Inﬂuence of Microbial Inoculation of Igneous Rock Phosphate-Amended Cow and Pig Manures on Vermidegradation and Nutrient Release

: Vermicomposting using Eisenia fetida has been shown to improve phosphorus mineralization from rock phosphate (RP). There is, however, a lack of information on the potential of integrating microbial inoculants like phosphate-solubilizing bacteria (PSB) during vermicomposting as a way of improving vermidegradation, and the release of nutrients from igneous RP-amended composts. This study evaluated the potential of using Eisenia fetida and Pseudomonas ﬂuorescence in enhancing the vermidegradation, and nutrient release in igneous RP- amended cow and pig manure-based vermicomposts at a C / N ratio of 30. Compost maturity, nutrient and phytotoxicity parameters were measured to determine vermicompost quality. Final vermicompost results showed that the pig manure treatments achieved greater maturity as indicated by lower C / N of 10.3, high humiﬁcation index (HI) of 7.6%, and humiﬁcation ratio (HR) of 12.89% in the treatment with E. fetida and P. ﬂuorescence . The inclusion of E. fetida alone in cow manure treatment resulted in the highest Olsen P of 2600 mg / kg, followed by the pig manure treatment with E. fetida only (2246.15 mg / kg). In conclusion, both E. fetida and P. ﬂuorescence are necessary in the vermicomposting of RP-enriched pig and cow manure for e ﬃ cient vermi-degradation and nutrient release.


Introduction
The major challenge associated with world population growth has been the generation of huge quantities of solid waste. Currently, the global production of solid waste is estimated to be 1.3 billion tons per year and expected to double by 2025, with the organic wastes fraction ranking the highest among the solid waste generated [1]. Furthermore, the intensification in animal production for meat, eggs and dairy products has also resulted in drastic increases in the amounts of animal wastes produced [2]. Recycling of nutrients from solid waste through composting and vermicomposting have been studied to reduce the inappropriate disposals of these organic wastes in agricultural lands [2,3]. Furthermore, there has been a growing realization of the importance of organic nutrient sources in improving overall soil quality. Vermicomposting is one technology that has gained momentum in recent years as having the potential for converting both organic and inorganic waste materials into nutrient-rich fertilizer sources [4,5].
During the vermicomposting process, microorganisms in the gut of earthworms get their nourishment from the ingested waste and earthworms to stimulate the activity of microorganisms in soils through their microorganism-enriched casts [4,6]. Both the earthworms and microorganisms play

Source of Materials
The study was performed at the University of Fort Hare research farm located in Alice Campus, Eastern Cape Province, South Africa. The white wastepaper used which was printed with black ink and not bleached, was collected from the University of Fort Hare, Alice campus and was shredded using a paper shredder. The cow manure used was collected from local kraals around the University of Fort Hare from cattle feeding exclusively on natural pasture. The cow manure was crushed to remove large lumps, air-dried, and stored under shade in a dry area. The pig manure was collected from a local piggery in Alice, where the pigs were fed exclusively on commercial feed. The pig manure collected had been mechanically squeezed to separate the solids from liquids, and these solids were then air-dried and stored under shade. Ground igneous RP was sourced from Phalaborwa located in the Limpopo province of South Africa. The igneous RP was supplied in a powder form which could pass through a 0.25 mm sieve, and its chemical properties were as follows: P 2 O 5 -40.3%,CaO-54.6%, MgO-0.26%, chromium-18.05 mg kg −1 , cadmium-1.2 mg kg −1 , copper-5.85 mg kg −1 , and zinc-13.22 mg kg −1 and lead-6.05 mg kg −1 . These chemical properties were provided through accredited methods and were availed to us by Forskor, Phalaborwa, South Africa. Selected chemical properties of the cow manure, pig manure and wastepaper used in this study are shown in Table 1. Mature E. fetida earthworms that had been feeding on a mixture of cow and wastepaper were obtained from the University of Fort Hare local wormery. Phosphate solubilizing bacteria were sourced from a local South African supplier Microbial Biological Fertilizer International (MBFi) (http://www.mbfi.co.za/biological-inoculants-2/). The vermireactors used were plastic truncated cone containers with a volume of 0.015 m 3 having perforations underneath and on the lids.

