{"id":310,"date":"2025-08-22T21:33:30","date_gmt":"2025-08-22T21:33:30","guid":{"rendered":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/?page_id=310"},"modified":"2026-03-12T16:16:57","modified_gmt":"2026-03-12T16:16:57","slug":"simulation-of-microbial-dynamics-and-differentiation-in-groundwater-using-drip-flow-reactors-and-multi-condition-plate-setups","status":"publish","type":"page","link":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/simulation-of-microbial-dynamics-and-differentiation-in-groundwater-using-drip-flow-reactors-and-multi-condition-plate-setups\/","title":{"rendered":"Simulation of Microbial Dynamics and Differentiation in Groundwater Using Drip Flow Reactors"},"content":{"rendered":"
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Simulation of Microbial Dynamics and Differentiation in Groundwater Using Drip Flow Reactors<\/p>\n

A Note from the Interns:<\/h1>\n

We had a lot of fun over our eight weeks at ISB. We made friends with the other interns, learned how to set up the reactors and were responsible for taking their daily measurements, learned all sorts of laboratory skills, and learned how to use R to code. It was incredible to have had the opportunity to co-run the experiment and be trusted in making decisions surrounding reactor flow rate and even deconstructing them on our own.<\/p>\n

We would like to think Dr. Nitin Baliga for hosting us in his lab over the past eight weeks and giving us this opportunity, and Claudia and the rest of the systems education experience team for guiding us through the internship and teaching us invaluable leadership skills. A huge thanks to Dr. Jacob Valenzuela and Dr. Chris Deutsch for guiding us through this experiment and teaching us all that we learned, we couldn\u2019t have done it without them.<\/p>\n

Project Background<\/h1>\n

The subsurface is a huge biosphere for microbial life. This experiment modeled a test site in Oak Ridge, Tennessee. Years ago during the Manhattan Project, this area was filled with heavy metals and nitric acid waste, resulting in very high levels of nitrate in the soil. The goal with our project was to see how the nitrate from this site impacts the subsurface microbial community behavior and composition. This project was particularly unique because unlike many studies where you can look at a site or take samples right from the test site, this is underground so you can\u2019t take repeated samples of the same spot without perturbing the environment and thus disrupting the communities.<\/p>\n

This experiment simulated the saturated zone, which allowed studying both the attached communities of microbes which is the dominant phase, and the planktonic communities which float in the liquid.<\/p>\n

More broadly this experiment could be applied to any sort of sub-surface research and the results from this specific experiment will reveal more about how microbes behave when excess nitrate from fertilizers such as in agricultural areas is input into the soil. Understanding these subsurface conditions will hopefully allow better management in the future.<\/p>\n

Drip-Flow Reactors<\/h1>\n

At ISB, Alice Cottrell-Steen and Elizabeth Gabbay worked under researchers Drs. Jacob Valenzuela and Chris Deutsch to study microbial stratification and metabolic interplay of key biochemical processes from a field-derived enriched groundwater community. They sterily set up four drip flow reactors in the the GC room, which is kept at 30 degrees C. Two reactors were at pH six, and two at pH seven to simulate the acidic conditions found at the field site. Media was pumped into and out of the reactors at a predetermined rate, flowing vertically from top to bottom to simulate the movement of water in the environment, and to ensure the microbes had access to new media. The reactors were injected with the EnCom that was shown above and let incubate for a couple days before the experiment officially started. The reactors also simulated the field site as their sediment and planktonic areas gave the microbes the opportunity to specialize as either attached or planktonic.<\/p>\n

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One thing that was really important to maintain over the course of the experiment was an oxycline throughout the reactor. This forms as microbes use up the oxygen, which is their preferred pathway for energy production, and after the oxygen is all used up, the microbes have to switch to using a different metabolism pathway where they use the nitrogen instead of the oxygen. The graph above shows the dissolved oxygen in milligrams per liter averaged over each week with more recent weeks, being darker lines and older weeks being paler lines. For the most part, the reactors maintained a proper oxycline. A number of other samples were taken as well, which required needle usage to maintain the sterile environment.<\/p>\n

