SWARM INTELLIGENCE IN BIOLUMINISCENT MICRO-ORGANISMS POPULATIONS

Author: Eduardo Mayoral González Advisors: Edward Keller [email protected] Mitchell Joachim AAR, 2009_2010 Collaborator: Oliver Medvedik Columbia University, GSAPP

Fig.1: Glowing dead prawns colonized by Pseudomonas Fluorescence bacteria

0. Abstract It analyzes the behavior of glowing micro-organisms populations and the relationships between them, There are a great number of bioluminescent micro- as well as the conditions that make them glow organisms that emit light for different purposes in better. To accomplish that purpose, two different nature. They form populations that show varied living forms are studied. The first one is Vibrio light emission features depending on the conditions Fischeri bacterium, and the second one Pyrocystis they live in and the relationships established Fusiformis, which is a unicellular alga. Different between the agents that shape these populations. living conditions are tested to observe their behavior and different geometries designed, to put This work explores the forms of swarm intelligence them inside and check how they work to extract these populations show and their living conditions, conclusions. to produce effective devices that emit light without consuming electricity. It does so taking advantage Keywords: bacteria, algae, bioluminescence, swarm of natural glowing mechanisms. intelligence, multiagent systems, quorum sensing.

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1. Glowing micro-organisms. Design possibilities. BioMario (Fig.3) is a design proposal developed by a team at the University of Osaka for the IGEM Bacteria can be genetically modified to glow using 2009 competition. It uses genetically modified GFP (Green Fluorescent Protein) (Fig.2). Then they glowing bacteria to display an image of the emit light after being exposed to it. They can also videogame character Mario. It consists of a set of show glowing properties through a very simple petri dishes containing glowing bacteria, but it chemical reaction: luciferin substrate + 02 could be the beginning for a billboard design. substrate + luciferase enzyme (expressed in bacteria), resulting oxyluciferin + light. The advantage of chemical glowing is that there is no need to first storage light to then emit it as in photoluminiscent based processes. Therefore it is easier to control when the population of bacteria glows or not inducing the inputs needed for the reaction to happen.

Depending on the density of the population and the way its agents communicate between them, the whole will behave differently. This is due to quorum sensing, which is a decision-making process used by decentralized groups of individuals to coordinate social behavior. Quorum sensing can function as a decision-making process in any decentralized system, as long as individual components have a means of assessing the number of other components they interact with. Then a standard response is given once a specific number of Fig.3: BioMario Namba K. Minamino T. Morimoto Y. (2009) components are detected. The bioluminescent There are other proposals for domestic furniture luciferase produced by certain kind of bacteria developed by the Symbiotic Bacterial Light Project would not be visible if it was produced by a single in Canberra, like Deep Green 1 or the Jellyfish cell. Using quorum sensing to limit the production Lounge that also deal with bioluminescent bacteria of luciferase to situations when cell populations are (Fig.4). The first one is a lighting structure that large, bacteria are able to avoid wasting energy has glowing bacteria inside tubes filled with water. and glow when the population reaches a significant The water moves producing bubbles and the number of agents. This form of swarm intelligence bacteria glow inside. The second one is a chair with is explored in this work to get population of micro- a screen, which is powered with bioluminescent organisms glowing brighter. bacteria that emits faint light for reading or chilling.

Fig.4: Deep Green 1 and Jellyfish Lounge Takayama K. and Fig.2: Genetically engineered glowing bacteria Nicholson J. (2004) 2

There are other bacteria and unicellular algae that 2. Manipulating Vibrio Fischeri and Pyrocystis glow naturally without being manipulated. They live Fusiformis for design purposes. in colonies or in symbiotic relationships with other living beings like corals or squids (Fig.5). That This work shows the analysis and results obtained opens up different design scenarios using these manipulating two different kinds of glowing micro- micro-organisms since there is the possibility to organisms. They were grown to test their think of designs where populations of glowing possibilities and build design devices that do not bacteria or algae are considered alone, or in consume electricity to emit light. The work digs symbiosis with other living systems that help them into the living swarm forms of intelligence they to subsist (e.g. providing them with nutrients). show, their living conditions, and the way they could be implemented in artificial geometries and structures for design purposes. It also presents some design proposals based on what was learnt. The two different species treated are Vibrio Fischeri and Pyrocystis Fusiformis.

