a series of experiments by AnneMarie Maes and Núria Conde Pueyo
This project is about research and finding solutions. We go all the way for the experiment!
Our manufacturing with nature is planned over several steps (2017-2018).
- Phase#01 starts at experimenting with raw materials (bacteria, growth medium) to create thin membranes and surfaces grown by a symbiotic community of bacteries and yeast cells (scoby skins).
- Phase#02a: In a second phase, this leather-like celluloseskin will be augmented with living technology. We will grow a biofilm with useful microbes for environmental sensing as well as with antibiotical properties (e.g. against Varroa - check info BlackBetty). We will engineer cells as a biosensors, and implement them on the leathery cellulose skin to sense environmental pollution. We will work with the skin cells as programmable material. A layer of bacterial biofilm will be added on top of the leathery skin. Specific bacteria can act as biosensors, they will change color when the bacteries sense a specific pollutant. The double-layered skin will behave as a bio-digital living system, the living matter becomes the monitoring technology.
- Phase#02b: In a parallel timeline to the previous experiments, we will 3D print simulation of skins with chitin (extracted from exoskeleton materials).
- Phase#03: Thirdly, we will grow a responsive, sensorial skin for Guerilla Beehives, via cell differentiation and tissue culture. Living exoskeleton cells from Apis mellifera are removed from the original context of the bee-body (or larva), to be used as raw material for growing an artificial exoskeleton/skin on a 3D-printed scaffold of bacterial cellulose. This will take place in the bioreactor.
RECENT POSTS ARE ON TOP OF THE PAGE
- rotative growth culture
- chitosan tests
- nano fiber (crystal) bacterial cellulose
- bacteria cultures from the beehive
- contamination problems
Núria's recipe for growing nanoparticle cellulose. The idea is to turn the kombucha a liquid solution of cellulose, by blending, grinding, shearing and applying an acid sulfurig treatment. Then mix the result with the chitosan. Núria already tried to apply some chitosan on top of the kombucha skin. They get attached but the chitosan makes the kombucha bend.
For 2 liters of water:
-8 gr Triptone
-8 gr Yeast extract
-10 gr Amonium Sulfate
-6 gr Potasium phosphate dibasic
-0.1 gr Magnesium Sulfate
-2.3 gr Acid Citric
Núria “the bacteria that I recovered from the samples are all the same and have no color at all…
That's why I started to look were we can find more bacteria or buy them. We can try a last hunting strategy from leaves (see LeaveCulture.pgn) or we can buy the one from the paper of the Solar Panel Microbiome at the German repository (see DSM-DeinococcusHopiensis.png and Novosphingobium Tardaugens.png). In principle, both will be pink/red.
Or… we can buy one at the Spanish Cataloge, also red one that is similar to the ones of the Solar Panel Paper and also have a lot of chances to be able to survive in the dry kombucha (see: CECT-DineoccocusRadiurans).
I also found a very interesting paper about how the LactoBacillus can be beneficial for the bees, since some species are able to avoid some other bacteria that kill the larvae. Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae.
Blue E-coli experiment and Red E-coli experiment: Neither the e.coli blue or red showed any apparent growth so, I went to the microscope and made some preparations scrubbing the surface of the kombucha skins. Well, no signal of the red or blue fluorescence or any signal of bacterial growth. As control to be sure that I was shooting the pictures in the correct magnification and light conditions, I checked the LB petri dish cultured e colis. And they are shining properly!.
We must conclude that the Lactobacillus is the spicy that lives happy in the acidic skin. We can try to clean the kombucha with some basic liquid to make sure that the pH is neutral when we culture the bacteria. Or we can keep working with Lactobacillus with X-gal.
conclusion for this experiment:
AM: As far as I understand from your explanation, only the Lactobacillus on a wet scoby skin gives a ‘good’ (visible) result. Does this mean we have to look out for another kind of bacteria to work with? Can we continue the experiment, or is this the end????
NCP: Yes, I've concluded that only Lactobacillus was able to survive on the kombucha-skin. The microscopic images have demonstrated that. Luckily the Lactobacillus not only grows, but also can change the colour clearly.
There are a lot of bacteria that can dye and probably be alive in kombucha skins (even a bit dried), the problem to perform those experiments is to obtain an alive and pure sample. But if we have a good candidate, then definitively I will try to obtain it and re-make the experiments. But for now, we know that Lactobacillus can be our backup plan.
I was doing a research to find a suitable species to grow in the outside, in not too much humid conditions.
Other taxa (families), different from Lactobacillus, that are resistant to those environmental conditions are: Deinococcus, Sphingomonas, Novosphingobium, and Hymenobacter.
