Robotic fabrication (KUKA Robot) of mycelium composites


#1

Hey! I am an architecture student, currently doing research at VUB on the robotic fabrication (using a KUKA Robot) of functionally graded biomaterials. Remarks or hints are always welcome, since I don’t have an extensive knowledge of Biology.


PROTOCOL PREPARATION OF THE FIBRE

  1. A desired amount of gelling agent is mixed with 100 ml distilled water.
  2. The mass of the gel to fill a jar is measured.
  3. The mass of the fibre to fill a jar is measured.
  4. A humidity of 80% (more or less) is needed for the living organism. Therefore, distilled water needs to be added to the fibres.
  5. The fibres, distilled water and the gelling agent are mixed in a bowl.
  6. Everything (filter bags) is autoclaved for 20 minutes at 121°C.
  7. The mixture needs to rest for 12 - 24 hours, in order to absorb the water and the viscous matter.

PROTOCOL INCUBATION

  1. The jar is filled with the desired amount of viscous matter and Trametes Versicolor (10% of the total mass).
  2. Everything is mixed by a spoon.
  3. The mixture is compressed by a spoon.
  4. The samples are placed in an incubator at 26°C.

LOGBOOK (15 - 22 Nov 2018)

During my first tests, three different % of fibres are compared (80% - 60% - 40%). The fibres were prepared on the 15th. In this case, 200% distilled water was added to the mass of the fibre (see protocol preparation of the fibre), because of the already small chopped fibres and the use of a viscous matter.

WC: chopped wood fibers
Psy: Psyllium Husk Powder
Tv: Trametes Versicolor

Test 1 (inoculated and tested on 19 Nov 2018): Print directly the viscous matter inoculated with mycelium

  • 80%WC_3%Psy_Tv
    During the preparation of the fibres, it became clear that this would be a too dry mixture. This was confirmed by trying to print it with a piping bag, only water was squeezed out.

  • 60%WC_3%Psy_Tv
    The created matter was between dry and gelly (more on the dry side), the fibres were able to stick to eachother. Similary to the 80%, only water was squeezed out.

  • 40%WC_3%Psy_Tv
    This mixture was gelly. Similar to the tests before, water was squeezed out. On the other hand, small pieces were able to be printed by squeezing the piping bag really hard. This mixture can maybe be optimised, since it is still really difficult to print.
    The result of printing, after 8 days (27 November 2018), is shown in the following pictures. It can be concluded that it is growing well. The back side seems still to be moist.

Test 2 (inoculated on 16 Nov 18, tested on 22 Nov 18): Pre-growth samples ( ± 6 days)

After 6 days, the growth process can be examined. It seems that both the 80% and 60% are able to grow well. On the other hand, the 40% is only colonized on the top side. It probably can’t go through the fibres because of the too much viscous matter.

80%WC_3%Psy_Tv

60%WC_3%Psy_Tv

40%WC_3%Psy_Tv

To test the printability, the matter was again mixed by a spoon and placed in a piping bag.

  • 80%WC_3%Psy_Tv
    This mixture was very dry, there came nothing out of the piping bag by squeezing it (even no water). So this mixture can’t definitely be used for printing.

  • 60%WC_3%Psy_Tv
    This mixture was less dry than the 80%, but still too dry to print. In contrast to the 80%, water was squeezed out.

  • 40%WC_3%Psy_Tv
    After being mixed, the matter was still gelly which is good. Unfortunately, the same results as “printing directly” became visible. Water was squeezed out and only small pieces were able to be print (still difficult).

CONCLUSION AND QUESTIONS

Both the 80% and 60% mixtures were thrown away, since they were too dry to be printed. I decided to keep the 40% to see how the growing process continues. Before placing them back in the incubator, the matter was mixed and compressed again by a spoon.

After some extra days of growing (result on 27th), the mixed 40% mixture seemed to have more or less the same results as before. The top side seemed again to be local colonized. On the other hand, it is able to grow (a little bit) through the matter.

