1 The Effect of Coniferous Tree Soil on Bacterial Growth Kirstyn Heino and Aleksandra Mijovic Abstract We tested whether soil in areas where there are coniferous tree populations will have more acidic soil and less bacterial growth than in areas with no coniferous tree growth. We found that there was more bacterial growth in soil taken from deciduous trees compared to that of soil taken around coniferous trees (P= 0.01). Keywords: Soil ph, Bacterial Growth, Coniferous and Deciduous Trees Introduction Soil is a very rich structure composed of many materials that influence the growth of microorganisms and their activity (Angers & Caron, 1998). Any changes to the soil s ph, water quantity and nutrients can effect plant growth and the microorganisms that live in the soil (Angers & Caron, 1998). In particular, soil bacterial communities are influenced greatly by ph (Chamier, 1987). Soil ph is a measure of how acidic or basic the soil is. The ph is determined by how many hydronium ions (H + ) there are in the soil (Skyllberg et al, 2001). ph can range from 0 to 14, with 7 being neutral. Any ph value below 7 is considered to be acidic and any ph value above 7 is considered to be basic. The ph of soil is very important for plant growth because it controls the chemical forms of nutrients in the soil (Skyllberg et al, 2001). In addition, the diversity of bacteria in soil is determined greatly by the ph of soil. Most bacteria will live in soil that has a ph range between 6.5 and 7.5 (Skyllberg et al, 2001). However, bacteria that are acidophiles will live in acidic soils that can range from 2 to 5. Acidophilic bacteria are able to live in these acidic conditions because they are able to continuously transport hydrogen ions out of their cells (Skyllberg et al, 2001). On the other hand, bacteria that require very basic environments are called
2 alkaliphiles. These bacteria will live in soils that have a ph range from 8 to 11. They are able to live in a basic environment because they transport hydrogen ions into their cells (Skyllberg et al, 2001). However, the most common is neutrophilic bacteria. Neutrophilic bacteria will grow in neutral soil, which is around a ph of 7 (Skyllberg et al, 2001). Soil ph is important because it controls how much nutrients, carbon and essential trace elements are available. In addition, bacteria in soil are essential to plant growth in various ways. Bacteria help with many chemical processes, including nutrient transformations and degradation of organic matter (Skyllberg et al, 2001). For that reason, any sudden changes to a specific soil s ph can affect bacterial growth. If the plant relies on bacteria for nutrient absorption, such as nitrogen fixation in leguminous plants, that will directly affect the plants growth. Soil ph is different depending on the type of surrounding vegetation, such as deciduous and coniferous trees. Pine trees are a type of conifer that has needles instead of the leaves and they grow best in acidic soil (Skyllberg et al, 2001). The acidity of soil surrounding pine trees is said to be caused by the pine tree needles that drop to the ground. However, it is caused by the amounts of nutrients that are taken up by the pine tree itself (Skyllberg et al, 2001). When nutrients are taken out of the soil, the soil can become acidic, making it less suitable for neutrophilic or alkaliphilic bacteria to grow. In addition, pine tree needles are slightly acidic, which can make the soil acidic. Skyllberg et al (2001) studied the effects of ph in acidic soils. The authors discovered that in areas where there was coniferous tree growth, such as a temperate forest environment, pine trees grew best in acidic soil (Skyllberg et al, 2001). As the needles decompose, they returned acid to the soil (Skyllberg et al, 2001). As a result, the ph of the soil surrounding coniferous trees decreased, thus becoming more acidic and creating less bacterial growth. They also studied soil nutrients and found that nutrients were lower in areas with acidic soil, which also contributed to why there was less bacterial growth. Factors including the soil s fertility, moisture, temperature, aeration, light and soil organic matter, also contribute to the activity and abundance of soil microorganisms (Skyllberg et al, 2001).
