03/31/2019 at 21:35 •
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!
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.  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)
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
03/31/2019 at 20:50 •
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.
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!
03/31/2019 at 18:58 •
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!
 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
 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
 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
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. 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 were 5:1 for volume solution:fecal pellets.
5. Solutions stirred at 250rpm (room temperature) for 12hrs.
6. The fecal pellets were washed with HCl (hydrochloric acid) before being placed in petri dishes and dried overnight at 60C
- "A total of 40 g freeze-dried cells was consumed by 50 g of mealworms
over a period of 16 days"
- "In total, 32 g of fecal pellets were obtained
after 16 days of feeding the cells"
- "The purity of the biologically recovered PHA granules seems to be primarily affected by the
presence of proteins"
- "The biologically recovered
PHA granules washed with water resulted in a purity of 89%. The PHA granules purity reached
100% when they were treated with 1% SDS and 1% SDS at 50 °C" 
Some additional methods of purification :
1. Just with distilled water
Fecal pellets were added into 100 mL of distilled water yielding the concentration of 10
This suspension was subjected to sonication (30 min) at 44 KHz
The mixture was stirred for 24 h at room temperature
Next was centrifugation at 8,000 rpm for 5 min at 4ºC. The supernatant was removed, and the
pellets were stirred in distilled water for another 3 h.
Another round of centrifugation at 8,000 rpm for 5 min (4ºC). "The supernatant was removed and the
recovered PHA was then dried at 50ºC in an oven until a constant weight was achieved" 
RESULTS: " purity obtained
by this method was less than 90%. Prolonged incubation of fecal pellets in distilled water could
be detrimental to the properties of the PHA because of the possibility that PHA hydrolysis could
2. Distilled water and NaOH 
So it's as above, but after the first centrifugation, the pellets were stirred in 0.05M NaOH for 1hr. RESULTS: Purity was >90% and "SEM micrograph of the washed fecal pellets showed that the PHA granules were spherical and were not surrounded by a mucus layer" 
So! You can see it is quite promising for extraction and purification of PHA without usage of nasty solvents! The best purity was from  with the granules reaching 100% purity when treated with 1% SDS and 1% SDS at 50 °C. In  they got >90% with NaOH but they didn't heat.
03/28/2019 at 20:40 •
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]. 
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" 
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" 
6. So that's it! In this paper  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!
 Cybernetic structured modeling of the production of polyhydroxyalkanoates by Alcaligenes Eutrophus
 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. Cells collected via centrifugation (22,220 × g for 20 min) 
3. Ok, so this is an interesting part. Normally, solvents would be used now. But we are instead wanting to use enzymes. As these will have no negative environmental impact.
4. "cell pellets were then lyophilized and stored at 4 ◦C" 
5. Now for the enzymes. This paper  used a variety. But I'll only mention papain and bromelein, since these are from plants, have no handling safety issues, and have no negative environmental impact. So the enzymes are suspended in buffers (phosphate and citrate respective )
6. PHB cells suspended to "25 ml (final concentration of 20 g l−1) in a specific buffer according
to the enzyme tested and transferred to a 125 ml Erlenmeyer flask. Then, 1 ml of enzyme suspension was added to achieve the desired enzyme mass per biomass. "  So PHB cells suspended in buffers matching those in which the enzymes are suspended.
7. Flasks agitated with rotary shaker at temps required for enzymes (40C and 50C) at 200rpm for variable time periods 
8. Next came checking on how cell lysis was progressing. This involved taking samples from the flasks, and diluting them, and measuring their absorbance at 560 nm (spectrophotometer) .
"Cell lysis was monitored by the decay of this absorbance at 560 nm,
in terms of the relative absorbance reduction (AR)" (See table in ). As you can see, this stage is to see how well each enzyme performs. So it's not super important for us at this point.
9. Enzymatic recovery of PHB part: Cells from the bacteria (8.25g mass) are suspended in buffer matching enzyme (as per parts 6 and 7). Then homogenised in a blender, before being passed through a sieve of 0.15mm. The aim being to eliminate cell agglomerates. More buffer is added to get volume to 330ml (final cell concentration of 25 g l−1)
10. This suspension was then transferred to 1L Erlenmeyer flask, agitated to 200rpm with shaker, and pre-incubated to desired temperature (see table in ).
11. Finally [!!] the enzyme is added 5.0mL, and samples withdrawn at various times. (see table in  but for bromelain this seems to be 6-12hrs?)
12. Next, the samples were centrifuged at 42,000 × g for 10 min (4 ◦C). The resultant cell pellets were then washed twice with a 0.85% saline solution.
[[ So we can see at this point what hardware we do and do not need! We need a number of flasks. Some of these need to be shaken. Some need to have capability for blending the suspensions in them. We need a sieve. And we need centrifugation devices built-into the workflow. ]]
13. Ok, so at this point we have completed recovery of PHB from the cells. But now it needs to be purified from the pellets!
So far everything has been nice and low-impact with the enzymes from plants, etc. but this purification stage seems to involve solvents such as 1,1-Dichloroethane or trichloromethane. Which is not good! We really can't use these chemicals! So I am stopped here until I find out more!
Update: So I did find something promising! In this paper: 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.
"We have found that some animals can readily feed on the dried bacterial cells that contain PHA granules. The digestive system of the animals is able to assimilate the bacterial cells but not the PHA granules which are excreted in the form of fecal pellets, thus resulting in partial recovery and purification of PHA. "
This was originally noticed by chance in rats (white fecal pellets after ingesting the cells), but it seems to apply to insects such as mealworms too! Per the paper above.