Agronomic Suitability of Biologically Produced PARP as a Source of Phosphorus for Maize Production

Phosphorus (P) and Nitrogen (N) deficiencies are recognized as the major constraint of agricultural productivity in developing countries including Zambia. While N deficits can be restored at least in part through the application of crop residues and manure, the restoration of soil P can only be achieved by use of phosphate fertilizers which are unaffordable by the small scale farmers. The aim of the study was to assess the availability of P for crop use from rock phosphate (RP), partially acidulated by acid produced by sulfur oxidizing bacteria. The RP was obtained from Keren Mining Limited at Sinda district, Zambia; the Pyrite rock (iron sulfur) and bacteria culture were obtained Nampundwe mine dump. The pyrite and PR were ground and mixed with the bacterial culture to produce the Partially Acidulated Rock Phosphate (PARP). A pot experiment was set up in a CRB design in a greenhouse on four soil types with four treatments of: soil alone (control), soil with GRP, soil with PARP and soil with super single phosphate (SSP) replicated four times. The results showed that the plant height, biomass yield and P uptake across the different P sources showed significant differences (p < 0.01); particularly, the PARP and SSP were significantly higher than the both control and the GRP. A comparison of PARP and SSP within each soil based on both biomass yield and P uptake showed no significant difference between them. Despite the fact that the RAE values based on biomass yield were in most cases higher than those base on the P uptake, the trend was the same. On average the PARP was >90% effective within and across the four soils indicating that the PARP is reactive and suitable for direct application for crop production.


Introduction
Most soils in Zambia are inherently low in P (3 -12 ppm) [1] and this seriously limits the productivity of maize which is the staple food crop. This deficiency is more severe in the high rainfall (800 -1200 mm) regions of the country (Region III) where predominant soils are the highly weathered Oxisols and Ultisols with high phosphorous fixing capacities [2]. Although judicious application of inorganic P fertilizers is recognized as the most effective method for alleviating P deficiencies, their high cost and inaccessibility limit their use to the majority of Zambia farmers, particularly the smallholder farms [3]. Application of farm yard manures and crop residues does add some limited quantities at farm level [4] [5] due to their low P content. For this reason, organic matter has been used mainly as sources of N, while inorganic fertilizers are used to supply P.
Zambia is endowed with a number of rock phosphate (RP) deposits of igneous origin that are associated with syenite and carbonatite related deposition. Syenite related RP deposits are found at Chilembwe in Eastern province and Mumbwa North, Central province. Carbonatite related RP deposits are found at Kaluwe, Rufunsa in Lusaka province, and at Nkombwa Hill in Northern Province [6].
Out of the four RP deposits, only two (Chilembwe and Mumbwa) hold promise of their exploitation for agricultural use [7]. The Chilembwe deposit constitutes four ore bodies associated with syenites varying in composition from mica syenites to monozonites, with the apatite rock in the form of massive lenses comprising apaptite, quartz, alkali feldspars, mica and amphibole with the P content varying 10% -12% P 2 O 5 estimated at 1.8 million tones [6] [8]. The Mumbwa North phosphate is associated with apatite and it is estimated at 1 Mt with the grade between 8% -12% [9]. The Nkombwa Hill RP deposits have the grade of 4.5% P 2 O 5 , but most of the phosphorus occurs as isokite (CaMg(PO 4 )) though beneficial for agricultural purposes, there is no effective beneficiation technology that exists [8]. The Kaluwe PR 0.5% -8.5% P 2 O 5 grade but the Niobium in these PRs as pyrochlore makes it technically and economically not suitable for phosphate extraction for agricultural purposes [10] [11].
Studies show that the agronomic effectiveness of direct application of indigenous ground rock phosphate (GRP) and its derivatives on major crops and representative soils is generally inferior to triple superphosphate (TSP) and single superphosphate (SSP), largely owing to the low solubility of RP of igneous origin [12]. Depending on soil and climate, it could take up to 4 years of annual application before rock phosphate treatments become as effective as super phosphate [13]. One method of reducing this long lag phase is to increase the solubility of rock phosphate by treating it with small amount of sulfuric acid or phosphoric acids. Such products, commonly known as partially acidulated rock phosphate (PARP), have not shown lag effects and have given high agronomic effectiveness [11]. Partial acidulation can be effected by industrial processing but this approach is capital intensive [14]. A possible alternative and probably less   These served as a source of inoculums for the bacteria which was used in the experiments to oxidize pyrite to sulfuric acid.

