Soil Enzyme Activity - Lab Report Example

 Soil Enzyme Activity Abstract Soil microorganisms have the ability to breakdown insoluble nutrients in the soil to produce extracellular enzymes. These are protein compounds produced inside microbial cells but exported externally to catalyze reactions that breakdown nutrients source to make it available. Alkaline Phosphatase is an example of such enzymes and function to cleave the phosphate molecule from the organic compounds such as nucleic acids and phospholipids. This paper aimed at measuring the amount of active enzyme in the soil samples by applying chromogenic substrate assay. Using soil samples with organic fertilizer, inorganic fertilizer, combined and unammended, alkaline phasphatase activity was tested under control of temperature, pH, ionic index and substrate concentration. Results show that combined fertilizer had the highest enzyme activity of 1.493 followed by organic fertilizer (1.148), inorganic fertilizer (0.529) and unammended (0.459) soil sample in that order. Combination of inorganic fertilizer and organic fertilizer provides efficient environment for the growth of soil bacteria hence production of more alkaline phosphatase enzymes thus resulting into high enzyme activity. These findings shows how an integrated fertilizer regime play an important role in altering the structure of the soil microbial composition, stimulates microbial growth and increases enzyme activity as compared to the inorganic fertilizer. Introduction Microorganisms play a vital role in biochemical transformation important for nutrient cycling in soils. The microorganisms in the soil are capable of breaking down insoluble nutrient sources in the soil and in the process produce extracellular enzymes. Extracellular enzymes are biochemical catalysts, protein compounds produced within the bacterial cell and exported out into the soil solution. While outside the cells, enzymes catalyze reactions that break down the structure of nutrient source in order to make it more accessible to the plants. The quantities of extracellular enzymes are therefore dependent on substrate concentration, soil microorganisms’ metabolic ability, soil environmental conditions such as temperature and pH and the number of microorganisms present in the soil (Lichtfouse, 228). Alkaline Phosphatase is an example of extracellular enzyme produced by many soil microorganisms and exported out into the soil solution. The main function is to eliminate the phosphate molecule from the organic compounds such as nucleic acids and phospholipids. This makes phosphate soluble hence can be easily absorbed by the cells. This is very important because phosphate is often the limiting nutrient for microbial growth in the soil. Measurements of soil enzyme activities are therefore useful indicators of soil biological activity. Many studies have also referred to soil enzyme activity as an index for soil health and quality of land (Verchot and Borelli, 629). Enzyme activity has also been used in studies that investigate the impact of human activities on the biochemical cycles in ecosystem. The purpose of this study is to measure the amount of active enzyme in the soil samples by applying chromogenic substrate assay. The principle behind Alkaline Phosphatase assay is that alkaline phosphatise catalyzes the conversion of a colourless para-nitrophenol phosphate to para-nitrophenol which has a bright yellow colour. The rate of enzyme activity in this experiment is calculated from the spectrophotometer readings from the amount of para-nitrophenol formed. The basic enzyme assay applied in this study is the addition of known amount of soil to a solution containing a standard concentration of substrate then measuring the rate at which substrate is converted to a product while keeping enzyme environmental factors such as temperature, ionic strength and pH constant. The results of this study will also be standardized by calculating the dry weight of the soils. Materials and Methods The materials used in this experiment were categorized s equipment, media and reagents and supplies. Equipment included: 370C incubator, Balance, Aluminium weighing dishes, scissors, 16x100mm test tubes, 5 ml pipettes and pumps, 440nm Spectrophotometer, drying oven and benchtop centrifuge. Media and reagents included: Buffer of pH 10, 0.5M CaCl2, para-nitrophenol Phosphate (PNPP) in buffer which served as the test solution and 2mM p-nitrophenol. Supplies included: Plastic flowerpots, markers, dry, sieved soil, filter paper and yeast extract. All