Attempts at growing hydrogen-oxidizing bacteria

[gerrit]

On 20 December I attempted to grow some hydrogen-oxidizing bacteria using a sample I gathered from the sea near our house. I used the following experiment setup:

• macro nutrient solution as described in HOB Culture Medium Ingredients - #7 by Martin
• 1mL/L Reef Trace micronutrient solution added
• Electrolysis intensity set at 10%
• CO2 sparging setup and experiment profile (sparge CO2 for 10s every hour) as per CO2 Sparging | AMYBO
• temperature set to 30 degrees Celsius
• inoculated with around 0.5ml of the saltwater sample

Here are the results, stopped at around 20 hours:

I increased the temperature to 35C around the 18-hour mark, but that did not have any measurable effect. OD was close enough to zero that it didn’t seem like anything was growing.

On 26 December I gathered another sample, this time from the woods on a hill near our house, and just downstream from a place called Martin’s Pond, which I thought was ideal

Experimental setup:

• same as above, but with temperature set to 20 degrees Celsius
• Electrolysis set to 20%
• inoculated with around 0.5mL of freshwater sample

Here are the results:

This time I ran the experiment for around 45 hours. Again, not much happened I increased the temperature to 25C about halfway through the experiment, but not much changed.

So far I’m assuming that neither of my water samples contained any hydrogen-oxidizing bacteria, but maybe someone (@Martin?) can tell me if there are any issues with my experimental setup. Let me know if there’s something I forgot to share.

[Martin]

Hmm, interesting. Let me confirm if I’m interpreting the data correctly. In the first experiment, you mentioned stopping it at around 20 hours, but I see readings extending to ~75 hours. You also noted raising the temperature to 35°C around the 18-hour mark, which aligns with a spike in the data; however, it seems to drop back to 30°C shortly after, was the drop after termination?

While the OD remains low, those step changes in OD are intriguing—do you have any thoughts on what might be causing them?

Regarding your seawater sample, I’m not sure we can rule out the presence of HOB just yet. The nutrient solution’s ionic strength might have been much lower than seawater, and adding just 0.5 ml of the sample could have caused a rapid osmotic shock, leading to cell lysis. If you decide to revisit this, it might be worth maintaining a consistent ionic strength or total dissolved solids (TDS). That said, watch out for chlorine production during seawater electrolysis, as it could disinfect the sample.

Did you follow the technique described in your Collecting Samples post? To mitigate the potential effects of ionic or temperature shock, it might help to start the experiment at the sample’s original temperature and gradually transition to your target growth conditions.

Another approach to minimise shock might be adding nutrients directly to the sample, rather than diluting it. For instance, you could test pure sample water in the Pioreactor vial, then mix sample water with minerals in a nutrient solution, gradually adding it throughout the experiment.

Of course, it’s entirely possible that the samples simply lacked HOB. You might find better results with soil or sediment samples, which are often richer in microbial diversity.

Given that you have multiple Pioreactors it may be worthwhile setting up a control, in which you use the same sample but heat sterilise it. Call me an optimist, but your ‘Take 2’ experiment did seem to have a little OD increase (possibly until a little after the point of temperature increase) comparison of the two may help show biological growth.

[gerrit]

I forgot to add a picture of what the vial looked like after completing the second experiment:

Looks like there may have been a very small amount of biofilm growing on the cathode? Or am I being too much of an optimist now?

@Martin

was the drop after termination?

Yes, the experiment itself was only running for 20 hours - doesn’t seem like I can truncate the part where it wasn’t actually running but still took readings.

@Martin

While the OD remains low, those step changes in OD are intriguing—do you have any thoughts on what might be causing them?

Noise? For the first experiment I also had the electrodes wired the wrong way for a minute or two at the beginning of the experiment, so there may have been a small amount of debris from the electrode in there.

@Martin

If you decide to revisit this, it might be worth maintaining a consistent ionic strength or total dissolved solids (TDS).

Good call. I’ll measure TDS in future (maybe pH too).

@Martin

Did you follow the technique described in your Collecting Samples post?

The seawater sample was collected from the waves, as it was pouring down with rain at the time and it was high tide. Maybe I’ll collect some sediment during a calm low tide next time. The freshwater sample was just the water itself - I tried collecting sediment below the water but the soil was pretty compacted.

@Martin

Another approach to minimise shock might be adding nutrients directly to the sample, rather than diluting it. For instance, you could test pure sample water in the Pioreactor vial, then mix sample water with minerals in a nutrient solution, gradually adding it throughout the experiment.

That’s an interesting idea - I guess you could do this manually or with the peristaltic pumps.

@Martin

Given that you have multiple Pioreactors it may be worthwhile setting up a control, in which you use the same sample but heat sterilise it.

Unfortunately I only have one platinum-coated anode at the moment, otherwise I could’ve utilised both Pioreactors.

I did a third experiment this past week with a sample I collected from a pond in another wood on 2 January. Experimental setup:

• same as oreviously, but with temperature set to 30 degrees Celsius
• Electrolysis set to 30%
• inoculated with around 3mL of freshwater sample

Again, nothing really happening after running it for about 70 hours. This time the sample was taken from a small pond with decaying leaf matter.

[gerrit]

Just tested the TDS and pH of my most recent experiment:

Test	TDS (ppm)	pH
Nutrient solution	1842	7.6-7.8
Water sample	115	6.6-6.8
Innoculated vial after experiment	1172	7.6-7.8

So it’s possible that osmotic shock occurred. Finding an HOB that tolerates osmotic stress pretty well would be cool! For my next experiment I think I’ll attempt a soil/sediment sample, as there’s probably a higher chance to find HOB in there.

[Martin]

@gerrit

For my next experiment I think I’ll attempt a soil/sediment sample, as there’s probably a higher chance to find HOB in there.

Yes, I think soil/sediment probably makes most sense. I’m thinking about getting a large enough sample that we can drain water and test it’s ionic strength to avoid osmotic shock.

@gerrit

Looks like there may have been a very small amount of biofilm growing on the cathode? Or am I being too much of an optimist now?

I can’t see in this photo if its bubbles or what could be biofilm. Is it just me or is your cathode bolt looking rather darker than the portion above the cap?

Also I notice the ‘rusty’ discolouration on your anode. I have just checked the specs of my original bolts and they were A2 like your 304SS bolts, so I have bought a set of these A4 316 bolts as I couldn’t find any 316L’s. I should probably have gone up a grade to duplex, the prices aren’t unreasonable, but M6 is the smallest, and the options I can find are either full thread hex head or cap head without full thread.

@gerrit

I guess you could do this manually or with the peristaltic pumps.

I was definitely meaning with the persitaltic pumps. Start with pure sample in the vial and operate in turbidostat mode feeding in your nutrient solution. If you didn’t see any rise in OD, you’d want to do a small manual pump activation to get the trace nutrients in - GPTo1 reckons you’d only want to wait 1-2 hours for this (which seems way too rapid IMHO) and is worried that the HOB may starve if left without trace nutrients for 6h.

[gerrit]

@Martin

Is it just me or is your cathode bolt looking rather darker than the portion above the cap?

That’s just the lighting I think. The part that gets submerged has corroded a bit, so I’ll replace it with a new bolt before my next experiment.

@Martin

Also I notice the ‘rusty’ discolouration on your anode.

Yes, it started getting worse when I had the LED intensity set to 20%, which is why I have gone back to 10% intensity. These three experiments total up to running it for around 135 hours, so it will be interesting to see how long it still holds up. It will also be interesting to see if using A4/316 bolts would prevent it from happening. I’m looking forward to seeing your experimental results!