Vibrio Fischeri (Fig.6) is a gram-negative rod- shaped bacterium found globally in marine environments, usually in temperate sub-tropical waters. It is heterotrophic (it commonly eats agar) and moves by means of flagella. It can survive on decaying organic matter. It has bioluminescent properties and is found predominantly in symbiosis with various marine animals. The bioluminescence of Vibrio Fischeri is caused by transcription induced Fig.5: Avatar James Cameron (2009) and Pyrocystis Fusiformis by population-dependent quorum sensing. Hence in symbiosis with a squid the luminescence is only seen when population density reaches a certain level. It is regulated by Therefore it is worth taking advantage of forms of autoinduction. An autoinducer is a transcriptional swarm intelligence shown by populations of glowing promoter of the enzymes necessary for micro-organisms (like quorum sensing), as well as bioluminescence and it must be present for other forms of intelligence that might take place glowing. Luminescence in Vibrio Fischeri appears to due to symbiotic relationships between these follow a circadian rhythm, meaning it is brighter organisms and other living systems. That would during nighttime than daytime. give rise to design proposals for lighting devices not based on electricity consumption. In addition we should consider the benefits of hybridizing these glowing living systems with artificially produced geometries and structures that might host them for design purposes.

Then it makes sense to think about how to manipulate these sort of living multiagent systems to create public lighting devices that do not generate artificial waste such as traffic lights, emergency lighting, faint illumination for parks or highways, or screens and billboards displaying information and light. All of them could work powered by populations of bacteria or algae.

Fig.6: Vibrio Fischeri 3

Vibrio Fischeri populations get the nutrients it needs thanks to the symbiotic relationship it establishes with animals. In return it helps animals to find mates, ward off predators, attract preys or communicate with other organisms, due to its bioluminescent properties.

Several tubes of Vibrio Fischeri were ordered to check their glowing properties. Some of the tubes contained populations of bacteria and others agar nutrients (Fig.7). To grow them you have to rub carefully the tubes containing bacteria with a spatula and introduce it in the ones containing agar. Then you remove the spatula and cap loosely the tube. They grow as far as they have nutrients and they start glowing in about four days. They were put inside an incubator (Fig.8-9) since they grow better if the temperature is between 18ºC and 27ºC. In ten days they consumed the nutrients and their bioluminescent properties started decreasing until they died. After a week, some cultures were placed in the fridge at 4ºC to keep Fig.8: Incubator them alive. Several subcultures were made during the process transferring a piece of culture from one tube to another one with nutrients.

Fig.7: Vibrio Fischeri and agar tubes Fig.9: Vibrio Fischeri tubes 4

Vibrio Fischeri does not glow much unless the Since they are organic living beings, they could be population reaches a very high density. Besides used to illuminate natural environments without they die if they run out of nutrients. Therefore they placing artificial lamps in the woods (Fig.11). Vibrio work well for faint illumination or in very high Fischeri performs chemical bioluminescence and concentrations as long as they have nutrients. follows a circadian rhythm, which makes it glow Vibrio Fischeri does not need to receive light for naturally at night. Since there is no need to have a glowing like photoluminiscent organisms because very powerful lighting source in the woods, Vibrio they chemically glow. These features should be Fischeri would perfectly work for highlighting considered for design when using these bacteria. remarkable spots or for signposting paths. A flexible structure made of natural rubber or other Vibrio Fischeri could be used for static commercial organic biodegradable material, could work to billboards (Fig.10). In that case, the bacteria would adapt its shape to organic natural forms and be make the images more visible at night but the attached to them. If the geometry of this structure where the bacteria would be structure is pixel based and it is provided with a implemented should reproduce the image very number of holes or cavities, it could host certain clearly. Otherwise the image would not be visible. amount of agar. Agar is a glucose substance Eventually the image would disappear but that extracted from red algae and it works pretty well might be an advantage for a commercial strategy. to feed Vibrio Fischeri. It would have to be replaced It would also take a while for the image to be to keep bacteria alive though. However the recognizable while the bacteria are growing. prototype could be designed in a way that releases agar or that includes either red algae or any sort of gelatin that might work as a nutrient source fro the bacteria.

