Effects of Pineapple Peel (PP) Composts on Soil Microbial Structure and Enzyme Activities

*1Inyang C.U, 2Nwaugo, V.O. and 3Ntukidem, E.

1

Dept of Microbiology, University of Uyo, Nigeria

Dept of Microbiology, Abia State University, Uturu, Nigeria

ABSTRACT The effects of Pineapple peel (PP) composts on soil microbial community structure and enzyme activities were evaluated in Uyo, Southern Nigeria . Two types of composts were produced, one pineapple peel (PP) compost and the other, PP supplemented with poultry droppings (PD). During the composting process, analysis of the composts showed that N03 had a range of 1.32-3.17 mg/g, PO4 had 2.84-3.91 mg/g and TOC 13.31-16.8 mg/g. pH was 8.41-9.2 while temperature was 30.4-30.8oC. Water content was 36.7-28.4%. Except for water content, all other parameters had higher value in the PD supplemented PP compost. Amendment

of soil with these composts, showed higher microbial populations in PD supplemented PP compost. The bacterial groups estimated were total

heterotrophic (THB), phosphate solubilizing (PSB), cellulose utilizing (CUB), nitrifying (NB) and coliform bacteria (CB). THB was the highest while CB was the least. Soil enzyme activities showed that dehydrogenase had the highest range of activity (18.62-28.31), followed by cellulase (10.71-22.31). Urease was more active in the PD supplemented PP compost (19.7) than the control soil (3.1). Alkaline

phosphatase followed the urease trend which increased in activity with increase in pH. Acid phosphatase did not show significant change in neither composts nor compost-amended soil. Results obtained suggest that addition of PD improved the PP compost quality for agricultural purposes. Key words: compost; soil quality, bacterial groups, enzymes , pineapple peel. * Corresponding author DR V.O Nwaugo Email, vonwaugo @ yahoo.com Phone: 238063494654.

INTRODUCTION The fertility of a soil depends extensively on the microbial community structure of that soil ecosystem. Soil microorganisms and their enzymes not only play active roles in soil fertility, as a result of their involvement in the recycling of nutrients but are also, sensitive biological indicators for soil quality evaluation (Shantikhsar et al, 2008: Nwaugo et al 2008a). Haran et al, (2006) and Karin (2006) stated that both the enzymes and the producing microorganisms are very sensitive to slight changes in the soil environmental conditions. Following the abuse, use and re-use of the soil for food production, soil easily loses its quality. It quickly becomes depleted in nutrients, resulting in the application of both organic and inorganic fertilizers to improve its quality (Nwaugo et al., 2008b, Nattipong and Alissara, 2006). Some of these additions to soil, affect the soil physicochemical parameters as well as the soil microbial community structure and functions, thereby improving or adversely affecting soil quality with time (Karin, 2006 Haran et al, 2008).

For sometime, the assessment of soil quality had been mainly by the use of physicochemical parameters. The nature of soil microbial community structure was a later addition (Nannipieri et al, 2002). Very recently, the use of soil enzymes functions in soil quality assessment has shown the soil situation more vividly as these enzymes are very sensitive to slight changes (Cookson, 1996: Nwaugo et al, (2008). The involvement of these enzymes in soil quality

assessment gives an indication of all forms of microbial community structure involving both culturable and unculturable micro-organisms. This becomes very important as Pelczar et al., (2003) and Humphries (2002) stated that only 3-5% of all microbial species are culturable in the laboratory. Many types of enzymes have been used as indicators of soil stress or changes. However, while some are produced by all microorganisms, a few are specific to particular organisms or induced by the presence of certain substances in the environment. In most developing countries, the use of compost as the main enhancer of soil fertility is still low. Where such exists, data concerning the effects on soil microbial community structure and enzyme functions are very scanty. Again, the sources and types of composting materials determine the quality of the compost produced. This study therefore was designed to assess the quality of pineapple peel compost and its effects on soil after amendment using soil microbial community structure and enzyme activities as parameters. Materials and methods; The study area was Uyo, a metropolitan community in southern Nigeria. It has tropical rainforest climate and lies between latitude 4o33and 5o33 N and

