In the Laboratory
A Qualitative Experiment To Analyze Microbial Activity W in Topsoil Using Paper and a Handmade Reflection Photometer Julius Kofi Agbeko Teacher Education Division, Accra Teacher Training College, Accra, Ghana Masakazu Kita* Faculty of Education, Okayama University, Okayama, Japan; *
[email protected] Topsoil is the topmost layer of a soil profile (the A horizon) that contains large amounts of bacteria and other microorganisms that help the soil support life. This makes topsoil one of the key components of the environmental chemical cycle (1, 2). Biological activities of microorganisms in the soil cause decay, making the soil rich in nutrients used by plants. Some of the microorganisms in the soil include bacteria, fungi, algae, protozoa, and viruses. Increased microbial activity facilitates the cycling of nutrients from organic matter through their conversion (i.e., following digestion and excretion) into forms readily taken up by plants (2, 3, 4). Most paper—including the blank, white copy paper used here—is made of wood cellulose. To improve the paper’s strength, well-dissolved starch is added to the wet pulp. The large starch molecules cause cellulose chains to adhere to one another, increasing the cohesion of the cellulose tissue (5, 6). To create good writing conditions (e.g., avoiding excessive ink absorption) the surface has to be sealed well: again, starch is used. Hence, it is clear that copy paper contains a significant amount of starch that can be reacted with KI ⫹ I2 solution to yield paper that has a deep blue color (4, 6, 7). Microorganisms in the process of feeding consume the starch in copy paper; when we exposed sheets of paper to microorganisms and then subsequently exposed these sheets to KI ⫹ I2 solution, the results did not yield the expected deep blue coloration. Remember that
We developed a unit on the qualitative analysis of microbial activities in topsoil using copy paper and a handmade cadmium sulfide–light emitting diode (CdS–LED) reflection photometer, applying the principles of combining complementary colors. This reflection photometer is inexpensive and simple to make. Students can use it for environmental sampling and analysis to obtain better understanding of the biological activities of microorganisms in various soil types in the students’ environment. This activity can be appropriately scaled for all levels of education in the area of environmental studies and environmental chemistry (4).
slight health hazard because continued exposure to this substance may cause skin irritation and irritation to the digestive tract. Potassium iodide and iodine solutions are also toxic if swallowed. Further health and safety notes may be found in the Supplemental Material.W Experiment To Determine Microbial Activity in Soil
Construction of the CdS–LED Reflection Photometer Directions for making a handmade reflection photometer are provided in the lab documentation of the Supplemental Material.W After construction, the reflection photometer was connected to the dry cells and the digital multimeter. The open end of the L-shaped polyvinyl chloride tube was placed on a sample sheet of paper as shown in Figure 1. The LED was attached; a yellow LED was used because yellow is complementary to the starch–iodine color. Calibration of the reflection photometer to different percentages of blue-purple color saturation was carried out according to principles described in the lab documentation of the Supplemental Material.W Experimental Procedures Different types of soil (from both sandy and mountainous landscapes, and from land used for gardening and agriculture—in our case, rice cultivation) were mixed into water and stirred completely. The mixture was filtered to obtain a soil solution. Sheets of white copy paper were dipped into the soil solution and removed after 0, 1, 2, 3, and 4 h intervals and then allowed to air-dry. Next, the dried sheets of paper were placed in iodine solution for ~10 s and allowed to airdry. The resulting depth of the blue color of the paper was measured as the resistance of the CdS sensor contained in the handmade reflection photometer. The same procedure was carried out for a blank solution that used deionized water.
Hazards Cadmium may cause eye or skin irritations and burns with exposure to large concentrations. The cadmium sulfide sensor is coated with glass: if the sensor is broken, it poses a www.JCE.DivCHED.org
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Figure 1. Schematic diagram of the reflection photometer setup.
