Doi: http://dx.doi.org/10.15446/abc.v20n2.42713

Artículo de investigación

In vitro DISSOLUTION OF ACIDULATED ROCK PHOSPHATE BY PHOSPHATE SOLUBILIZING MICROORGANISMS

Disolución in vitro de rocas fosfóricas aciduladas por microorganismos solubilizadores de fósforo

Ángela Patricia MORENO QUEVEDO¹, Nelson Walter OSORIO VEGA¹, Octavio Augusto GONZÁLEZ MURILLO².

¹Universidad Nacional de Colombia, Calle 59A. n.º 63-20, of. 14-208. Medellín, Colombia.
²Sobiotech S.A.S. Km 25 Autopista Medellín–Bogotá. Guarne, Colombia
For correspondence. apmorenoq@unal.edu.co

Received: 20 March 2014; Returned for revision: 16 September 2014; Accepted: 24 October 2014. ]]> Associate Editor: Francisco José Martínez Pérez.

Citation / Citar este artículo como: Moreno Quevedo AP, Osorio Vega NW, González Murillo OA. In vitro Dissolution of acidulated rock phosphate by phosphate solubilizing microorganisms. Acta biol. Colomb. 2015;20(2):65-71. doi: http://dx.doi.org/10.15446/abc.v20n2.42713

ABSTRACT

The low availability of phosphorus (P) in the soil and the high cost of P fertilization are factors that limit agricultural productivity. A biotechnological alternative for to handle this problem is to use soil microorganisms capable of dissolving rock phosphate (RP), thus improving its effectiveness as a P fertilizer. This study was carried out with the objective of determining the effectiveness of Aspergillus niger –As-, Penicillium sp. –Pn-, Bacillus sp –B-. and an unidentified actinomycete –At- in the in vitro dissolution of two partially acidulated rock phosphates. The treatments consisted of 2x16 factorial arrangement [2 levels of RP: either Boyaca RP or Norte de Santander RP; 16 levels of inoculum: an uninoculated control, individual inoculations (with As, Pn, B, At), dual inoculations (AsPn, AsB, AsAt, PnB, PnAt, BAt), triple inoculations (AsPnB, AsPnAt, AsBAt, PnBAt), and quadruple inoculation (AsPnBAt)]. Each treatment was replicated three times. It was found that the microbial effectiveness in the in vitro dissolution of RP depended on the type of RP, the composition of the inoculum used and the interaction of both factors. The best results were obtained with the Norte de Santander RP and A. niger used alone. When this fungus combined with the other microorganisms, its capacity to dissolve RP was significantly reduced.

Keywords: Aspergillus niger, Bacillus sp., biofertilizer, phosphate, Penicillium sp.

RESUMEN

La baja disponibilidad de fósforo (P) en el suelo y el costo de la fertilización fosfórica son limitantes para la productividad agrícola. Una alternativa biotecnológica para manejar este problema es mediante el uso de microorganismos del suelo capaces de disolver rocas fosfóricas (RP) y así mejorar su efectividad como fertilizante fosfórico. Con este fin se realizó un ensayo para determinar la efectividad microbial en la disolución in vitro de dos RP (Norte de Santander y Boyacá) parcialmente aciduladas. Los tratamientos consistieron en un arreglo factorial 2x16 [2 niveles de RP: Boyacá o Norte de Santander; 16 niveles de inóculo: Un control no inoculado, inóculos individuales (Aspergillus niger –As-, Penicillium sp. –Pn-, Bacillus sp. –B-, y un actinomiceto no identificado –At-), inóculos dobles (AsPn, AsB, AsAt, PnB, PnAt, BAt), inóculos triples (AsPnB, AsPnAt, AsBAt, PnBAt), e inóculos cuádruples (AsPnBAt)]. Cada tratamiento tuvo tres replicas. La efectividad en la disolución in vitro de RP fue dependiente del tipo de RP, tipo de inóculo y la interacción de ambos factores, teniendo mejores resultados con la RP del Norte de Santander y A. niger sólo. Cuando este hongo se combinó con otros microorganismos su capacidad para disolver RP se redujo significativamente.

Palabras clave: Aspergillus niger, Bacillus sp., biofertilizante, fosfato, Penicillium sp.