longitude 5o

. Pineapple peels used in the study were

obtained from both orchards and market places where the fruits are processed for sale. The peels were composted using the pit method with regular turning for twelve (12) weeks. Two types of pineapple peel composts were produced; one was pineapple peel alone and the other was pineapple peel (PP) supplemented with poultry droppings (PD). Amendment of the soil was done according to Nattipong and Alissara (2006) at the rate of 1kg of the compost type to 10m2 of the farmland soil. The compost was evenly spread and mixed with the 0-10cm of the top soil using hand operated (manual) plough. Soil sampling After soil amendment, the soil was allowed to set for two weeks before sampling for analysis. The top 5-10cm of each soil type amended with each compost type were sampled using shipreck augar and sterile universal sample bottles. All samples were analyzed within 2hr of collection for biological parameters and 2-3 days for physicochemical parameters.Soil samples for physicochemical analysis were stored in the refrigerator (4 oC) until required for use. Physicochemical parameters analysis; A few physicochemical properties of the composts and soil samples were determined using various standard methods. These were pH, temperature,

organic carbon, nitrate and phosphate contents which were assessed according

to AOAC (2005). The moisture content was determined by drying to constant weight method according to the same AOAC (2005). Microbiological analysis; Prevalence of various bacterial species in the soil was determined by the use of various culture media, specific for the group. This was done after ten-fold serial dilution according to Chesebrough (2002) using the spread plate technique. Five bacterial groups were assessed. These were total heterotrophic (THB), coliform (CB), phosphate solubilizing (PSB) cellulose utilizing (CUB) and nitrifying bacteria (NB). Tryptone Soy Agar was used for THB, McConkey Agar for CB. Phosphate medium (US Patent, 2003) and modified mineral salt Agar were used for PSB and NB respectively. Cellulose agar was used for CUB. Soil enzyme activities; The activities of the soil enzymes were determined using soil samples sieved with 0.5 sieves after drying at room temperature for 24 hours. The enzymes whose activities were determined include dehydrogenase, urease, cellulase and the phosphatases (acid and alkaline). Dehydrogenase was determined as described by Alef (1995) which involved the use of 0.25% aqueous triphenyl tetrazolum chloride (TTC). The formed triphenyl formazon was measured at 485nm. The urease activity was estimated according to Nannipieri et al (1997) using the colorimetric method involving urea amendment of soil. The result was expressed as mg NH4-Ng-1 dry soil 24 h-1. The activity of the cellulase was determined according to Deng and Tabatabai (1994) involving the carboxyl methyl cellulose (CMC) amendment of soil.

Both acid and alkaline phosphatases activities were determined using the methods of Tabatabai (1997) in which p-nitrophenyl phosphate was used in the soil amendment. The reactions were stopped and read at 410nm at pH of 6.8 (acid) and 11.5 (alkaline). The results were expressed as umol-p-nitrophethol Results; Results of the physicochemical properties are shown in Table 1. In the compost, temperature was above 30.0 C while in soil (both compost amended and control) it was below 30.00C. The difference was only significant (P = 0.05) in control soil which had 28.70C. pH had its highest value in PD supplemented PP compost (9.2), followed by the PP + PD compost amended soil (8.4) while the least was in the control soil which was acidic (6.3) (Table 1). The difference observed in the pH values was quite significant ( P = 0.05). P04, N03 and TOC, ranged from 2.41-3.91 mg/g, 0,21-1.34 mg/g and 9.7216.86 mg/g respectively with the highest values in the PP+PD compost. Statistical analysis showed significant difference (P = 0.05). In terms of C/N ratio, the PP+PD compost had the best ratio of 10.79 while the least was in contrl soil (26.77), followed by the soil amended with the PP compost (20.08). Lowest values were in soil (control) (10.71). The acid phosphatase enzyme was the least effected as it remained fairly the same in all the samples (soil and compost) analyzed with a range of 3.123.59 umol.p-nitrophenol. On the other hand, alkaline phosphatase showed statistically significant variation in its activities (P = 0.05). Its activities correlated positively with the observed increase in pH. Alkaline phosphatase had its highest