the supplies, media and reagents were prepared and stored according to the manufacturers’ instructions and standard operating procedures before being used in this experiment. Procedures Constructing Microcosms 300ml of soil was measured using a glass beaker and then appropriate amount of fertilizer was weighed and mixed with the measured soil. A circle of a filter paper that fits the bottom of a flower pot was then cut and then placed at the bottom of the flower pot. The soil-fertilizer mix was then poured into the flower pot until it was ½ inch from the top before being watered until it was almost moist. Four soil microcosms with different fertilizer treatments were created as shown below: Treatment Fertilizer Yeast Extract Organic 0.0 g 1.6 g Inorganic 0.2 g 0.0 g Combined 0.2 g 1.6 g Unamended 0.0g 0.0 g Phosphatase Assay Two 2-grams portions of my group’s soil sample was weighed and poured into screw-cap tubes labelled “test” and “soil blank” and an extra screw-cap tube labelled “reagent blank”. 5ml of 0.5 M CaCl2 solution was then poured into each of the test tubes using a pipette and shaken well. In the tubes labelled “test” and “reagent blank”, 1ml of PNPP was added using a pipette and shaken well to ensure consistency of the mixture. 1ml of phosphate buffer was then added into “soil blank” tube using a pipette to serve as a control. All the three test tubes were then incubated at 370C for 1 hour. After one hour, 4 ml of the liquid from each of the tubes was carefully drawn and taken into 16x100mm test tubes and labelled accordingly. The test tubes were then centrifuged at 2500rpm for five minutes then 1 ml of the resultant supernatant transferred into clean cuvettes. Before reading for the absorbance, the wavelength of the spectrophotometer was set to 440nm and absorbance set to zero using “soil blank” tube and the results recorded in the table 1. The absorbance of the prepared standards was then read by setting absorbance to zero with corvette containing 1ml of CaCl2. The absorbances for each of the prepared standards were then read and a standard curve drawn by plotting absorbance against concentration. Water Content Analysis was done as follows: The aluminium dish was weighed and weight recorded as the weight of empty aluminium dish. Approximately 10grams of soil sample was then weighed into the aluminium dish and the exact weight recorded. The soil samples in the aluminium dish were then placed in an oven at 1000C overnight and left to cool in desiccators then dry samples were weighed and actual weight recorded. The results were then presented in table. Results Figure 1: Standard Absorbance Curve Y = 0.079x From the standard curve above: Concentration (x) = Absorbance (y)/0.079 Table 1: Spectrophotometer Readings and Enzyme Activity Absorbance Net Absorbance (test – reagent blank) Concentration of p-Nitrophenol Enzyme Activity Organic fertilizer (test) 0.278 control (reagent blank) 0.147 0.131 1.658 1.148 Inorganic fertilizer 0.089 control (reagent blank) 0.029 0.06 0.759 0.529 Combined 0.279 control (reagent blank) 0.111 0.168 2.127 1.493 Unamended 0.122 control (reagent blank) 0.068 0.054 0.684 0.459 The above table shows the absorbance, concentration of p-nitrophenol and the calculated enzyme activities for organic fertilizer, inorganic fertilizer, combined and unammended soil samples. The figures of concentration of p-nitrophenol shown above were calculated using the standard curve and are expressed in moles in 6mls of the solution. Enzyme activities expressed in the table above were calculated by dividing the concentration of p-nitrophenol by the number of hours and dividing the results by the dry weight of soil sample (table 3). This reports enzyme activity as unity (U) per gram of dry soil. One unit of enzyme activity (U) is defined as the amount of enzyme that is able to convert 1 mole of substrate to product in one minute. Table 2: Standard Curve: Concentration Vs Absorbance Concentration 2.0 mM 1.0 mM 0.5 mM 0.25 mM 0.125 mM 0.063 mM absorbance 0.153 0.09 0.043 0.017 0.009 0.005 Table 2 above shows the figures of absorbance readings by the spectrophotometer against standard concentrations. These readings were used in drawing of the standard curve (figure 1) which generated a standard curve of equation; Y= 0.079x and was used to calculate back the concentrations for different soil samples using their respective absorbance. Table 3: Water Content Analysis Sample Organic Inorganic Combined unamended Dish weight 2.606 2.599 2.63 2.646 Wet weight with dish 12.659 12.63 