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.