Treatments, Experimental Design, and Experimental Establishment
The experiment was factorial with four factors, i.e., manure type (cow and pig manure); earthworm presence (with or without); PSB presence (with or without) and time (0, 2, 4, 6 and 10 weeks). This factorial design gave eight treatments as listed below, which were laid in a completely randomised design with three replications.
(1) Cow manure (CM) with no E. fetida or P. fluorescence (Control) (2) Pig manure (PG) with no E. fetida or P. fluorescence (Control) (3) CM with E. fetida alone (4) PG with E. fetida alone (5) CM with P. fluorescence alone (6) PG with P. fluorescence alone (7) CM with E. fetida plus P. fluorescence (8) PG with E. fetida plus P. fluorescence The cow and pig manure used in this study, were mixed with the shredded paper to adjust the C/N ratio to 30:1, as recommended by Mupondi, [18] whilst the RP was then added at 2% (w/w basis) to the organic waste materials as recommended by Unuofin and Mnkeni, [13]. The total weight of the mixture that was used was maintained at 3 kg (dry weight), with no extra feed being added during the experiment.
Before adding the earthworms and the P. fluorescence, the mixtures from all the treatments were allowed to pre-compost for 1 week to moisten the mixture and to eliminate volatile toxic gases [19].
After the pre-composting, the earthworms and the P. fluorescence were introduced in the respective treatments, and the substrate was adjusted to a moisture content of 60-70% as recommended by Mupondi, et al. [5]. Mature clitellate earthworms were introduced at 22.5 g-worm/kg dry substrate as recommended by Unuofin and Mnkeni, [13] while the P. fluorescence was inoculated at 50 mL/kg substrate having 1 × 10 9 CFU/mL as described by Das,et al. [20] and Busato, et al. [16]. Samples were collected at 0, 2, 4, 6 and 10 weeks, and oven dried before being ground using a mechanical grinder with a 1 mm sieve. The ground vermicompost samples were then analysed for several chemical and biological parameters as described below.

Total C and N Determination
The total C and nitrogen were determined on dry, ground samples using a LECO Truspec CN auto analyser [21].

Humic and Fulvic Acids
The humic and fulvic acid fractions in the composts were extracted using a method described by Sánchez-Monedero, et al. [22]. A 0.1 M NaOH solution was used as an extraction ratio of 1:20 (w/v), with the extracts being centrifuged at 8000 rpm equivalent to 8.2 × 10 3 g (relative centrifugal force). The supernatant was then divided into portions with one half being analyzed for total extractable carbon fraction (C tEX ). The other half was acidified using H 2 SO 4 to a pH of 2 and then centrifuged at 8000 rpm after coagulation. After centrifuge, the supernatant was used for the analysis of fulvic acid (C FA ). The C concentrations in the supernatants were determined using the dichromate oxidation method, with the concentration of the humic acid (C HA ) fraction being calculated as the difference between (C tEX ) and (C FA ). Humification ratio (HR; Equation (1)) and Humification index (HI; Equation (2)) which are indices used for the evaluation of humification level in the compost were calculated as presented in the equations below; where C is total carbon in the vermicompost samples determined by dry combustion [21]: 2.3.3. Inorganic Extractable Nitrogen (Ammonium, Nitrate, and Nitrite) The mineral nitrogen was extracted using 0.5 M potassium sulphate (1:10 w/v) [23]. 50 mL of the 0.5 M potassium sulphate was added to 5 g sample, shaken for one hour in a reciprocal mechanical shaker at 180 rpm and then filtered using Whatman No. 2 filter paper for colorimetric analysis. The ammonium and nitrate concentration were determined using a UV/Vis spectrophotometer (Helios Gamma, Thermo Spectronic, Cambridge, United Kingdom), after the development of a blue color using salicylate-nitroprusside colorimetric method for ammonium-N, and a yellow color using 5% salicylic acid in concentrates sulphuric acid for nitrate and nitrite-N [24].