Preparation of Solid Ferrous Iron-Silica Gel Medium
The Ferrous iron-silica gel medium was prepared according to [18]. To obtain a ferrous iron-silica gel the following salts were dissolved together in 250 ml of distilled water a flask; 6.00 g (NH 4  The 9 K medium of Silverman and Lundgren solution was used in multiplying the bacterial culture. The 9 K Solution was prepared by mixing 3.0 g (NH 4 ) 2 SO 4 , 0.1 g KCL, 0.5 g K 2 HPO 4 , 0.5 g MgSO 4 •7H 2 O and 0.01 g Ca(NO 3 ) 2 in 700 ml of distilled water in a 1 L flask according to [19]. 100 mls of 9 K solution and solution B from above were sterilized separately and mixed in 250 ml Erlenmeyer flasks making the 9 K medium of Silverman and Lundgren.

Culturing of the Bacteria
The water samples collected from Nampundwe Mine was used in the culturing of the bacteria using the [20] method. The sulfur oxidizing bacteria was identi-

Incubation of Pyrite and Solubilization of the Rock Phosphate
To oxidize the pyrite using sulphur oxidizing bacteria according to [21], 100 g of ground pyrite was placed into 50 ml capacity beakers. The bacteria culture prepared from the preceding phase was diluted five-fold. Appropriate amounts of these dilute solutions were added to the pyrite to bring the moisture content to 21 percent by weight and kept 30˚C in the darkness for six weeks. Five samples from each treatment were randomly selected every week for analysis of pH and total acidity over a period of six weeks. The FeS 2 + bacterial culture + nutrient solution produced the strongest acidity among the three combinations (see Table 2). To determine the pH, 10 g of sample was mixed with 50 ml of distilled water, the suspension shaken for an hour after which the pH of the suspension was measured using a digital pH meter. The total acidity of the sample was determined on a 25 ml aliquot filtrate of the suspension by titration with 0.1 N NaOH.
The four rock phosphates above were evaluated for water soluble P. The rock phosphate from Sinda was selected for the production of PARP in this experiment because it produced the highest water soluble P content under the treatment of FeS 2 + bacterial culture + Nutrient Solution ( Table 2). The bulk samples of the selected rock phosphate were crushed and ground to produce Ground Rock Phosphate (GRP). Portions of GRPs were treated with the acid produced from the above procedure to produce the Partially Acidulated Rock Phosphate (PARP) which was used as a source of P for maize in the pot experiment. Every week, five beakers from each treatment were removed and analysed for water-soluble P. The water soluble P, was determined from 1 g moist sample extracted with 100 mls distilled water, shaken for 30 min and filtered. The concentrations of water soluble P in the filtrate was measured by UV/Visible Spectroscopy as described by [22].

Greenhouse Experiment
The soils described above were used and were characterized by low pH and P content, as observed in Table 3. The soils were composite samples from 0 -20 cm soil depth in each soil.
A pot experiment to test the agronomic effectiveness of the PARP produced from the above experiments as a source of P for maize was conducted under greenhouse conditions. The treatments consisted four sources of P

Setting Up of the Pot Experiment
Amounts of SSP or PAPR, GRP to give 300 mg P kg −1 (0.9 g) soil were weighed out and mix thoroughly with three (3)

Determination of P Uptake by the Crop and Other Agronomic Parameters
Plant tissue analysis was used to determine P uptake. The P content of maize dry matter was determined from 1 g dry ground plant material using the dry ashing method described by [23]. The 1 g of dried ground plant material was boiled in 1% nitric acid, ignited and cooled. The residue dissolved in 20 ml 1 N nitric acid.
The solution was heated for 20 minutes on a hot plate, cooled, filtered and diluted to 100 ml. One milliliter of sample was used to develop color and P determined calorimetrically on a Spectrophotometer at 882nm. Agronomic parameters of Plant height, the number of leaves, leaves with typical symptoms of p deficiency (purple appearance on the edges of the leaves), were measured weekly from the date of thinning to the date of harvest.

