Effects of NPK Fertilizer on Soil Enzymes and Micro Biota1

CHIOMA, Okorea, OGECHUKWU, Mbanefoa, BRIGHT, Onyekwere b, ASSUMPTA, Ugenyia, AGAPTUS, Ozurumbaa, ULOMA, Nwaehiria and CHIMAHHALAM, Akueshia

a Biology Department, Federal Polytechnic Nekede Owerri, Imo State, Nigeria, Africa. E-mail: chiomakore@gmail.com
b Chemistry Department, Federal Polytechnic Nekede Owerri, Imo State, Nigeria, Africa

Abstract Soil enzymes are involved in various biochemical processes and play a vital role in maintaining soil fertility. Soil samples were collected from Federal Polytechnic Nekede School farmland in Owerri West Local Government Area of Imo State, Nigeria. The collected samples were further processed and analyzed using standard biochemical and microbiological procedures over a sampling period of three (3) weeks to determine the effect of NPK (Nitrogen, Phosphorus, Potassium) fertilizer in the ratio 20:10:10; on soil micro biota and enzymes. The result revealed that microbial population of the 3rd week had the highest viable bacteria count (4.5 × 105) followed by the 2nd week (3.8 × 105) and least is the 1st week ( 2.4 × 105). Similar trend was also observed in the viable fungal count 2.2 × 105, 1.4 × 105 and 0.8 × 105 in the 3rd, 2nd and 16th weeks respectively. Pseudomonas sp, Salmonella, Proteus, E.coli, Klebsiella, Staphylococcus, Aspergillus, Penicillium, Phycomycetes and Rhizopus species were isolated from the sampling area over the sampling period. The enzyme activity of the soil treated with 12.5ml, 10ml, 7.5ml and 5ml of fertilizers also revealed the effective utilization and breakdown of the chemical component of the fertilizers. This is upheld by the result obtained on the effects of the fertilizer on catalase, dehydrogenase, acid phosphatase and alkaline phosphatase enzyme activities. The statistical analysis showed that there is no significant difference in the catalase, acid and alkaline phosphatase enzyme activity, but a difference in the dehydrogenase enzyme activity at α = 0.05 degree of significance. This study therefore reveals that NPK fertilizer has great effect on the soil microbial and enzyme activity which are vital components in the maintenance of soil health and fertility.

Keywords Micro Biota, NPK Fertilizer, Soil Enzymes

1  Introduction

Soil enzymes are involved in various biochemical processes and play a vital role in maintaining soil fertility and plant yields (Nannipieri et al., 2003). Changes in the activity and diversity of soil enzymes may reflect changes in soil quality. It has been shown that different soil management practices affected the structure and activity of soil enzymes (Monreal et al., 1998). Intensive cultivation, which leads to depletion of soil nutrients, is a major constraint on food production. To overcome the rapid decline amendments such as fertilizer, manure, compost and animal dung that contain nutrients are applied to the soil to improve and maintain crop yield. Fertilizer is any organic or inorganic material of natural or synthetic origin which is added to a soil to augment the level of or supply certain elements essential for plant growth. Any amendment to the soil to enhance soil fertility is therefore a fertilizer (Martens et al., 1992; Graham et al., 2005). However, while many chemical reactions do take place in the soil, the nitrogen conversions occur primarily through the action of soil enzymes. Efficient fertilization provides plants with nutrients at appropriate proportions and quantities which enable maximum yield increase of crops with high biological and technological quality (Seymour, 2002). Fertilization should also gradually improve soil fertility. It should be mentioned that soil fertility, decisive for proper development of cultivated plants and crop yield, depends on many factors, the most important being nutrients contents (Okore et al., 2012), physical and chemical properties and metabolic activity of many micro enzymes implicated in metabolic processes and energy flow. Although biomass of all micro enzymes living in soil constitutes only several percent of organic matter content, they play an important role in the functioning of entire ecosystems because, due to their enormous biochemical and biogeochemical activity, they can exert crucial effects on dynamics of multidirectional microbiological processes (Paul, 1996). Micro enzymes are part of all natural ecosystems, the biocenosis are significant and essential for biochemical elements, responsible for the entirety of biogenic element transformation in soil environment, and which exert critical effects on biochemical activity, ecological stability, and biological productivity of many field, forest and grassland ecosystems. They are involved in biochemical transformations of mineral fertilizers, particularly NPK fertilizers, synthesis of biologically active substances (amino acids, vitamins, antibiotics, toxins) and nitrogen fixation from the air. They regulate element circulation in soil environment and make them assimilates for plants. Phosphatases have been extensively studied in soil, as shown by some reviews (Nannipieri et al., 2002, 2003 ; Bunemann, 2011; Quiquampoix and Mousain 2005; Tabatabai 1982; Sarathchandra et al., 2001; Saggar et al., 2000). They catalyze the hydrolysis of ester–phosphate bonds, leading to the release of phosphate (P), which can be taken up by plants or microorganisms. A high enzyme activity level and soil structural stability represents a high state of soil health. Conversely, low soil health is associated with soils having both low soil enzyme activity level and structural stability (Monreal et al., 1998). The present study was undertaken to determine microbial community structure in a short term fertilizer application experiment.