I know some from the scientist of Valencia that sequenced the microbioma of solar panels: http://www.microbe.net/2015/10/29/like-a-cat-on-a-hot-tin-roof-microbiome-of-solar-panels/
Deinocuccusis red and can stand really high UV radiation. It has been genetically modified to clean radioactive pools. https://en.wikipedia.org/wiki/Deinococcus_radiodurans
Sphingomonas and NovosphingobiumUnderlined Text are yellow. They are also capable of live in soils and poor enviroments and can resist pesticides and even “break” some of them. https://microbewiki.kenyon.edu/index.php/Sphingomonas_sp.,_agents. They look very nice in the petridishes: http://www.microbeworld.org/component/jlibrary/?view=article&id=14215
Another crazy idea would be use Gloeocapsa magma, but they need Calcium carbonate to survive. https://en.wikipedia.org/wiki/Gloeocapsa_magma.
Looking around I also found a project named Holobiont urbanism. The data is presented in an artistic way, but I think they give accurate information about the microbiome around bees. http://microbiome.nyc/taxonomy.html. I think they found some Pseudomonas, and Pseudomona can do biofilms, produce cellulose and are also producers of beautiful colours (NOT use P. areuginosa, which is very pathogenic).
to do for the Cellulose skin/bacteria experiment:
- Make a new experiment with the lactobacillus or change to other bacteria (get them in a lab or buy them).
- Or we can follow the protocol of this paper (environmental bacteria) and obtain bacteria from the “environment”. Therefore we can just put some dry kombucha cellulose skin outside at Valldaura and afterwards cultivate the samples.
- We can liquify the cellulose skin solution and make it more clear (see transparent mix/film grown by AM) and mix it with chitosan 6% or 9% (still working in which protocol will be successful)
05.01.2017: experiment #01: growing nanofiber cellulose in heated fishtank
I compare the growth of BC in 3 different mediums: 1.BC mixed to pulp and growing in sterilized sugarwater with Acetic citric (started 5/1/17); 2.BC as mother, growing in sterilized sugarwater with Acetic citric and a bit of herb tea (started 17/1/17); 3.BC as mother growing in sterilized sugarwater with red vinegar (started 17/1/17).
The first culture is in an unheated tank (but next to the electric radiator), the last 2 are heated with a thermistor for fishtanks (Furious Fish).
Result of this experiment: with the 'nanofiber' experiment the BC was growing slowly into a film, but the film never made it into a solid skin. The water is slowly evaporating (as the container is next to the heating) but the BC stays in loose pulp. I will let it dry out even more (today = 26/1, it is growing yet for 20 days) and check the result once back in BCN (end of February).
The other 2 skins are slowly growing - I hope that they will form a film at the end!
clear and liquid) from the first experiment I did at home with mixed kombucha skin and citric acid + sugar + water. The skin that was growing was not really solid. It stayed rather mashed pulp but it looked like a film as all the water evaporated. I left it 10 days ago, and I am very curious to see what happened to it when I come back in 2 weeks. Maybe it became a film after all, or maybe it is still thick pulp. Aniways, it is completely transparent …
20.02.2017: results of experiment #01: growing nanofiber cellulose in heated fishtank
The 'nano-fiber experiment' never made it into a film. After 2 months, the result is still clear, loose and very sticky. It looks like the xylinum bacteria and the yeast cells never consumed the sugar. Maybe I killed them by mashing them up to pulp with the mixer?
The transparent cellulose: OK, this was growing, but it did not grow into a skin, it are still all loose particles. I wonder if I can start new growth containers with it, adding for now a bit of tea, so that they finally will grow into a skin. Now the substance is beautifully transparent (see photos), but it is more sticky than glue and has rather the substance of pulp instead of a solid sheet. Also, it is extremely sticky, it seems that the bacteria did not at all digest the sugar …
I think the main reason for not growing into a solid skin, is the low Temperature in the growing room and, secondary, the change in recipe, as I was using acetic acid instead of vinegar or instead of adding a bit of kombucha growth starter medium, and also the absence of tea.
01.03.2017: continuarion of experiment #01: I decided to stop this failed experiment, and to modify it with the regular and known ingredients. I added some regular tea, a kombucha starter pilz and some vinegar to the remains of the former experiment. They are still a sugary, sticky, tranparent paste … I hope a new skin will now grow out of it.
21.01.2017: experiment #02: growing nanofiber Bacterial Cellulose around wooden object in stirrer
This experiment is set up to study if BC will grow around an object that is submerged, instead of forming a film on top of the growth medium (dfr. stoof of Jannis Huelsen). I prepare 1,5 sterilized sugarwater (100gr sugar for 1 l water), I add 6 ml of Acetic acid (Ph=±3) and I mix the BC mother (±50gr) to pulp and add it to the growth medium. I place the beaker on a magnetic stirrer and start the experiment. After 2 hours fibers of BC start fixing themselves to the wooden object. The first 2 days I stir continuously. Later I stop by moments. After 3 days a film is yet forming on top.
