Enhancing rock phosphate integration rate for fast bio-transformation of cow-dung waste-paper mixtures to organic fertilizer
© The Author(s) 2016
Received: 6 April 2016
Accepted: 9 October 2016
Published: 16 November 2016
Rock phosphate (RP) addition in cow-dung waste-paper mixtures at rates above 2% P has been reported to increase the rate of bio-transformation and humification of organic waste mixtures during vermicomposting to produce organic fertilizer for organic farming. However, the optimization of RP for vermicomposting was not established. The objective of this study was to determine the optimal amount of RP integration rates for effective bio-transformation of cow-dung waste-paper mixtures. Arrays of RP integration degrees (0, 0.5, 1, 1.5, 2, and 4% P as RP) were thoroughly mixed with cow- dung waste-paper mixtures to achieve an optimized C:N ratio of 30 and allowed to vermidegrade following the introduction of earthworms at a stocking mass of 12.5 g-worms kg−1. The bio-transformation of the waste mixtures was examined by measuring C:N ratios and humification index (HI) and per cent ash and volatile solids. Application of 1% P as RP resulted in fast bio-transformation and maturation of cow-dung waste-paper mixtures. A scanning electron microscopy (SEM) was used to evaluate the morphological properties of the different vermicomposts affected by rates of RP showing the degree of degradation of initial compacted aggregates of cellulose and protein fibres in the mixtures at maturity. A germination test was used to further determine phytotoxicity of the final composts and microbial biomass assessment. The final vermicompost (organic fertilizer) had a C:N ratio of 7, MBC of 900 mg kg−1 and HI of 27.1%. The RP incorporation rate of 1% P of RP investigated is therefore, recommended for efficient vermidegradation and humification of cow-dung waste-paper mixtures. However, higher rates of RP incorporation should be considered where greater P enrichment of the final vermicompost (organic fertilizer) is desired.
Vermicomposting has proved to be a suitable technique of processing biodegradable organic wastes and converting them to organic fertilizers, because of its low cost and the large quantity of wastes that can be processed (Lim et al. 2016; Wu et al. 2014). Generally, the vermicompost which is produced from vermicomposting process can enhance soil fertility physically, chemically and biologically (Lim et al. 2015). However, the nutritional values of some compost/vermicompost products are erratic and determined principally by the types of the substrates used and the degree of composting (Matiullah and Muhammad 2012; Mupondi 2010; Yan et al. 2012). In order to increase the acceptability of vermicompost products as sources of nutrients or as growing media, it is essential to increase their P contents. Biswas and Narayanasamy (2006) found that it is possible to increase the total P content of straw compost from 0.37% in control to 2.20% by adding 4% P as RP, while (Yan et al. 2012) also increased the total P of rice straw vermicompost from 0.392 to 0.82% by adding 2% P as RP. Matiullah and Muhammad (2012) also increased the total P content in poultry litter from 0.3 to 1.02% by adding 4% P as RP.
In addition to increasing the P contents of composts, the inclusion of RP has also been shown to improve the humification of organic wastes. Singh and Amberger (1990) reported that organic solid waste compost inoculated with RP not only increased its P content but also enhanced the humification of the resultant vermicomposts. Similarly, Mishra (1992) obtained highly humified compost from plant wastes composting by adding 25% Mussoorie RP. This prompted (Mupondi 2010) to investigate the effect of RP incorporation on the vermicomposting of cow-dung paper-waste mixtures. He investigated rates of RP incorporation ranging from 2 to 8% P as RP. The results showed that the vermicomposts were highly humified and had high total P and N contents as well as available P and N contents compared to the control. All rates of RP incorporation >2% P improved humification to more or less the same extent suggesting that lower rates of RP incorporation could possibly being effective in improving the vermidegradation of cow dung waste paper mixtures. This study, therefore, investigated the effectiveness of using low RP application rates of less than 2% P or 4% P in improving the vermidegradation of cow-dung paper-waste mixtures.
The objectives of this study were to determine: (1) the minimum amount of rock phosphate necessary for efficient bio-transformation of cow-dung paper-waste mixtures, and (2) the effects of the rate of RP application on total P, Bray-1 extractable P, microbial biomass index, Ammonium and nitrite-nitrate levels and germination index of the vermicompost (organic fertilizers).