Data Analysis
Analysis of variance (ANOVA) was performed to determined significant differences in DM yield and P uptake between the various treatments. The ANOVA was based on factorial arrangement in a randomized complete block design in which the effects of replication, P source were accounted for as sources of variance using the Genstat discovery Edition computer software.

The Effectiveness of the Oxidizing Bacteria Culture for PARP Production
The result of the experiment for incubation of pyrite and bacteria culture are presented in the table and Figure 1. Figure 1 shows the pH values obtained from the treatments over the period of six weeks.
The results in the table shows the pH values obtained from the treatments of the pyrite incubation. The combination treatment of FeS 2 + Bacterial culture + Nutrient solution produced the highest acidity, pH = 2.3 (which is closer to the pH of sulfuric acid) compared to 3.6 and 4.3 in FeS 2 + bacterial culture and FeS 2 respectively at the end of 6 weeks period. Agricultural Sciences  the soil as can be seen from the letters showing the differences in Table 5 and A comparison of soils P uptake among the soils show that there was more P uptake in the Kasama soils than all other soils. The P uptake in Kasama soil (p = 0.40% P, highest) was 30% higher than the P uptake in Mporokoso soil (p = 0.28%, the lowest) in the PARP treatment while in the SSP treatment, the P uptake was 17.5% higher in Kasama soil (p = 0.40%) compared to Mporokoso soil  The agronomic suitability of the biologically produced PARP as a source of P biomass yield while the RAE based on P uptake ranged between 6.9% in Mporokoso soil to 12% in Mufulira both indicating a very low P uptake effectiveness (see Table 5). Table 5. Mean value of plant height, P up taken biomass yield and the RAE % among sources of P (control, GRP, PARP, and SSP) in the four types soils (same letter in a column were not significantly different at (p < 0.05)).

Comparison of P Uptake by the Maize among Soil Treatments
The control in all the four soils from the different districts had the lowest percentage of phosphorous in the above ground dry matter weight. It is not surprising that the lowest level of percentage phosphorous were observed in the control treatments since the soils used in this study were strongly acidic on average of <4.5 pH (Table 3) with P ranging from 2 -3 ppm. Similarly, in the GRP the P uptake was slightly increased because of ground rock phosphate (GRP) application. However, the GPR is known for its slow P release when applied directly to the soil as a fertilizer. In this study it was observed that P uptake was only 0.06% -0.08% P/pot in GRP compared to the 0.33% -0.40% P in the PARP and SSP. This low concentration of P in GRP treatment limited the P uptake and growth of these potted plants because of its relative insolubility [24]. Mapiki and Singh [1] also found that direct application of Chilembwe PR to all the test crops was generally ineffective as a P source. In addition, the soil pH of <4.5 in the soils was equally low to cause aluminium toxicity. Al toxicity is usually characterised by an inhibition of P uptake and translocation with the immobilisation of P on and in plant roots [25]. Large concentrations of Al can severely restrict root have both been shown to reach a minimum value at pH 5.5 [26]. However, the P uptake in the PARP and SSP treatments were significantly higher (p < 0.05) than the control and the GRP treatments in spite of the above soil acidic conditions.
The P uptake from PARP and SSP treatments with readily available phosphorus in soils were not significantly different (p < 0.05). The two treatments had the highest levels of P uptake across the four soils in spite of the low pH and their associated potential Al and Mn toxicities and P deficiency. The negative effect of soil acidity could have been overcome by the large concentration of P provided by its dissolution of the two fertilizers. It has been observed that maintaining an adequate soil P status reduces the effects of soil acidity on plant growth [25] [27] by its liming effect [25]. From the results the PARP was an effective fertilizer comparable to SSP in providing P for the growth of the plant regardless of the soil type. This result can be explained by the fact that these two fertilizers provided prompt release of phosphorus, making it more available to the plants. The higher concentration of P in the plant was attributed to a higher P content in the soil provided by the PARP and the SSP fertilizers. Phosphorus solubilizing bacteria play role in phosphorus nutrition by enhancing its availability to plants through release of P from the PR by solubilization and mineralization [28] [29].
PAPRs may offer an economic means [3] of enhancing the agronomic effectiveness of indigenous PR sources that may otherwise be unsuited for direct application.