Olsen Extractable P
A solution of 0.5 M sodium hydrogen carbonate that had been adjusted to a pH of 8.5 using 1 M sodium hydroxide was used for extraction of Olsen P [25]. From the compost sample, 2.5 g was weighed and then extracted with 50 mL of 0.5 M NaHCO 3 solution and was shaken on a horizontal shaker for 30 min at 180 rpm. After shaking at this time, the sample extracts were filtered through Whatman TM No.42 filter paper, and the filtrates were adjusted to a pH of 5 with 2.5 mL sulphuric acid before the analysis. The P in the solution was analysed using the colourimetric ascorbic acid method. The ascorbic acid was dissolved in an ammonium molybdate solution that was prepared by mixing 2.5 M sulphuric acid with ammonium molybdate and potassium antimony tartrate. For the analysis of P, 10 mL aliquot was mixed with 8 mL of the ascorbic reagent in a 50 mL volumetric flask and filled to the mark with deionized water. The absorbance was measured at 880 nm after 10 min of standing, using UV spectrophotometer (Helios Gamma, Thermo Spectronic, Cambridge, United Kingdom), that had been calibrated using standards with the concentration range of 0-1.2 mgP/L treated in the same manner as the samples for the colourimetric ascorbic acid method [26].

Crop Phytotoxicity
Phytotoxicity was determined using a seed germination test on the samples collected at the end of this study. Liquid extracts were prepared from the two vermicompost mixtures with distilled water (1:10 w/v) as described by Ravindran, et al. [27]. Three test crops, i.e., tomato (Solanum lycopersicum), spinach (Spinacia oleracea L.), and radish (Raphanus raphanistrum subsp. sativus) were used for seed germination bioassay, and were sourced from freshly packed Starke Ayres seeds products. Two layers of filter papers were placed in a sterilized plastic petri dish, as 10mL of the liquid was added. In addition to different treatments, deionized water was used as a control. On top of the filter paper, ten seeds of each crop species were placed, and the Petri dishes used were then incubated at 25 degrees Celsius. After 5 days, the relative seed germination (RSG; Equation (3)) and relative root elongation (RRE; Equation (4)), germination index (GI; Equation (5)

Earthworm Biomass
During vermicomposting, the E. fetida biomass was measured at 0, 2, 4, 6 and 10 weeks. At each sampling date, the composting mixture was emptied on to a plastic sheet, and 10 earthworms per pot were randomly removed manually and weighed. The weighted values of earthworms were determined as live weight after they were picked using the hand-sorting method and removal of any adhering material. After that, the earthworms were returned together with the composting mixture into their respective containers.

pH and Electrical Conductivity (EC)
The pH and the EC were determined in the water at the ratio of 1:10 (w/v) using a pH meter equipped with a glass electrode (Crison Instruments, Barcelona, Spain) [13].

Data Analysis
The data analysis was carried out with repeated measures analysis of variance (ANOVAR) and where sphericity assumption could not be met, then the Greenhouse-Geisser [28] was used as correction of the p-value. All data were analyzed using JMP version 14.0.1 statistical software (SAS Institute, Inc., Cary, NC, USA). Microsoft Excel (Microsoft, Washington, USA) was used for descriptive analysis and plotting of graphs.

Effects of E. fetida and P. fluorescence Presence on Vermidegradation Efficiency
There was a significant interaction (p < 0.0001; Table 2) between time, earthworm presence, PSB inoculation and manure type, indicating that the effects of these factors were not consistent on changes in C/N ratio when averaged across the vermicomposting period. After 10 weeks of vermicomposting, the pig manure treatment with both E. fetida, and PSB showed the lowest C/N ratio of 10.3, followed by the cow manure treatment with both E. fetida and PSB with the C/N ratio of 12.1 ( Figure 1). By contrast, the cow and pig manure controls treatments only reached C/N ratios of 15.2 and 14.5, respectively. Table 2. Repeated measure analysis (ANOVAR) for changes in biochemical properties of rock phosphate enriched cow manure and pig manure waste paper mixtures inoculated with E. fetida and P. fluorescence after 10 weeks of vermicomposting.  The inconsistent changes in humification index (HI) and humification ratio (HR) observed in this study are explained by the significant interactions between the various factors as indicated in Table 2 above. For HI, the significant interaction (p < 0.0001; Table 2) between time, earthworm presence, PSB and manure type was observed. For cow manure-waste paper RP-enriched treatments, the highest HI at week 10 was observed in the control treatment, followed by the treatment with P. fluorescence only, with HI values of 4.3 and 3.7, respectively ( Figure 2). For the pig manure-waste paper RP mixtures, the treatment with both E. fetida and P. fluorescence showed the highest percentage increase of 78.82%, with the control treatment showing the lowest percentage increase of 0.5%. Unlike the HI, changes in HR were significantly (p < 0.01) influenced by manure type, PSB presence and earthworm presence, with the significant interaction between all three factors indicating the inconsistent influence of these factors on changes in HR (Table 2). It was interesting to observe that after 10 weeks of vermicomposting there were no differences on HR between all cow manure-based treatments with the highest HR being the treatment with both E. fetida and PSB. However, for the pig manure-based treatments, there were significant differences between treatments, with the E. fetida plus P. fluorescence treatment showing the highest final HR of 12.89, followed by treatment with PSB only (9.60) and the treatment with E. fetida only (7.97) while the control had an HR of 7.75 ( Figure 3).