The stirrer is running for 1 week (with a stop now and then). The current brings the strains of fibers together around the wooden object. There was an invasion of ants in the beaker, and now all dead ants are stirred together with BC strains. Tomorrow (27/1) I'll switch the stirrer off and I'll leave the experiment growing till end of February when I come back to BCN.
20.02.2017: results of experiment #02: growing nanofiber Bacterial Cellulose around wooden object in stirrer
The cicular growing around an object in the stirrer: as long as I was in Barcelona, I kept the stirrer rotating and I controlled the room temperature. When I had to leave to Brussels, the room was not heated anymore and I had to switch off the stirrer - this made the growing stop at that point. So, again, I think that the biggest problem was that the ambient temperature was too low. I made this conclusion because there was no continuous growth at the surface, after the stirring was stopped (which, in a normal situation, should have taken place.)
22.01.2017: experiment #03: casting with chitosan
I will do some casting tests with different chitosan concentrations
⇒ in a small mold in which we can paint layers of the the liquid mix with a brush
⇒ with a small nozzle syringue to simulate 3D printing
After the problems with the viscosity of the Chitosan/Acetic acid preparation, the hope to be able to work with the material became nearly unexistent. I did some tests with the 3% and with the 6% concentrations aniways, and surprisingly they are 'workable' with a seringue of 10ml (nozzle 2mm). The 3% solution is rather liquid and spreads open after designing a line with the seringue, the 6% if more difficult to handle (you need to put much more power to suck the material into the seringue) but once it is in, you can push it out and it even spreads around (but not so much as the 3% liquid).
Both solutions dry after 1 night (the thinner the sooner) and they loose ± 80% of their thickness as water evaporates.
It IS possible to work with Chitosan! But you need the correct working conditions. 3% Chitosan is very liquid and leaves an ultra thin film. 6% Chitosan needs to be heated to work with. But once you spread it with the syringue, it starts flooding out. So, for a real neat print, we need the 9% or 12% solution - but this is only workable with an electrical extruder. It does not work with the syringue anymore.
The negative design in the plasticine, which I used for the casting Chitosan test, was not ideal as 1. it was too subtle, and 2. the plasticine mixes with the Chitosan - so there is no neat cast.
Also: when drying, the Chitosan shrinks in the length which makes the casted form turn around its axis.
To do the experiments with the bacteria, we worked at the lab of the PRBB.
We have following bacteria: 1. Lactobacillus plantarum, 2. a regular yohurt lactobacillus, 3. yeast for bread (fungi), 4. modified e-coli Cyan and Red. In this experiment we will try to find out if the WET or DRY (both, or one of both, or none of both) Bacterial Cellulose skin is a good growth medium for one of these bacteria. If so, than we can let them grow into a film that covers the cellulose skin. Purpose is to get advanced environmental warning from the bacteria changing color if they sense a specific threat.
We will do different experiments:
- we'll monitor the growth of the bacteria with food / and without food
- we'll monitor the growth of the bacteria on wet BCskin and on dry BC skin.
Every bacteria has its own specific food:
- MRS (broth for the lactobacillus)
- YPD (food for the yeast)
- LB (food for the e-coli).
We use double petri dishes to make monitoring easy; first we prepare the food/non-food petridishes.
- we cut little pieces of BCellulose skin and put them in the half petrisdishes: 1 petridish for the 2 lactobacillus specimen; 1 petrisdish for the yeast; 1 petridish for the RED e-coli; 1 petridish for the CYAN e-coli.
- first we paint the skin with x-gal: if the bacteria are happy in their medium, they will change color with this compound.
- second we put the respective food (200 microliter for each) on the skin (1 side with food, 1 side without food)
- thirdly we add the respective bacteria to their food/non food.
This experiment is done on WET skin. Later we will repeat this experiment also on DRY BC skin (once the skin is dry).
We put all prepared petri dishes in the 37°C-room and we'll wait for the result.
Núria Condé Pueyo, on the results of the experiment dd. 18/01/2017: I made a new solution of chitosan at 9% and 12% using the stirrer machine at 70ºC and 800-900 rpm. I let the mixing go for 5 hours. Then I poured it inside some small petri dishes. The Chitosan alone or mixed with some glycerol (half and a half) or with a bit of Sodium Alginate 4%. I also put some on top of a wet Kombucha piece. I let these samples dry at room temperature. I took some pictures the day after but it was not dry at all. Finally, they took three days to dry. The chitosan pieces have bended, the 12% ones at more extend than the 9% ones, including the kombucha skin. The samples that contain glicerol stick to the bottom of the petri dish and are not bended but they are very thin. The same happens with the “stamps” that we painted with 3% chitosan at Hangar (on 18/1/17). You can see in the pictures that it is dry, but all are too fragile to be removed without breaking.