40%WC_3%Psy_Tv, mixed again by a spoon


LOGBOOK (21 - 27 Nov 2018)

Since a lot of water was squeezed out during the first tests, only 100% of distilled water was added to the fibres. Only the 60% and 40% of chopped wood fibres were remade (on the 21th), since the 80% was too dry.

During the preparation of the fibres, the structure of the 60% seemed the same as the previous 80%. So it can be assumed that this mixture won’t work. On the other hand, the 40% seemed to have a good composition (viscous and not aqueous).

Test 1 (inoculated and tested on 22 Nov 2018): Print directly the viscous matter inoculated with mycelium

  • 60%WC_3%Psy_Tv
    As assumed, the mixture was very dry, there came nothing out of the piping bag by squeezing it (even no water).

  • 40%WC_3%Psy_Tv
    The results of this matter were good. By using the piping bag, no water was squeezed out and the printing itself went easier. But, the structure still needs to be optimised, since only small pieces were able to be print (no continuous piece of material, pieces were falling apart).

The result, after 5 days (27 Nov 2018):

Test 2 (inoculated on 22 Nov 18, tested on 27 Nov 18): Pre-growth samples ( ± 5 days)

  • 60%WC_3%Psy_Tv
    The mixture seemed to grow well. As can be seen on the picture, the matter is very dry when mixed again by a spoon, nothing came out of the piping bag (even no water). It was even not possible to push the material till the end of the piping bag. So this matter can’t be used to print.

  • 40%WC_3%Psy_Tv
    Compared to the previous mixture of 40% fibres (200% distilled water), this one seemed to grow better. On the other hand, the matter wasn’t viscous anymore, only water was squeezed out when using the piping bag. It can be concluded that this composition can be used to print directly (still needs optimisations) but not after it is being pre-growth.

CONCLUSION AND QUESTIONS

The 60% mixture can’t be used to print (directly or pre-grow), since it is too dry. The 40% can’t be used to print after it is being pre-growth. On the other hand, the mixture has good results to print directly. Optimisations are still needed in order to print in a fluent way.


CHANGING THE METHOD

From now on, I decided to stop testing the printability of the pre-growth samples. Nevertheless, I am still making the pre-grow samples in order to analyse the growth. After all the previous tests, it became clear that the best method to fabricate a mycelium composite is to print it directly (a substrate which is inoculated with mycelium).

In the previous tests, I already made a few substrates which looked to have a good viscosity. Since I wasn’t able to print it fluently with the piping bag, I was suggested to test the already made substrates with different diameters of the bag. In that way, I could conclude if the fibres were too big what would cause the difficulty to print. I decided to not do these tests since the piping bag already has a diameter of ± 5mm, and the printhead of the KUKA robot is limited to this size.

Therefore, I decided to make substrates with different percentages of a certain gelling agent or another kind of gelling agent in order to see the consequences on the printability.


LOGBOOK (6 - 13 Dec 2018)

In the following tests, 40% of chopped wood fibres, 100% of distilled water and different percentages of Psy are used (5% - 10% - 20%).

During the preparation of the fibres (on the 6th), the matter made with 5%Psy looked to have a good composition (more viscous than the 3% and not aqueous). On the other hand, the substrates made with 10% and 20%Psy were not able to be mixed homogeneously since the gelling agent was one cohesive matter.

Pre-grow samples (inoculated on 10 Dec 2018): to analyse the growth

40%WC_5%Psy_Tv

40%WC_10%Psy_Tv

40%WC_20%Psy_Tv

Test (inoculated and tested on 13 Dec 2018): Print directly the viscous matter inoculated with mycelium

  • 40%WC_5%Psy_Tv
    The substrate was still viscous after 7 days and was able to mix well with the mycelium. Compared to the previous test with only 3%Psy, the matter was able to stick to each other but the printing itself was still difficult.