Effects of E. fetida and P. fluorescence Presence on Inorganic Nitrogen Content
There was a significant interaction (p < 0.001; Table 2) between time, PSB, and manure type, indicating the effects of these factors were not consistent on changes in ammonium concentration when averaged across the vermicomposting period. The ammonium content was higher on the pig manure treatments than cow manure treatments, and the highest ammonium content in the pig manure was observed in the treatment with PSB only (333.54 mg/kg), followed by the treatment with both E. fetida plus PSB (253.85 mg/kg). Unlike the cow manure treatments were the ammonium content at the end of vermicomposting showed the same order of magnitude, the ammonium content in the pig manure treatment at the end of vermicomposting varied remarkably as it ranged from 187.85 mg/kg to 333.54 mg/kg. Among the cow manure treatments, the control showed the highest increase of 37.47%, while the ammonium content in the treatment with both E. fetida and PSB decreased from 41.31 mg/kg to 36.00 mg/kg (Figure 4). Across all the treatment combinations, the highest increase in ammonium concentration of 40.34% was observed in the pig manure treatment with PSB only. Regarding the nitrate concentration, the changes during vermicomposting were observed because of significant interaction (p < 0.001; Table 2) between time, earthworm presence and manure type. Opposite to the ammonium content, the cow manure relative treatments, showed the highest nitrate content compared to the pig manure treatments. However, the nitrate content increased for all the treatments except for the control treatment that showed a decrease from 275.92 mg/kg to 103.67 mg/kg ( Figure 5). Among the treatments, the highest increase was observed in the treatment with both E. fetida and PSB with the percentage increase of 72.85% followed by the treatment with E. fetida only with the percentage increase of 63.22%. In all the pig manure relative treatments, the nitrate content decreased including the controlled treatment with no E. fetida and PSB.

Effects of E. fetida and P. fluorescence Presence on Olsen Extractable P
A significant interaction (p < 0.001; Table 2), between time, earthworm presence, PSB and manure type was observed, indicating the effects of these factors were not consistent on changes in Olsen P when averaged across the vermicomposting period. At week 6, the pig manure-waste paper RPenriched treatment with E. fetida only had the highest P level of 3837.21 mg/kg amongst all the other treatments, followed by the pig manure and waste-paper RP-enriched treatment with both E. fetida and PSB (3051.2 mg/kg; Figure 6). At week 10, the Olsen extractable P for cow manure and waste-paper RP-enriched treatments ranged between 1150 and 2600 mg/kg and between 558.97 and 2246.154 mg/kg for pig manure and wastepaper RP-enriched treatments. When these are compared to the control treatments of both cow and pig manure-waste paper RP-enriched mixture, the cow and pig manure-waste paper RP-enriched mixture with E. fetida only showed a percentage difference of 54.44% and 75.1%, respectively. The cow manure RP-enriched treatments with P. fluorescence only, and both E. fetida and P. fluorescence peaked at week 6 (2933.33 mg/kg and 3342.636 mg/kg), while the treatment with E. fetida only reached its highest peak at week 10 (2600 mg/kg).