Location description, earthworms and wastes utilized
The vermicomposting experiment was performed in an enclosed, shaded yard at the University of Fort Hare’s Teaching and Research farm located 32°46′S and 26°50′E in the Eastern Cape Province of South Africa under an ambient mean temperature of 25 °C. Paper waste used for the study was collected from the University printing press (Xerox) and Faculty offices, while RP was obtained from Phalaborwa in Limpopo Province, South Africa, which is granitic in nature. Eisenia fetida (E. fetida) earthworms used in the study were collected from a local wormery at the University of Fort Hare’s Teaching and Research farm. Cow dung was obtained from Keiskammahoek Dairy Project located about 60 km North East of the University of Fort Hare.
Phosphorus enhancement of vermicomposts with RP
Rock phosphate was incorporated into cow dung waste paper mixture at 6 rates of 0, 0.5, 1, 1.5, 2 and 4% (elemental P basis had the following chemical properties: P2O5-40.3%; CaO-54.6%, MgO-0.26%; cadmium-2.2 mg kg−1, chromium-18.05 mg kg−1, copper-5.85 mg kg−1, lead-6.05 mg kg−1 and zinc-13.22 mg kg−1) as ground RP with the aim of improving the P contents of the mixtures. Each was thoroughly mixed with enough feedstock (5 kg on dry weight basis) and prepared by mixing 2.16 kg of shredded paper and 2.84 kg of cow dung to achieve an optimized C:N ratio of 30. The resultant mixtures were loaded into worm boxes whose capacity measured 0.50 m × 0.40 m × 0.30 m (length × width × depth) with an exposed top surface area of 0.2 m2 in a well-ventilated farm building at an average mean ambient temperature of about 25 °C. Mature E. fetida earthworms were introduced into the vermin-reactors at a stocking density of 12.5 g worms kg−1 feed established by (Unuofin and Mnkeni 2014) as optimum for the bio-conversion of cow-dung paper-waste mixtures. Treatments were arranged in a completely randomized experimental design (CRD) with three replications. Moisture content in each box was brought to 80% by sprinkling water (Reinecke and Venter 1985) and maintained at this level by regular sprinkling throughout the 6 weeks of vermicomposting period.
Sample collection was carried out on days 0, 14, 28, 42 and 56 of vermicomposting and analysed for volatile solids (where ash content is a complementary parameter of VS) total C, total N, organic and inorganic phosphorus.
The resultant vermicompost samples were analysed for volatile solids (VS), ash content, total carbon and total nitrogen, pH, extractable phosphorus P and humic substances. These vermicompost samples were first air-dried until constant weight was achieved and subsequently pulverized (<2 mm) to offer a uniform sample for analysis. The volatile solids (VS) were determined as sample weight loss of previously oven-dried (at 105 °C) samples following ashing at 550 °C for 4 h in a muffled furnace (Ndegwa et al. 2000) while total nitrogen (N) and carbon (C) were determined using a Truspec CN Carbon/Nitrogen analyser (LECO 2003). The pH was determined in a vermicompost-water suspension (1:2.5) with a pH/Conductivity meter as described by (Ndegwa et al. 2000).The suspensions were allowed to stand for 1 h after constant shaking using a mechanical shaker at 230 rpm for 30 min prior to pH measurement.
Microbial biomass carbon
Morphological assessment of the resultant vermicomposts
Scanning electron microscopy (SEM) images of the samples were taken using a scanning electron microscope model JOEL (JSM-6390LV, Japan). Briefly, the samples were oven-dried and ground to pass through a 2 mm sieve. A small representative portion of the samples was coated with gold and mounted on SEM. Samples were then imaged by scanning them with a high-energy beam of electrons in a raster scan pattern.
Data reported herein are the means of three replicates (n = 3). Statistical analysis was done using the repeated measures analysis of variance (ANOVAR) since sampling was done non-destructively. Fisher’s protected least significant difference (LSD) test at P < 0.05 was used for means separation. All statistical analyses were done using JMP® Release 10.0 statistical package (SAS Institute, Inc., Cary, North Carolina, USA, 2010)
Effects of RP rate on volatile solids contents
Repeated measures ANOVAR for C:N ratio, HI, Total P, Total N, Bray-1 extractable P, MBC, VS and Ash contents, NH4-N and NO3-N
Added RP rates
Added RP rates × time
Total P (g Kg−1)
Total N (%)
Bray 1P (mg Kg−1)
MBC (mg Kg−1)
NH4 +-N (mg Kg−1)
NO3 −-N (mg Kg−1)
NH4 +:NO3 −
Effect of added RP application on %Ash conversion in relation to %VS
RP application rates (%)
23 ± 1.0
77 ± 1.0
32 ± 0.01
68 ± 0.01
23.33 ± 0.5
76.66 ± 0.5
40 ± 1.0
60 ± 1.02
22.66 ± 1.2
77.33 ± 1.2
42.33 ± 3.21
57.67 ± 3.24
23.33 ± 0.5
76.66 ± 0.6
48.66 ± 0.5
51.37 ± 0.6
23 ± 1.0
77 ± 1.01
44.66 ± 1.54
55.37 ± 1.54
23 ± 1.0
77 ± 1.01
44.66 ± 1.54
55.37 ± 1.55
The volatile solid decreased significantly with time at each added RP rate, with the 1% P rate of RP application resulting in consistently low VS content levels, however, the absolute control where no RP was added had the highest VS levels (Table 2). The other rates of RP application resulted in intermediate low VS contents which followed the order 4% P ≈ 2% P < 1.5% P > 0.5% P (Table 2). Final per cent VS content ranged from 50 to 60% in vermicompost where RP was added, while in the control, where no RP was added, the VS content was 68% (Table 2).