activity in PP +PD compost with the highest pH of 9.2 and least activity in control soil (2.77) with the least pH (6.3). Table 2 shows the bioloads of the various compost types and soil samples according to the amendments made. The THB was the most abundant group in all the samples assessed with PP+PD compost having the highest (3.9x107cfu/g) followed by PP+PD compost amended soil (3.7xcfu/g). The least occurring bacterial group was the coliform bacterial, which had a range of 1.2x10 2cfu/g (pp compost)- of 3.4x104 and 2.9 x 102 3.3 x 103 cfu/g (soil control). CUB and PSB which ranged from 2.7x104-3.9 x104 (control soil PP+PD compost) respectively were not statistically different from each other in all the samples analyzed (P =0.05). NB was lowest in PP compost (1.1x102cfu/g0 but increased to 2.4x102cfu/g in the PP +PD compost amended soil. Other sample had values between there figure. Generally, higher bacterial prevalence were observed in the soil amended with the two compost types, but the PP+PD compost caused higher increase than PP compost. Values are shown in Table 2. The results of the enzyme activities shown in Table 3 followed the pattern of THB except the urease. Dehydrohenase had its highest activity in soil amended with PP+Pd COMPOST (31.86) which had the highest THB. Its lowest activity was in the control soil (16.72). Urease activity was highest in the PP+PD compost (9.7), followed by PP compost (7.24) but lowest in control soil (3.74). Cellulose activity was statistically high in the PP+PD compost (22.31), followed

by the soil amended with the PP+PD compost (19.62) while the least was in test soil (10.71) (P=0.05). DISCUSSIONS AND CONCLUSION Results obtained in this study indicated that all the parameters determined in the physicochemical section were significantly increased by soil compost

amendment. The high TOC and PO4 observed in compost, which reflected in the amended soil, occurred as a result of the release of these nutrients from the composted material - pineapple peels. Chrost and Siuda (2006) and Karin (2006) had earlier reported similar increase in soil following organic matter incorporation. Nwaugo et al., (2008a) also observed that soil impacted with Palm Oil Mill Effluent (POME) had an increased nutrient content. In the same vein, the significant change in pH of both compost and compost amended soil could have been caused by the metabolism of the little protein in the pineapple peel or from the poultry droppings. This metabolism resulted in the release of NH3 which then dissolved in the available moisture to cause the increase in pH values. Nwaugo et al, (2008c) and Karin (2006) also reported that soil impaction with organic materials caused increase in pH values. This study therefore agrees with these researchers that composting releases plant nutrients from the composted materials which when incorporated into the soil, caused corresponding increase in the soil nutrients. Analysis of the physical and chemical parameters measured showed some significant differences in the effects of composting pineapple peel alone and incorporating poultry droppings into it. All the parameters showed higher

values in the poultry droppings amended pineapple peel compost. A similar observation had earlier been reported by Nattipong and Alissara (2006) and Tam and Wong (1995). Pineapple peel contained mainly carbohydrates while incorporation of poultry droppings added the much needed nitrogen source to have a more balanced medium in favour of better C/N ratio. The moisture content which showed highest values in the PP compost decreased in the amended soil samples. Pineapple fruit is a high moisture fruit (HMF) hence its waste had high moisture too. However this moisture was taken up by the impacted soil to decrease the observed soil moisture. Observation in the bioloads of the various bacterial groups analyzed showed increase of all groups except the coliforms. The coliforms were adversely affected by high temperature and pH. Nattipong and Alisssara (2006) reported that during composting, temperature could rise up to 60-70oC which could kill non-thermophiles, like the coliforms. Similarly, these coliforms survive more under slightly acidic or neutral pH values (Chessbrough, 2003) but in the study the pH became alkaline hence the change in coliform prevalence. The increase in PSB and CUB above the soil (control) values could be attributed to the compost effect. The composting process caused the release of microbial utilizable nutrients, resulting in their proliferation. The bacterial proliferation was more pronounced in the PD amended PP compost. This observation buttresses the earlier one in the physicochemical parameters and agrees well with Nattipong and Allissara (2006) who reported that swine manure encouraged higher bacterial growth in cassava pulp compost. Nwaugo et al,