Fig.10: Commercial Vibrio Fischeri billboard Fig.11: Natural park illumination system using Vibrio Fischeri 5

Vibrio Fischeri could also be used to create ambient lighting for interior or exterior spaces at night. It could be embedded in modules able to be aggregated to form 3D usable structures (Fig.12). These structures would then emit faint light generating different ambient conditions around them. The bacteria could be placed in removable small containers attached to the modules so that they can be replaced (Fig.13), or the modules could be filled with nutrients inside.

Fig.13: Petri dish containing Vibrio Fischeri

Besides bacteria there are unicellular algae that also glow naturally in the dark. This is the case of Pyroscystis Fusiformis, a species of dinoflagellates that lives in sea water (Fig.14). Dinoflagellates are marine unicellular planktonic organisms. They perform photosynthetic metabolism, heterotrophic metabolism or both. Pyrocystis Fusiformis is a mixotroph, meaning that it conducts both photosynthetic and heterotrophic metabolism. Fig.12: 3D usable glowing structure using Vibrio Fischeri

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According to the “burglar alarm theory” Pyrocystis Fusiformis emits light to attract attention to its predator. Whenever a predator approaches a population of dinoflagellates, it moves the water exciting them. Hence they glow and the predator is illuminated increasing the chances that it is itself preyed upon.

To start cultivating Pyrocystis Fusiformis, four 50ml bags containing dinoflagellates (Fig.15) were ordered along with nutrients and vitamins for them to grow (Fig.16). Artificial salty water was created using distilled water and adding marine salts to it (35gr of salts for every liter of distilled water) (Fig.17). Then nutrients, minerals and the dinoflagellates were poured in the water in 1:3 proportion; 10ml of minerals and 10ml of vitamins for 1500ml of dinoflagellates in 500ml of salty water. They were put inside the incubator at 25ºC with a timer that controlled a lamp to light them in cycles of 12h on and 12h off (Fig.18-19).

Fig.15: 50ml Pyrocystis Fusiformis bags Fig.14: Pyrocystis Fusiformis

Pyrocystis Fusiformis produces bioluminescence in a circadian rhythm. It photosynthesizes during the day and produces bioluminescence when mechanically or chemically stimulated at night. It emits blue-green light from microsources found evenly distributed throughout the cytoplasmatic layer surrounding the large central vacuole. During the day the microsources migrate from the cell´s periphery to a spherical region distal to the nucleus. During their migration from the periphery they are replaced by chloroplasts. This results in a lack of bioluminescence. The microstructures return to the periphery at night, and produce bioluminescence. Fig.16: 10ml of minerals and nutrients bags 7

The nutrients helped the dinoflagellates to grow and the fluorescent lamp provided them with light to make the photosynthesis to achieve their own circadian rhythm for glowing.

The first culture did not glow at all so a sample was watched under the microscope to check it. It did not glow because the water was contaminated by an organism that was killing the cells (Fig.20- 21). More dinoflagellates were ordered and the same protocol was repeated to fill two containers of one liter each. This time the lighting cycle was modified to 17h on and 7h off. The bacteria started glowing in three days (Fig.22). After a month the containers were split and more nutrients, vitamins and artificial salty water were added to the new Fig.17: 35gr of marine salts containers. As a result six containers of one liter each were glowing (Fig.23).

Fig.18: First Pyrocystis Fusiformis culture

Fig.20: Contaminated Pyrocystis Fusiformis cells

Fig.19: Light timer Fig.21: Healthy Pyrocystis Fusiformis cells

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Different kinds of geometries were tested to check dinoflagellates´ glowing behavior. The first one was a pixel-pocket structure that would allow each pixel to glow independently if it is excited by movement (Fig.24). It was made injecting 10ml of Pyroscystis Fusiformis in each pixel-pocket. It worked pretty well and each pixel could be excited to glow.

