Effect of Long-term Deposition of Phosphate and Nitrogen on Pphosphatase Activity in Moss - Lab Report Example

EFFECT OF LONG-TERM DEPOSITION OF PHOSPHATE AND NITROGEN ON PHOSPHATASE ACTIVITY IN MOSS 1.0 Introduction Mosses belong to phylum Bryophyta, class musci and family sphagnaceae (Barbour & Billings 2000). Mosses grow mostly on moist walls, shaded tree sides and even in sidewalks. They share common characteristics of phylum Bryophyta with other plants belonging to this phylum. Mosses are a few cells thick, lack true root, leaves and stem system. They are small and require water for fertilization. During breakdown of exposed substrata, mosses release nutrients, which are used by complex plants that succeed them. They have a dense surface cover that aids in absorbing water from the ground as well as preventing soil erosion (Hubers & Kerp 2012). These plants are very important in the ecosystem as they create a significant buffer system for other plants around them (Speck 1941). Plants from this family can also be a distinct indicator of the quality of the environment surrounding them, as most of them can be sensitive to the moisture present in the atmosphere (Small 1933). Mosses and liverworts contain several secondary metabolites being investigated for various agricultural, phytochemical, and pharmacological products. This experiment was aimed at comparing phosphatase enzyme rate in moss species Hypnumjutlandicum, having received prior treatment of nitrogen and phosphate. Two different hypotheses were tested on two different variables, the nitrogen and the phosphate. The first hypothesis was that phosphate will increase the rate of the phosphatase activity and the second was that nitrogen would decrease the rate of the phosphatase activity. 2.0 Materials Rubber gloves Goggles 15 test tubes Moss Spectrophotometer Deionized water Enzyme substrate (10mM nitrophenyl phosphate) Timer 30ml of 0.2M NaOH (30ml divided into 6 test tubes, 5ml each) Paper and pencil to note down the results 2.1 Method 2.1.1 Sample preparation The test tubes containing different moss sample nutrient treatment were labeled to avoid contamination of the solutions. About 2 cm of each moss sample was placed in each of the labeled test tubes followed by 2.5 ml of deionized water and the contents of the test tubes mixed. A total of 2.5 ml of 10 mm nitrophenyl phosphate (NPP) was then added to the mixture and the stopwatch started to record time. The mixtures were left to stand for thirty minutes at room temperature to ensure that the enzyme substrate does not denature since enzymes have a working optimum temperature beyond which they denature. The test tubes were shaken at an interval of five minutes. 2.1.2 Calibration A solution of 5 ml of 0.2M NaOH was placed in each of the six labeled test tubes. A pipette was used to draw 0.5 ml of the NPP/moss/ water assay solution into each of the test tubes containing 5 ml of NaOH the test tubes were then shaken for the mixture to combine. A shade of yellow was used as an indicator of a complete reaction. The test tubes were compared and ranked in terms of color. The Absorbances of these solutions was then measured at a wavelength of 410 nm using a spectrophotometer. Water was used as blank. The calibration curve obtained was used to convert the absorbance into a concentration of nitrophenol. Finally, the reaction rate was calculated and expressed in mole nitrophenol per minute per sample. The experiment was repeated ten times in order to get sufficient and reliable results. Sample labels Nutrient treatment 0 0 (water) 60N 60N 120N 120N P 7.5P 60N+P 60N+7.5P Blank Blank (no Moss) 3.0 Results 3.1 Qualitative data i. The solutions turned yellow on the addition of moss assay. The degree of color change varied from one test tube to the other. ii. The different moss samples looked the same, even though, some had been submerged in phosphate and some in nitrogen. iii. The most opaque solution had the highest absorbance level at 0.449 nm while the palest solution had the lowest absorbance level at 0.047nm 3.2 Qualitative data Table 1: Enzyme assay data collected from various groups, and various mosses that had been submerged in either Nitrogen, Phosphorus or both for a period of time. Group Blank Control 60N 120N P 60N+P SHONNY 0.000 0.010 0.007 0.013 0.003 0.002 ZCL 0.000 0.008 0.019 0.017 0.006 0.008 Lunch 0.005 0.124 0.198 0.197 0.135 0.074 Luke 0.020 0.196 1.015 0.837 0.225 0.306 YJL 0.000 0.130 0.043 0.510 0.112 0.050 JBEK 0.003 0.047 0.102 0.148 0.006 0.051 LESJ 0.004 0.045 0.001 0.004 0.022 0.022 W 0.011 0.016 0.018 0.023 0.093 0.013 JDTS 0.000 0.011 0.014 0.011 0.003 0.004 ECS 0.016 0.008 0.008 0.014 0.005 0.007 MARK 0.001 0.014 0.010 0.033 0.006 0.012 Table 2: The Mean and STDEV calculated from the data in Table.1 Blank Control 60N 120N P 60N+P Mean 0.005 0.055 