(2008c) also reported a similar situation in cattle market waste impacted soil in Okigwe. Generally the THB were predominantly higher in all cases both in the composts and amended soil types. Nwaugo et al, (2008b) and Pelczar et al, (2003) agreed that THB is a group of all culturable bacteria while the other groups are only fractions of this THB. Again, these other bacterial groups, equally constitute part of the THB. hence will be smaller than the THB. However, the similarity between CUB and PSB could be attributed to the pineapple peel content. The major source of soil cellulose is plant material and pineapple peel is a plant material. In addition, pineapple has considerable phosphate content, hence could encourage PSB growth. . In this study, all the enzymes assessed showed highest activities in the poultry droppings (PD) supplemented pineapple peel (PP) compost impacted soil. Similarly the pineapple peel compost supplemented with poultry dropping had higher enzyme activities than the PP compost.. PP+PD compost amended soil also had higher soil enzyme activities while PP compost amended soil had lower values. Dehydrogenase activity was closely tied to the values of the bioload of the THB. This enzyme is an integral part of the living bacterial cells and is involved in the oxidation of organic matter (Makoi and Ndakidemi, 2008). It is produced by all microorganisms hence a good indicator of changes in soil. In this study, dehydrogenase had the highest activity in all the compost and amended compost

soil types. The conditions in the PD supplemented PP compost

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favoured more bacterial growth which then reflected in the dehydrogenase production and activities. The PP compost also caused higher dehydrogenase activities in soil amended with it above the control soil. Urease activity was observed to be higher in the compost with poultry droppings. This observation agrees well with Saliha et al, (2005) and Shahinrokhsar et al, (2008) who stated that there was high urease activity in soil with high urine (NH3) odour as observed during the composting process. This was more in the poultry dropping supplemented compost than the pineapple peel compost. Urease is responsible for the hydrolysis of urea with consequent release of NH3. This observation further explains the high pH change observed in the PP + PD compost and compost impacted soil. Nwaugo et al, (2008c) had earlier reported high urease activities in cattle market waste and POME impacted soil types. Cellulase activities closely followed the trends in bioloads of THB and CUB. Results indicated that high bacterial load samples showed high cellulase activities indicating more oxidation of the cellulose contained in the compost materials. However, higher cellulose per gram could be found in the pineapple peel compost but this did not induce higher cellulose activities above the poultry dropping supplemented compost. This is because increase in carbon utilization is accompanied by increase in Nitrogen consumption too (Pelczar et al, 2003). The utilization of carbon and Nitrogen results in the proliferation of the microbial cells observed in PP +D compost and compost amended soil. This observation agrees with Prescott et al (2003) that higher C/N ratio encourages better

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microbial proliferation. The increased proliferation is synonymous with increased metabolic activities. The least affected enzymes are the phosphatases which are important in the hydrolysis of esters and anhydride of phosphoric acid (Makoi and Ndakidemi, 2008). Since these are both phosphatases (acid and alkaline), they operate in both conditions to metabolize the phosphates. However, observations in this study agree well with Nwaugo et al (2008a) Ndakidemi, (2006) and Wright and Reddy (2001) that pH is an important factor in phosphatase activities. The alkaline phosphatase showed increased activities with the increase in pH values while that of the acid phosphatase did not show much change. This suggests that alkaline phosphatase is more sensitive to pH change within the range observed in this study. The presence of uric acid in the PD, in addition to the acidic nature of the composting material PP could have accounted for the observations in the acid phosphatase. In conclusion, observations in this study suggest that supplementing plant material in composting process with animal/poultry wastes has better effects on soil microbial and enzyme activities than composting plant materials alone. This means that increased biogeochemical transformations favourable to agricultural activities will be experienced in the amended soil. REFERENCES Alef, K (1995). Dehydrogenase activity; In Alef K and Nanniperi, P (Eds). Methods in applied Soil Microbiology and Biochemistry. Academic Press Inc San Diego C.A 218-230.