Phytotoxicity Test
Each test crop evaluated showed a significant (p < 0.05) response to the eight treatments on relative seed germination (RSG), relative root elongation (RRE) and germination index (GI) percentage ( Table 3). The RSG ranged between 43.3% and 100% in spinach; 96% to 112% in radish and between 48% to 88% in tomato (Table 3). Among the different treatment combinations and crops, the RSG with 100% and above were observed in the cow manure-waste paper RP-enriched control treatment for spinach with the RSG of 100% compared to the pig manure-waste paper RP-enriched control treatment with RSG of 43.3 %. In radish the highest RSG was observed in the cow manure-waste paper RP-enriched control treatment with RSG of 112% compared to pig manure-waste paper RP-enriched control treatment with the RSG of 96.3%. The RRE for all the treatments ranged between 78.2% and 129%, 44% and 108%, and 71.4 % and 143.1% for spinach, radish, and tomato. RRE of 100% and above were recorded in three treatments in spinach, two in radish and six in tomato. In spinach, the highest RRE was observed in the cow manure-waste paper RP-enriched treatment with E. fetida only, with the RRE of 129% compared to both RP control treatments with RRE of 78.2% and 89%. In radish, the highest RRE of 108% was observed in the pig manure-waste paper RP-enriched treatment with P. fluorescence only, compared to both RP-enriched control treatments with the RRE of 48.7% and 67.1%. However, in tomato the highest RRE of 143.1% was observed in the pig manure-waste paper RP-enriched treatment with P. fluorescence only compared to the cow manure, and pig manure-waste paper RP-enriched control treatments with RRE of 129% and 103%. The GI across all the treatment combinations ranged from 38.5% to 94.5% for spinach, 49% to 112% for radish and 50.2% to 77% for tomato. The GI of 80% and above was recorded in two treatments for spinach with the highest GI in the cow manure-waste paper RP-enriched treatment with P. fluorescence only with the GI of 94.5% compared to the cow and pig manure-waste paper RP-enriched mixture with GI of 78.2% and 38.5%. In radish, three treatments showed a GI of 80% and above, with the highest GI observed in the pig manure treatment-waste paper RP-enriched treatment with P. fluorescence only with the GI of 112% compared to the control treatments of cow and pig manure with GI of 55.2% and 64.7%.

The Effects of Manure Type and P. fluorescence Presence on E. fetida Biomass
There was a significant effect on the two-way interaction between time and manure type implying that the effect of manure type was not consistent when averaged across the vermicomposting time ( Table 2). There were significant differences (p < 0.05) on E. fetida biomass between cow and pig manure (Table S1; Figure S1). On average, pig manure-waste paper RP-enriched mixture resulted in a maximum of 7.45 g of E. fetida biomass compared to 4.2g in cow manure-waste paper RP-enriched mixture, representing a 44% difference between the two manures. The E. fetida biomass peaked at week 6 for all the treatments, and thereafter either decreased or remained constant. Generally, E. fetida biomass was higher in the treatment where no P. fluorescence was added compared to where P. fluorescence was added, but this effect was not statistically significant.

The Effect of E. fetida and P. fluorescence Inoculation on Changes in pH and EC
There was a significant interaction (p < 0.038; Table 2) between time, earthworm presence, PSB inoculation and manure type, indicating that the effects of these factors were not consistent on changes in EC when averaged across the vermicomposting period. There was a significant difference (p < 0.05) in pH between the two manure types (Table 2). However, neither earthworm presence nor PSB inoculation significantly influenced changes in pH in this study. Generally, there was minimal variation in the pH of the two manure types throughout the vermicomposting period ( Figure S2). For the cow manure treatments, pH ranged from 8.52 to 8.99 while the pH for the pig manure treatments ranged from 7.21 to 7.73. There were significant differences (p < 0.01; Table 2) on the average EC between the cow and pig manure mixtures ( Figure S3). The highest increase from week 0 to week 10 was observed in the cow manure control treatment (1.28), followed by the pig manure treatment with an EC value of 1.09. Also, the presence of PSB significantly (p < 0.01) influenced the changes in EC, with the average EC in cow manure with PSB being 1.92, and that for pig manure being 1.52. At week 10, in all the treatment combinations in both cow and pig manure, the EC was significantly higher compared to values at week 0.

