Using pH and Cell Growth to Measure the Urease Activity of Sporosarcina pasteurii in Stuart’s Urea Broth with Bromothymol Blue

Presenter Information

Olivia Wright
Terri N. Ellis

Faculty Sponsor

Dr. Terri Ellis

Faculty Sponsor College

College of Arts and Sciences

Faculty Sponsor Department

Biology

Location

SOARS Virtual Conference

Presentation Website

https://unfsoars.domains.unf.edu/2021/posters/using-ph-and-cell-growth-to-measure-the-urease-activity-of-sporosarcina-pasteurii-in-stuarts-urea-broth-with-bromothymol-blue/

Keywords

SOARS (Conference) (2021 : University of North Florida) – Archives; SOARS (Conference) (2021 : University of North Florida) – Posters; University of North Florida -- Students -- Research – Posters; University of North Florida. Office of Undergraduate Research; University of North Florida. Graduate School; College students – Research -- Florida – Jacksonville – Posters; University of North Florida – Undergraduates -- Research – Posters; University of North Florida. Department of Biology -- Research – Posters; Honorable Mention Award

Abstract

Honorable Mention Winner

Sporosarcina pasteurii can perform microbially-induced calcite precipitation (MICP)—when bacteria hydrolyze urea and precipitate calcium carbonate crystals. This has potential applications in biocementation, though there are many barriers to implementation. One way to overcome these barriers is to measure cell growth and urease activity of S. pasteurii. Because urea hydrolysis increases the surrounding pH, urease activity could be measured using Stuart’s Urea Broth with the pH indicator bromothymol blue. Additionally, a standard curve was generated to quantify pH change, and cell growth of S. pasteurii was measured before and after urea hydrolysis. It was found that that higher concentrations of bacteria resulted in higher pH values and a faster pH increase, indicating higher urease activity. It was also found that cell growth declines during urea hydrolysis, and the growth media seems to influence this decline. These findings suggest that higher cell concentrations should be used in MICP applications to produce the highest urease activity and that the cell growth of S. pasteurii may not increase with urease activity.

Comments

Audio Presentation Transcript:

Hello everyone, my name is Olivia Wright, and I am presenting my research topic, “Using pH and Cell Growth to Measure the Urease Activity of Sporosarcinia pasteurii in Stuart’s Urea Broth with Bromothymol Blue”.

[Introduction]
So, Sporosarcina pasteurii is a bacterium that can perform MICP—microbially-induced calcite precipitation. This happens when the bacteria hydrolyze urea, and the carbonate ions from that process react with environmental calcium to precipitate calcite. This process has potential in biocementation applications to stabilize soils and fill in concrete cracks. However, there are barriers to implementing this process on a large scale, and one way to overcome some of these barriers is to know more about the urease activity and the cell growth of S. pasteurii and be able to measure it.

[Objectives]
The objectives of this experiment were to measure S. pasteurii’s urease activity as pH increase, use a standard curve to quantify pH change, and determine S. pasteurii’s cell growth during urease activity. Because urea hydrolysis raises the pH of the environment, we used pH change to measure urease activity, and that was determined by using Stuart’s Urea Broth with the pH indicator bromothymol blue.

[Methods]
After growing S. pasteurii in an incubator at 37 degrees Celsius, we placed the bacteria in Stuart’s Urea Broth and suspended enough cells to get the optical density to 0.6. To measure urease activity afterwards, different concentrations of the bacteria were placed into blank tubes of Stuart’s Urea Broth, then placed into a well plate, and read in a spectrophotometer. This plate was read every hour for 5 hours, and at 24 hours, which was considered maximum urease activity. To measure cell growth, different concentrations of the bacterial broth from before were placed into blank tubes of Stuart’s Urea Broth. These tubes were incubated for 24 hours at 30 degrees Celsius. Bacterial cells from each concentration were plated onto BHI plus 2 percent urea plates both before and after incubation. These plates were then incubated for 48 hours at 37 degrees Celsius. This entire process was performed with both Stuart’s Urea Broth and BHI + 2% urea broth.

[Results]
After doing the urease activity assay and using the standard curve to quantify pH change, the results were plotted out onto a line graph, seen in Figure 2. The graph shows that the highest concentration of bacteria—500 microliters—showed the fastest increase in pH, and showed the highest increase in pH, more than the other concentrations. This indicates that a high concentration of cells results in higher urease activity.
Next, we looked at the pH of the solution before incubation—at zero hours—and after 24-hour incubation, seen in Figure 3. At 24 hours was considered the bacteria’s maximum urease activity. This graph shows that the highest concentration of bacteria—500 microliters—showed the largest difference in pH over 24 hours and reached the highest pH after 24 hours. This indicates that a high concentration of bacterial cells results in the most urease activity within 24 hours, compared to lower concentrations.

[Results con’t]
Lastly, we looked at cell growth over 24 hours, which is seen in Figure 4. One thing we noticed was that despite being dark blue which indicates urease activity, the Stuart’s Urea Broth tubes were not turbid. However, some of the BHI + 2% urea tubes were turbid. This difference in cell growth in the broth could have had an effect on the cell growth on the plate 24 hours later, as indicated by the bottom pictures in Figure 4. At the lowest concentration of bacteria, in Stuart’s Urea Broth, there was no cell growth seen 24 hours later, while for BHI broth over here, there was slightly less cell growth seen 24 hours later. In both cases, cell growth seemed to have declined over 24 hours, but declined more with the Stuart’s Urea Broth than with the BHI broth.

[Conclusion]
In conclusion, urease activity was measured as the change in pH and could be quantified using a standard curve. Higher concentrations of bacteria not only had faster urease activity, but also had a higher urease activity over 24 hours. Regarding cell growth, a slight decline was seen after 24 hours of growth in BHI broth, while a large decline in cell growth was seen after 24 hours of growth in Stuart’s Urea Broth. To explore more of S. pasteurii’s urease activity and cell growth, future experiments can include refining the pH standard curve for more accurate pH measurement and determining the exact maximum urease activity for the different bacterial concentrations, since the peaks happen somewhere between 5 hours and 24 hours. Other future directions can include determining cell growth in BHI plus 2 percent urea broth and quantifying bacterial growth over time.

[Acknowledgements]
I would like to acknowledge that funding from UNF’s Transformational Learning Opportunity Grant was used to support this research. Additionally, I would like to thank Michael Durnin and Dr. Terri Ellis for their past work on this subject.

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/