A Co-Op Micro-cosm? Population Dynamics as Bacterial and Microalgal Species Interaction in Unfavorable Conditions

Armando Flores Author

07/29/2021 Added

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Bioenergy REU presentation about algal and bacterial co-cultures. In addition, we monitor community changes as we applied an external pressure of restricted nutrients.

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[00:00:00.760]Hello, my name is Armando Flores,
[00:00:03.050]and I am presenting the research
[00:00:04.580]I've been working on these last few months.
[00:00:07.920]Before I begin,
[00:00:08.790]I'd like to give some context to my project
[00:00:12.160]and how it relates to the topic of bioenergy,
[00:00:15.940]specifically to what my project works with,
[00:00:19.030]which is Algal Biofuels.
[00:00:21.450]And these algae bioenergy represent a third generation
[00:00:26.795]with several advantages over previous generations,
[00:00:29.490]such as the lower environmental impact
[00:00:31.240]and quicker cultivation times.
[00:00:33.020]However, before we were able to scale it up
[00:00:35.280]to meet growing energy demands,
[00:00:37.860]we need to overcome a few challenges
[00:00:40.570]such as a low natural growth density in algae,
[00:00:44.500]and there are two approaches to this.
[00:00:46.100]One is through genetic modification,
[00:00:47.730]but this is a very difficult process
[00:00:49.750]and has only been completed successfully a few times,
[00:00:53.050]and another approach is with microbial symbiosis.
[00:00:57.330]As you may guess, my project relates
[00:00:59.160]to developing and monitoring symbiosis
[00:01:03.553]between algae and bacteria.
[00:01:05.530]And one of the candidates
[00:01:06.570]that we're particularly interested in
[00:01:09.160]is Methylobacterium which
[00:01:10.990]is a very common plant beneficial organism,
[00:01:15.285]in that it's able to accomplish
[00:01:18.510]a lot of the nitrogen mineralization for plants
[00:01:22.730]and make nitrogen available to them.
[00:01:25.670]As well as produce certain plant hormones
[00:01:28.070]that promote the growth and development of plant cells.
[00:01:31.840]And as you can see by this diagram on the left,
[00:01:34.908]it's proposed that there's an interaction
[00:01:37.080]between this Methylobacterium and species of algae.
[00:01:43.520]In addition to the literature,
[00:01:44.950]we also have some background
[00:01:46.730]with the work done in our lab previously,
[00:01:49.970]with additions of bacteria strains to Arabidopsis plants,
[00:01:55.570]and to their root system, specifically.
[00:01:57.650]And we could see an increase of greenery
[00:01:59.750]as well as root length with the addition of E. Coli
[00:02:03.240]and LP, which is a strain of Methylobacterium.
[00:02:06.620]And then we can also see on the right side,
[00:02:09.189]how different strains of Methylobacterium
[00:02:11.521]encourages the growth of algae in plates.
[00:02:16.120]With this background,
[00:02:17.350]I can describe my projects in which our first project
[00:02:21.770]was focused on studying the interaction
[00:02:23.910]between our model algae, Chlorella sorokiniana,
[00:02:27.600]and different bacteria strains
[00:02:29.010]under restricted nutrient conditions.
[00:02:31.490]And our second project focused on following
[00:02:34.980]how an external pressure affects species diversity
[00:02:37.850]and abundance of wild communities,
[00:02:40.720]with a side objective of isolating
[00:02:43.610]and identifying wild algae and bacteria from the local area.
[00:02:48.060]Moving onto our first set of experiments
[00:02:50.380]in which we conducted Liquid Co-culture on 24-Well Plates.
[00:02:55.640]We started with primary cultures
[00:02:57.290]of our algae and bacteria strains,
[00:03:00.100]and we placed them into the wells
[00:03:02.022]with the red highlighted wells being our experimentals,
[00:03:06.540]in which we sought to test out
[00:03:09.690]this proposed interaction model in the upper right corner.
[00:03:15.550]And hypothesize that neither the LP
[00:03:18.947]nor the Chlorella would do well in these medias.
[00:03:23.130]But when combined together,
[00:03:24.960]they would be able to outperform our positive controls,
[00:03:27.070]which are then the next set of columns.
[00:03:30.150]And looking at the first set of data, at 600nm,
[00:03:33.450]our optical densities of the cultures
[00:03:36.220]show that the Co-culture had the highest density overall
[00:03:40.580]while the experimental LP did not do well.
[00:03:44.260]However, surprisingly, the experimental Chlorella
[00:03:47.200]did very well compared to the positive control.
[00:03:49.950]And you could see how the addition of Peptone
[00:03:54.100]not only encouraged growth of experimental,
[00:03:57.040]but in this bottom graph,
[00:03:59.112]it also encourages the growth
[00:04:00.940]of the positive control, Chlorella.
[00:04:04.650]And we can see this again at 740nm
[00:04:07.320]with our Algal density readings
[00:04:09.772]in which Co-culture had the highest density overall
[00:04:13.590]with the experimental Chlorella
[00:04:16.350]being able to overtake the positive control
[00:04:18.884]in the first set.
[00:04:20.630]But in the second set of experiments, or trials,
[00:04:25.590]the positive control was able to overtake the experimental
[00:04:29.920]with the addition of Peptone.
[00:04:32.290]And then lastly, looking at our florescence of chlorophyll,
[00:04:35.190]you see that our experimental Chlorella