2  Materials and Methods

Soil samples from 0-15cm depth were collected at random from Federal Polytechnic Nekede farmland. The soil samples were bulked and sieved.

2.1  Preparation of samples composite

10g of soil samples were weighed out and placed into five different test tubes labeled A, B, C, D and E. Then 250mg of NPK fertilizer was diluted in 1ml of water which gave the concentration of NPK fertilizer as 250mg/ml. Then 12.5ml, 10ml, 7.5ml, 5ml and 0ml of the fertilizer solution, were added to the test tubes respectively. Test tube E was used as the control because no fertilizer was added to it.

2.2  Preparation of extract for enzyme activity

100ml of phosphate buffer with pH 7.4 was added into 1g of each soil sample from A,B,C,D,E in a test tube and homogenized gently and properly. The soil suspension was filtered with cheese cloth and the filtrate centrifuge at maximum speed of 700rpm for 10 minutes such that the supernatant was obtained. Then the supernatant was subjected to catalase, dehydrogenase, acid and alkaline phosphatase tests by methods of Tabatabai, 1982.

2.3  Characterization and identification of microbial isolates

The characterization and identification of the microbial isolates were based on the morphology, biochemical test and their ability to hydrolyze cellulose. The fungal isolates were identified by microscopic and macroscopic techniques described by Barrow and Feltham (2003).

2.4  Plate count and enumeration

Duplicate plates of each dilution were used to achieve a plate count of between 30 and 300 colony forming unit (CFU) as described by Cheesbrough, (2005). This quantitative determination of bacterial and fungal populations was carried out by standard or viable plate count method using a colony counter.

2.5  Purification and preservation of isolates

The bacterial isolates were picked with a wire loop based on their morphological appearance. The picked colonies were sub cultured unto freshly prepared nutrient agar plates for purification. They were further incubated for 24 hours at 37 C. After incubation pure cultures were stored in bacterium preservers (cryo-preserver) in a refrigerator. Fungal isolates were sub cultured unto freshly prepared sabraund dextrose medium using needle tease method.

2.6  Preparation of standard inocula

The method of Dubey and Maheshawari (2004) was adopted for the preparation of the standard inocula of bacteria. Isolates from the stock cultures were revived by picking two balls each from the cryo-preserver and dropped into 5ml of nutrient broth in test tubes and incubated at 37 C for 24 hours. The revived isolated were further sub-cultured into sterile prepared nutrient agar plates for another 24 hours, at 37 C. The fresh cultures of bacteria were used as standard inocula for the different morphological and biochemical tests.

2.7  Statistical analysis

An analysis of variance of the data was made at α = 0.05 degree of significance using the SPSS software.