INTRODUCTION

Phosphorus (P) deficiency in tropical soils is a factor that limits plant productivity (Oberson et al., 2006; Cramer, 2010). For this reason, it is necessary to apply high doses of soluble fertilizers to reach the necessary concentrations of P in the soil solution (Narsian and Patel, 2000; Vassilev et al., 2012). One problem of intensive agriculture is that the fertilizers applied are expensive and their efficiency is very low (Vassilev and Vassileva, 2003; Paiva-Coutinho et al., 2012), making it unsustainable. Because of this, there is a pressing need to identify sustainable alternatives that may allow for a more efficient use of P for cultivated plants (Consensus Statement Declaration, 2011). Rock phosphate (RP) is a locally available and cheap alternative, but its reactivity is low due to its slow dissolution and release of P (Yusdar et al., 2007; Hamdali et al., 2010). On the other hand, it is known that the dissolution of RP improves when microorganisms capable of dissolving it are added to the system. This is the case with phosphate solubilizing microorganisms (PSM) (Sahu and Jana, 2000; Whitelaw, 2000; Galindo et al., 2011; Paiva-Coutinho et al., 2012). There have been various reports on the dissolution of RP with individual inoculates (of just one species), but little is known about the effectiveness of microbial consortia (Xiao et al., 2008; Oliveira et al., 2009; Saber et al., 2009; Singh and Reddy, 2011). Additionally, existing reports on the subject have focused on one particular type of RP, which has limited the results to very specific effects (Singh and Reddy, 2011; Habte and Osorio, 2012; Xuan Yu et al., 2012). Studies have also traditionally used non-acidulated RP, despite the fact that there are acidulated RPs available in the market whose solubility could potentially be further improved by inoculation with PSM.

MATERIALS AND METHODS

The PSM (Aspergillus niger -As-, Penicillium sp. -Pn-, Bacillus sp. -B-, and an unidentified actinomycete -At-) were obtained from the Laboratory at the Sobiotech S.A.S. (6°15´07´´ N 75°25´42´´ W, altitude 2117 m) (Guarne, Antioquia, Colombia). For this study, the microorganisms were multiplied separately for three days at 28 °C in Petri dishes using the medium YMA whose composition (g/L) was: yeast extract 4, malt extract 10, glucose 4, agar 20. The medium was autoclaved at 120°C, 0.1 MPa, and 15 min. The colonies were removed from the surface of the agar with a sterilized loop and suspended in sterilized, deionized water and maintained in the refrigerator at 4 °C for later use.

Later, 1 mL of each suspension containing 10⁶ colony forming units (CFU) per mL was aseptically transferred into 250 mL Erlenmeyer flask containing 75 mL of the selective medium, previously sterilized in an autoclave (120°C, 0.1 MPa, 15 min). The medium consisted of 10 g glucose, 1 g NH₄NO₃, 1 g NaCl, 0.2 g CaCl₂.2H₂O, 0.4 g MgSO₄.7H₂O and 3.5 g RP per liter (Osorio and Habte, 2001). The RP used came from commercial mines in the country, specifically in the departments of Norte de Santander and Boyaca. These rocks were partially acidulated prior to use with concentrated sulfuric acid in the industry (Hammond et al., 1980). After that, the RP were passed through a 250 µm sieve and kept in a 500 µm sieve. The RP´s had a P content of 12 %, and upon preparing an aqueous suspension of 10 % the concentration of soluble P was 1.74 and 1.45 mg L^-1, respectively. The Erlenmeyer flasks were incubated at 28 °C in an orbital agitator at 100 rpm for seven days.

After the incubation period, 50 mL of the suspension was centrifuged at 3220 x g for 15 minutes. The supernatant was filtered through Number 42 filter paper. The pH of the solution was measured directly with a selective H⁺ electrode. The concentration of P in the solution was determined using the molybdenum-blue method (Murphy and Riley, 1962). With the soluble P data obtained from this process, the RP dissolution effectiveness factor (RPDE) was calculated by dividing the concentration of soluble P by the quantity of total P in the RP and expressing this as a percentage [RPDE (%) = (soluble P/P in the RP) x 100].

The biomass of the microorganisms inoculated was collected through centrifugation, washed with distilled water and dried in an oven at 70°C for 12 hours. Microbial growth was expressed as dry microbial material produced per Erlenmeyer and was determined as weight lost after incineration at 500°C for five hours. This method was selected in order to avoid overestimation of weight due to the adherence of the microorganisms to the RP (Reyes et al., 1999; Habte and Osorio 2012; Mendes et al., 2014).

A completely randomized experimental design was employed, the treatments consisted of 2x16 factorial arrangement [two levels of RP: either Boyaca RP or Norte de Santander RP; 16 levels of inoculum: an uninoculated control, individual inoculations (with As, Pn, B, At), dual inoculations (AsPn, AsB, AsAt, PnB, PnAt, BAt), triple inoculations (AsPnB, AsPnAt, AsBAt, PnBAt), and quadruple inoculation (AsPnBAt)]. Each treatment was replicated three times.