[00:04:37.160]had the highest fluorescence reading overall
[00:04:40.520]with the co-culture and the positive control
[00:04:44.780]having a very similar reading
[00:04:47.247]across the days of the experiment.
[00:04:51.460]And we believe this is contributed to the co-culture
[00:04:54.700]having both bacteria which contained carotenoids,
[00:04:58.710]which block light and the experimentals' high fluorescences
[00:05:02.270]contributed to the stress of the chloroplasts.
[00:05:06.980]In our next set of experiments
[00:05:08.230]we looked at solid co-cultures
[00:05:10.230]in which we utilize an Agar Overlay,
[00:05:13.420]and again, very similar primary cultures.
[00:05:16.640]We mixed our top agar with our algae media
[00:05:21.620]and placed it over the top of bottom media
[00:05:24.820]with the only nitrogen source being Peptone.
[00:05:27.710]And once we allowed that to settle,
[00:05:29.950]we placed in spotted with our bacteria candidates,
[00:05:34.090]which was Methylobacterium strain,
[00:05:36.210]which we call LP and E. Coli,
[00:05:38.500]and then we allowed that to grow over a week.
[00:05:41.410]And as you can see from these results,
[00:05:43.800]the bacterias perform very similarly
[00:05:47.220]in promoting algae growth.
[00:05:49.270]And we believe that's attributed to
[00:05:51.980]a common nitrogen metabolite
[00:05:54.750]created by both bacteria strains.
[00:05:58.130]In our experiments for our community project,
[00:06:02.650]we collected wild samples from a nearby waterway,
[00:06:07.160]which included algae, water, and soil samples.
[00:06:10.610]And we would place that into a flask of BBM,
[00:06:13.300]which placed a pressure on the organisms within the sample.
[00:06:17.920]We would allow that to grow over a course of a week
[00:06:19.890]before streaking it out on a plate of either BBM or MEA,
[00:06:23.490]and then allow the plate to grow and pick up colonies
[00:06:27.770]and isolate them into their own liquid co-cultures.
[00:06:31.180]And we would reap the flask passages
[00:06:34.618]multiple times over the summer.
[00:06:37.240]And starting with the flask passages,
[00:06:39.070]you could see on the left, how as we continued our passages,
[00:06:42.830]the flask became more green.
[00:06:45.580]And with our microscope photos,
[00:06:47.870]you can see how the species diversity decreased,
[00:06:50.731]and it became more algae-centric
[00:06:53.390]with Algal Abundance growing.
[00:06:55.990]And we even found Chlorella species
[00:06:58.550]as identified by their round morphology.
[00:07:02.400]And we see this trend repeat itself
[00:07:03.970]with our plating on the left BBM,
[00:07:06.317]and our right, MEA.
[00:07:08.230]And as mentioned earlier,
[00:07:10.560]the abundance of certain species went up
[00:07:12.580]all the diversity of the overall community went down.
[00:07:16.310]And at the end of our passages,
[00:07:17.840]we found pink bacteria and algae
[00:07:20.780]living close together on the plate.
[00:07:23.320]And we believe this to be our Methylobacterium species
[00:07:27.060]as well as our Chlorella species.
[00:07:30.750]From my first set of experiments,
[00:07:32.050]we saw that algae be denser at the co-culture,
[00:07:35.120]but we're unsure if this is attributed
[00:07:36.720]to the actual symbiosis between the bacteria and algae,
[00:07:39.630]or if it's the pH level for the media,
[00:07:42.230]or the type of nitrogen available.
[00:07:45.210]In the second set of experiments we saw
[00:07:46.800]that both bacteria increased the density
[00:07:49.450]of the algae, but we believe
[00:07:51.860]it's due to a common nitrogen metabolite
[00:07:55.230]produced by both bacterias
[00:07:57.060]rather than the proposed plant hormones,
[00:08:02.640]that Methylobacterium is able to produce.
[00:08:04.710]And in our community passages,
[00:08:06.750]we found that our top picks for bioenergy,
[00:08:09.470]Chlorella and Methylobacterium were found at the end,
[00:08:12.910]from all of our collected samples.
[00:08:15.470]And this is the good sign that these candidates
[00:08:20.280]are good for bioenergy in terms of robustness
[00:08:23.410]and ability to adapt to different environments.
[00:08:28.390]So in conclusion, we found that our hypothesis
[00:08:32.220]on the nitrogen source available to the algae was incorrect
[00:08:37.480]and the other variables may play critical role
[00:08:39.670]in determining algae density.
[00:08:41.870]In addition to that,
[00:08:42.750]we found that our picks of bio energy,
[00:08:45.470]the Chlorella and Methylobacterium, are good symbiosis pair,
[00:08:51.500]as they were found together at the end of the passaging
[00:08:55.040]and from each sample we collected.
[00:08:57.550]So future steps include, IDing our samples
[00:09:01.410]and determining any changes in the community.
[00:09:04.440]Exploring that nitrogen source utilization
[00:09:08.180]by our algae.
[00:09:09.013]Further, follow interactions between the different bacteria
[00:09:12.400]and our algal candidate.
[00:09:14.890]I would like to finish the presentation
[00:09:16.190]with an acknowledgement
[00:09:17.260]of my lab group that I worked with over the summer,
[00:09:19.450]as well as the funding provided by NSF
[00:09:23.010]to proceed with this research.
[00:09:25.327]And to thank you for listening.

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A Co-Op Micro-cosm? Population Dynamics as Bacterial and Microalgal Species Interaction in Unfavorable Conditions

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