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AOAC (2005) Association of Analytical Chemists USA Analysis.

Official Methods of

Chessbrough, M (2003). Laboratory Manual for Tropical Countries Vol,II Microbiology ELBS edition Tropical Health technology and Butterworth, London. Chrost, R.J and Siuda, W. (2006) Microbial Production, Utilization and enzymatic degradation of organic matter in the upper trophogenic layer in the pelagial zone of lake along a eutrophication gradient. Limnol Oleanogr. 51(apart 2) 749-762 Cookson, P and G.L Lepiece, (1996) Urease enzyme activity of soil of the Bantinah region of Sultanate of Oman J Acid Environ 32:225-238. Deng, S.P and Tabatabai, M.A (1994) Cellulase activity of soils Biochem. 26: 1347-1354. Haram, S. Logendra, s. Saskar, M, Bratanora, M and Raskin,I (2000) Characterization of Arabidopsai acid phosphate promoter and regulation acid phosphatase expression. Plant Physiol. 124:615-626. Humphriers, P.A (2002). Effects of long term metal Contamination of the structure and function on microbial communities in soils. PhD. There environmental toxicology Texas Technology University, Texas, U.S.A. Karin, N. (2006)Impact of organic waste residue on structure and function of soil bacterial communities Ph.D Thesi faculty of Natural Resources and Agricultural Sciences Swedish University of Agricultural Sciences Uppsala, Swedish. Soil Biol

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Li, H Zhang, T. Zhang, C.G and Chem, GX (2005) Effects of petroleum containing waste water irrigation on bacterial diversities and enzymatic activities in a paddy soil irrigation area. J. Environ. Qual. 34:1073-1080. Makoi, J.H R and Ndakidemi, P.A (2008) Selected soil enzymes. Examples of their potential roles in the ecosystem Afri J. Biotect. 7(3):181-191. Nannipieri, P., Kandeler, E and Euggiero, P (2002) Enzyme activities and microbiological and biochemical processes in soil. In, Burns RG and Dick, R.P (Eds) Enzymes in the environment activity, ecology and applications. Dekken New York. 1-33 Nannipieri, P., Crecanti, B, Bianchi, D and Bonmati M (1985) Fractionation of hydrolase humus complexes by gel chromatography. Boil. Feit Soils 1:2529.

Nattipong K and Alissara, R (2006) Effect of cassava pulp and swine manure compost on growing plants in Greenhouse J Water Environm. Technol. 4(1): 9-32. Nwaugo, VO, Onyeogba , R.A , Obiekezie, S.O and Ugbogu. O.C (2007). Effect of petroleum produced (information) water on some farmland soil bacteria species Egbema Rivers State. Int. J. Biotechnol and Allied Sc. 1(1):32-36. Nwaugo, V.O. Etok, C.A Obiekeze, S.O and Chinyere, G.C (2008a). Evaluation of the effects of Okigwe cattle market wastes on the surrounding agricultural soil parameters Bio-Res 6 (11) 367:370.

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Nwaugo, V.O Chinyere, G.C and Inyang, C.U (2008b) Effects of Palm Oil Mill effluents (POME) on soil bacterial flora and enzyme activities in Egbema. Plant Prod. Res. J 12:10-13. Overing, J.D, Wecks, H.H, Wilson, Z.P Sullivan S. and Ford, R.D (1995) Soil survey of Okalosa Country, Florida, U.S Department of Agriculture National Resources Conservation Service U.S.A. Onyeagba, R.A; Nwaugo, V.O.; Obiekezie, S.O and Verla, A.W