  • 40%WC_10%Psy_Tv
    As mentioned above, it was not possible to have a homogeneous substrate (parts were gel, other ones were fibres). On the other hand, the substrate was able to be mixed with mycelium quite easily. During the printing, the matter was able to stick to each other, but it was still not possible to print it in a fluent way.

  • 40%WC_20%Psy_Tv
    I didn’t test this substrate, since it seemed a dry mixture that contained out of clumps of gel and clumps of fibres. So it can be assumed that there will come nothing out of the piping bag.

LOGBOOK (12 - 13 Dec 2018)

I decided to test another kind of gelling agent in the following tests, Xanthane Gum and Locust Bean Gum. Since I wasn’t familiar with this kind of gelling agent, I made two substrates with the same percentages (3%Xan and 6%Locust) of gelling agent but with two different percentages of fibers, namely 40% and 60%. Again 100% of distilled water is added.

I also remade the 40% chopped wood fibres with 5%Psy in order to compare the result with the previous test. In the previous test the substrate was 7 days old when I tested the printability, and in this test the substrate is just 1 day old.

During the preparation of the substrates (on the 12th), 3%Xan and 6%Locust created a very viscous matter, but it seemed to be 1 cohesive clump. So it was logic that trying to mix it with 60%WC was difficult (result: dry mixture), and with 40%WC it went easier.

WC: chopped wood fibers
Psy: Psyllium Husk Powder
Xan: Xanthane Gum
Locust: Locust Bean Gum
Tv: Trametes Versicolor

Pre-grow samples (inoculated on 13 Dec 2018): to analyse the growth

60%WC_3%Xan6%Locust_Tv

40%WC_3%Xan6%Locust_Tv

40%WC_5%Psy_Tv

Test (inoculated and tested on 13 Dec 2018): Print directly the viscous matter inoculated with mycelium

  • 60%WC_3%Xan6%Locust_Tv
    The substrate was able to be mixed well with the mycelium, which was surprising due to the clump of gelling agent. On the other hand, the structure of the mixture seemed dry, therefore I didn’t test it. It can be assumed that there will come nothing of the piping bag.

  • 40%WC_3%Xan6%Locust_Tv
    After being mixed with mycelium, the substrate is very viscous, compared to the 60%. But as the previous tests, the matter is able to stick to each other but still difficult to push the material out of the piping bag.

  • 40%WC_5%Psy_Tv
    The mixture has the same result as the 7 days old substrate during the printing itself (still viscous). Compared to the 40%WC_3%Xan6%Locust_Tv, the structure looked more viscous, so pushing the material out of the piping bag went easier, but still not good to print.

CONCLUSION AND QUESTIONS

It can be concluded that the best percentage of chopped wood fibres is 40, since the 60% gave bad results. The both gelling agent, Psyllium Husk Powder and Xanthane Gum in combination with Locust Bean Gum, had good results concerning the viscosity.

I have noticed that testing the printability using a piping bag isn’t the best option. Therefore, I am going to remake the substrates (5%Psy, 10%Psy and 3%Xan6%Locust) and test them using a syringe for nutrient measurement.


LOGBOOK (19 Feb 2019)

By rechecking my calculations, it became clear that I wasn’t using the correct densities of the materials (chopped wood fibres and psyllium husk powder). Before continuing the research, I decided to repeat some previous tests that had the best results (40%WC_3%Psy, 40%WC_5%Psy and 40%WC_10%Psy).

In order to progress faster, I tested directly the substrate (the fibres and the gel), without being autoclaved (no mycelium added), with a syringe.


40%WC_3%Psy


40%WC_5%Psy


40%WC_10%Psy

I had hoped to have more or less the same results as before, but it was clear that these substrates aren’t suitable to print. No matter was able to be pushed out of the syringe or only water was squeezed out.


CHANGING THE METHOD

I changed the calculation method into:
% chopped wood fibres (WC) + % gelling agent (Psy) + % H2O + % mycelium = ± 100 %

The percentage of mycelium will always be 10% of the sum of the masses of chopped wood fibres, gelling agent and distilled water. In order to achieve the best viscous substrate, the percentage of distilled water will be altered in function of the percentage of fibres.