The data obtained was statistically analyzed using analysis of variance and the Duncan multiple range test to determine the separation of media. The level of significance (p) was ≤ 0.05. For all cases, the statistical analysis was performed using the statistical program Statgraphics Centurion XV (StatPoint, Inc., Herdon, Virginia, EE.UU.).

RESULTS

Microbial effectiveness in the in vitro dissolution of partially acidulated RP was affected by the treatment. The pH of the medium was significantly reduced (p<0.05) by both individual and combined inoculation (As: A. niger, At: actinomycete; B: Bacillus sp.; Pn: Penicillium sp.) (Fig. 1). The control treatment (uninoculated) had a pH of 5.09, which was significantly greater than the pH of all of the inoculated samples. Significantly lower pH values were obtained when the medium was inoculated with As alone (3.58) or in combination with other microorganisms (3.63 – 3.74). Inoculation with the Bacillus sp. (B) resulted in the highest pH value (4.53) of the inoculated treatments (Fig. 1). On the other hand, neither the type of RP nor the interaction of RP x microorganism significantly affected the pH of the medium.

The concentration of soluble P was significantly affected by the treatments (RP x inoculum). The concentration of P was significantly greater (p<0.05) when the Norte de Santander RP was used (P: 42.3 mg L^-1) than with the Boyaca RP (P: 35.2 mg L^-1) (Fig. 2), independent of the effect of inoculation with the microorganisms.

The concentration of soluble P (mg L^-1) in the control treatment (uninoculated) was 17 mg L^-1. After inoculation with A. niger alone (As), this significantly increased to 93.5 mg L^-1, which represents a 5.5-fold increase. This was the highest value detected for this variable (Fig. 2). Inoculation with As in combination with the other microorganisms presented soluble P values that fluctuated between 39.5 and 76.5 mg L^-1 (significant differences were found among these). These were significantly greater than the control, but lower than with inoculation with as alone. Inoculation with other microorganisms did not produce significant differences from the uninoculated control (Pn: 20.7 mg L^-1, B: 12.8 mg L^-1, At: 7.5 mg L^-1). Combined inoculation with these last two microorganisms did not differ from the control either.

In regards to the interaction of RP type and the type of microorganism(s) used as inoculates, there were significant differences found in the concentration of soluble P (mg L^-1) in some cases. For example, with the Boyaca RP, the best results were obtained with the B, PnxB, and PnxAt inoculations. For the Norte de Santander RP, the best results were seen with Pn, AsxPn, AsxAt, AsxPnxB, AsxPnxAt, and AsxBxAt. With the rest of the inoculates, there were no significant differences according to the type of RP used (Fig. 2).

Upon comparing the variables of soluble P concentration and the pH of the medium corresponding to each type of microorganism(s) used as inoculates, an inverse relationship was found between the variables. That is to say, at lower pH values, there was a greater concentration of soluble P. The lowest pH and the highest concentration of soluble P occurred when the medium was inoculated with just As. This was followed by the group of treatments that included As in the inoculation in combination with other microorganisms. Inoculation with the microorganisms Pn, B, At and combinations of the three did not show differences with the control in regards to the concentration of soluble P, despite a decrease in pH (∼4.0) (Fig. 3).

On the other hand, the microbial dry mass (mg) was significantly different depending on the type of RP used; a better result was obtained with the Boyaca RP (27.8 mg) than with the Norte de Santander RP (23.2 mg), independent of the type of microorganism used as an inoculate (Fig. 4).

The microbial dry mass was also affected by the type of microorganism applied to the medium. Upon examining the dry mass for each of the individual inoculates, the following order was found: As 36.1 mg > Pn 21.0 mg > At 14.7 mg > B 9.0 mg. In general, the treatments that included As in combination with other inoculates had a greater mass (25.3 – 38.3 mg), while the other combined treatments resulted in a smaller mass (Fig. 5). The results indicate that the interaction of type of RP with type of inoculate did not have a signficant effect on the variable of microbial dry mass.

DISCUSSION

The results obtained allowed us to clearly demonstrate the hypothesis that microbial effectiveness in dissolving partially acidulated RP depends on the type of inoculate used, the type of RP and the interaction of both factors. The dissolution of RP by microorganisms through acidification has been well documented. This acidification is caused by the extrusion of protons associated with the assimilation of ammonium or the production of organic acids (Kang et al., 2008; Sharan et al., 2008; Ben Farhat et al., 2009; Oliveira et al., 2009; Habte and Osorio 2012; Selvakujmar et al., 2013). Evidence of these mechanisms in the present study is seen in the decrease in pH from an initial value of 5.09 to 3.58 with the use of A. niger. In regards to this fungus, there are authors who speak of the production of organic acids, particularly citric, oxalic and gluconic acids, as its dissolution mechanism (Sperber, 1958; Illmer and Schinner, 1995; Reyes et al., 1999; Seshadri et al., 2004).

