Enhanced solubilization of rock phosphate by Penicillium bilaiae in pH

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Abstract: Little is known about how pH-buffering capacity affects phosphorus (P) solubilization by Penicillium bilaiae. This study compared solubilization of rock ...
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Enhanced solubilization of rock phosphate by Penicillium bilaiae in pH-buffered solution culture M. Takeda and J.D. Knight

Abstract: Little is known about how pH-buffering capacity affects phosphorus (P) solubilization by Penicillium bilaiae. This study compared solubilization of rock phosphate (RP) by P. bilaiae in nonbuffered (pH 5.0) and buffered (pH 7.0) media. Fungal growth reached the stationary phase around day 12 and was slightly enhanced in the buffered medium. The fungus reduced solution pH from 5.0 to 4.1 in the nonbuffered medium and from 7.0 to 4.9 in the buffered medium by day 12. Phosphorus concentrations increased after day 9 more in the buffered than in the nonbuffered media (53 and 5 mg P·L–1, respectively, on day 12). On day 12, higher concentrations of citric and oxalic acids were detected in the buffered (2.0 and 1.2 g·L–1, respectively) than nonbuffered media (0.5 and 0.04 g·L–1, respectively). Solubilization of RP was simulated without P. bilaiae in solutions equivalent to the nonbuffered and buffered cultures of P. bilaiae grown with RP. After a 24 h incubation, the P concentrations were of similar magnitudes to those observed in the P. bilaiae culture (18 and 47 mg P·L–1, respectively, in the nonbuffered and buffered media). Under increased pH-buffering conditions, the enhanced production of citric and oxalic acids led to significant RP solubilization. Key words: phosphorus solubilization, pH buffering, Penicillium bilaiae, citric acid, oxalic acid. Résumé : On sait peu de choses de la façon dont le tamponnage du pH affecte la solubilisation de phosphore (P) chez Penicillium bilaiae. Cette étude a comparé la solubilisation du phosphate naturel de P. bilaiae en milieu non tamponné (pH 5,0) et tamponné (pH 7,0). La croissance fongique a atteint la phase stationnaire autour du 12ième jour et était légèrement augmentée en milieu tamponné. Le champignon a réduit le pH de la solution de 5,0 à 4,1 en milieu non tamponné et de 7,0 à 4,9 en milieu tamponné au jour 12. La concentration de phosphore a davantage augmenté après le jour 9 en milieu tamponné que non tamponné (soit 53 et 5 mg P·L–1 respectivement, au jour 12). Au jour 12, des concentrations plus élevées d’acide citrique et d’acide oxalique ont été détectées dans le milieu tamponné (respectivement 2,0 et 1,2 g·L–1) que dans le milieu non tamponné (respectivement 0,5 et 0,04 g·L–1). La solubilisation de phosphate naturel était stimulée en absence P. bilaiae dans des solutions équivalant à des cultures non tamponnées et tamponnées de P. bilaiae cultivées en présence de phosphate naturel. Après une incubation de 24 h, les concentrations de P étaient à des niveaux similaires aux niveaux retrouvés dans une culture de P. bilaiae (18 et 47 mg P·L–1, en milieux non tamponnés et tamponnés respectivement). Sous de meilleures conditions de tamponnage du pH, la production plus élevée d’acide citrique et d’acide oxalique résulte en une solubilisation significative du phosphate naturel. Mots clés : solubilisation du phosphore, tamponnage du pH, Penicillium bilaiae, acide citrique, acide oxalique. [Traduit par la Rédaction]

Takeda and Knight

Introduction Management of soil phosphorus (P) is one of the biggest agronomic challenges facing organic farmers in Saskatchewan. Few high P-containing amendments are certified for use on organic farms. Among those that are allowed are some sources of rock phosphate (RP). However, commercially available RP is composed of sparingly soluble apatite minerals (i.e., calcium phosphate minerals), of which the P is generally Received 11 May 2006. Revision received 13 July 2006. Accepted 17 July 2006. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 14 December 2006. M. Takeda1 and J.D. Knight.2 Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada. 1

Present address: Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences, 1–1–1 Yayoi, Bunkyo-ku, University of Tokyo. 2 Corresponding author (e-mail: [email protected]). Can. J. Microbiol. 52: 1121–1129 (2006)