In order to progress faster, the substrate will be tested directly on the flow rate without being autoclaved (no mycelium added). Once a good mixture achieved, the next step will be to test if it is as easy as before to print the mixture with a syringe after being autoclaved. Simultaneously, the ability to grow mycelium on the substrate will be tested.

NEW PROTOCOL PREPARATION SUBTRATE

  1. Measure the needed mass of fibre.

  2. Measure the needed mass of gelling agent.

  3. Mix the fibres with the gelling agent.

  4. Add the needed amount of distilled water and mix everything.


LOGBOOK (26 Feb 2019): % H2O = 2 x % WC

The substrates (see new protocol substrate) are made and tested directly on flow rate using a syringe (syringe of 150 ml).


LOGBOOK (27 Feb 2019): % H2O = 2 x % WC & % Psy = % Corn starch

For this test, I decided to add an element in order to improve the substrates. Once again, the substrates are made and tested directly on flow rate using a syringe (syringe of 150 ml).

The only mixtures that were able to be tested are:

In general, adding corn starch isn’t the best way to improve the mixtures since it results in a very cohesive matter. Therefore, I will try to optimize during the next tests by “playing” with the amount of distilled water.


LOGBOOK (3 Mar 2019): % H2O = % WC

The substrates are made and tested directly on flow rate using a syringe (syringe of 150 ml).

It can be concluded that when the percentage of distilled water is equal to the percentage of the fibres, the mixtures are not suitable to be printed. The substrates are too dry, since nothing comes out of the syringe. For the next test, the percentage of water has to increase.


LOGBOOK (3 Mar 2019): % H2O = 1,5 x % WC

The substrates are made and tested directly on flow rate using a syringe (syringe of 60 ml).


LOGBOOK (5 Mar 2019)

For this test, I decided to remake the best substrates and autoclave them in order to check if they have the same viscosity as before.

Selection of the substrates:
% H2O = 2 x % WC: 15%Psy, 12%Psy & 10%Psy
% H2O = 1,5 x % WC: 12%Psy & 10%Psy

% H2O = 2 x % WC

% H2O = 1,5 x % WC

After this test, it can be concluded that the substrates have less viscosity after the autoclave. It is even difficult to break down the gel into pieces, since it is one cohesive matter. Therefore, the best way to make a viscous substrate will probably be by autoclaving a mix of dry fibres and dry gelling agent. Water will be autoclaved as well, but separate. Afterwards, the gel will be made in a sterile flow. It can be assumed that this will also be the best way to incubate mycelium, in order to achieve a homogeneous mix.


LOGBOOK (8 Mar 2019): Growth samples

The aim of this test is to search for the best way to incubate mycelium. Since mycelium seeds are too big which results in blocking the printhead of the KUKA Robot.

Method 1: blending mycelium seeds with distilled water
Steps:

  1. Measure the needed mass of fibre.
  2. Measure the needed mass of gelling agent.
  3. Mix the fibres with the gelling agent.
  4. Autoclave the mix.
  5. Autoclave distilled water.
  6. Blend the needed amount of mycelium seeds with the needed amount of distilled water.
  7. Make the gel in a sterile flow by mixing everything (mix of fibres and gelling agent with the mix of seeds and water)

Test:

The lack of growing is due to problems of the used strain of Trametes Versicolor. In general, not all the seeds were able to be blended, since the blades of the blender are located too high.

Method 2: blending grown mycelium with distilled water
Steps:

  1. Measure the needed mass of fibre.
  2. Measure the needed mass of gelling agent.
  3. Mix the fibres with the gelling agent.
  4. Autoclave the mix.
  5. Autoclave distilled water.
  6. Blend the needed amount of mycelium with the needed amount of distilled water.
  7. Make the gel in a sterile flow by mixing everything (mix of fibres and gelling agent with the mix of mycelium and water)

Test:

In this test, Granoderma is used instead of Trametes Versicolor. This method needs some optimization as well. Blending grown mycelium with distilled water results in a paste. This can maybe influence the viscosity since the gelling agent needs water in order to become a gel.