The Effect of P. fluorescence Inoculation on the E. fetida Biomass
Earthworm growth is a biological indicator that can be used to monitor the vermicomposting process [29]. The higher earthworm biomass observed in pig manure compared to cow manure-waste paper RP-enriched vermicompost could be due to differences in the availability of nutrients in the mixture [30]. Generally, pig manure treatments maintained higher levels of NH 4 -N and Olsen extractable P than cow manure-based treatments ( Table 1) and possibly other nutrients that were not analysed. This implies that higher nutrients concentrations in pig manure could have explained at least partially the much higher earthworm biomass treatments observed in pig manure treatments than in cow manure-based treatments, as the pH, EC, nitrates and ammonium in the pig manure showed favourable conditions for the growth of earthworms ( Table 1). The lack of significant differences after the inoculation with P. fluorescence in both cow and pig manure-based RP vermicompost shows that the cow and pig manure enriched RP is enough for the growth of E. fetida during vermicomposting.
The weight loss of earthworms in both cow and pig manure vermicompost mixture recorded between week 6 and week 10 ( Figure S1) could be attributed to a possible decline in water-soluble carbon in the vermicompost mixtures with time [18]. The rapid degradation of organic waste during vermicomposting could also result in the loss of N due to leaching, among other things, which in turn could according to Bernal, et al. [31] affect the E. fetida growth. In a related study, de Lima Rodrigues, et al. [32], observed a decrease in earthworm density at the end of vermicomposting using cattle manure mixed with tannery sludge that had been amended with rock powder. In their study, the decline of earthworm was attributed to the decrease in the availability of the substrate in all the treatments at the end of the vermicomposting.

Effects of E. fetida and P. fluorescence on Changes in pH and EC During RP-enriched Vermicomposting
No clear treatment effect on differences in pH were observed in either cow manure or pig manure treatments. These findings corroborated previous findings by Yan, et al. [14] and Unuofin, et al. [33], who also reported that changes in pH during RP-enriched manure-waste paper mixtures vermicomposting using E. fetida to be minimal and with no consistency. The slight variations in pH could be due to the application of RP and P. fluorescence, which is likely due to the compost buffering system, which resulted in slight changes in pH [33,34].
Electrical conductivity (EC) is regarded as a good indicator of the safety and agricultural suitability of vermicompost mixtures [35]. However, the magnitude of EC in all the treatment combinations was less than 4 mS/cm, and thus, its use is unlikely to result in salinity hazards [36]. The observed increase in EC at the end of vermicomposting for each manure type was most probably due to the release of phosphate and soluble salts like ammonium, caused by the decomposition of the readily biodegradable organic mixture by earthworms and microbes [37]. The results of EC agree with the findings of other researchers who also observed increased EC levels at the end of vermicomposting [38][39][40][41]. The increased EC in the present study can be explained by the degradation of organic matter [38], and the release of soluble and inorganic salts like ammonium salts, phosphates and nitrates [42]. Therefore, the increased EC is an indication that the vermicomposting process has accelerated the mineralization of organic matter and have transformed insoluble particles into soluble forms [43].

Effects of E. fetida and P. fluorescence on Humification Parameters During RP-enriched Vermicomposting
The C/N ratio reflects the decomposition of the waste material and is a widely used maturity indicator for monitoring vermicomposting [2]. The loss of organic carbon in the final RP vermicompost mixture is because of high CO 2 emission due to mineralization of carbon from the substrate through respiration by both earthworms and microbes. Also, an increase in the production of nitrogenous excreta can increase N content resulting in the decreased C/N ratio [44,45]. The findings of C/N in the present study confirms the findings of Unuofin and Mnkeni, [13] and Malafaia, et al. [46] which observed decreased C/N ration during cow manure RP-enriched vermicomposting and during vermicomposting of different tanning sludge mixed with cattle dung. The results for C/N ratio (Figure 1) proved that the incorporation of E. fetida only, P. fluorescence alone and in combination, accelerated the vermi-degradation of RP-enriched cow and pig manure-waste paper mixtures. This shows that the degradation was faster when both E. fetida and P. fluorescence were added and that the mixtures can be used to enhance the rate of biodegradation of cow and pig manure-waste paper RP mixtures using E. fetida. A C/N ratio of less than 20 is believed to indicate a progressive degree of the solubilization of organic matter and satisfactory degree of compost maturity [47,48]. In all the treatment combinations at the end of vermicomposting, the C/N ratio in the present study ranged from 10.3 to 15.8 which is less than 20, suggesting that compost maturity was achieved in all treatment combinations.

