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Preliminary ideas for the bioreactor

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 03/28/2019 at 20:400 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: 

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 20 min) [2]

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" [2]

5. Now for the enzymes. This paper [2] 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 [2])

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. " [2] 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 [2]

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 [2]). 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 [2]).

11. Finally [!!] the enzyme is added 5.0mL, and samples withdrawn at various times. (see table in [2] 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.

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