The Effect of Coniferous and Deciduous Soil on Bacterial Growth in Jackson Park
Katrina Teresi, Melissa McLean, Jamie Hoernke, and Anna Botiva
††††††††††† We tested whether there would be fewer colony forming units of bacteria in acidic soil near coniferous trees compared to more neutral soils near deciduous trees. Most species of bacteria require neutral pH levels for growth and maintenance of other biological processes. There was a statistically significant difference between the pH levels of coniferous and deciduous soils (P = 9.4 x 10-6) and the number of colony forming units encountered on each condition (P = 5.8 x 10-3). These findings are consistent with current research in the field of soil biology.
Keywords: pH, acidiphile, neutrophile, alkaliphile, coniferous, deciduous.
††††††††††† Soils consist of a wide range of microbes that aid in biological processes such as nutrient cycling of Phosphorous, Nitrogen, and Carbon, and converting organic matter into energy for plants (Fitter, et al, 2005). This makes the microbial community a critical part of Earthís ecosystems. There are many characteristics of soil that affect whether or not certain types of bacteria can grow comfortably in a given area (Barcenas-Moreno, et al, 2011). One of them is moisture content. All bacteria need at least some water to grow and maintain their basic biological functions. Another factor required for bacterial growth is nutrient content (Eskelinen, et al, 2009). Nutrients are required for the maintenance of cell structures and metabolic pathways. Individual microorganism species also have an optimal temperature at which they can grow and survive. One of the biggest influences of bacterial growth and colony formation is environmental pH (Fierer & Jackson, 2006).
††††††††††† The concentration of H+ ions in an area is what defines measured pH (Londo, et al, 2006). The more H+ ions that are present, the more acidic a substance tends to be. The pH scale has a range of 0 to 14, with 7 representing chemical neutrality. Some bacterial species have adapted to survive under very specific pH ranges (Fierer & Jackson, 2006). In addition to bacterial growth, soil pH is critical for the growth of plants because it determines which chemical form that available essential nutrients can take (Eskelinen, et al, 2009). Because of this, any sudden changes in soil pH levels can drastically affect the ability of certain plant species to grow. Essential nutrients for plant growth, such as Phosphorous, are most readily available in slightly acidic soils (pH 6.5) (Skyllberg, et al, 2001). A pH range of approximately 6 to 7 is where most nutrients are soluble and readily available for uptake by the plant. Factors that influence soil acidity include the types of plants and microorganisms that grow there, rainwater leaching away more basic ions (like Calcium), and the formation of acids from the decay of organic matter or the oxidation of artificial fertilizers (Eskelinen, et al, 2009).
††††††††††† Some bacteria have adapted to grow well in acidic environmental conditions (Fierer & Jackson, 2006). However, a vast majority of species thrive near a neutral pH, and their intracellular environments will be within one unit of pH 7. These bacteria are known as neutrophiles. The few bacteria that can live in acidic pH ranges between 2 and 5 are called acidiphiles. The acidiphiles can survive such extreme conditions because they have adapted through random genetic mutations to have efficient proton pump systems, which can take excess H+ ions and move them out of the intracellular environment (McGowan, et al, 1994). This raises their internal pH back up to the more neutral levels required for most of their biological functions, such as metabolism. Alkaliphiles, bacteria that live above a pH of 8, have proton pumps that work in the opposite direction, moving protons into their intracellular environment in order to lower pH. True alkaliphiles are rare in soil samples, because soil pH does not often rise very far above 8 (Fierer & Jackson, 2006).
††††††††††† Some plants have also adapted to grow particularly well in acidic soils. These include coniferous trees, like various pine species (Ovington, 1953). Pine trees are capable of leaching more nutrients from the surrounding soil than deciduous trees, and this leads to an acidic soil pH (Skyllberg, et al, 2001). While the needles from pine trees are slightly acidic, they contribute only minimally to low soil pH when they fall off the tree and decompose. This reduction in nutrient available and low pH in areas surrounded by coniferous tree growth limits the number of bacterial species that can thrive there (Fierer & Jackson, 2006). Deciduous trees grow preferentially in neutral soil, and have not adapted to tolerate low pH values (Ovington, 1953). Areas surrounding deciduous trees tend to be more nutrient-dense because when their leaves fall, they decompose and release a broader array of micronutrients compared to pine needles (Skyllberg, et al, 2001). These characteristics should theoretically allow for the largest category of bacteria (neutrophiles) to grow in soil surrounded by deciduous trees. According to Fierer and Jackson, soil pH is one of the best indicators of both diversity and richness of bacterial species.
