccharides. However, specific hardware equipment is needed to extract brown algal oligosaccharides from algae and put them into use in the actual environment. We known from the interview that the existing hardware equipment - fermenters are too complex and expensive, so we decided to develop a new one, which is easy to use.
After the investigation, we had our first attempt:
This hardware involves smashing down the seaweed, providing the fermentation environment, and finally breaking down the bacteria to release alginate oligosaccharides:
For smashing, we are inspired from the meat grinder that can efficiently divide the seaweed to fine fragments.
For fermentation, we used a normal fermentation tank with the engineered bacteria, which helps to decompose the bacteria. Temperature is monitored by Arduino programme and displayed an alarming system.
Consequently, the alginate oligosaccharides produced by bacteria can be completely released and applied as fertilizers.
turn on the meat grinder and put the washed seaweed in, wait for the meat grainier to crush the seaweed into fragments.
Add the engineered bacteria into the fermentation tank beforehand, then put it under the output pipe of the meat grinder, the crushed seaweed will drop into the tank, the temperature of seaweed is monitored.
add the Arabinose for the right time.
wait and take the substances in the tank when fermentation is about finished.
To evaluate the significance of our findings, a experiment was conducted as following.
Experimental Methods
To evaluate the effectiveness of the engineered E.coli with gene Bgls/SRRz(cellulase gene) and AL2/SRRz(alginate lyase gene) in the fermentation of seaweed, we set two test groups:
Group 1: Control: no bacterial strain inoculated
Group 2: Bgls/SRRz+AL2/SRRz bacterial strains
Washed the seaweed thoroughly and dried it under 65 degree Celsius for 2 hours, measured 1000g dried seaweed.
Soaked the 1000g dried seaweed then crushed it, added it to 20L thermos flask. Subsequently, the following was added: 5 L of LB medium (containing 100 μg/mL Ampicillin) and 10 % (v/v) of engineered bacterial strain (OD600 = 0.6). According to the in vitro test results of SRRz lysis strain, 0.1 mM L-arabinose (which cannot be consumed by E. coli) was added to induce bacterial lysis and allow the engineered bacteria to continue growing. Fermentation was carried out at 37℃ and 150 rpm.
A tissue culture film seal was used to ensure the supply of oxygen. Every 48 hours, 50% of the fresh LB medium (supplemented with 100 μg/mL Ampicillin) was replaced inside the ultra-clean workbench. After two weeks, the seaweed in the fermentation bottle was filtered and washed, then dried to constant weight at 65°C.
The degradation rate was calculated by comparing the weight of the seaweed before and after treatment. All experiments were repeated three times, and data will be presented as mean plus SD.
The degradation rates of the two groups is shown below: we can find that the seaweed with modified bacteria digested has much higher degradation rate of 60%, compared to that of the control(0%). This means our modified bacteria has a significant effect on the fermentation of seaweed.
In our hardware, we used the Arduino uno board, temperature sensor, and LED to detect temperature and send alarms when the temperature has exceeded the previously set temperature thresholds. If the temperature is outside the set upper and lower limits over a certain amount of time, the LED will turn green, otherwise, the LED will stay green. In real applications, alarms other than LED can also be used, such as radio alarms and screen alarms. This code just shows the feasibility of using Arduino to detect and alarm temperatures during fermentation.
Testing And Feedback of the First Generation
Since the completion of our model, our team has been conducting customer research and collecting product feedback:
The noise and energy consumption generated by machines was too large.
Inconvenient (need to move by hand)
Low efficiency (non-automated production process)
Low quality of results (non-automated production makes control variables more difficult to control).
Driven by these feedback, we improved the initial product.
The Final Generation Introduction
Based one the first attempt and the feedback, we brainstormed and finally decided to
employ 3D modeling tools to create our ultimate hardware generation.
Functional Components
(Labelled Blueprint)
Smashing Machine:
The Smashing Machine serves the purpose of breaking down algae into debris through the utilization of the spiral stirring knife(4) including two sets of blades: a spiral blade and a cutting blade. As algae enters the machine through the funnel(1), the spiral blade propels it towards the spinning cutting blade, resulting in finely cut algae pieces. The debris is then directed to the thermos flask(6) via an extension tube connected to the Smashing Machine.
thermos flask(6):
The thermos flask(6) combines both mixing by blender(8) and temperature control by temperature detection panel(7) functionalities into one system.
Blender(8):
Inside the flask, a blender, powered by a bottom-mounted battery, ensures continuous mixing. The blender's structure consists of an axle connected to two swinging arms.
Temperature detection panel(7):
Located at the flask's base is a built-in heating coil. Additionally, an internal thermometer is integrated into the flask. These two components are linked through a control panel located on the exterior of the flask, allowing users to monitor and adjust the tank's interior temperature in Celsius. Real-time temperature monitoring is available on the panel, and users can set temperature limits for alarms as well as adjust temperature settings.
Powder Dosing Machine:
The Powder Dosing Machine enables precise dispensing of various powders into the Fermentation Tank, such as Bacteria powder and arabinose. The titration vessel(9) of the machine is easily interchangeable for refilling or changing the type of powder being dispensed. The Powder Dosing Machine features a weight sensor(10) that determines the quantity of powder, measured in grams, being dispensed by monitoring changes in the mass of the powder in the container.
Escherichia coli (E. coli):
Engineered E. coli is programmed to release necessary enzymes, alginate lyase, and cellulase, to break down brown algae and produce alginate oligosaccharides. Furthermore, the E. coli incorporates the SRRz lyase gene as a promoter activated by arabinose. This enables the addition of arabinose into the Fermentation Tank to induce lyase activity in the E. coli.
Physical Soundproof Device:
This is installed on the crushing procedure in the first part. The number of gears between components is increased so that less energy leads to more effective rotation. The energy loss rate is reduced to less than 30%.
(Final 3D model)
Manual Instructions:
Preparation of Brown Algae:
The initial step involves placing brown algae into the Smashing Machine. The algae undergo processing until they achieve the desired consistency, resulting in the production of debris, which is collected within the Fermentation Tank.
Introduction of Escherichia coli (E. coli) Bacteria:
E. coli bacteria are introduced into the Fermentation Tank through a precision Powder Dosing Machine.
Temperature Regulation:
Precise temperature control is maintained at a consistent 35 degrees Celsius within the Fermentation Tank.
Arabinose Addition:
Once the designated temperature of 35 degrees Celsius is reached, arabinose is introduced into the Fermentation Tank using a specialized Powder Dispenser.
Incubation at Constant Temperature:
The Fermentation Tank is held at a constant temperature of 35 degrees Celsius for a duration of 48 hours.
Here is the raw data of our 3D Modeling, which can be copied in the future if needed.
Our team conducted several experiments and tests on the hardware, figuring out some parts that we needed to refine. Based on the customer research and the feedback, we improved our device which later became more efficient. The detailed description mentioned above indicates the utility and functionality of the hardware. On a macro level, by building our hardware, we achieved the feasibility of generating our product in a simple way. If our hardware launches into the market, it can also bring the idea of synthetic biology to the public, letting more people acknowledge the influences of bio-engineering techniques. Our hardware will eventually become either a reference or further support for later teams in the following years within the iGem community.
Future teams could reach out to us via email (igemthinkershenzhen@gmail.com) if they have any problems.