3 However, deciduous trees grow best in neutral soil (Angers & Caron, 1998). When deciduous trees lose their leaves they lose nutrients. Coniferous trees will usually adapt to low nutrient levels in the soil, while deciduous trees cannot. Since deciduous trees grow best in neutral soil, we can infer that there may be more bacterial growth, just because the majority of soil bacteria grow best in neutral soil. Based on this research, we hypothesized that soil surrounding coniferous trees will be more acidic and have less bacterial growth, while soil surrounding deciduous trees will be more basic and have more bacterial growth. If the hypothesis that bacteria growth in coniferous tree soil is less, one would predict it is due to pine needles adding more acid to the soil and causing a lower ph, which may not be optimal conditions for bacteria to grow. Materials and Methods On October 29, 2012, we collected our soil samples at 20 different locations in the Milwaukee Area (Table 1 and 2). We collected 10 pine tree soil samples and 10 non-pine tree soil samples. Samples were taken from horizon A topsoil, of the various sites using a garden shovel and plastic spoons. Samples were placed into Ziploc bags and labeled according to their location. Samples were left outside overnight and then transported to Alverno College in Milwaukee, WI, for testing. To prepare our samples for plating, we gathered two 250 ml beakers to mix our samples, distilled water, ph strips, sterile cotton swabs, and 20 nutrient agar plates that were prepared in advanced, by Alverno College s Microbiology Lab Department. First, using a balance, we weighed out 10 grams of soil and mixed it with 10 ml of distilled water in a beaker to make a slurry. Next, we took the ph strips and recorded the ph of each sample (Table 1 and 2). After the samples were thoroughly mixed, we labeled 20 nutrient agar plates according to soil location and type. Then, we used a plastic 1 ml pipette to drop four drops of slurry onto the plates. We used a sterile cotton swab to gently streak the plates so that the agar was lightly covered with the soil slurry. We placed the lid of the Petri plate ajar to let the samples dry before inverting them for incubation. Each of the samples were prepared following these steps.
4 After all the plates were completely dried, we inverted them and placed them into a 35 C incubator for 24 hours before results were read and recorded. After 24 hours the plates were placed into a cold room for 24 hours before results were read. This was done to stop the growing process. The bacteria were growing rapidly and we did not want confluent growth on the plates. Bacterial counts were determined by counting the number of colonies present in a quarter of the plate and multiplying that number by four. We eliminated soil clumps on the plates or areas that were not covered by the sample slurry. The counts from each sample group were averaged, and then the bacterial plates were autoclaved and discarded. Data was analyzed using a 1 tailed, type 2 dependent T-Test on Excel for Windows Results Bacterial growth on agar plates with non-pine tree soil was significantly greater than bacterial growth on agar plates with pine tree soil (P=0.01). The mean of the pine tree soil bacterial colonies was with a standard deviation of The mean of the non-pine tree soil bacterial colonies was with a standard deviation of The mean of the pine tree soil ph was 8.5 with a standard deviation of The mean of the non-pine tree soil ph was 5.8 with a standard deviation of
5 Table 1. Record of pine tree soil samples location, ph, and average number of bacterial colonies found on plates Soil Sample Location of Sample ph of soil Number of Bacterial Pine sample Colonies Boerner Drive Hales Corners, WI Boerner Drive Hales Corners, WI Boerner Drive Hales Corners, WI South 92 nd street, Hales Corners, WI South 92 nd street, Hales Corners, WI Seminary Woods, St. Francis WI Seminary Woods, St. Francis, WI Lenox, Milwaukee, WI backyard Lenox, Milwaukee, WI backyard Crawford, St. Francis, WI, backyard (Blue Spruce) Table 2. Record of non-pine tree soil samples location, ph, and average number of bacterial colonies found on plates. Soil Sample Pine Location of Sample ph of soil sample Number of Bacterial Colonies 1 Twelve Bridges Condominiums (backyard), Greenfield WI (Oak) 2 Twelve Bridges Condominiums (backyard), Greenfield WI (Oak) 3 Twelve Bridges Condominiums (backyard), Greenfield, WI (oak) 4 Wood Haven Bridges (backyard), Greenfield, WI 5 Wood Haven Bridges (backyard), Greenfield, WI 6 Seminary Woods, St. Francis WI (Oak) Seminary Woods, St. Francis, WI (Maple) 6 1,025 8 Lenox, Milwaukee, WI backyard (under pear tree) 9 Lenox, Milwaukee, WI backyard (under Maple Tree) 5 1,