Every day 1mL of gas from the headspace of the reactors was sampled to run through the gas chromatograph to test for nitrous oxide. The presence of nitrous oxide would indicate that the reactors were using the nitrate for energy, but not fully denitrifying it such that it becomes nitrogen gas. It\u2019s important that it gets fully broken into nitrogen gas because nitrous oxide is a greenhouse gas. No nitrous oxide was detected in the headspace over the course of the experiment, which indicates that either the microbes who are metabolizing anaerobically either were able to complete the full denitrification pathway, which is unlikely, microbes could have been working together to complete the denitrification pathway, or the microbes could have been relying on the carbon and amino acids present in the media to metabolize.<\/p>\n

Plate Experiment<\/h1>\n

They also configured 48 well plates at differing pH values, nitrate concentrations, and oxygenation states to further understand the relationship between environmental conditions and microbial growth. Two plates contained sediment to allow analysis of attached communities and two were sediment free to allow analysis of planktonic communities. Alice and Elizabeth took regular optical density measurements of the planktonic plates. They allowed the plate experiment to run for three weeks to give the microbes time to specialize according to their unique condition. Throughout the experiment, they washed the sedimentary plates with Phosphate Buffered Saline (PBS) to ensure that the final measurements only represented microbes attached to the sediment. This experiment was a novel instance of using plates to measure attached microbial growth. At the end of the plate experiment, the two took bulk protein assays to demonstrate the presence of metabolic activity and performed 16S rRNA sequencing to understand the condition preferences of the EnCom.<\/p>\n

Phylogenetic Trees<\/h1>\n

In order to analyze the data they generated, Alice and Elizabeth learned the coding language R to create phylogenetic trees with the 16S sequencing data, which allowed them to visualize the roles and genetic proximity of the members of the enriched community. By identifying which species are more prevalent in the different conditions, we can better understand the specific role they play in subsurface conditions and better predict the impact changes in nitrate and oxygen levels may have on the behavior of microbial communities.<\/p>\n

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After the conclusion of Alice and Elizabeth's internship, Dr. Valenzuela and Dr. Deutsch completed the 16S analysis of the reactors. While Lysinobacillus dominated the reactors, when you examine the microdiversity disregarding Lysinobacillus, the stratification across planktonic and attached communities, between pH 6 and pH 7, and down the oxycline becomes apparent.<\/p>\n

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Final Presentation<\/h1>\n

https:\/\/docs.google.com\/presentation\/d\/1s48Ec-CAJgz3zBztK-BOT6Elp2z-bXoO_AhE666aPDs\/edit?slide=id.g37b68f46c20_1_267#slide=id.g37b68f46c20_1_267<\/a><\/p>\n<\/div>\n<\/div><\/div><\/div><\/div><\/div>","protected":false},"excerpt":{"rendered":"

Simulation of Microbial Dynamics and Differentiation in Groundwater Using Drip Flow Reactors A Note from the Interns: We had a lot of fun over our eight weeks at ISB. We made friends with the other interns, learned how to set up the reactors and were responsible for taking their daily measurements, learned all sorts of […]<\/p>\n","protected":false},"author":108,"featured_media":659,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-310","page","type-page","status-publish","has-post-thumbnail","hentry"],"_links":{"self":[{"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/pages\/310","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/users\/108"}],"replies":[{"embeddable":true,"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/comments?post=310"}],"version-history":[{"count":33,"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/pages\/310\/revisions"}],"predecessor-version":[{"id":726,"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/pages\/310\/revisions\/726"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/media\/659"}],"wp:attachment":[{"href":"https:\/\/baliga.systemsbiology.net\/see-interns\/hs2025\/wp-json\/wp\/v2\/media?parent=310"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}