Both methods need optimization and should be remade, when having a good strain of Trametes Versicolor, in order to evaluate the best grow method.


LOGBOOK (12 Mar 2019): 3D printer

The tested substrates:
% H2O = 2 x % WC: 15%Psy, 12%Psy & 10%Psy
% H2O = 1,5 x % WC: 12%Psy & 10%Psy

The substrates were made at 11 am (not autoclaved and no mycelium added) and tested at 2 pm. All the substrates had less viscosity so none of them was able to be printed using the 3D printer. Even not the 10%Psy with % H20 = 2 x % WC.

Until now, the substrates were made and tested directly. For the next test I will take into account the time between making and testing the substrates. The percentage of psyllium needs to decrease and the percentage of water needs to increase.

The following substrates will be made during the next test:
% H2O = 2 x % WC: 7%Psy, 6%Psy & 5%Psy
% H2O = 2,5 x % WC: 7%Psy, 6%Psy & 5%Psy


Creative Commons License

Master Thesis by Ichelle Nieberding, Under supervision of Elise Elsacker, Prof. Lars De Laet and Prof. Eveline Peeters / Vrije Universiteit Brussel / Department of Architectural Engineering and Bioengineering Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.


3D printing with Mycelium / Algae
#2

Nice! In order to visualize better what you’re doing: what does such a “piping bag” look like? In my head you’re working with bagpipes :smiley:


#3

This is what I want to say with “piping bag”! :smile:
To test the printability, I first mix the substrate (= fibres + distilled water + gelling agent) with mycelium and place it afterwards in the bag.


#4

What are you using as gelling?

a lot of gels, and that includes starch/cellulose water mixtures, are non newtonian. so the way you apply pressure will have an effect on how they flow.

KUKA runs on hot tip printing or how is the mixture cured post printing??


#5

I am using Psyllium Husk Powder (Psy) as gelling agent (3%). The KUKA Robot that I am using, contains a multi-printhead (3 extruders with a plunger) which is not designed to be warmed up.


#6

Got it, I did some reading on the chemical composition of your gelling agent. and uses.

3% seems like the common propoertion for food and drug applications but does mixtures typically include more starch that binds easier to the glucosides than the cellulose. have you added starch to the samples, it is not ideal due to yeast contamination but might work. Otherwise you might want to increase the amount of gelling agent get less clumpiness.
or preteat it with citric or acetic acid make it more reactive.


#7

The reason why for the first experiments an amount of 3% was chosen is because it showed a higher stiffness compared with 6%. Yet, those tests should be performed again to confirm it.


#8

Thanks for the ideas! :slight_smile:

I didn’t added yet starch to the samples, I will do it later!
But I already tested with an increased amount of gelling agent (5%, 10% and 20% Psy).
Both the 5% and 10% had good results, the matter was able to stick more to each other, but the printing itself was still quiet difficult and not fluent. I didn’t test the 20%, since it was no homogenous mixture (lots of clumps). I also tested another kind of gelling agent: 3% Xanthane Gum in combination with 6%Locust Bean Gum, which had good results with the 40% chopped wood fibres. I am still in the progress to find a way to print it fluently. I think the piping bag isn’t a good option to test this, therefore I am going to use a syringe during the next tests in order to be able to push more uniformly.

I have updated my “blog”, so you can read more in details how the testing of these substrates went! :slight_smile:


#9

Happy New year and best of luck.

how careful are you on particule size control?? this might have a big effect on how it flows. are you doing separation by size or just trusting that the break is even on your wooddust?


#10

Sorry for answering so late!

I have updated my blog, under the title “NEW PROTOCOL PREPARATION SUBSTRATE” there is a picture of the used chopped wood fibres. These are already very small, so it is not necessary to do a separation by size.