6 ph Number of Bacterial Colonies 10 Crawford, St. Francis, WI, backyard (Under Birch Tree) Pine Soil Type Non-Pine Figure 1. Mean (+/- S.D.) of the number of bacterial colonies in pine and non-pine tree soil Pine Soil Type Non-Pine Figure 2. Mean (+/- S.D.) of the ph values in both the pine and non-pine tree soils
7 Discussion The data did support the hypothesis that soil in areas where there is coniferous tree growth will have less bacterial growth than in areas with no coniferous tree growth. The data was similar to the findings of Skyllberg et al (2001), who found that coniferous tree soil can have less bacterial growth because the pine tree needles make the ground more acidic, which is usually not an optimal ph for bacteria to grow. Bacteria will most likely grow at a neutral ph. Although the hypothesis was supported, there were limitations to this experiment. Based on our data, there was more bacterial growth in the soil surrounding deciduous trees, but the ph values for each location for both soil types was very similar. The soil ph of the coniferous trees seemed to be more basic, while the deciduous tree soil was more neutral. This could be because the soil that we sampled for this experiment was exposed to a lot of water because days prior to experimentation, it was very rainy. This could have caused leaching or other disruptions to the soil. During experimentation we checked the ph of each soil sample twice, to be sure that we had an accurate reading. Both of the soil types could have had very similar readings because the ph strips could have been old or there could have been fertilizer in one of our soil samples that we were unable to identify. In addition, we also changed our incubation from 48 to 24 hours because our bacterial growth was high after 24 hours. The increased bacterial growth after 24 hours might be due to contamination, but we performed sterile procedure while inoculating the plates. If we were to repeat this experiment in the future we would have collected soil samples are different locations. We would collect our samples at different sites far away from the city, in some local forest were we knew for sure that the soil was natural, without any fertilizers being added, which could interfere with our ph readings and or bacterial colony count. We would have also tried a different method for plating. Some of the plates were easier to count than others. If we had incubated the plates for longer than 24 hours, there might be confluent growth on our plates, making it very difficult to discern which soil type had the most growth and the least.
8 Through these improvements, we believe we would have been able to find more information about soil ph and bacterial growth. In particular, we would like to learn more about the types of bacteria that live in Wisconsin soils. This further research will help us gain insight into what are optimum conditions for different soil bacterial communities and how soil ph can fluctuate just by the vegetation it surrounds.
9 LITERATURE CITED Angers, D.A. and Caron, J. (1998). Plant induced changes in soil structure: Processes and feedbacks. Biogeochemistry, 42, Retrieved September 10, 2012, from JSTOR Database. Chamier, A. (1987). Effect of ph on Microbial Degradation of Leaf Litter in Seven Streams of the English Lake District. Oecologia, 71(4), Retrieved September 11, 2012 from JSTOR Database. Fierer, N. and Jackson, R.B. (2005). The diversity and biogeography of soil bacterial communities. Proceedings of the National Academy of Sciences of the United States of America, 103, 3, Retrieved September 9, 2012, from JSTOR Database. Skyllberg, U., Raulund-Rasmussen, K. and Borggaard, O.K. (2001). ph buffering in acidic soils developed under Picea abies and Quercus robur-effects of soil organic matter, absorbed cations and soil solution ionic strength. Biogeochemistry, 56, 1, Retrieved September 10, 2012 from JSTOR Database.