Homebuilt bioreactor

[Arkos Vähämäki]

www.biotechniques.com/BiotechniquesJournal/2008/August/An-automated-home-built-low-cost-fermenter-suitable-for-large-scale-bacterial-expression-of-proteins-in-Escherichia-coli/biotechniques-45258.html

What equipment and knowledge would be required not just to build this but use it to produce something? Could I as a hobbyist with some studies in biochemistry be able to pull it off?

[Avery louie]

You could pull it off but you would need to learn some things about hose fittings and electronics.

--A

On Apr 7, 2014 9:46 AM, "Arkos Vähämäki" <rkos....@gmail.com> wrote:

www.biotechniques.com/BiotechniquesJournal/2008/August/An-automated-home-built-low-cost-fermenter-suitable-for-large-scale-bacterial-expression-of-proteins-in-Escherichia-coli/biotechniques-45258.html

What equipment and knowledge would be required not just to build this but use it to produce something? Could I as a hobbyist with some studies in biochemistry be able to pull it off?

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[Open BioLab Graz]

Well, what they call 'home built' is actually an assembly of special lab equipment, costing several 1000$.

Depends on what you want to do. You can grow E.Coli in shaking flasks, adding a stirrer, pH measurment and air supply to reach higher concentrations is not a too big deal. But building a totally sterile system to grow less strong bacteria/funghi is a little different and ways more expensive.

[John Griessen]

On 04/07/2014 01:07 PM, Open BioLab Graz wrote:
> building a totally sterile system to grow less strong bacteria/funghi is a little different and ways more expensive.

What sterilization methods are practical for a reactor with ports for stirring, pH,
conductivity, gas bubbler, and a big cleanout opening? Is autoclaving necessary,
or is UV light workable?

[Nathan McCorkle]

In industry/academia as much as possible is autoclaved, otherwise
alcohol or phenol can be used to sterilize chemically, ozone (HEPA
filter blowing past an ionizer), or gamma radiation (this is how
sterile plastic culture flasks are sterilized). UV is used in
biocontainment hoods but its efficacy is argued (or so I've heard here
on this list). UV is sold at camping stores and for home well-water
treatment too, so it might not be 100%, but it does seem to cut down
enough that our immune systems can handle the rest (you don't have
that liberty in a bioreactor with no immune system).

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--
-Nathan

[John Griessen]

On 04/07/2014 02:31 PM, Nathan McCorkle wrote:
> otherwise
> alcohol or phenol can be used to sterilize chemically,

Seems like expensive cleanup required often.

ozone (HEPA
> filter blowing past an ionizer),

Sounds good for low costs.
Suggests an ozone generator/electroporation HV module!


or gamma radiation
never mind that...

[SC]

Hi Arkos,

You may want to consider what the "something" is you would like to produce before making the tools to produce it.  You may likely be able to use something very simple.

(Although I get the love of groovy equipment, believe me.)

Stacy

 

[Nathan McCorkle]

Seems no more complex than a beer fermenter with some automation
added, which I'm sure there are 100s of hobbyist brewers on various
other forums that you could talk to or look to for cheaper ways to
proceed.

You'll also have a hard time with the size of the main vessel, so I'd
start smaller for initial experiments. Being able to fit everything in
a pressure canner would make things much faster.

Depending on the strain you're growing, you could take advantage of
some natural culture-produced disinfectants... yeast has ethanol, etc,
or you could do what industry/academia does and use antibiotic
resistance genes, and add antibiotics to the culture media. There are
some food-grade antibiotics available, such as Nisin.


As for what to do with it, a desired/favorite protein is personal
choice, so you'll have to decide that on your own!

If you get this working, you should be able to add on more modules
that would slowly expand the capabilities to be able to do more and
more experiments (growth curves, time dependent chemical analysis).

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[Nathan McCorkle]

On Mon, Apr 7, 2014 at 1:49 PM, John Griessen <jo...@industromatic.com> wrote:
> On 04/07/2014 02:31 PM, Nathan McCorkle wrote:
>>
>> otherwise
>> alcohol or phenol can be used to sterilize chemically,
>
> Seems like expensive cleanup required often.

Yeah, certain things like RNAses are hard to kill without some
powerful weapons though. I'm not sure but I'd imagine X-ray or gamma
would be stronger/as-effective as DEPC for RNAses, but I could be
wrong. I'm on the fence on my opinion of ozone vs DEPC. Maybe I'll
look it up later... or someone here knows.

>
> ozone (HEPA
>>
>> filter blowing past an ionizer),
>
> Sounds good for low costs.
> Suggests an ozone generator/electroporation HV module!

Probably!

>
>
> or gamma radiation
> never mind that...

this is actually probably one of the most effective! which is why the
big industry uses it.

Or just use the above HighVoltage circuit to produce X-rays too, and
make sure to use a metal box for the whole setup!

[Cathal Garvey]

I suspect bleach or ozone will do for destroying RNases; we normally
avoid such things because they also kill RNA! ;)

Why are we worrying about RNAse, by the way? It contaminates all and
sundry, yes, but if you're producing protein for work on RNA, your RNAse
cleanup happens after bioreacting, surely?

