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• Things to do list!

  Neil K. Sheridan • 03/31/2019 at 21:35 • 0 comments

  1. Obtain 1kg of Polyhydroxyalkaonate granules is ~£215 from sigma aldrich , then test if these can be made into 3d-printing filament using e.g. an extruder like the filabot.

  2. If ok with making filament, then try some 3d prints! e.g. just a water holder for drinking!

  CHECKPOINT PASSED! [yes/no]

  3. Obtain Cupriavidus necator culture (e.g. 185,- €) and experiment with the growth and PHA accumulation phases using protocols from papers mentioned earlier. You will need to invest in various analytic tools!

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  Q. What should Ralstonia eutropha be fed with?

  Q. Is wild-type of an engineered type best?

  Q. Which PHA is the best for 3d-printing? Depending on what we are printing of course!

  A. [1] describes PHAs as 

  Properties	Extrusion Temp	Pros	Cons
  Several copolymers, brittle and stiff	~160C	UV-stable, stiffness	Elasticity, brittle

  So I'm not sure, since there are such a wide variety in the family! 

  Two of the common members of the PHA family I have encountered so far:

  - Poly(3-hydroxybutyrate) aka P(3HB)

  - P(3HB-co-3HHx)

  References:

  1. Pakkanen, J., Manfredi, D., Minetola, P., & Iuliano, L. (2017). About the Use of Recycled or Biodegradable Filaments for Sustainability of 3D Printing. Smart Innovation, Systems and Technologies, 776–785.doi:10.1007/978-3-319-57078-5_73

• 3d-printing with PHA filaments

  Neil K. Sheridan • 03/31/2019 at 20:50 • 0 comments

  So there isn't a whole lot of point to the project, if we can't make filaments for 3d-printing from PHA!

  Useful papers list:

  Recent Advances in 3D Printing of Aliphatic Polyesters.

  https://www.ncbi.nlm.nih.gov/pubmed/29295559

  ======

  1kg of Polyhydroxyalkaonate granules is ~£215 from sigma aldrich 

  I've seen one PHA filament, called Z-PHA from zortrax. I guess the Z is because of their company name? (datasheet here https://cdn.zortrax.com/wp-content/uploads/2018/11/Z-PHA_Technical_Data_Sheet_eng.pdf Tg=62.58C) I've seen some PLA/PHA blends. So that's kinda promising. 

  Maybe a good starting point would be for me to go buy the granules, and try making filament from them using a filabot!

• Extracting and purifying PHAs using mealworms

  Neil K. Sheridan • 03/31/2019 at 18:58 • 0 comments

  In the first log I investigated the methods of growing bacteria, and extracting ,then purifying their PHA granules into usable plastics! A major roadblock is the usage of toxic solvents in the extraction and/or purification process.  This means you need handling processes, and a recycling method for the solvents. Not really so great for a personal plastic-producing bioreactor! 

  So!  An interesting way to overcome this is using mealworms (or other insects) to eat the pellets containing PHA that have come from the bacteria. The bacteria digest the majority of the non-PHA cellular components, but they do not digest the PHA! This is passed right through them, emerging as poop!

  Useful papers:

  [1] Murugan, P., Han, L., Gan, C.-Y., Maurer, F. H. J., & Sudesh, K. (2016). A new biological recovery approach for PHA using mealworm, Tenebrio molitor. Journal of Biotechnology, 239, 98–105.doi:10.1016/j.jbiotec.2016.10.012 

  [2] Ong, S. Y., Zainab-L, I., Pyary, S., & Sudesh, K. (2018). A novel biological recovery approach for PHA employing selective digestion of bacterial biomass in animals. Applied Microbiology and Biotechnology, 102(5), 2117–2127.doi:10.1007/s00253-018-8788-9 
  

  [3] Ong, S. Y., Kho, H.-P., Riedel, S. L., Kim, S.-W., Gan, C.-Y., Taylor, T. D., & Sudesh, K. (2018). An integrative study on biologically recovered polyhydroxyalkanoates (PHAs) and simultaneous assessment of gut microbiome in yellow mealworm. Journal of Biotechnology, 265, 31–39.doi:10.1016/j.jbiotec.2017.10.017 

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  About mealworms

  Now, they are not really worms! They are beetles, and resemble worms in their larval period. The larval period lasts from 90-114 days (10-14 larval instars) [not ref]. This is the stage at which they will be eating the pellets containing PHA. As far as I could see, mealworm breeders feed them hormones to keep them in the larval stage for longer. However, they won't stay in that stage for ever! So we have to work out what to do with them afterwards! Unless you are a fan of having 1000s of beetles as pets?

  The best outcome I can think of, is for us to eat the mealworms before they reach the end of their larval period. Obviously it would need a change in diet, to make sure we are not eating PHA too. Although PHA is thought to be non-toxic in humans (it's used for medical impants). That would be great, because not only are we then producing plastic, we are also producing protein source! There is, of course, an ethical issue with using insects like this. 

  The whole eco-system pertaining to mealworms can be completely self-contained. I.e, some mealworms can be allowed to progress to pupal period (~6 days at 35C) and then to adults. As adults, they will happily reproduce (70-100 eggs per female). The eggs hatch after 7-14 days, then we have more mealworms to eat the pellets containing PHA! The adults live for 30-60 days. I'm not sure if they reproduce for entire lifetime.