Effect of rate of RP application on the pH of cow-dung paper-waste vermicompost mixtures
RP application rates (%)
Initial pH of cow-dung paper-waste mixtures
Final pH of cow-dung paper-waste mixtures
8.87 ± 0.01
7.98 ± 0.01
8.85 ± 0.01
7.81 ± 0.02
8.82 ± 0.01
7.33 ± 0.01
8.83 ± 0.01
7.64 ± 0.02
8.84 ± 0.01
7.68 ± 0.03
8.85 ± 0.01
7.77 ± 0.01
Effect of RP rate on the carbon to nitrogen ratio
Effect of rate of RP application on the vermidegradation time of cow-dung paper-waste mixtures as reflected by C:N ratio
Treatment % P as RP
Regression equations (C:N ratio VS Incubation time in days)
Days required to maturity (i.e. at C/N ratio = 12)
% Improvement in reducing time to vermicompost maturity
y = −0.0026x2 − 0.2072x + 30.8
y = 0.002x2 − 0.1234x + 31.0
y = 0.0066x2 − 0.7714x + 29.229
y = 0.005x2 − 0.6458x + 31.6
y = 0.0059x2 − 0.7038x + 30.4
y = 0.007x2 − 0.7646x + 29
P > F
Effect of RP rate on humification parameters
Effects of the rate of RP on MBC
Changes in the morphological structure of cow-dung-waste paper mixture during vermicomposting
Effects of RP rate on Nitrite-N NH4 +-N and NH4 +:NO3 − ratio
The ammonium-N (NH4 +-N) and the ratio of NH4 +:NO3 − in cow-dung-waste paper vermicompost followed the same trend whereby they both increased up to day 14 with no striking differences in added RP, thereafter; they decreased linearly up to day 28. Beyond day 28, sharper and significant decreases were observed with time at each added RP rate (Fig. 5b, c). The NH4 +-N and the NH4 +:NO3 − ratios of the waste mixtures both increased significantly with time at each rate of added RP. The control, where no P was added, had the highest NH4 +-N and the NH4 +:NO3 − ratio, while 1% P rate of RP application resulted in consistently the lowest contents (Fig. 5b, c). The other rates of RP application followed the order: 4% P ≈ 2% P ≈ 1.5% P > 0.5% P (Fig. 5b, c). Final NH4 +-N contents and the NH4 +:NO3 − ratio values ranged from 12 to 4 mg kg−1 and 0.8 to 0.1 where RP was added, respectively (Fig. 5b, c). In the control where no RP was added, the NH4 +-N content and the NH4 +: NO3 ratio were 16.4 mg kg−1 and 0.9, respectively (Fig. 5b, c).
Effect of rate of RP on the phytotoxicity level
Effect of added RP on the phytotoxicity of cow-dung waste-paper vermicomposts
The effect of RP rate on total P, Bray 1 extractable P
Effects of added RP on Total P and Bray-1 extractable P contents of the final cow-dung paper-waste vermicomposts
Total P (%)
Bray 1 P (mg kg−1)
Increase in Extractability of P (%)
Effects of RP rate on selected maturity
One of the main aims of this study was to explore the possibility of enhancing the bio-transformation of cow-dung-waste paper mixtures with lower rates of RP integration.
The observed significant decrease in volatile solids contents with time, at each rate of added RP (Table 2), indicated degradation of organic matter (OM) in the vermicomposting mixtures, as reported by Mupondi (2010). This trend was confirmed by the maturation parameters used to monitor the stabilization of the vermicomposts, namely: Humification index (HI) (Fig. 2), microbial biomass carbon (MBC) (Fig. 3), Nitrite-N (Fig. 5a), Ammonium-N (Fig. 5b), and ratio of NH4 +:NO3 − (Fig. 5c) and SEM imagery (Fig. 4a, c).