--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com

[Nathan McCorkle]

They're just what I saw as the hardest thing to kill, not really a worry when dealing with a cellular system.

[John Griessen]

On 04/08/2014 10:13 AM, Nathan McCorkle wrote:
> I suspect bleach or ozone will do for destroying RNases; we normally
> avoid such things because they also kill RNA! ;)

I'm just asking for ideas about sterilization
for bioreactors. Using a water solution seems
very good for letting a concentrated disinfectant
diffuse into places between seals and surfaces
then rinse away in a flow bench clean air flow.

Ozone might be difficult to tell what it has touched
well enough for long enough to sterilize. A plus
for ozone is being a gas, it dissipates easily.

I can't think of a good way to sterilize a reusable
bioreactor without a flow bench -- by simply flushing
disinfectants through it. Seals would always trap
material traces and when pressure and temperature changed,
the trapped material could be exposed to the interior again.
So sterilizing a bioreactor means disassembly, cleaning, reassembly
while surrounded by filtered air.

[Nathan McCorkle]

Or you just add a ton of chemical sterilizer that your organism is
resistant to. Not sure how to do this better without moving to an
animal model (which has an immune system, aka active cleaning). Maybe
another reason to move towards stripping down an animal's
brain/nervous system.

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[CodonAUG]

Idea of how to handle cleaning without a hood:

Pour some 70% alcohol into the vessel.  Rubberband a textile/cloth/paper over the mouth of the container (such as you would for autoclaving).  Then swish the alcohol around all over the insides and then let it dry.  The textile/cloth isn't permeable to microorganisms but it is permeable to water/alcohol (similar to sterilization pouches and swatches).

[Sebastian Cocioba]

You could pump in hot water with soap first, then 10% bleach, then a
few more hot distilled water rinses. Maybe even include a sealed
germicidal UV bulb inside the reactor. There are several plastics that
lets UV through. Polypropylene is one of them. If your reactor isn't
under pressure it could be an easy hack. The UV also produces ozone, I
know this because of the smell coming out of my UV sterilization
cabinet. The reactive oxygen species will do some added damage.

I worked at a blood lab for a while and the sanitation system for all
the sampling machines was just a long sip of dilute bleach through the
tubes. If you limit the orifices and gasketed parts in the fermentation
area you could avoid all those crevasses too. In case bleach is too
alkaline for routine use, quaternary ammonium salts work too. Phytotech
sells a low pH alternative to bleach IIRC its called NaDCC. Else
Calcium hypochlorite bleach is less harsh.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: John Griessen
Sent: 4/8/2014 2:22 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] low cost bioreactor -- sterilizing methods

On 04/08/2014 10:13 AM, Nathan McCorkle wrote:
> I suspect bleach or ozone will do for destroying RNases; we normally
> avoid such things because they also kill RNA! ;)

I'm just asking for ideas about sterilization
for bioreactors. Using a water solution seems
very good for letting a concentrated disinfectant
diffuse into places between seals and surfaces
then rinse away in a flow bench clean air flow.

Ozone might be difficult to tell what it has touched
well enough for long enough to sterilize. A plus
for ozone is being a gas, it dissipates easily.

I can't think of a good way to sterilize a reusable
bioreactor without a flow bench -- by simply flushing
disinfectants through it. Seals would always trap
material traces and when pressure and temperature changed,
the trapped material could be exposed to the interior again.
So sterilizing a bioreactor means disassembly, cleaning, reassembly
while surrounded by filtered air.

[Nathan McCorkle]

Not all uv is ionizing, it has to be less than about 250nm

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/-1968054983803593570%40unknownmsgid.

[Sebastian Cocioba]

I meant the UV from the germicidal lamps/tubes. I've seen short tubes that are in that range. I believe any germicidal UV lamp must be in that range to be effective. Im sure it won't work with party lights :P

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: Nathan McCorkle
Sent: 4/9/2014 9:13 PM
To: diybio
Subject: RE: [DIYbio] low cost bioreactor -- sterilizing methods

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2B82U9LjHG0PpsGRmc21Djk6y%3DLbgtvnfdCLV71UiYkxUYGRBA%40mail.gmail.com.

[John Griessen]

On 04/09/2014 07:52 PM, Sebastian Cocioba wrote:
> You could pump in hot water with soap first, then 10% bleach, then a
> few more hot distilled water rinses. Maybe even include a sealed
> germicidal UV bulb inside the reactor. There are several plastics that
> lets UV through. Polypropylene is one of them. If your reactor isn't
> under pressure it could be an easy hack.

I'm wanting to have mild pressures -- how else could it evolve into a
pushbutton beer machine as well as bioreactor?

The UV also produces ozone, I
> know this because of the smell coming out of my UV sterilization
> cabinet.

Is your cabinet's ozone from UV, or from the electronics driving a
mercury vapor lamp UV source?

> I worked at a blood lab for a while and the sanitation system for all
> the sampling machines was just a long sip of dilute bleach through the
> tubes. If you limit the orifices and gasketed parts in the fermentation
> area you could avoid all those crevasses too. In case bleach is too
> alkaline for routine use, quaternary ammonium salts work too. Phytotech
> sells a low pH alternative to bleach IIRC its called NaDCC. Else
> Calcium hypochlorite bleach is less harsh.