3  Results

Table 1: Effects of fertilizer on catalase enzyme activity
 Volume (ml) of the fertilizer per 10g of soil sample
Week interval0 (control)57.51012.5
1st week0.2750.3010.2310.5900.500
2nd week0.2810.4000.2310.5850.550
3rd week0.2790.4400.4110.5800.450
Table 2: Effects of fertilizer on catalase enzyme activity
 Volume (ml) of the fertilizer per 10g of soil sample
Week interval0 (control)57.51012.5
1st week0.1000.0070.1130.3010.459
2nd week0.0900.0190.2601.0201.550
3rd week0.0870.0050.1030.0110.240
Table 3: Effects of fertilizer on acid phosphatase enzyme activity
 Volume (ml) of the fertilizer per 10g of soil sample
Week interval0 (control)57.51012.5
1st week0.2700.3000.3500.3010.280
2nd week0.2500.3030.3670.2510.291
3rd week0.2650.3600.3590.3510.350
Table 4: Effects of fertilizer on alkaline phosphatase enzyme activity
 Volume (ml) of the fertilizer per 10g of soil sample
Week interval0 (control)57.51012.5
1st week0.2700.3000.3500.3010.280
2nd week0.2500.3030.3670.2510.291
3rd week0.2650.3600.3590.3510.350
Figure 1: Effect of fertilizer on catalase enzyme activity
Figure 2: Effect of fertilizer dehydrogenase enzyme activity
Figure 3: Effect of fertilizer on acid phosphatase enzyme activity
Figure 4: Effect of fertilizer on alkaline phosphatase enzyme activity
Figure 5: Percentage occurrence of isolates through the weeks
Table 5: Mean total heterotrophic microbial count (cfu/ml)
Weeks\CountsX105 TVBCX105 TCCX103 TVFCX102 TCEMBX102 TSSCX102 TPC
1st WK2.
2nd WK3.
3rd WK4.
TVBC = Total Viable Bacterial Count
TCC = Total Coliform Count
TVFC = Total Viable Fungal Count
TCEMB = Total Count on EMB
TSSC = Total Salmonella Shigella Count
TPC = Total Pseudomonas Count

4  Discussion

The result of the study showed that fertilizer application does not inhibit the soil enzyme and microbial activities. The catalase activity increased from the 1st week to 2nd, then to the 3rd week in the control, 5ml and 7.5ml NPK fertilizer application. The reverse was observed with higher 10ml and 12.5ml NPK fertilizer application where there was decrease in catalase activity from the 1st to 2nd then to the 3rd week. The dehydrogenase activity for all levels of NPK fertilizer application decreased from the 1st week to the 2nd week and increased again from the 2nd week to the 3rd week. The acid and alkaline phosphatase activity recorded the same results for all levels of NPK fertilizer application. The trend showed a decrease in the control from the 1st week to the 2nd week and then the 3rd week; in the 5ml and 12.5ml applications there was increase from the st week to the 2nd week then to 3rd week of applications; 7.5ml NPK applications rates increased from the 1st week to the 2nd week and decreased on the 3rd week while the 10ml NPK application decreased from 1st week to the 2nd week and decreased on the 3rd week. The result of this work was therefore in correlation with earlier reports that showed that fertilizer enhance soil enzymes activities. For instances Albiach et al., (2000) showed that 10mg term application of fertilizers positively influences soil available nutrient and results in increased microbial proliferation. Ranigaraj et al., (2007) also found that incorporation of fertilizer increases the available phosphorus (P) status at maximum level. The different isolates include Pseudomonas, Salmonella, Proteus, E.coli, Klebsiella, Staphylococcus, Aspergillus, Penicillium, Phycomycetes and Rhizopus. The Pseudomonas, Staphylococcus and Rhizopus showed the highest occurrence of 100%. The mean total heterotrophic microbial counts in cfu/ml in all the media used showed increase from the 1st to the 2nd and then to the 3rd week. The result of the bacterial and fungal enumeration at the different period suggests that the applied doses were well tolerated by the resident micro biota. Evidently, none of the application rates affected the overall population of soil bacteria and fungi. Also, the frequency of occurrence of bacterial and fungal species present in the table indicates a little or no change in the relative abundance of the different species after organic fertilizer treatment. The results of this study indicate that long term fertilizer application was well tolerated by the native soil micro-biota since there were no discernable changes in the overall population of the soil micro biota. The statistical analysis showed that there is no significant difference in the catalase, acid and alkaline phosphatase enzyme activity, but a difference in the dehydrogenase enzyme activity at α = 0.05 degree of significance.


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Chioma, O; Ogechukwu, M.; Bright, O.; Assumpta, U.; Agaptus, O.; Uloma, N.; Chimaghalam, A. (2014): Effects of NPK Fertilizer on Soil Enzymes and Micro Biota. In: Planet@Risk, 2(4), Special Issue on One Health: 249-253, Davos: Global Risk Forum GRF Davos.

This article is based on a presentation given during the 2nd GRF Davos One Health Summit 2013, held 17-20 November 2013 in Davos, Switzerland ( http://onehealth.grforum.org/home/)