Vermicomposting decreased pH of the mixtures from 8.87 ± 0.01 in control to 7.33 ± 0.01, of different RP application rates, respectively. This is in accordance with the results of (Atiyeh et al. 2000; Venkatesh and Eevera 2008; Raphael and Velmourougane 2011). pH decrease may be due to the nitrification process experienced by the composting process, accumulation and reduction of organic acids from microbial metabolism, production of fulvic and humic acids during decomposition or as a result of NH3 volatilization during composting process (Albanell et al. 1988; Hanc and Vasak 2015; Cáceres et al. 2006).
The C:N ratio results (Fig. 1) confirmed that incorporation of RP accelerated the vermidegradation of the cow-dung-waste paper mixtures in that by day 42, the C:N ratio of the waste mixtures had declined from 30 when the experiment was initiated (day 0) to less than 14 where RP was incorporated, compared with 18 (where it was not). The results further revealed that the improved degradation was fastest when RP was added at a rate of 1% P, which allowed compost maturity to be achieved within 33 days. This indicated that a rate lower than the lowest rate of 2% P as RP used by Mupondi (2010) could be used to improve the rate of biodegradation of cow-dung-waste paper mixtures using E. fetida. The possibility of using lower rates of RP incorporation to enhance the vermidegradation of cow-dung-waste paper mixtures and (possibly) other waste will mean less transportation costs to places far from the Limpopo Province of South Africa where RP is mined.
The rate of C:N ratio decline was fastest between days 14 and 28 and slowest after day 42, thereby explaining the observed interaction between significant RP rate and time (Table 1). Bernal et al. (2009) and Lim et al. (2014) reported that the decline in C:N ratio could be attributed to the decomposition of organic matter as a result of microbial action. This is supported by the MBC data (Fig. 3) which shows that microbial activity was the highest on day 14 followed by day 28. This is a period coinciding with the sharpest decline in C:N ratio at each rate of added RP. This is further supported by the nitrate and ammonium data (Fig. 5a, b) which shows that ammonification and nitrification, both microbially-mediated processes, were most intense during this period. Ammonification is produced before nitrification. In this process, ammonia is converted into nitrite and nitrate during the nitrification process, for which mainly two different groups of microorganisms are responsible, the ammonia oxidizing and the nitrite-oxidizing bacteria. The nitrate is further assimilated into organic material or reduced to nitrogen oxides by denitrifying bacteria or completely into ammonia by the process of nitrate ammonification, mainly operated by fermentative microorganisms. The MBC increased with the rate of RP application, thus suggesting that RP had a stimulatory effect on microbial activity. Mupondi (2010) reported a similar trend of highest microbial activity on day 14 followed by day 28 during the vermicomposting of dairy manure-paper waste mixtures, as well as enhanced increase in available P. The author ascribed this increase in available P to the presence of high microbial populations, as shown by large amounts of microbial biomass C and P at the early vermicomposting stage. This increase, the author further noted, could be as a result of an increase in phosphate-solubilising microorganisms. Also the gut of the earthworm usually produces a considerable amount of alkaline phosphatase which is essential enzyme excreted through cast deposition and involved in biogeochemical cycle of phosphorus in soils (Lim et al. 2011). Similarly, Bhattacharya and Chattopadhyay (2002) reported an increase in phosphate-solubilising microorganisms while vermicomposting fly-ash amended cattle manure.
The increase in the HI with time indicated that the vermidegradation increased humification of the cow-dung-waste paper mixtures. This transformation was influenced by the rate of RP application and interestingly, it was highest at the 1% P rate of RP application. This intensification of degradation of the mixtures was further confirmed by the images from the scanning electron microscopy (Fig. 4a, c) that showed RP application intensified the degradation of cow-dung-waste paper mixtures and that the 1% P rate of RP application resulted in consistently greater vermidegradation of the waste mixtures at each sampling date than with the other RP treatments. This was further reflected in higher segregation of the waste mixture aggregate particles. According to Lim and Wu (2015, 2016) the SEM image of the vermicompost showed a distinct physical appearance that was more scattered fragmented and smaller in nature as compared to the initial waste mixtures. Therefore, the SEM images observed in this study were similar to Lim and Wu (2015, 2016) and showed that degradation of the cow dung-waste-paper mixtures was intensified with time at each rate of RP application. The wide differences in the extent of degradation, at different rates of RP application observed on days 14 and 28 (pH 20, which coincided with the period of maximum microbial activity (Fig. 3), is further proof that the observed improved vermidegradation of waste mixtures mixed with RP could be related to its stimulatory effect on soil microorganisms. The RP constituent responsible for this stimulation remains to be established.