Fig.22: Population of Pyrocystis Fusiformis glowing

Fig.23: Third Pyrocystis Fusiformis culture Fig.24: Pixel-pocket structure 9

Based on this prototype there is the possibility to think about a screen or billboard that emits light and at the same time, displays information like images or text (Fig.25). It can be done controlling the movement of each pixel individually to be on or off. The pixels could be excited by a mechanical device or by sound waves. This sort of geometry could be used for façades or for commercial purposes. The advantage of this system regarding billboards made of static glowing bacteria like Vibrio Fischeri is that the information displayed can change according to the movement of the pixels. However there is the need to think about a mechanism to move the pixels so that they glow. Fig.26: Surface-like geometry for Pyrocystis Fusiformis

The third trial explored the possibility of growing Pyroscystis Fusiformis in a denser element than water. To do so, hydrogel was fabricated mixing water and powder in different proportions. The containers used for that experiment were three petri dishes and a tube (Fig.27). All of them were put in the incubator under a light source controlled by a timer (Fig.28). Having a gel-like glowing material, could be useful to fabricate pieces of glowing furniture that could adapt to the shape of the body when laying or sitting on them. However none of the prototypes worked out and no glowing effects were observed.

Fig.25: Pixel based screen using Pyrocystis Fusiformis

A second kind of geometry was filled in with Pyrocystis Fusiformis to test its behavior. The volume injected in this geometry was also 10ml but in this case, instead of a cubic volume, it was more superficial (Fig.26). The idea was to design a transparent surface to divide spaces able to glow when excited by movement. However out of three prototypes placed in the incubator (Fig.28) only one was glowing and not very much. Apparently dinoflagellates glow better in 3D containers. Fig.27: Containers with Pyrocystis Fusiformis in hydrogel 10

Fig.30: Light system for highways using Pyrocystis Fusiformis

We can conclude that populations of dinoflagellates glow better if their density is higher. Moreover they need certain amount of 3D space to glow properly since they only perform faint glowing inside superficial containers. A volume containing 10ml is enough for them to glow but the glowing starts to be quite visible using at least 200ml containers. Moreover the bigger the container, the longer they last. They do not survive in hydrogel, at least not in the one that was fabricated for this experiment.

Designing a prototype that emits light without Fig.28: 1l containers, hydrogel containers, 200 ml bags and consuming electricity taking advantage of 10ml screen-like geometries filled with Pyrocystis Fusiformis Pyrocystis Fusiformis glowing properties, means The fourth prototype developed was a 200ml bag the recognition of a living form of wealth translated filled with Pyroscystis Fusiformis (Fig.29). Its into an architectural outcome. Besides controlling glowing properties did not differ much from the the density of a population of dinoflagellates helps ones the one liter containers show. It achieved the to regulate its quorum sensing mechanisms. That same brightness as one of these original means manipulating the kind of swarm intelligence containers. It was also placed inside the incubator a population of dinoflagellates shows to improve its (Fig.28). The fourth prototype geometry can be glowing properties; and therefore the architectural used for highways signal lights. The dinoflagellates outcome. Furthermore there is a way to achieve a inside could be excited either by the movement of design that is not only consuming energy to glow cars activating a device that is communicated with but transforming the one that uses into something the lighting source, or just by the wind (Fig.30). else besides light.

This kind of prototype would be a bar field where each bar contains salty water with populations of Pyrocystis Fusiformis (Fig.31-33). This bar field would be moved by the wind. This movement would excite the bacteria inside the bars and they would glow. At the base of each bar, a device could be placed to use the kinetic energy produced by the movement of the bars for different purposes depending on where the prototype is placed.

Fig.29: 200ml bags filled with Pyrocystis Fusiformis 11

It could be built in public plazas, rooftops or building façades (Fig.31). In the two first cases the kinetic energy could be used to warm the ground, for pavement lighting, or to set up an electricity network with plugs that could be used by people (Fig.34). In the third case it could be transformed into electricity to supply the building.

Fig.32: Glowing Bar Field plaza prototype

Fig.31: Glowing Bar Field prototype variants

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Fig.33: Glowing Bar Field plaza prototype

Fig.34: Glowing Bar Field plaza prototype detail 13