Comparison of Above Ground Biomass Yield of Maize among Soil Treatments
Similarly, the biomass yield (47.7 -49.1 g/pot range) in PARP and SSP treatments followed a similar trend in all the four soils from the different districts, however, the soil with GRP (21.6 -24.3 g/pot range) was significantly different (p < 0.05) from the control (10.2 -10.9 g/pot range) as shown in Table 5 and Figure 2(b). This shows that grinding rock phosphate and mixing it to the soil will enhance crop growth but not as effective as water soluble P fertilizer like the SSP or the PARP because of its slow dissolution and does not promptly release phosphorus, for plant growth and biomass yield. On the other hand, the PARP has been observed to be as effective as water soluble P fertilizer (the SSP) through many studies conducted internationally [30]. The soluble P fractions in these two fertilizers stimulated root growth and facilitated greater exploitation of P enriched soil thereby producing higher amounts of plant biomass. The soils in the Control and the GRP were heavily affected by the low pH (<4.1) at which Al and Mn toxicity (pH 5.5 is the threshold for soil acidity [25] [31] is prevalent and phosphorus fixation eminent, creating detrimental conditions for crop growth hence the observed low biomass yields in the two treatments. The fact that there was no significant difference among the soils with regard to biomass yield shows that the soils did not have other underlying factors that seriously negatively affect the plant growth other than the P availability.

The Agronomic Effectiveness of the GRP and PARP
The agronomic effectiveness of GRP and PARP were measured relative to SSP (a water soluble P fertilizer), referred to as Relative Agronomic Effectiveness (RAE).
The RAE values for GRP and PARP were calculated from the ratio of the marginal increase in Biomass yield or P uptake from the P sources in comparison to SSP ( Table 5). The RAE based on biomass was higher than the one based on P uptake in both GRP and PARP phosphorus sources. based on biomass yield were in most cases higher than those base on the P uptake, the trend was the same. However, the P uptake may be a more sensitive indicator of relative agronomic performance of P sources than biomass yield because P uptake reflects the differences in internal efficiency of P supplied to plant by different P sources [32] [33] also reported that phosphorus-uptake (acquisition) efficiency resulted in higher yield increase as compared to internal P-utilization efficiency in rice [34]. Based on P uptake, the application of PARP was 100% as effective as SSP in Kasama soil, 94% in Mufulira soils, 85% in Mansa soil and 79% in Mporokoso soil with an average of 90% effectiveness. Similiarly, Khau et al. (2009) [29] also found out that the use of phosphorus solubilizing bacteria on PR increased P uptake. This indicates that PARP is a reactive phosphate rock form that is suitable for direct application and its outstanding performance at all sites is consistent with the available P provided by its application. According to FAO [30] and Chien [35], PAPRs may offer an economic means of enhancing the agronomic effectiveness of indigenous PR sources that may otherwise be unsuited for direct application as the PAPRs are cheaper than fully acidulated Water Soluble Phosphates fertilizers because less acid and energy is required per unit of P in its production.

Conclusion
This study shows the importance of the local rock phosphate as a source of phosphorus in maize plant in acidic soils. The biologically produced PARP significantly (p < 0.01) enhanced both P uptake and biomass yield of maize compared to the control and the GRP. The GRP is slow in dissolving and does not promptly release phosphorus, for plant uptake, growth and biomass yield and therefore not suitable for direct application. The relative agronomic effectiveness of PARP in relation to SSP based on P uptake and biomass yield was >90% suggesting that PARP is as good as SSP fertilizer and therefore a suitable alternative source of P for direct application in the soil for crop production. In addition, PAPRs may offer an economic means of enhancing the agronomic effectiveness of indigenous PR sources of P.