HI and HR
Both the HI and HR increased during vermicomposting, which is in agreement with the findings of Mupondi, et al. [5] who vermicomposted cow manure-waste paper mixture at different RP rates using E. fetida. In all the cow and pig manure-waste paper RP-enriched treatments, except for the pig manure control treatment, the humification Index (HI) increased with time consistent with observations in other studies where the cow manure-waste paper RP-enriched vermicompost was tested using E. fetida [13]. In their study, the found an increased HI during cow manure-waste paper RP-enriched vermicompost, which indicated that all treatment combinations resulted in a high degree of humification as also indicated by the C/N values. These HI findings further showed that the presence of E. fetida and P. fluorescence enhanced the maturation of the vermicomposts. This is likely due to the increased rate of decomposition of the organic substrate mixture by the E. fetida which produce numerous bioactive humic substances, and the increased humification level in organic matter is mostly associated with the higher agricultural value of the final vermicompost. In the present study, all the treatments at week 10 had HI values greater than 3.5% with the highest value observed in the treatment with both E. fetida and P. fluorescence in the pig manure-waste paper RP-enriched mixture. According to Bernal, et al. [31] a mature compost is expected to have a HI of greater than 3.5%. Therefore, the results of the present study shows that the earthworms, microorganisms and the composition of humus are closely related to soil fertility [49], and in this case the mixture can be used as supplement for nutrient deficient soils.
In all the treatment combinations, the HR was greater than 7, which, according to Bernal, et al. [31] signifies a matured compost mixture. The presence of E. fetida and P. fluorescence showed greater HR, followed by cow manure-waste paper RP-enriched treatment with both E. fetida and P. fluorescence. However in most studies, the HI and HR at the beginning of vermicomposting is usually the same in all the treatments, and the initial difference in this study among the treatment is likely due to differences in chemical composition of vermicompost mixtures.

Effects of E. fetida and P. fluorescence on the Ammonium and Nitrates During RP-Enriched Vermicomposting
During vermicomposting, the nitrogen balance is usually regulated by the nitrification process which enhances the quality of vermicompost. The high nitrate content of cow manure RP-enriched vermicompost compared to low ammonium content indicated that the process of ammonification and nitrification was not hindered. Similar observations were reported by Mupondi, et al. [5] who reported decreased ammonium, with a corresponding increase in nitrate content during RP-enriched cow manure vermicomposting. However, they associated the increased nitrogen mineralization with increased biodegradation and P availability from the added RP because of higher biological sorption. Based on Nigussie, et al. [50], the high nitrate content implies low nitrate loss in the vermicompost mixture. Additionally, the use of E. fetida alone which showed high nitrate content compared to other treatments in cow manure RP vermicompost can be explained by the presence of earthworm, which is well known for its impact of increasing nitrate content [51]. The different trends in both ammonium and nitrate of the cow manure and pig manure can be explained by their initial concentration of ammonium and nitrate (Table 1). For instance, it was noted by De Guardia, et al. [52] that, the nitrification ability is an intrinsic property of the waste since the rate of nitrification is dependent on the waste type.