  =======

  Summary of the Murugan et al. protocol [1]:

  1. 50g of mealworms (age 30-35 days) were fed 5% of their body weight/day, for 16 days, with freeze-dried C. necator cells (these are the bacteria they make the PHA). Freeze-drying is quite an expensive procedure, and I don't know if it is required if the cells are being fed immediately after being taken from the bacterial colony [?]. 

  2. The fecal pellets from the mealworms were collected [how I don't know!], then sieved using a 0.5 mm mesh. From what I have seen looking at mealworm farms, trays have nylon netting bottoms, so the poop falls thru, whilst the feed and worms do not (see e.g. https://www.instructables.com/id/Mealworm-Farm/) 

  3. Pellets dried at 60C overnight

  4. Pellets purified using water and 1% (w/s) sodium dodecyl sulfate (SDS). Note that SDS doesn't have any major toxicity issues. It's even used in cosmetic products. They tried this without heating, and with heating at 50C. The resulting solutions...

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• Preliminary ideas for the bioreactor

  Neil K. Sheridan • 03/28/2019 at 20:40 • 0 comments

  So to begin with I am going through experimental papers and getting a general idea of the kinda workflow to cultivate and then extract the PHA/PHB from the bacteria! I.e. what energy inputs are needed (gases, nutrients), and what is needed to extract and purify the polymers..

  === Using PHB-producing strain R. eutropha DSM545 ==

  Bacteria info: https://en.wikipedia.org/wiki/Cupriavidus_necator

  1. Now in the first paper I looked at, the R.eutropha comes as an Inoculum and needs to be reactivated. This was done using a growth medium consisting of  5.0 g.l^-1 of peptone and 3.0 g.l^-1 of meat extract [no idea what meat extract!]. 

  2. Next the innoculum is prepared for the bioreactor conditions with a mineral medium [3.5 g.l^-1 of Na₂HPO₄, 1.5 g.l^-1 of KH₂PO₄, 3.0 g.l^-1 of (NH₄)₂SO₄, 0.06 g.l^-1 of ammonium ferric citrate, 0.01 g.l^-1 of CaCl₂.2H₂O, 0.20 g.l^-1 of MgSO₄.7H₂O, 10.0 g.l^-1 of glucose and 1.0 ml.l^-1 of a trace element solution]. [1]

  3. These first two steps were performed in a rotary shaker at 30°C and 200 rpm. Each lasting 15hrs.

  4. Now the bacteria is added to the bioreactor (32C pH 7.0) and left to grow with the following medium: "1.29 g.l^-1 of KH₂PO₄, 1.83 g.l^-1 of (NH₄)₂SO₄, 0.05 g.l^-1 of ammonium ferric citrate, 0.02 g.l^-1 of CaCl₂.2H₂O, 0.55 g.l^-1 of MgSO₄.7H₂O, 15.0 g.l^-1 of glucose, 15.0 g.l^-1 of fructose and 2.0 ml.l^-1 of a trace element solution" [1]

  5. Next we have the stage in which we trigger the bacteria to accumulate the polymers we want! So here it is supplied with the following medium: "51.3 g.l^-1 of glucose, 51.3 g.l^-1 of fructose and 45.6 g.l^-1 of propionic acid" [1]

  6. So that's it! In this paper [1] they used the following analytic methods to monitor how the bacteria was getting along: 

  • cell mass concentration (dry-weight method), 
  • intracellular polymer content in HB and HV units (gas chromatography) 
  • sugar concentration (liquid chromatography – HPLC) 
  •  nitrogen concentration (Kjeldahl method) 
  •  acids (occasionally present) concentration (gas chromatography)

  The bioreactor used could monitor things like dissolved oxygen. It was the Braun BIOSTAT Ed Fermenter W/ DCU Controller & Gas Flow Regulator. This is quite an old one (since the paper is from 1999) so maybe they are on ebay for lowish cost, for me to have a look at? The new ones are obviously hugely expensive i.e. 10k+ Euros.

  * I'm still not sure about the gases used. But the interesting take-home bit from 5 is that this bacteria will sequester polyhydroxyalkanoate (PHA) or PHB plastics when exposed to excess amounts of sugar substrate. Hence the massive increase in sugars given during stage 5. Kinda like humans will sequester fats when they are being well-fed!

  [1]  Cybernetic structured modeling of the production of polyhydroxyalkanoates by Alcaligenes Eutrophus

  [2]   Enzymatic recovery and purification of polyhydroxybutyrate

  produced by Ralstonia eutropha (2006)

  Ok, so the next bit I looked at is how we get the polymers out of the bacteria cells after they have finished making them. Poor things went to so much trouble and then we steal their things!

  1.  First of all the bacteria are exposed to heat ~95C to denature their genetic material and protein, and to destabilize their outer membranes. This will also importantly denature PHB depolymerase. That's the enzyme that will go ahead and degrade the polymers we want. So we want to get rid of that! I'm not entirely sure on the times for heat exposure or the actual temps, so that would require further research. These are also kinda old papers, but they are giving me a good idea of the workflow required! [2]
  

  2. Cells collected via  centrifugation  (22,220 × g for...

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