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In the Laboratory
is the difference in their resistance values and that of deionized water as measured by the CdS–LED reflection photometer. Conclusions
Figure 2. Curves showing the extent of reflected light from paper dipped in different solutions of various soil types and water.
Results and Discussion
Determination of Microbial Activity in the Soil Based on the calibration for the depth of the blue color described in the lab documentation of the Supplemental MaterialW, the CdS–LED reflection photometer was used to measure the qualitative amount of microbial activities in soils collected from different sites in Okayama prefecture, Japan, as shown in Figure 2. In general, microbial activity in the different soil samples increased with increased exposure time, as observed by the resistances measured by the CdS–LED reflection photometer when white copy paper was dipped into soil solution, dried, and then dipped in KI ⫹ I2 solution to develop color. For use in the classroom, 4 h is the suggested time duration to expose the copy paper to the soil solution. The data shown in Figure 2 indicate that there are microorganisms in all the soils used in the experiment and that microbial activity in the garden soil is more pronounced relative to the other soil types. Microbial activity in the other soil samples decreases in this order: soil from a rice field, soil from a mountainside, and sandy soil collected from an Okayama University sports field, respectively. This observation confirms the fact that garden soil contains more microorganisms, hence more nutrients available to plants, than any other soil type—even soil used for agriculture. The color effects from the starch–iodine reaction diminishes with time, yet remain within the level of experimental error. From Figure 2, it is obvious that microbial activity measured by the CdS–LED reflection photometer in absolute terms is not only the extent of microbes’ activities in the soil as starch is soluble in water; the amount of starch in the copy paper also decreases with time. This is shown by the gradual decrease in resistance (values in MΩ) over time of the copy paper dipped in deionized water. It can be confirmed that the actual extent of microbial activity among the soil samples
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Copy paper contains starch used to strengthen the paper and prevent ink from running; consumption of the starch in copy paper can be used to determine the qualitative extent of microorganisms’ activity in different soil types. The handmade CdS–LED reflection photometer with yellow LED is preferred for measuring the iodine–starch reaction that yields a deep blue color. This handmade device can be constructed simply and inexpensively (total cost is less than $4), and can be used by students for environmental sampling and analysis. We also recommend that the handmade CdS–LED reflection photometer constructed with a white LED can be used to determine levels of environmental pollution manifest as particulates, dust, and smoke in air using white paper as the collecting medium. We are confident that students (at all levels of education) who conduct this experiment will gain a better understanding of the presence and significant contribution of the biological activities of soil microorganisms to environmental health. In addition, this experiment will also expose students to some of the practices and challenges of environmental research, as well as the importance of soil science. W
Supplemental Material
A number of materials related to this paper are available in this issue of JCE Online. They include: a longer version of the paper with additional figures, a table, and directions for constructing the reflection photometer; notes for the instructor; and instructions for students. Literature Cited 1. Chemical Storylines, 2nd ed., Burton, G., Holman, J., Lazonby, J., Pilling, G., Wadddington, D., Eds.; Heinemann: Oxford, UK, 2000; pp 182–189. 2. Manahan, S. E. Environmental Chemistry, 8th ed.; Academic: London, 2004; pp 135–163. 3. Harrison, R. M. Understanding Our Environment, 3rd ed.; Royal Society of Chemistry: Cambridge, UK, 1999; pp 199– 235. 4. Kita, M.; Takeda, K. Naruto University of Education Forum for Classroom Research 2003, 75, 59–73. 5. Chemistry and Technology, 2nd ed., Moore, C. O., Tuschhoff, J. V., Hastings, C. W., Schanefelt, R. V., Eds.; Academic Press: San Diego, CA, 1984; pp 575–291. 6. Morales, M. D.; Escarpa, A.; González, M. C. Starch/Stärke 1997, 49 (11), 448–453. 7. Spiro, T. G.; Stigliani, W. M. Chemistry of the Environment, 2nd ed.; Prentice Hall: Upper Saddle River, NJ, 2003; pp 292– 296.
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