††††††††††† We hypothesized that there would be fewer colony forming units (CFUs) of bacteria and a lower pH in the soil surrounding coniferous pine trees and more CFUs and a higher pH in soils surrounding deciduous trees. We based this hypothesis on the affect that pine trees have on lowering soil pH, and the limited ability of most bacteria to live at acidic pH values. This hypothesis is falsifiable in the event of increased bacterial growth or elevated pH in the coniferous soil condition, decreased growth or acidic pH in the deciduous soil condition, or if there is no significant difference between conditions.
Methods and Materials
††††††††††† The samples for this experiment were collected on October 19, 2013 at approximately 1500 from Jackson Park (37th Street, Milwaukee, WI). 20 soil sites were selected at this location, 10 at the base of coniferous trees, and 10 at the base of deciduous trees (Tables 1 and 2). The coordinates for all sampling sites were obtained using the curb directly north of the 37th Street sign on Forest Home Avenue. An orienteering compass and a Keson 50 m measuring tape †(model number OTR50mm) were used to record exact site locations for both the coniferous and deciduous soil samples (Tables 1 and 2). 20 sterile sample collection cups were labeled (ie. Pine 1, Pine 2, non-pine 1, etc) and brought to the site on 37th street. An egg-sized amount of soil was gathered from each site, placed in the sample collection cup, and returned to the laboratory at Alverno College (3400 S 43rd Street, PO Box 343922, Milwaukee, WI 53234-3922).
††††††††††† 20 separate 50 mL beakers were gathered and labeled in the same manner as the sample collection cups. Approximately 0.1 g of soil was weighed out from each sample container and placed in the corresponding beaker. At this point, 10 mL of distilled water was added to each 50 mL beaker and stirred until a soil slurry was obtained. Litmus paper was used to make a pH reading, and the results of this were recorded (Tables 1 and 2).
††††††††††† 20 nutrient agar plates were obtained and labeled the same way as the sample collection cups and beakers. 20 sterile streaking swabs were also gathered. One swab per sample was used to streak the soil slurry onto the corresponding nutrient agar plate. The plates were then wrapped in parafilm (to further guard against contamination), inverted, and allowed to incubate at 25 C for 48 hours. After the incubation period, colony forming units were counted and recorded for each plate (Tables 1 and 2). The pH and colony forming unit data was analyzed using a 2-tailed, paired T-test in Microsoft Excel 2010.
††††††††††† The difference between pH values for coniferous and deciduous soil types was statistically significant (Fig. 1, P = 9.4 x 10-6). The mean for the coniferous soil groupís pH was 6.84, and there was little variation between values (S.D. = 0.1174). The mean pH for deciduous soils was slightly higher at 7.31, and there was also not a great deal of variation between values (S.D. = 0.1197). The difference between the number of colony forming units that developed in the coniferous and deciduous soil samples was also statistically significant (Fig. 2, P = 5.8 x 10-3). The mean for CFU data was 181 colonies for the coniferous soil agar plates (S.D. = 42.4), and the mean for the deciduous soil agar plates was 261.8 colonies (S.D. = 54.7).
Table 1. Location, pH, and Colony Forming Units for 10 Coniferous Soil Samples. All samples use the curb directly north of the 37th ST sign on Forest Home Avenue in Jackson Park as an origin point.
Table 2. Location, pH, and Colony Forming Units for 10 Deciduous Soil Samples. All samples use the curb directly north of the 37th ST sign on Forest Home Avenue in Jackson Park as an origin point.
Figure 1. Mean (+/- S.D.) pH values for coniferous and deciduous (non-coniferous) soils samples.
Figure 2. Mean (+/- S.D.) number of colony forming units for coniferous and deciduous (non-coniferous) soil samples.
††††††††††† The data supported our hypothesis. Soil samples from test sites with coniferous tree growth had both a lower pH and lower levels of bacterial growth than deciduous soil sites. These results are consistent with the findings of Fierer and Jackson, who uncovered a consistent relationship between the number of colony forming units and soil pH (2006). According to their species abundance curves, low pH soils had low levels of diversity in bacteria species, regardless of what type of ecosystem the soil samples were collected from. Species diversity peaked at a pH of 7, and began to decrease again as soil conditions became more basic. Their results are consistent with the findings of our experiment, which indicated that bacterial growth is lower at low pH and highest at a neutral to slightly basic pH (pH 7-7.4).
††††††††††† In spite of our supported hypothesis, there were several issues in this experiment that could have impacted the final results. While we tried to stay away from the more heavily cultivated areas of Jackson Park, it is still possible that there could have been some chemical runoff from fertilizers used in certain areas. This could have altered the pH of our soil samples, and, if the level of runoff was uneven through the park, could have impacted some of our sampling areas more than others. If this chemical runoff altered the pH, it is possible that the number of bacterial colonies could have been affected as well.