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unavailable for plant uptake. Exacerbating the problem is the fact that soils in the agricultural region of Saskatchewan are predominantly calcareous. In calcareous soils, soil pH is buffered between 7.3 and 8.5 depending on the amount of CaCO3 present (Lindsay 1979). The resultant low H+ and high Ca2+ activities enhance the precipitation of orthophosphate ions contributing to the low availability of P for plant uptake. In general, field applications of RP do not increase available P for plant uptake (Kucey 1987; Kucey and Leggett 1989). Penicillium bilaiae is a fungus originally isolated from soil in southern Alberta. The fungus enhances solubilization of RP in solution culture (Kucey 1983; Asea et al. 1988). Penicillium bilaiae acidifies the growth medium and releases citrate and oxalate into the medium. Thus, it was suggested that P. bilaiae solubilizes apatite minerals by excreting H+ (acidification) and (or) by producing organic acid anions that reduce Ca2+ activity in solution through complexation with Ca2+ (Asea et al. 1988; Cunningham and Kuiack 1992). Jumpstart™ (Philom Bios Inc., Saskatoon, Sask.) is a com-

doi:10.1139/W06-074

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mercial product containing P. bilaiae that is marketed in Canada and elsewhere. It enhances soil and fertilizer P uptake but does not replace the need for P fertilization (Philom Bios Inc. 2006). The long-term application of Jumpstart™ on organic land would eventually lead to the depletion of the soil P reserve. In related research, we are investigating the co-application of RP and P. bilaiae in field trials as an organic P management strategy to increase the amount of P available for plant uptake. However, the role that the high pH buffering of calcareous soils plays in this process is largely unknown. The overall objective of this study was to determine if pHbuffering capacity affected RP solubilization by P. bilaiae. To minimize the confounding effects that rapid fungal growth would have on P solubilization, we identified the stationary growth phase of P. bilaiae under the experimental conditions and performed intensive measurements of organic acid production at this stationary phase. Specific objectives were to (i) quantify solution P concentration, fungal growth, acidification, and the production of organic acids in nonbuffered and buffered media simulating pH conditions occurring in acidic and calcareous soils and (ii) quantify P solubilization in culture when major organic acids produced by P. bilaiae were included in the absence of the organism.

Materials and methods Culture preparation A stock solution of P. bilaiae spores (ATCC strain No. 20851: 7.5 × 108 spores·mL–1 in 0.2% v/v Tween 80 + 10% v/v glycerol solution) was provided by Philom Bios Inc. The P. bilaiae stock solution was subdivided into 1.5 mL vials and stored at –70 °C until used. Prior to inoculation, 1 mL of the P. bilaiae stock solution was diluted 102 times in sterile Milli-Q water (18 mΩ·cm–1). In each experiment, a viable spore concentration was determined to be 2.4 × 108 to 2.5 × 108 colony forming units·mL–1. Determination of optimal 3-(N-morpholino)propanesulfonic acid concentration for subsequent experiments 3-(N-Morpholino)propanesulfonic acid (MOPS; C7H15NO4S; Sigma-Aldrich, St. Louis, Mo., USA) was selected as the buffer reagent based on its pKa of 7.2 at 25 °C and the absence of P in its structure. The pKa of 7.2 is close to the naturally occurring pH values in calcareous soils in Saskatchewan (Rostad et al. 1987). A range of MOPS concentrations (0, 0.05, 0.1, 0.14, and 0.18; all mol·L–1) were tested to determine the optimal concentration of MOPS for the subsequent solution culture experiments. All five media contained the following (mmol·L–1) 83.3 glucose, 1.71 NaCl, 11.6 KNO3, 4.14 MgSO4·7H2O, and 0.68 CaCl2·2H2O (modified from Asea et al. 1988), prepared with Milli-Q water. The pH was adjusted to 7.0 with 5 mol·L–1 NaOH (0.05 mol·L–1 NaOH for the control, 0 mol·L–1 MOPS solution). Each MOPS concentration was replicated three times. Idaho RP (Soda Springs Phosphate Inc., Soda Springs, Idaho (all m/m): 0.74% S, 11.7% P, 0.22% K, 0.34% Mg, 27.9% Ca, 0.61% Na, 0.64% Fe, 0.40% Al, 0.02% Mn, 0.01% Cu, and 0.13% Zn) was ground and sieved to a particle diameter between 106 and 250 µm. A 0.1 g sample of the ground RP was wrapped in aluminum foil and dry