I propose to check different proportions glycerol-chitosan and also to remake the tests with alginate, as this time I didn't get it well dissolved, because 4% is a too high concentration and it needs a protocol to be dissolved similar to the one of chitosan. I will do it in parallel next time.
Chitosan experiment: (see also experiment 'casting with chitosan' at the bottom of this page)
We can conclude that 3 and 6% are a bit too liquid to work with. And that for 9 and 12% we must find a way to keep the solution warm (around 65Cº). Next steps require more expertise to prepare the solution homogeneously and to prepare the 3D printer or syringue so that they are able to extrude the hot solution.
- Mix the chitosan in a proper way with a 4% alginate solution to make the chitosan more flexible and make it dry slower.
- Spread chitosan+glycerolor alginate on a cellulose skin and check if the result is water proof.
- Make a series of combinations of 9% chitosan + different acetic concentrations/ glycerol percentage / alginate percentage, and check which result is better for our purposes.
Chitine/Chitosan: state of things
The Chitosan (90% deacetilised Chitine) is ordered at the supplier in Barcelona but it did not arrive yet.
Printing Chitosan with the seringue fixed on the DIY bioprinter will (for now) not be possible, as the seringue is too big for the printer. Alternatives can be created, ex. the tool to print ceramics.
We decide not to focus (for the moment) on printing the Chitosan with the 3D bioprinter, but instead to cast the Chitosan in a 3D printed mold and to freeze-dry it. Mold preferably in latex for easy detachment of the object.
We can cast the Chitosan in different layers. The fastness of the drying will give the final object different physical properties.
As a positive form, we can use the formerly 3D prints (fablab) of the scaled Guerilla Beehive, as a negative form we can make a latex cast of it.
Microbial cellulose skin: state of things
We decide to grow a more pure cellulose skin than the ones we have for now.
Núria will try to grow crystal-clear cellulose: this is a pure cellulose from which the fibers are reduced to a nanoscale. This makes the cellulose stronger for use. The shape of the molecules is as a prism, and the bonding is organised as fractals in a snowflake. This makes the resulting skin transparent and after polishing clear as crystal glass. (nanocellulosecrystals
We will also do a growing test of microbial cellulose in a beaker in which the microbial cellulose-pulp is continuously stirred in the liquid, as such it will attach on the (wooden?) object submerged in the beaker (cfr. stool).
I also do a (more simple) experiment at home:
I try a new recipe without green tea and with Acetic acid instead of vinegar. As a starter, I use a small piece of scoby that I thoroughly mix to pulp before adding it to the sterilised sugar water. (Ratio's: 1500ml water, 200gr critalised sugar, 2 teaspoons of Acetic acid (4gr Ac. Citric/2liters water), superfine pulp of formerly grown microbial cellulose). Start of the experiment: Thursday January 5th.
Check: only after 1 week (thursday 12/1) a very shallow film is starting to grow. The mass still looks transparent.
Growing a biofilm inside/ouside the beehive, on a layer of microbial cellulose. The microbial cellulose support serves as growth medium for the bacterial colonies. Outside: we will focus on bacteria that can sense the environmental threats - they change color under specific circumstances. Inside: we can try to develop a colony of bacteria that reflects the healthy microbiome of the bees; these bacteria should give the bees a 'good feeling' in their home, a kind of 'wellness', and make them at once more resistant to natural ennemies as varroa mites or other bee-pathologies. After development of the bacterial colonies, the cellulose skin acts as a crust that crumbles under the ever developing new layers of bacteria.
pictures, from top left, clockwise:
01. bio 3D printer with syringe for agar or chitosan
02. ToDo list
03. Effects of different drying processes on the material propoerties of Bacterial Cellulose
04. experiment to make pure nano-fiber BC got infected with mold
05. maybe because the PH was too low (should be around 4, and is here ± 2)
06. experiment with stirrer and magnet, with pieces of cellulose in beaker and 3D-object in suspension in medium
10. tests with bacteria: e-coli and other, reacting with different colors
I also have more kombucha skin growing over the little plastic cube, it is still too thin to say but, I am afraid that we need to change the way we are agitating the culture. I think is better to let the culture quiet and then make the piece turn over itself in the surface of the liquid. I will try to make a “rotation” machine for the kombucha 3D growth, to see that works better.
to do for the skin-growing-around- object experiment:
Try to make a rotating system for the object to grow cellulose skin around it.
The Chitine Experiment
⇒ model of beeswax foundation to grow in Chitine and in Bacterial Cellulose
The Living Cells Experiment
Presentation in the bioreactor