††††††††††† One methodological issue that could have affected our results had to do with the sterile cotton swabs we used to plate the bacteria after making the soil slurries. Each swab likely absorbed a slightly different volume of the water and soil slurry. A higher plated volume of slurry could have held a higher number of bacteria than a lower volume. In the future, it might be better to use a very small volume micropipette to first plate an even amount of the slurry and then streak the plate with a sterile inoculating loop.
††††††††††† One issue we noticed with our coniferous soil agar plates was that many of them had a large amount of what appeared to be a light gray, spongy mold growing alongside the bacteria. This mold species, in order to increase its own growth, could have siphoned off some of the essential nutrients contained in the agar. This would have decreased the amount of available nutrients for the bacteria and potentially limited its growth. If this did happen, it could have artificially decreased the number of colonies we had in our coniferous soil plates compared to the deciduous agar plates (which contained no mold) and produced a statistically significant difference where one did not truly exist. The mold could have been the result of contamination in the lab, or it could have been naturally present in the coniferous soil areas. It would be necessary in future experiments to develop control methods for this and determine the moldís source.
††††††††††† While the influence of the mold could have decreased bacterial growth on the coniferous soil, the actual pHs of the samples could also have increased bacterial growth. Some of the coniferous soil samples in this experiment were perhaps closer to neutral than truly acidic pH values (2-5) that acidophilic bacteria prefer. The average pH of the coniferous soil plates was well out of the pH 2 to 5 range with an average of 6.84. This could have provided optimal conditions for a wider array of bacteria to grow on these plates than one would typically expect in more truly acidic soil conditions. The deciduous soilís pH average was only slightly higher at 7.31. It is possible that rainwater reduced differences in natural pH by evenly spreading any chemical substances on the group. Also, it is possible that the coniferous and deciduous trees that we selected for our soil samples were too close to one another. Close proximity could mean that things like pine needles or other types of deciduous leaf litter made it into the areas where the opposite type of soil condition was located. In future experiments, it would be interesting to separate our sampling sites by greater distances and compare results to see if it made a difference in pH values. It would also be interesting to attempt to identify different bacteria species to see if the actual pH values at these locations impacted the microbial diversity in the area.
Barcenas-Moreno, G., Rousk, J., Baath, E. (2011). Fungal and bacterial recolonisation of acid and alkaline forest soils following artificial heat treatments. Soil Biology and Biochemistry, 43: 1023-1033. Retrieved from ScienceDirect on September 15, 2013.
Eskelinen, A., Stark, S., Mannisto, M. (2009). Links between plant community composition, soil organic matter quality and microbial communities in contrasting tundra habitats. Oecologia, 161: 113-123. Retrieved from JSTOR on November 3, 2013.
Fierer, N., Jackson, R. (2006). The Diversity and Biogeography of Soil Bacterial Communities. Proceedings of the National Academy of Sciences of the United States of America, 103(3): 626-631. Retrieved from JSTOR on November 3, 2013.
Fitter, A. Gilligan, C., Hollingworth, K., Kleczkowski, A., Twyman, R., Pitchford, J. (2005). Biodiversity and ecosystem function in soil. Functional Ecology, 19(3): 369-377. Retrieved from JSTOR database on November 3, 2013.
Londo, A., Kushla, J., Carter, R. (2006). Soil pH and Tree Species Suitability in the South. Southern Regional Extension Forestry, 2:1-4. Retrieved from http://www.Isuagcenter.com/NR/rdonylres/3E784F3F-Ob26-44E9-958D-3C31CB911EFD/69963/SoilpH.pdf
McGowan, C., Cover, T., Blaser, M. (1994). The proton pump inhibitor omeprazole inhibits acid survival of Helicobacter pylori by a urease-independent mechanism. Gastroenterology, 107(5): 1573-1578. Retrieved from ScienceDirect on November 5, 2013.
Ovington, J., (1953). Studies of the Development of Woodland Conditions under Different Trees: Soils pH. Journal of Ecology, 41(1): 1573-1578. Retrieved from JSTOR on November 3, 2013.
Skyllberg, U., Raulund-Rasmussen, K., Borggaard, O. (2001). pH Buffering in Acidic Soils Developed Under Picea abies and Quercus robur: Effects of Soil Organic Matter, Adsorbed Cations and Soil Solution Ionic Strength. Biogeochemistry, 56(1): 51-74. Retrieved from JSTOR database on November 3, 2013.