Close

Extracting and purifying PHAs using mealworms

A project log for Plastic-producing personal bioreactor system

Cultivate a [safe!] bacteria that accumulates PHA, then recover the PHA granules, purify, and use for 3d-print filament!

Neil K. SheridanNeil K. Sheridan 03/31/2019 at 18:580 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 

======

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 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

Results:

-  "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" [1]

===

Some additional methods of purification [3]:

1. Just with distilled water

Fecal pellets were added into 100 mL of distilled water yielding the concentration of 10
g/L. 

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" [3]

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
occur" [3]

2. Distilled water and NaOH [3]

So it's as above, but after the first centrifugation, the pellets were stirred in 0.05M NaOH for 1hr. [3]

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" [3]

So! You can see it is quite promising for extraction and purification of PHA without usage of nasty solvents! The best purity was from [1] with the granules reaching 100% purity when treated with 1% SDS and 1% SDS at 50 °C. In [3] they got >90% with NaOH but they didn't heat. 

Discussions