0.130 0.164 0.056 0.050 STDEV 0.007 0.065 0.299 0.270 0.075 0.088 Fig 1: A graph of Mean values of the Enzyme assay data, including the Blank, Control, 60N, 120N, P and 60N+P. The results obtained in this experiment shows that moss that had been submerged in 120N had the highest average absorbance level at 410 nm with the highest concentration of nitrophenol and the largest phosphatase activity mmol NP per 30 minutes (fig. 1). The lowest average absorbance level at 410nm was recorded for the blank. The blank recorded the lowest concentration of nitrophenol and the smallest phosphatase activity mmol NP per 30minutes. Since it contained no enzyme substrate, the light was able to shine right through it hence the lowest absorbance value. The average activity of phosphorus was 0.056 mmol NP per 30 minutes, while that of 120N was 0.164 mmol NP per 30 minutes. Therefore, Nitrogen is more effective in increasing phosphatase activity than phosphate. It can be deduced from this graph that phosphorus is not as effective as nitrogen when it comes to increasing phosphatase activity. Based on these results, the nutrient content of the soil can be measured by comparing the levels of phosphorus and nitrogen present in the soil. The overlap of error bars in the graph is a clear indication of statistical significance. The 60N and 120N are significant with each other because their error bars overlap. The blank and control, P and 60N+P are significant with each other because their error bars overlap. Wholistically, the results are not statistically significant. Fig 2: A graph of STDEV values of the Enzyme assay data, including the Blank, Control, 60N, 120N, P and 60N+P. The error bars between 60N and 120N, P and 60N+P, and blank and control overlap with each other (fig. 2). These results show a statistical significance with each other, but as a whole the results do not show statistical significance. Fig 3: A graph of activity values of the Enzyme assay data, including the Blank, Control, 60N, 120N, P and 60N+P. 4.0 Discussion The use of phosphate to increase phosphatase activity was ineffective. These results compared well with other studies (Mukunda et al., 2008). In the research conducted by Mukunda, the phosphatase activity in Sphagnum stem ranged from 110 to 600 nmol pNP g dwt-1s-1 for the green stems. In this experiment, the phosphate group had a phosphatase activity of 0.003 NP for every 30 minutes in the leaves of Hypnumjutlandicum. The difference between the parts of the plants could significantly alter the results. In the research conducted by Mukunda, the moss samples that were treated with NH4 and NO3 displayed the highest levels of phosphatase activity. In this experiment, the moss samples that had been treated with 60N and 120N displayed the highest phosphatase activity. These results, therefore, do not support the first hypothesis of this study ‘’nitrogen will decrease the rate of the phosphatase activity.’’ During Makunda’s research, the overall effect of nitrogen was stimulatory and typically increased phosphatase activity by around 30%, results which compare well with those from this experiment. Comparing the mean control value of the moss phosphatase activity at 0.055 to the mean value of 120N at 0.164 in this experiment, there is an increase of 33.54%. The results obtained here are more reliable as they are similar to the results of other studies that have been conducted on mosses. The phosphorus decreased the phosphatase activity in the mosses just like PK reduces phosphatase activity in the green tissue Mukunda et al., 2008). The results obtained here, shows a decrease in the phosphatase activity in the moss on the addition of phosphorus. These results do not support the second hypothesis, ‘’phosphate will increase the rate of the phosphatase activity.’’ The slight deviation of the results from this experiment and the previously performed experiments could be because of factors such as: i. Lack of a scientific method involved in ensuring all the pieces were of equal quantity. ii. Contaminants present in the pipettes used Reference list Barbour, MG & Billings,WD 2000, North American terrestrial vegetation, Cambridge University Press, Cambridge, United Kingdom. Hubers, M Kerp, H 2012, ‘Oldest known mosses discovered in Mississippian strata of Germany, Geology vol. 40 no. 8, pp. 755. Mukunda, P Kebekka, REA Sheppard, L Leith ID & Jonson D 2008, ‘Long-term nitrogen deposition increases phosphorus limitation of bryophytes in an ombrotrophic bog’, Plant Ecology, vol.196, pp.111-121. Small, JK 1933,Manual of Southeastern floraUniversity of North Carolina Press, Chapel Hill, North Carolina. Speck, FG 1941, ‘A list of plant curatives obtained from the Houma Indians of Louisiana Primitive Man’Quarterly Bulletin of the Catholic Anthropological Conference, 14,pp. 49-75. Read More