12.645 12.786 Wet weight of soil 10.053 10.031 10.015 10.14 Dry weight with dish 11.271 11.202 11.18 11.58 Dry weight of soil 8.665 8.603 8.55 8.934 Water content 1.388 1.428 1.465 1.206 Figure 2: Comparison of Absorbance, Concentration of P-Nitrophenol and Enzyme Activity Figure 2 above shows the comparisons of absorbance, concentration of p-nitrophenol and enzyme activity between different soil samples; organic fertilizer, inorganic fertilizer, combined and unammended. Discussions The objective of this study was to measure the activity of enzyme in various samples of soil. The results show that soil samples with organic fertilizer, inorganic fertilizer, combined and unammended soil have various enzyme activity levels. The enzyme activity levels for soil samples with organic fertilizer, inorganic fertilizer, combined and unammended are 1.148, 0.529, 1.493 and 0.459 units per gram of dry soil respectively. Combination of inorganic and organic fertilizer shows the highest enzyme activity while the unamended soil shows the least activity. Enzyme activity is influenced by substrate concentration, pH, temperature, ionic index and enzyme concentration. In this experiment, substrate concentration, temperature, pH, ionic index were kept constant and controlled. It is also important to note that the concentration of substrate was the same since the substrate used in this study; Para-Nitrophenol Phosphate (PNPP) was added in equal amounts to each sample. The differences in measured enzyme activity are therefore as a result of enzyme concentration and not any other factor affecting enzyme activity. These results show that combination of inorganic fertilizer and organic fertilizer provides efficient environment for the growth of soil bacteria hence production of more alkaline phosphatase enzyme. As stated by Simon and Czako (317), enzymes are costly to make thus are highly regulated and their production is done when necessary. These findings shows how an integrated fertilizer regime play an important role in altering the structure of the soil microbial composition, stimulates microbial growth and increases enzyme activity as compared to the inorganic fertilizer. In this regard, the growth of bacterial is highly influenced by the fertilizer regime supplied. However, the use of organic fertilizer such as manure is better than inorganic fertilizer as indicated by enzyme activity measured in this experiment. Addition of organic fertilizer to the soil sample has the capability of increasing the Gram-negative bacteria responsible for the production of alkaline phosphatase as compared to inorganic fertilizer (Qin, Hu and Oenema, 4). Alterations in the soil microbial counts resulting from different types and combined organic and inorganic fertilizers show that they can be justifiably used for soil fertility. According to Lichtfouse (228), microbial biomass changes are an important parameter in providing a faster and clearer response to the application of organic and inorganic fertilizers to the soil and finally affect its effective and potential fertility. The results show that the highest enzyme activity was recorded in combined inorganic and organic fertilizer while the least was recorded in non amended soil samples. This is an indication that a mixture of organic and inorganic fertilizer is useful for the growth of microorganisms and phyto-hormones, improves air and water relationships, increases the amounts of organic matter hence results into high mineralization processes and enzymatic activity of the soil microorganisms (Zhang and He, 102). The application of soil enzyme activity assays has been widespread in areas where commercial fertilizers have been used to supply essential nutrients for adequate crop production. Soil enzyme activity measurements are therefore important because it is an indication of agricultural soil quality. References Lichtfouse, E. 2010. Genetic Engineering, Biofertilisation, Soil Quality and Organic Farming, Dordrecht: Springer. Qin, S, Hu, C, and Oenema, O. 2013. 'Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication', Plos ONE. 8, (3):1-6 Simon, T. And Czako, A. 2011. Influence of Long-term application of Organic and Inorganic Fertilizers on Soil Properties. Plant Soil Environment. 60, (7):314-319 Verchot, L, and Borelli, T. 2005. 'Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils', Soil Biology And Biochemistry. 4: 625-633 Zhang, H, and He, Z. 2014. Applied Manure and Nutrient Chemistry for Sustainable Agriculture and Environment. Dordrecht: Springer. Read More