Narsian and Patel (2000), Reddy et al., (2002), Barroso and Nahas (2005) and Mittal et al., (2008) reported that relative efficiency in the dissolution of RP depends on the type of RP and the type of microorganism, as well as the quantity and quality of the acid secreted into the medium. Chuang et al., (2007) report that, regarding A. niger, when Ca-P was used as the source of P, they detected gluconic acid; when the phosphoric source was Fe-P or Al-P, they detected oxalic acid. Goenadi et al., (2000) and Bojinova et al., (2008), meanwhile, detected principally citric acid, among other acids.

When the RPDE of the individual inoculates was examined, the following order was found among the microorganisms evaluated: As 21.9 % > Pn 4.8 % > B 3.0 % > At 1.8 %. In the uninoculated control, the RPDE was 4.0 %. Based on this, it is worth highlighting the high effectiveness of A. niger in dissolving RP. These results corroborated those obtained by Achal et al., (2007) and Mittal et al., (2008), who found that Aspergillus spp. increased soluble P by dissolving tricalcium phosphate and RP (4.7 y 248 mg L^-1, respectively). Similarly, Saber et al., (2009) reported that the maximum in vitro dissolution of RP was obtained with A. niger (67 mg L^-1), followed by Penicillium spp. (46.2 mg L^-1). In contrast, Jayasinghearachchi and Seneviratne (2006) found that Penicillium spp. produced significantly greater RP dissolution than Aspergillus spp., which does not agree with our results, likely due to differences in microbial species and laboratory conditions (shaking, type of RP, time of incubation, among others).

On the other hand, Reyes et al., (1999) and Yadav and Tarafdar (2003) found a negative correlation between microbial dry mass and the concentration of soluble P obtained from the dissolution of RP. This coincides with the results obtained in the present study, since the RP with the greatest dissolution (Norte de Santander RP) generated a lower microbial dry mass than that found with the Boyaca RP. These authors suggest the presence of an H⁺ pumping mechanism in the dissolution of small quantities of RP, which allows for improved biomass development. They also suggest that the microbial isolates with a lower biomass can transfer carbon in order to produce a higher quantity and diversity of organic acids that dissolve more RP, which leads to a drainage of C and could explain the lower biomass (Mathews et al., 2000).

It is clear that combining a microorganism that is efficient at dissolving RP, such as A. niger, with other less efficient or inefficient microorganisms (Pn, B and At) significantly reduces the microorganism's capacity to dissolve RP, with respect to its individual effect.

It is possible that the lower concentration of soluble P is due to (i) competition for the C source (glucose) and other nutrients (p.e., Ca, Mg, Fe, etc.) and/or (ii) that the other microorganisms are consuming the P that has been released by A. niger. Regardless of what the reason is, it is evident that the potential use of these co-inoculations in the presence of cultivated plants is not ideal, given that they produce less soluble P. It is preferable to use the most efficient microorganism by itself. This result could be further investigated in the future in order to test this conclusion.

The fact that the RP used in this study was acidulated is very important. The uninoculated controls presented soluble P values of 17 mg L^-1, while in other studies using the same methods, the values have been ∼1 mg L^-1 (Osorio and Habte, 2001). The acidulation of RP has been proposed as a way of improving the effectiveness of RP as a fertilizer (FAO, 2007). Our results suggest that the RP solubilizing microorganisms can further accelerate the dissolution of this material, thus improving its capacity to release P for cultivated plants. In the review performed of the relevant literature, and in other recent reviews (Daza, 2010; Londoño, 2010; Habte and Osorio, 2012), no reports were found that studied the microbial dissolution of previously acidulated RP; for this reason, we believe this is the first study performed.

Future investigations should center on the current sustainability of agricultural systems, focusing particularly on the RPDE of A. niger under field conditions and in combination with viable, simple and easily applied biotechnological alternatives, such as micorrhyzal fungi that positively impact the interaction of plant and soil in different plant species.

The microbial effectiveness in dissolving partially acidulated RP depends on the type of inoculum used, the type of RP and the interaction between the two factors. In general, Aspergillus niger was more effective in dissolving partially acidulated RP. The coinoculation of A. niger and other microorganisms evaluated did not generate better benefits in dissolving partially acidulated RP. The use of phosphate solubilizing microorganisms can further accelerate the dissolution of partially acidulated RP.

ACKNOWLEDGEMENTS

We would like to thank the Universidad Nacional de Colombia and Soluciones Biotecnológicas y Agroambientales (Sobiotech) for the support they provided during our investigation. We thank the English translation and grammar corrections made by Marguerite Cawley.

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