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autoclaved at 121 °C for 20 min. The 0.1 g sample of autoclaved RP and 1 mL of diluted spore solution were added aseptically to 50.0 g of sterile medium. Sample flasks were destructively sampled 12 days after incubation on a rotary shaker (150 r·min–1) at room temperature (21 ± 1.5 °C). Each sample was filtered through Whatman No.1 filter paper. Residue on the filter paper, containing a mixture of fungal and RP material, was oven-dried at 70 °C for 48 h and then weighed. The filtrate was filtered a second time through a 0.20 µm membrane filter. The second filtrate was used for pH and solution P determinations. Solution pH was measured with a pH–ion–conductivity meter (Accumet™ Model 50, Fisher Scientific, Pittsburgh, Pa., USA). Solution P (orthophosphate ions) was measured by the colorimetric molybdate blue method (Industrial method No. 94-70W; Technicon Industrial Systems, Tarrytown, N.Y., USA) with a Technicon AutoAnalyzer™ II. RP solubilization time-course experiment Nonbuffered and buffered media contained the same nutrient components, in the same quantities, as described for the determination of optimal MOPS concentration. The pH of the nonbuffered medium was adjusted to 5.0 with 0.1 mol·L–1 HCl. The buffered medium was prepared in 0.1 mol·L–1 MOPS, and the pH was adjusted to 7.0 with 5 mol·L–1 NaOH. A 0.1 g sample of autoclaved RP and 1 mL of diluted spore solution were added aseptically to 50.0 g of sterile medium. Nonbuffered and buffered media containing only RP were included as control treatments to account for abiotic dissolution. For the control treatments without P. bilaiae, 1 mL of sterile Milli-Q water was added instead of diluted spore solution. All flasks except those collected on day 0 were shaken on the rotary shaker at 150 r·min–1 and at room temperature (21 ± 1.5 °C). Flasks were destructively sampled at each sampling time. Because of space limitations on the rotary shaker, three replicate samples were collected after 0, 6, 9, 12, and 18 days of incubation (i.e., days 0, 6, 9, 12, and 18), and four replicates were collected after 15 days of incubation (i.e., day 15), for treatments containing P. bilaiae. For the control treatments without P. bilaiae, four replicates were collected only on day 15. Dry masses, pH, and solution P were determined in the same manner described for the determination of optimal MOPS concentration. Concentrations of oxalic and citric acids were determined by high-performance liquid chromatography (HPLC) for the samples collected on days 9, 12, 15, and 18. Samples collected on days 0 and 6 were not analyzed by HPLC because the high concentrations of inorganic anions interfered with the measurement of these organic acids. The filtrate sample was diluted five times with Milli-Q water prior to HPLC analysis to minimize the interference of impurities. The HPLC analysis was performed with a SUPEL COGEL C-610H ion exclusion column (30 cm × 7.8 mm internal diameter: Sigma-Aldrich Ltd., Oakville, Ont.) equipped with an SP8800 ternary HPLC pump (SpectraPhysics Inc., San Jose, Calif.). The column temperature was maintained at 30 °C. Organic acids were detected by a Spectroflow 757 Absorbance Detector™ (Applied Biosystems, Ramsey, N.J.) at 210 nm. A 20 µL sample was injected into © 2006 NRC Canada

Takeda and Knight Fig. 1. Dry mass (a), solution pH (b), and phosphorus (P) concentration (c) in nonbuffered (䊊) and buffered (䊏) media containing rock phosphate and Penicillium bilaiae. Error bars represent standard deviation (n=4 on day 15, and n=2 or 3 on the other sampling days). ***, **, and * indicate significant differences at p ≤ 0.001, p ≤ 0.01, and p ≤ 0.05, respectively, between nonbuffered and buffered media analyzed for incubation days separately.

a mobile phase, 0.1% v/v H3PO4, pumped at a flow rate of 0.5 mL·min–1. A mixture of organic acid standards containing oxalate, citrate, malate, succinate, formate, and acetate (Bio-Rad Laboratories, Hercules, Calif.) was run through the HPLC before and after the analysis of samples on each day. Acids were identified by retention time. Composition of P. bilaiae exudates For each medium (nonbuffered and buffered), four treatments were established: (i) control, containing neither RP nor P. bilaiae; (ii) RP, containing only RP; (iii) P.b., containing only P. bilaiae; and (iv) RP + P.b., containing

1123 Fig. 2. Oxalic acid (a) and citric acid (b) concentrations in nonbuffered (䊊) and buffered (䊏) media containing rock phosphate and Penicillium bilaiae. Error bars represent standard deviation (n=4 on day 15, and n=2 or 3 on the other sampling days). ***, **, and + indicate significant differences (p ≤ 0.001, p ≤ 0.01, and p ≤ 0.10, respectively) between nonbuffered and buffered media analyzed for incubation days separately.