The seed germination index is a more direct indicator of both vermicompost and compost maturity as it directly tests whether the finished vermicompost can inhibit plant growth or not when used as a growth media. The over 80% GI observed for all test crops in this study indicated that addition of P as RP to cow-dung-waste paper mixtures in the presence of E. fetida resulted in vermicompost that was free of phytotoxins, according to (Zucconi et al. 1981; Tam et al. 1998). These results are also in agreement with those of Bustamante et al. (2001) which showed that a germination index of ≥80% indicated the disappearance of phytotoxins in composts.
Effects of RP rate on the P enrichment
The total P content of 2.31% P in the final cow-dung-waste paper vermicompost, where RP was added at a rate of 4% P, is consistent with results of Biswas and Narayanasamy (2006) who reported increase in the total P content of straw compost from 0.37% in control to 2.20% by applying 4% P as RP. Yan et al. (2012) also reported an increase in the total P of rice straw compost from 0.392 to 0.82% through application of 2% P as RP. More significantly, however, in the present study, Bray 1 extractable P increased from a low 80 mg P kg−1 where no RP was added to 207 mg P kg−1 where RP was added at a rate of 4% P (Table 5). This contributed to the increase in the extractable P and, thus, implies significant increases in the effectiveness of vermicompost in providing available P.
The improvement in the extractability of P with the rate of RP application (Table 5) mirrored an increase in microbial biomass with the rate of RP application (Fig. 3), thereby suggesting that among the micro-organisms stimulated by added RP were phosphate bacteria which facilitated the mineralization, hence, extractability of P from the added RP. The P release is mediated by phosphate enzymes produced by micro-organisms in the earthworm guts and those in the earthworm casts (Yan et al. 2012). The observed increase in humic acids reflected by the humification index or polymerization index (HI or PI) (Fig. 2) could also account for the enhanced mineralization and dissolution of P. According to Singh and Amberger (1990), humic acids can absorb significant quantities of calcium ions and release an H+ ion, which further facilitates the dissolution of RP. In addition, the functional groups in humic acids such as carboxylic and phenolic groups can also chelate Ca++ ions, thus providing a driving force for the mineralization and dissolution of P from RP.
The increase in Bray 1 P up to day 42 and its decline thereafter (Fig. 6) suggested that after 42 days, the mineralized P underwent precipitation reactions, hence the decreased extractability. Sequential extraction showed that the H2O-Pi fraction made the largest contribution to the total inorganic P extracted, thus suggesting that most of the mineralized P, during the vermicomposting of the cow-dung-waste paper mixtures enriched with RP, would be available for plant uptake. These results indicate that vermicomposting cow-dung waste-paper mixtures enriched with phosphate rock improves the solubilisation of the Phalaborwa RP used in this study and thus improved its fertilizer value. According to Edwards et al. (2010), RP is an acceptable source of P for organic agriculture, but its use is limited by its slow rate of P release. The high total P and extractable P contents observed in the cow-dung waste-paper vermicomposts enriched with RP point to their potential as organic P fertilizers. Future studies should explore this potential.
The results of this study have confirmed that incorporation of rock phosphate improves the biodegradation of cow-dung-paper-waste mixtures. The cause of the influence of P is as a result of the gut of the earthworm which produces a considerable amount of alkaline phosphatase, which is an essential enzyme excreted through cast deposition and involved in biogeochemical cycle of phosphorus in soils. The result further revealed that optimal vermidegradation can be achieved with the application of 1% P as RP. It also showed that the final C/N ratio at 14, 28 and 33 days of vermicomposting were 20, 17 and 12 respectively. Hence, at this rate of RP incorporation, the vermicomposting mixtures required only 33 days to reach maturity. The improvement in vermicomposting occurred mostly between days 14 and 28. Although a 1% P rate of RP application is all that was needed for fast maturation of the vermicompost, higher rates of RP application are necessary for an enhanced P fertilizer value of the resultant vermicompost. Therefore, higher rates of RP incorporation may be necessary where final composts with higher P contents and, thus, better P fertilizer value are desired. Future studies will need to examine the agronomic value of these composts, find out the reason why 1% treatment was the one with the best degradation of the OM. Nevertheless, the results of the present study have shown that cow-dung waste-paper vermicomposts enriched with phosphate rock have potential as organic fertilizers which would be acceptable in organic farming.
FOU conceived of the study, participated in its design and sequence alignment, performed the statistical analysis and drafted the manuscript; MS participated in the sequence alignment and drafted the manuscript; ENC participated in the coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Funding was provided by the Directorate of Research and Innovation Centre of Walter Susulu University, Mthatha, South Africa.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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