[curiouscat]

Hey Padford, thanks for posting. Always interesting to see a real problem from a scientist in another field.

+1. Even, sans the "a real problem from a scientist in another field"

My perception is that we have a very high ratio of ( pedagogic / real ) posts on the forums.

[padford]

Hi Arkon,
I would love to have an autoclave available to me, it's what I've always used in the past. Unfortunately, I'm trying to grow algae in what is best described as a field lab with limited facilities and availability of materials to source. Artificial seawater (ASW) is quite a common practice now for culturing algae, especially for lab purposes. If you get precipitates forming after autoclaving ASW it might be that you need to split some of salts into separate solutions and combine after autoclaving. Massive pain in the proverbial behind though!

[padford]

opsomath,
My pleasure! I've never tried forums before and I'm enjoying the experience. I'm sure most of your biology friends will be like me, used to having the luxuries of autoclaves and filter sterilisation!

Re. magnesium and calcium hydroxide... that's a welcome suggestion. I'm not sure of the exact concentrations of Mg and Ca in the seawater but it is real seawater that has been only mildly treated (low ppm chlorination) before reaching me. Is there any tests I could try here to remove it from the glassware? I tried performing my own 'ocean acidification' experiment today by bubbling some of the precipitate in seawater with pure CO2, with no joy. Not sure how low I got the pH but I'm fairly confident would have been low enough to start removing CaCO3.

To everyone else proposing hydrogen peroxide, I'm liking this more and more. Highly effective but short residence time, degrading quicker with heat... it could be the solution. The pH shift would be a concern, but seawater has a nice built in, albeit limited, buffering capacity (A bold statement to make to a forum of chemists!). My big concern now is about the health and safety aspect of H2O2 in the work place (more reading!).

In my reading today about commercial chemical sterilisers (both H202 and NaClO based), they all seem to add something that increases the residence time of these chemicals in solution, presumably to increase their effectiveness at reducing biological contaminants. I'm wondering if this is the case for the NaClO I have been provided with as it is the same stuff they use to keep their pipes from fouling. I need to find out more about my source I think! For now I have picked up some standard household bleach from the supermarket to see if I have the same problem with that. Least I can be (more) confident of the contents.

Thanks everyone for the help, more ideas are welcome!

[Borek]

Re. magnesium and calcium hydroxide... that's a welcome suggestion. I'm not sure of the exact concentrations of Mg and Ca in the seawater but it is real seawater that has been only mildly treated (low ppm chlorination) before reaching me. Is there any tests I could try here to remove it from the glassware?

More or less that's what I had on mind when asking about hydrochloric acid - it should work perfectly for this type of contamination. If that's the problem, bubbling CO₂ was probably a step in the right direction, you just didn't wait long enough - it works wonders, but on a geological scale  .

[curiouscat]

My big concern now is about the health and safety aspect of H2O2 in the work place (more reading!).

H2O2 at low conc. is fairly safe. At higher conc. organic impurities can cause fast decomposition, and runaway reactions. Especially without stabilizers.  The liberated O2 can be major an explosion hazard.

Mostly though, these are significant risks for large quantities and / or very high conc. If you stay below 30% and 5 Litres I suppose the risks are very manageable. As such H2O2 at low conc. is GRAS I believe, so not much of an acute / chronic toxicity hazard.

I'm no expert though. An MSDS might be more reliable.

[opsomath]

A brief conversation with my boss (NOAA phytoplankton researcher for like 20 years) provided the following:

1) He suggested microwaving as an alternate sterilization technique.

2) I know sterile filtration doesn't look practical to you now, but consider that you will probably need to do some kind of filtration anyway to keep from randomly introducing funk into your medium.

3) He looked like I had just started chanting Nazi slogans when I mentioned that you were interested in chemical solutions. Apparently that is Not Done.

[curiouscat]

3) He looked like I had just started chanting Nazi slogans when I mentioned that you were interested in chemical solutions. Apparently that is Not Done.

What's the goal of your experiments?

[padford]

3) He looked like I had just started chanting Nazi slogans when I mentioned that you were interested in chemical solutions. Apparently that is Not Done.