both RP and P. bilaiae. Each treatment was replicated five times and the experiment was duplicated in time. The addition of RP and P. bilaiae was performed as described for the determination of optimal MOPS concentration. Each flask represented one replicate. All flasks were shaken on a rotary shaker at 150 r·min–1 at 22 ± 0.5 °C for 12 days. Because fungal growth (Fig. 1a) and citric acid production (Fig. 2b) reached a maximum on day 12 in the time-course experiment, 12 days was chosen as the incubation period in this experiment. Sample filtration and measurements of dry mass, solution pH, and solution P concentration were carried out as described for the determination of optimal MOPS concentration. Organic acids were analyzed in detail by gas chromatography (GC). The filtrate samples were diluted five times and alkalized with NH4OH (final concentration of 2% m (NH3 )/v), in which organic acids existed in anionic forms. Organic acids were extracted first with anion exchange resin membranes (Western AG Innovations, Saskatoon, Sask.) and secondly with diethyl ether, and derivatized to the tertbutyldimethylsilyl ester by reacting with N-methyl-N-(tertbutyldimethylsilyl) trifluoroacetamide (MTBSTFA, SigmaAldrich). Organic acid extraction and derivatization generally followed the methods developed by Gillespie (2003), which © 2006 NRC Canada

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are based on those reported in Kim et al. (1989) and Szmigielska et al. (1997). The GC analysis of organic acids was performed with a Hewlett-Packard model 5890 Series II gas chromatograph equipped with a Zebron ZB-5 capillary column (30 m × 0.32 mm internal diameter × 1.0 µm film; Phenomenex, Torrance, Calif.). The column temperature was programmed as follows: initial temperature, 60 °C; initial time, 2 min; temperature increase rate, 4 °C·min–1; final temperature, 280 °C; final time, 2 min. The injector and flame ionization detector temperature was 300 °C. Helium at a pressure of 186 kPa was used as the carrier gas. The GC peaks were integrated by PeakSimple™ for Windows (SRI Instruments, Torrance, Calif.). A 1 µL derivatized sample was injected with a Hewlett-Packard model 7673 injector. Formic (BDH Chemicals, Poole, UK), acetic, butyric, lactic, glycolic, oxalic, malonic, maleic, succinic, fumaric, malic, trans-aconitic, and citric acids (all from Sigma-Aldrich) were used as external standards and methylmalonic acid (SigmaAldrich) as an internal standard for the derivatization procedure. Butyric acid was not detected in any samples and is not discussed further. Organic acids were identified by retention time. Abiotic RP solubilization Concentrations of Ca, K, Mg, Na, Al, Cu, Fe, Mn, and Zn in the culture filtrates were analyzed by atomic absorption – emission spectrometry (SpectrAA 220 Atomic Absorption Spectrometer, Varian Australia Ltd., Victoria, Australia). Nitrate (NO3–) was analyzed by a modified Griess–Ilosvay method (Keeney and Nelson 1982). Chloride was analyzed by the Hg (II) thiocyanate method (Adriano and Doner 1982), and sulfate (SO42–) by a turbidimetric method (Wall et al. 1980). Results of the chemical analyses of culture filtrates (Table 1) were used to develop media for abiotic RP solubilization. Magnesium, K, Na, SO4, and Cl were selected as the inorganic components of the media. Other inorganic components were detected at negligible concentrations and were not included. Chloride was used as a counter ion of Na and K because of its negligible complexation ability with Ca (National Institute of Standards and Technology 1998). The concentration of Na measured in the buffered medium was high because the pH was initially adjusted to 7.0 with NaOH. Thus, Na was added to both media prior to pH adjustment at the concentration measured in the nonbuffered medium. The base composition of both media was as follows (mmol·L–1): 5.01 MgSO4·7H2O, 10.7 KCl, and 2.38 NaCl. The buffered medium was prepared in 0.1 mol·L–1 MOPS. Idaho RP was ground to a diameter between 106 and 250 µm. The ground RP was dry autoclaved at 121 °C for 20 min to maintain consistency between the culture and abiotic experiments. Based on the chemical analysis of the P. bilaiae cultures (Table 2), oxalic acid (Sigma-Aldrich) and citric acid (Sigma-Aldrich) were added in concentrations of 37.6 and 463.5 mg·L–1, respectively, in the nonbuffered medium and in concentrations of 1233 and 2034 mg·L–1, respectively, in the buffered medium. Malonic, maleic, succinic, fumaric, malic, and aconitic acids were also detected in the culture filtrates but in limited amounts (