Effects of E. fetida and P. fluorescence on the Olsen Extractable P During RP-enriched Vermicomposting
Olsen P is a very important chemical property as it represents the mineralized portion of organic P in the vermicompost mixture. In the vermicompost experiment conducted by Getachew, et al. [53] to investigate the potential of E. fetida and Eudrilus eugeniae (Debrezeit and Keshmando) on the decomposition of various organic waste through the production of nutrient-rich vermicompost and worm biomass, the Olsen P content in the treatment with E. fetida was high compared to Olsen P produced by the two local earthworms. The release of P in these treatments can be explained by the earthworm gut phosphatase and P solubilizing microorganisms in the worm cast [54]. Their results agree with the findings of the present study, were high Olsen P was observed in the cow and pig manure RP-enriched treatment with E. fetida only compared to control treatment. In another study, Schaik, et al. [55] conducted an experiment, where they investigated the practicality of vermicomposting septic tank waste using E. fetida. In their study, they noticed an increase of Olsen P in all the treatments during vermicomposting, until day 89 after which a steady state was reached. The observed increase in P in the vermicomposting mixtures due to the enhanced dissolution and the mineralization of P from RP could have been facilitated by organic acids released during organic waste decomposition [5,14,33]. The release of the organic acids during organic waste mixture decomposition is facilitated by E. fetida, and the presence of P. fluorescence. The organic acids have carboxylic acid and the phenolic groups that can chelate with Ca 2+ ions from the apatite minerals in RP thereby enhancing the dissolution and the mineralization of P from RP [34] (Ca 10 (PO 4 ) 6 (F) 2 + OH − + H + → 10 Ca 2+ + 6PO 4 3− + 2F − + H 2 O). The observed increase in humic acid reflected by the high HI in week 10 ( Figure 3) could also have contributed to the improved mineralization and dissolution of P. Furthermore, the increased Olsen P in cow and pig manure RP-enriched mixture with E. fetida only like decreased C/N ratio, might be due to the accelerated humification that is promoted by earthworms during vermicompost [44,56]. This is because these humic acids also have carboxylic acid and the phenolic groups like those found in simple organic acids, which have the ability of chelating Ca 2+ ions from the apatite minerals in RP and thus enhance the dissolution and the mineralization of P from RP [34].

Effects of E. fetida and P. fluorescence on the Phytotoxicity of Vermicomposting Cow and Pig Manures Mixtures Enriched with RP
It is generally accepted that for reliable and complete characterization of compost quality for safe use in agriculture, it is crucial to also evaluate compost stability [57]. Incomplete composting is known for its effect of limiting the degradation of phytotoxic synthetic compounds and intermediates [58]. It is, therefore, essential for vermicomposts to be evaluated for their phytotoxic properties. The GI index of more than 50% was observed in most treatments, and according to Jodice, [59], this indicates matured compost with low levels of phytotoxicity. The GI of less than 50% in radish plant, that was planted using pig manure RP vermicompost with both E. fetida and P. fluorescence, indicates higher phytotoxicity [60]. The high phytotoxicity observed, could be because of high ammonium content observed in the treatment (Figure 4), since radish is considered sensitive to high ammonium content. For instance, in the study conducted by Olivera Viciedo, et al. [61] on the response of radish to difference ammonical nitrogen concentrations in absence and presence of silicon, observed highest sensitivity in treatments without silicon. In the cow manure treatment with E. fetida only, the highest germination observed agree with findings of Bernal, et al. [62] who found increased GI value of radish from between 24 to 120 h of germination using cow manure vermicompost using E. fetida. Therefore, the results showed low levels of phytotoxic substances.

Conclusions
The addition of both E. fetida and P. fluorescence during cow and pig manure-waste paper RP-enriched vermicomposting, significantly influenced biodegradation, and nutrient release, though the effects of these factors were not consistent between the two manures evaluated in this study. However, there was greater humification and P release from the RP-enriched pig manure than from the cow manure. This was reflected in greater reduction of the C/N ratio, higher humification parameters (HI and HR), and higher levels of Olsen extractable P. Inoculation with P. fluorescence further enhanced the maturation of the vermicomposting mixtures as well as the release of P from these mixtures. This indicated that inoculation with P. fluorescence could be used to optimize the vermicomposting of RP-enriched animal manure-waste paper mixtures and possibly other waste mixtures as well.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/10/1587/s1, Figures S1-S3: Changes in earthworm biomass (E. fetida), pH and EC during vermicomposting of cow and pig manure amended with rock phosphate and inoculated with P. fluorescence, Table S1: Repeated measure analysis (ANOVAR) for changes in earthworm biomass in rock phosphate enriched cow manure and pig manure waste paper mixtures inoculated with P. fluorescence after 10 weeks of vermicomposting.