We developed artificial, technological evolution and used it to design functional ecosystems consisting of up to three forms of living technology, namely, artificial chemical life, living microorganisms, and complex chemical reaction networks for the purpose of improved treatment and cleanup of wastewater for energy generation. The goals of this project are i) develop a general, robotic platform, which by using artificial evolution can optimize the performance of a physicochemical or microbial system and its environment and ii) use the robotic platform to evolve improved microbial fuel cells in terms of robustness, longevity, or adaptability. The robot evolutionary platform will take the form of an open-source 3D printer extended with functionality for handling liquids and reaction vessels, and for obtaining feedback from the reaction vessels either using computer vision or task-specific sensors in real-time. The robot platform will optimize parameters such as the environment, hydraulics or real-time interaction with experiments (for instance, timing of injection of nutrients, removal of metabolic products, stirring, etc.) to maximize a desired functionality. Initially, we investigate processes such as fluid-structure-interaction driving bio-aggregate structure and in turn metabolic activity as well as the interaction of nanoparticles and bacterial cells by analyzing the outcome of the evolutionary process using state-of-the-art imaging techniques. We then seek to exploit synergies between these technologies to significantly improve the ability of the living technology, in the form of optimized microbial fuel cells, to cleanup wastewater. Overall, this is a cross-disciplinary project involving state-of-the-art chemistry, imaging, robotics, artificial life, microbiology and bio-energy harvesting for the purpose of enhancing our understanding of living technologies and how to best design and exploit groundbreaking bio-hybrid systems.
Objective 1: The first objective has three sub-objectives:
- Liquid-handling robotic platform: to develop the liquid-handling robotic platform to a level of functionality and robustness that it can be used to achieve objectives 2 to 5. We produced the robotic platform to make it robust enough for non-evolutionary experiments and make a copy available to all partners. The current prototype is based on the RepRap open-source 3D printer whose printing head is modified to handle liquids instead of plastics. The robotic platform is further extended with a gripper for manipulating petri dishes. Under the working area of the robot a camera is placed for automatic recording and simple tracking of elements of interest in the on-going experiment in the petri dish. The platform also has a simple graphical user-interface that allows the experimenter to specify which objects or colours to track and to visualize the performance of the current tracking system. The first is to mature the mechatronics to make it work reliably and prepare it for functional extensions needed to reach the other objectives. The software infrastructure will also be matured and prepared for further extension.
- Liquid-handling robot for evolutionary experiments: The robotic platform will be extended with functionality to facilitate evolutionary experiments. The exact requirements will be decided as part of work package 2. A key element will be to provide the robotic platform with sensors that allow it to measure the fitness of a given experiment. The camera system will provide visual monitoring and feedback for macroscopic characteristics. However, it may also be relevant to monitor the energy production of the experiment (in case of microbial fuel cells) and hence the robotic platform may have to place conductive probes in the petri dish to monitor current output. An attractive alternative is to deposit the cells on conductive ITO-coated glass. We also will combine the liquid handling functionality with the original functionality of printing 3D structures out of plastics to allow for construction of complex, physical environments inside the reaction vessel. The evolutionary robotic platform will be developed incrementally throughout the project in response to the needs of the consortium.
- Standalone liquid-handling robot: the evolutionary robotic platform will become a standalone system reducing the lab space it takes up, allowing for control and monitoring of the evolutionary experiment from any web-enabled device, and making it possible to remotely update the platform’s software. This objective is targeted at upgrading the electronics and software infrastructure to increase user-friendliness and maintainability. It is also the last step taken within the context of the project to mature the evolutionary robotic platform for commercial exploitation. iv) Evaluation of system robustness, reproducibility and evolutionary benchmark: the robotic platforms will be evaluated with respect to the number of manipulations that can be done within a certain range of tolerances exploring the mean time to failure (MTF) and also optimization of the stage trajectory to minimize wear and to evaluate the maximum number of repeat processes that can be done within a certain time period. The practical limits of the robot-facilitated evolution will also be determined in terms of number of parameters, time for each experiment, and speed of reactions.
Objective 2 (Microbial ecosystems): Microbial ecosystems are formed within the aforementioned Microbial Fuel Cells, which can be seen as selective pressure systems. Inside these MFCs, only the organisms that can directly or indirectly contribute to power generation can survive, and this depends on the establishment of an ecosystem, which can support these communities. In essence, facultative or strictly anaerobic microorganisms that can respire through the anode electrode – and hence generate electricity – will dominate, but depending on the nutrient source, other organisms capable of first-stage digestion might proliferate to break down the difficult substrate and produce metabolites (second-stage digestion), which can be the feedstock for the electricity producers. This would be an excellent example of an ecosystem with energy extraction being the main driver.
Objective 3 (Microbial biofilm): The complex biofilm structure will be imaged and characterized in 3D. This will allow a deeper understanding of the fluid-structure-interaction of biofilm systems since the development of the biofilm structure influences mass transport as well as mass transfer processes and vice versa. The correlation of the biofilm structure development and the system performance (power output) will allow an optimized operation of the MFC. This correlation will be used to mature the evolutionary platform by “printing” inoculation to force the optimal structural development with respect to development duration, costs and performance of the MFC.
Objective 4 (Artificial chemical life): Three different approaches will be explored in the device: i) We will use the formation of protocell droplets with various compositions and characterize the resulting chemotaxis and informed response. ii) Embedding small molecule kinetic networks will be embedded that can be fed using an external food source and can show adaptive behaviour. iii) Parts (i) and (ii) will be used to drive the assembly of nanoparticle catalysts to increase the efficiency of microbial fuel cell outputs
Objective 5 (Synergies): Explore synergies between the four underlying technologies for next generation microbial fuel cells. i) More robust communities that have been exposed to chemical enhancement or inhibition. ii) Microbial communities better tailored for specific substrates and hence applications (e.g. urine therefore better toilet MFCs)