Haha, I had the same reaction when I was told the intended method! My background is lab not mass cultivation, so I'm not familiar with the techniques for sterilising such large volumes of water.  With a larger budget and better infrastructure, UV seems to be the way to go. Autoclaving is impossible and filtration with a small pore size is impractical so it was decided to use a single method that would work at all scales of culturing we will be working with... chemical was the proposal (NaClO, specifically). I guess it depends on your background, but I sympathise with your boss's reaction!

What's the goal of your experiments?

We're trying to find a low cost, low carbon footprint and effective solution to marine algae mass cultivation (for biofuel, mariculture feedstocks, pharmaceuticals etc) in the desert... Ambitious but, for my sins, I decided to sign up because it is actually feasible and could be a game changer! No conflict with freshwater and land use and could be scaled up to really make a dent on fossil fuel use.

[curiouscat]

What's the goal of your experiments?

We're trying to find a low cost, low carbon footprint and effective solution to marine algae mass cultivation (for biofuel, mariculture feedstocks, pharmaceuticals etc) in the desert... Ambitious but, for my sins, I decided to sign up because it is actually feasible and could be a game changer! No conflict with freshwater and land use and could be scaled up to really make a dent on fossil fuel use.

My concern is how valid is chemical treated seawater as a surrogate for pristine sea water.

Say, you get results in NaOCl treated water, can you trust them to scale to non-treated water? If the end use is based on untreated water (or do you plan massive production scale UV) why not do the tests on untreated water?

[padford]

Curious Cat,
The need for sterilisation is purely related to reducing biological contamination risks, i.e. anything that could reduce the biomass yield of the desired algae that you are trying to grow. For closed systems like cultures in the lab, and larger scale photobioreactors, this risk needs to be reduced as effectively as possible, otherwise you will continually need to revert back to scaling from clean algal innoculum on a regular basis.

If you are also using large open ponds for cultivation, then the risk of contamination is greatly increased as they are exposed to environment. However, by ensuring that at least the large volume of algal feedstock you put in the pond is as clean as you can make it, this risk is also reduced. Shortening the growth period in the pond also helps with limiting this risk, especially if the initial feedstock is at a level of biomass that it would have to be Darwin's monster to take over!

[Arkcon]

I'd suggest UV sterilization then, its appropriate for scale-up, as it is commonly done with large scale hydroponics.  It should be easy to set-up in small-scale as well.  The problem is, some organic carriers of important inorganic salts may be destroyed by sterilizing UV and precipitate out.  This can be a problem with chelators for iron, the iron ending up precipitating on the submerged quartz sleeve.  That said, you don't have to kill all microbes, just beat back the titer count of unwanted algae while your preferred ones flourish and crowd them out.  In fact, I'd even consider filtration, you don't have to remove the smallest bacteria, just most of the blue-green algae.  And for small scale, you can still autoclave water, transport it, then sterile filter your prepared seawater.

[padford]

UV is my recommendation as well. I was trying to look into how much it would restrict flow rates and, therefore, filling time as there is presumably an optimal flow rate past the UV tubes to effectively sterilise but then realised having a large capacity holding tank and sterilising there before pumping to the photobioreactors and ponds would equally effective. That will be the mark two system now though.

As an update, the algae is growing fine in the untreated seawater (curiouscat was asking) and household bleach (5.25%) treated seawater is not producing the precipitate when aerated. That problem seems unique to the sodium hydrochlorite solution I was supplied. I will test the toxicity of the household bleach treated seawater with the algae today and tomorrow.

Looks like the solution is to just not use the sodium hypochlorite. It'll bug me that I might never know what caused the precipitation and why it was toxic... but then maybe some things just aren't meant to be known by mere mortals! (That said, if anyone has a eureka moment on this I'd still love to know).

I appreciate everyone's input on this so far, its been a good discussion.

[curiouscat]

To make the situation weirder I'll wager if you try again with the bad hypochlorite this time it may not kill. Experimental variation.

[curiouscat]

UV is my recommendation as well. I was trying to look into how much it would restrict flow rates and, therefore, filling time as there is presumably an optimal flow rate past the UV tubes to effectively sterilise but then realised having a large capacity holding tank and sterilising there before pumping to the photobioreactors and ponds would equally effective.

What sort of flow rates are your goal? How many and how large are your reactors and how often will they get filled.

[Borek]

Just a random thought... Are you sure it wasn't calcium hypochlorite?