[padford]

Hello Chemists!
I have a problem that I hope someone with expertise can help me with.

I am currently trying to use sodium hypochlorite (8%, although I have suspicions it is lower than this based on smell comparisons with household bleach) to sterilise seawater prior to using it for growing cultures of microalgae. I'm trialling it on small 5L volumes of seawater. Initially I added the hypochlorite to a final conc. of 800ppm and left it to degas chlorine by passive diffusion. I found this took a considerable amount of time so have now added aeration through a 0.2 micron air filter. In doing so, I am now finding a white precipitate forming on the glassware. It is a fine crystal that is pretty well cemented onto the glass. My first thought was calcium... I know its a pain to dissolve when making artificial seawater, so might be the first to precipitate out in other situations?

I've used the sterilised seawater after 40hrs of degasing, via aeration, and it does not sit well with the algae either. I immediately notice some kind of chemocline () when adding the sterilised seawater to the microalgae culture. I can only compare this to haloclines you see in the ocean (I'm a marine biologist, sorry). However, salinities of both liquids are the similar (approx. Algae 45ppt vs seawater 48 ppt).

I had previously noticed small amounts of precipitate forming without aeration, but assumed it to be particulate rather than anything more sinister.

Any thoughts? Google searches for seawater and sodium hypochlorite bring up a wealth of info on electrolytic formation of the latter from the former, but nothing on what I've observed.

In summary:
What is the precipitate?
Why would the sterilised seawater be toxic?
Are there any steps I can take to resolve the above?

Best regards,
Padford

[Borek]

No real answers to your problem, just some more questions and guesses.

Is it a real seawater, or a synthetic one?

Hypochlorite solution has a high pH (makes it stable), so you introduce not only chlorine, you also change pH of the solution. Have you checked it after aeration?

Have you checked if the precipitate dissolves in a strong acid (like hydrochloric)? Doesn't even have to be concentrated, something 1M will do. If it dissolves, chances are it is a hydroxide, or some kind of carbonate/basic carbonate.

[padford]

Hi Borek,
Thanks for the swift reply. Its a natural seawater, that has been filtered and pre-treated at a chemical plant before it comes to me through the same pipeline. As far as I have been able to find out, it has only had a 'light' chlorination before coming to me. I don't have much more information on it than that, but I do know it is hospitable to microalgae as it has grown nicely in it before (without my own sterilisation).

The pH of the sodium hypochlorite treated water after aeration is at pH 8.05, pre-treatment the seawater is at pH 7.9-pH 8.1 (samples vary from the seawater feed). Both pHs are comfortable for the algae.

I don't have access to HCl (field lab facilities) but could bubble the solution with CO2 to see if the precipitate dissolves. Is that sufficient?

[Borek]

I don't think CO₂ could change much.

But it occurred to me (having these fish tanks in the past finally pays off ). Where do you see the precipitate? On the glass over the water level? If you are aerating water, bursting bubbles wet the glass surface over the water level and it dries out there, so perhaps what you observe is just an effect of aeration, not related to hypochlorite?

[padford]

Unfortunately the precip is on the base and sides of the glass, below the waterline.

[curiouscat]

When you say "notice some kind of chemocline" what exactly do you mean?

Why would the sterilised seawater be toxic?

Are you sure the sterilization makes it toxic? Or is it already toxic.

Can you try growing some in a 5L sample that was not subject to your sterilization procedure?

Can you try growing some in water that got the Hypochlorite treatment but no aeration (exact same water; it may have changed since your last test)?
 
Can you try growing some in water that got just aeration but no hypochlorite?

Let's try to isolate the offending factor. It could be the seawater, the chemical or the aeration. Or some combination.

Could the ppt be a red herring? Are you sure the ppt has anything to do with algal death?

Also, if as you report the algae grew fine even without your sterilization is there a specific reason you want to sterilize the water?

[padford]

Hi curiouscat,
Thanks for your reply. I've tried to address your questions in turn, hope it helps!

When you say "notice some kind of chemocline" what exactly do you mean?

When I add the algae culture to the sterilised seawater there is the same mixing you see in haloclines, although I know it is not due to a difference of salinities in this case. I can't describe it any better than that... its looks like it almost has a viscous appearance at the boundary where the two liquids mix. My thinking was if its not salinity-driven could it be some other difference in properties between the two liquids (another chemocline for example)? This is my best attempt at a biologist trying to be a chemist!

Are you sure the sterilization makes it toxic? Or is it already toxic.

The same seawater source has been used to grow algae without sterilisation on several occasions.

Can you try growing some in a 5L sample that was not subject to your sterilization procedure?

This is currently happening. I will report back!