(2008).Phytoremediation of Pb and Zn polluted soil with Chromolegna odaranta and Talium triangulare in Ishiagu mining area, Ebonyi, State Nigeria. 32nd Annual Conference of Nigeria Society for Microbiology, Abia State University Uturu, Oct. 10-13th, 2008. Pelczar, M, Chan EI.S and Kriegi N.R (2003) Microbiology of the soil and atmosphere; concepts and applications in microbiology McGraw Hills, Inc. U.S.A. 772-873 Prescott L.M, Haley J.P and Klein, D.A (2003) Microbiology 6th edition 234-576 McGraw Hill United State of America. Saliha, B.B, Krishnakumar, S, Saravanan, A and Natarajan, S.K (2005) Microbial and enzyme dynamics in distillery spent wash treated soil.Res. J. Agric Biol. Sc 1 (2). 166-169 Shanhinrokhsar, P, Vahed, H.S and Haghdad, A. (2008) Evaluation of some paddy soils Properties on urease enzyme activity. Conference on International Research on Food Security, Natural Resources Management and Rural Development University of Hohenhein, October, 7-9,2008.

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Tabatabai, M.A (1997). Soil enzyme In: Page, .C Miller P.H and Kenny, D.R Methods of soil Analysis parts 2 chemical and microbiological properties Soil Science Society of America. Madison W.I. Tam, N.F.Y and Wong Y.S (1995) Spent litter as fertilizer for growing vegetables. Biores Technol. 53: 151-155. Wright, AL and Reddy, K.R (2001) Phosphorus loading effects on extracellular enzymes activity in everglades Wetland Soil, SSSAJ. 65.588-595. TABLE 1: Physicochemical parameters of the compost and soil amended with these composts Parameters Temp C TOC mg/g NO3 mg/g PO4 mg/g Moisture content % pH C/N Ratio
o

Pp compost 30.4 13.31 1.32 2.84 28.4 8.4 10.08

PP + DD compost 30.8 16.8 3.17 3.91 26.74 9.2 5.31

Soil + PP compost 29.6 9.7 1.24 2.76 26.2 7.2 7.82

Soil PP + PD 29.8 12.8 2.21 3.28 24.11 8.1 5.79

Soil alone 28.7 12.28 1.4 2.41 10.42 6.3 8.77

TABLE 2: Values of enzymes activities determined in the compost study Bacteria group THB CB CUB Pp compost 3.2X10 + 0.10 1.2X102 + 0.2a 2.7X 104 + 0.2a
5 a

PP+PD compost 3.9X107 + 0.3b 1.5X102 + 0.3a 3.9X104 +0.4b 16

Soil alone 4.7X10 + 0.5 3.4X104+ 0.5b 2.2X104+ 0.6C

6 C

Soil

PP+DP Soil +PP+PD 3.7X107 + 0.9e 1.6X103 + 0.9e 3.6X104 + 0.0e

compost 8.7X105+ 0.7d 1.0X103+ 0.7c 3.1X104+ 0.8b

PSB NB Key THB CB CUB NB PP

2.7X102 + 0.2a 1.1X102 + 0.2a

3.1X103 + 0.4b 1.8X103 +0.3b

2.7X103+ 0.7C 2.1X103+ 0.4C

2.4X103+ 0.8c 1.7X103+ 0.3b

3.3X103 + 0.9d 2.4X103 + 0.9e

- Total Heterotrophic Bacteria - Coliform Bacteria - Cellulose Utilizing Bacteria - Nitrifying Bacteria - Pineapple Peel PD - Poultry Dropping

Figures followed by the same alphabets are not significantly different but those followed by different alphabets are significantly different.

TABLE 3: Bioloads of various bacterial groups in composts and composts amended soil sample (cfu/g) Enzyme Dehydrogenase Urease Acid phosphatase PP compost 18.62 7.24 3.12 PP+ PD Soil alone 16.72 3.74 3.27 2.97 10.71 Soil+PP compost 23.91 4.58 3.31 3.67 14.41 Soil +PP+PD 31.86 5.74 3.43 4.12 16.62

compost 28.31 9.7 3.59

Alkaline phasphatase 3.10 4.62 cellulase 19.24 22.31 Values are means of three times sampling

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