I really appreciate all these liquid disinfectant ideas Sebastian!
I've been thinking of smooth flexing rubber seals with flow all along them
when stretched to open a port for cleaning. Then you compress them to seal
and keep a port closed. That would be how to seal sensors as well as in/out pipes.
The max pressure/vacuum could be low, but still close to one atmosphere easily, maybe two.

On 04/09/2014 08:16 PM, Sebastian Cocioba wrote:> I've seen short tubes that are in that range. I believe any germicidal UV lamp

must be in that range to be effective

So, I guess the UV is the cause of the ozone when it's hard UV < 250nm...

Thanks for the idea generating.

[Mega [Andreas Stuermer]]

For what I heard UV is very effective. But.

It penetrates hardly. e.g. Google uv-a , uv-b and uv-c. the latter is shielded by the atmosohere. uv-b damages another layer of the eye than uv-a because it only penetrates the surface laxer.

What if you cycle the water and have a pipe with a uv light inside?

[Nathan McCorkle]

On Wed, Apr 9, 2014 at 11:04 PM, Mega [Andreas Stuermer]
<masters...@gmail.com> wrote:
> For what I heard UV is very effective. But.
>
> It penetrates hardly. e.g. Google uv-a , uv-b and uv-c. the latter is shielded by the atmosohere. uv-b damages another layer of the eye than uv-a because it only penetrates the surface laxer.

Yes but if its ionizing that doesn't matter, then you just gotta have
a fan to circulate it around. In case there are low pressure zones,
you'd want to alternate between a circulating and letting the gas sit
and diffuse into corners, I'd pulse the fan during the night time.
Also, with high levels of ozone you really need to start thinking
about human health concerns as far as breathing it in, it will burn
your lungs. So when you are done cleaning it, you'll want to pump
fresh air in and the ozone outside through a vent. want to get in and
do work you would need this to be in a ventilated room and let the
ozone gas do its space-filling-gas. Of course that vent needs a
moisture trap and/or a one-way valve (they sell these for bathroom
exhaust ductwork, so a Y pipe in the ceiling doesn't send humid air to
your bedroom).

>
> What if you cycle the water and have a pipe with a uv light inside?

I've seen exactly that for sterilizing well water (pumped from the
ground) for houses.

[Cathal Garvey]

Important question is how *deep* it sterilises water. UV of any type is
absorbed efficiently by water, so I think most UVC sterilisers tend to
pump water in a narrow jacket around a strip-bulb; just immersing the
bulb in a larger vessel might not cut it. If you wanted that effect, you
might have to design your vessel for that purpose; long and narrow,
sized to fit a UVC bulb and only as wide as UVC can penetrate.
Accounting, of course, for the usual degradation in output for those
bulbs; you'd have to assume the lumen output of the bulb after a year or
two of use in your design.

As far as ionising, does UVC ionise open air, or does it ionise surfaces
(i.e. metal) which go on to ionise air? That is, will you get ozone in
an air-filled glass vessel without metal or another UV-ionising surface?

And speaking of which, aren't there "self-sterilising nanomaterial
surfaces" now? I wonder are they attainable yet in the DIY market as
inner-surfacants for your projects? I imagine a metal container could be
coated with titanium dioxide without crazy difficulty, and IIRC titanium
dioxide is highly light-ionising, even by blue light; you might even
save the cost of the TiO2 treatment by avoiding the need for a UVC bulb
and yearly replacements. :)

[Nathan McCorkle]

I've heard some pretty bad stories about flow cytometers getting nasty
contamination that is hard to get rid of, and they pretty much rely on
liquid disinfectants. Upon googling, I found this which sounds
positive, and also brings to light a good point about bleach (NaClO)
that is isn't shelf-stable in regards to concentration (it the
chlorine off-gases), I'm not sure how quickly it changes though:
https://lists.purdue.edu/pipermail/cytometry/2005-February/028230.html

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--
-Nathan

[John Griessen]

On 04/10/2014 01:04 AM, Mega [Andreas Stuermer] wrote:
> cycle the water and have a pipe with a uv light inside?

Yes, and all the seals and passages need to become sterile...

On 04/10/2014 01:35 AM, Nathan McCorkle wrote:> Yes but if its ionizing that doesn't matter, then you just gotta have

> a fan to circulate it around.

UV germicidal strength to generate ozone could be a good low effort way to sterilize
if it flows past all seals and parts.

On 04/10/2014 03:14 AM, Cathal Garvey wrote:> IIRC titanium

> dioxide is highly light-ionising, even by blue light

Good one!

Thanks for all the ideas. Making vessels small enough so the tubes and sensors
and container can all be autoclaved in a pressure cooker with permeable wraps on tube ends
still has big value -- if you can use that method, you don't have to fiddle with it as much,
flowing here and there, the heat gets everything. But autoclaves or pressure cookers
are dangerous space-hogging extra equipment steps... We'll see if I/we can come up with something
that passes a sterility test for less effort.