Can you try growing some in water that got the Hypochlorite treatment but no aeration (exact same water; it may have changed since your last test)?

Have done previously but without aeration it was very difficult to remove the final chlorine gas from the seawater before adding the algae. Either the sterilised seawater (without aeration) was toxic or chlorine remained in solution. My observations suggests that the precipitate forms without aeration, but that aeration greatly accelerates this.
 

Can you try growing some in water that got just aeration but no hypochlorite?

The previous attempts at growing in non-sterilised seawater were aerated. This is common practice in algae culturing, with only a few species disliking it.

Could the ppt be a red herring? Are you sure the ppt has anything to do with algal death?

Algae are typically tolerant to a range of salinities (although how much of a range is species-specific). They are certainly tolerant to the differences we're talking about here.

Also, if as you report the algae grew fine even without your sterilization is there a specific reason you want to sterilize the water?

Sterilisation is required for keeping clean mono-cultures and reducing sources of biological contamination (be it other algae strains, bacteria, fungi or protozoan predators) that could be present in the seawater used. Typically, autoclaving is used, but that is beyond the resources I have available to me in the field.

[curiouscat]

Thanks!

As an aside, would hydrogen peroxide work for you?

It is easy to carry to field, quite cheap and the residual gas problem is eliminated.

Not entirely sure, but just a thought. 

Portable UV sterilization might be another neat option at the 5 L scale. (in case the algae remain finicky about the chemicals). I've seen marine ship-board units that were sterilizing thousands of gallons an hour but would fit inside a car sized skid. With invasive species being a big scare those units are going to become commoner on ships. The Coast Guard wants to be pretty sure any ballast water you dump into the great lakes is squeaky clean.

Of course, I mean smaller versions of that. Not sure how good or expensive those hand held wands are.

e.g. http://www.steripen.com/ultraviolet-light/proven-technology/

[padford]

No problem, I appreciate you taking part and always willing to discuss the wonders of microalgae.

Re. Hydrogen Peroxide... Do you know off hand what residuals would be left after adding to seawater? (I will look into this myself too!). I only ask because, if there is no residual gas, presumably that means it all stays in solution? Does it break down in some other way?

Appreciate everyone's efforts by the way! First time using a forum

[curiouscat]

No problem, I appreciate you taking part and always willing to discuss the wonders of microalgae.

Re. Hydrogen Peroxide... Do you know off hand what residuals would be left after adding to seawater? (I will look into this myself too!). I only ask because, if there is no residual gas, presumably that means it all stays in solution? Does it break down in some other way?

Appreciate everyone's efforts by the way! First time using a forum

It ought to normally end up as H2O and O2 but not sure if there's any unwanted oxidizing reactions with any other sea water salts.

[padford]

I've proposed UV units to echelons above me for the next project as I need to find a neater solution all round for this. For now, I need to find a solution that can be perfected at 5L and then scaled to 3000L! Chemical was an obvious choice at the time.

H2O2 seems like a rather neat solution however, especially if water and oxygen is all that's left over. My only work with H2O2 in the past is related to light and thermal stress in algae. Its a by-product of safety valves in photosynthetic pathways that needs to be removed before it disrupts protein synthesis/repair pathways and DNA replication (more algae knowledge for you!).

If it has a short enough residence time to work with algae later on, but long enough to kill everything initially in the seawater, then that sounds rather good. I'll definitely look into this!

[Arkcon]

I'm late to this discussion, but I did want to add my experience:  When culturing algae in college, the procedure was to collect ocean water, and store it in a dark tank.  Before use, it was steam autoclaved to kill all spores before being used to culture algae.  I asked why it was done this way, and the professor said there was no way to account for all micronutrients in synthetic seawater, and yes, dormant spores had to be killed before use.   We never experienced any precipitation after autoclaving, nor failure of algae to grow.

[Borek]

Note: commercial hydrogen peroxide is usually acidified with a phosphoric acid that stabilizes the solution. That means you will both lower pH of the sea water and add a nutrient. Doesn't have to be a problem - but you already didn't  expect problems from sodium hypochlorite.

[curiouscat]

Note: commercial hydrogen peroxide is usually acidified with a phosphoric acid that stabilizes the solution.

Non-standard unstabilized grades are available albeit expensive. I don't know the size of the associated risks.

[opsomath]

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

My guess is that you're seeing precipitation of magnesium or calcium hydroxide, since hypochlorite is always a very basic solution. Do you know what the Mg(II)/Ca(II) concentrations of your NSW is?

What kind of algae will you be growing? I work with some biologists who grow microalgae in seawater all the time, so I can ask some real experts about their means of sterilization.