Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown with P adsorbed to goethite

ARTICLE IN PRESS Microbiological Research 160 (2005) 177—187

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Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown with P adsorbed to goethite Esther Hoberg, Petra Marschner, Reinhard Lieberei Institute for Applied Botany, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany Accepted 11 January 2005

KEYWORDS Adsorbed P; Bacteria; Ph; P supply; Organic acids

Summary The actinomycete Gordonia sp. and the bacterium Pseudomonas fluorescens Pf-5 were grown in liquid media (pH 6.5) with phosphate adsorbed to the Fe-oxide/hydroxide goethite (Goe-P) and with soluble phosphate (0.1 mM or 1.0 mM P as KH2PO4). The two isolates showed distinct differences in their physiology. The pH of the medium was increased by Gordonia sp. by 1.1–1.7 units while it was decreased by P. fluorescens by 1.4–2.4 units. In all treatments the concentration of organic acids in the media with Gordonia sp. was up to 10 times lower (0.4–10.9 mmol L 1) than in media with P. fluorescens (33.4–84.4 mmol L 1). Gordonia sp. produced five different organic acids in varying amounts depending on P source and time. In contrast, P. fluorescens exuded mainly citrate and only small amounts of two to three other organic acids irrespective of P source or time. & 2005 Elsevier GmbH. All rights reserved.

Introduction Although the total amount of P is high in many soils, phosphate availability to plants and microorganisms is often low due to poor solubility of most P compounds and high P fixation capacity. Soil P can be organic and inorganic, with organic P varying between 20% and 80% of total P (Schachtman et al., 1998). The poorly available inorganic P fraction in

soil occurs as phosphate anions that are adsorbed to soil minerals, such as iron (Fe) and aluminium (Al) oxides, Al silicates and calcium (Ca) carbonates, or, depending on soil pH, as poorly soluble precipitates of calcium phosphates (Ca-P) in alkaline soils and iron and/or aluminium phosphates (Fe-P, Al-P) in acid soils (Richardson, 2001). Plants and microorganisms use similar mechanisms to increase P solubility and thus availability

Corresponding author. Present address: Soil and Land Systems, Faculty of Sciences, School of Earth and Environmental Sciences,

The University of Adelaide, South Australia 5005, DP 636, Australia. E-mail address: [email protected] (P. Marschner). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.01.003

ARTICLE IN PRESS 178 (Schachtman et al., 1998). In order to acquire P from the mineral fraction plants and microorganisms may alter the pH, which leads to changes in the solubility of Fe-P, Al-P, Ca-P as well as that of Fe/Al-oxides and hydroxides (Kim et al., 1997; Illmer and Schinner, 1995; Illmer et al., 1995). Moreover, they may exude low-molecular weight organic acids either constitutively or in response to P deficiency. Organic acids can carry varying negative charge, depending on dissociation properties, number of carboxylic groups and pH. Organic acid anions increase P availability by complexing metal cations such as Fe and displacing phosphate anions from adsorption sites (Jones, 1998). Microorganisms solubilizing poorly soluble phosphates in vitro have been isolated from many soils. In most cases they readily solubilize Ca-P, while they have only a limited capacity to solubilize FeP/Al-P (Banik and Dey, 1983; Whitelaw et al., 1999). The solubilization of Ca-P is mainly due to a pH decrease, while organic acids play a major role in release of available P from crystalline Al-P (Whitelaw et al., 1999). After inoculation with P solubilizers, plant growth and P uptake was increased in some cases (Gaind and Gaur, 1991; Wahid and Mehana, 2000; Peix et al., 2001) but there are also reports that they had no effect (Laheurte and Berthelin, 1988). Inoculation with P solubilizers may not stimulate plant P uptake if P release by the microorganisms does not exceed their own demand (Laheurte and Berthelin, 1988). Many P solubilizing microorganisms produce plant growth promoting factors such as auxins, thereby increasing plant growth even without affecting P availability to the plant (Leinhos and Vacek, 1994). Arbuscular mycorrhizal fungi (AM fungi) can increase P uptake by plants (Toro et al., 1997) and when plants are co-inoculated with AM fungi and P solubilizing bacteria, AM fungi can increase the P solubilization by the bacteria and the bacteria can stimulate AM colonization (Toro et al., 1997; Toro et al., 1998). However, soil microorganisms can also compete with plants for available P; a large proportion of P added to soil may be immobilized by the soil biomass (Schachtman et al., 1998; McLaughlin et al., 1988). The relative importance of the above mentioned mechanisms to increase P availability may depend on the microbial species, which in turn could affect the capacity of the microbial species to enhance P uptake by the plant. The inoculation of plants with effective P solubilizers may be a cost-effective alternative to inorganic P fertilizer application particularly in soils with high P fixation capacity where added P quickly becomes unavailable. In

E. Hoberg et al. order to achieve this, more microbial species have to be studied regarding their P mobilizing effects. The aim of this study was to characterize a newly isolated actinomycete and compare it with a ubiquitous soil bacterium, Pseudomonas fluorescens. We determined the response of the two microorganisms, Gordonia sp. and P. fluorescens to different P forms in terms of cell growth, pH and organic acid concentration in the medium. We used a P form commonly found in soils but rarely used in other P solubilization experiments. Most of the previous studies have been conducted with Ca-P and in some cases Fe/Al-P. However in many soils, adsorbed P forms are at least as important. We selected P adsorbed to goethite, a common iron oxide/hydroxide in European soils.

Material and methods Preparation of goethite and loading of goethite with P The iron oxide/hydroxide goethite was synthesized according to Schwertmann and Cornell (1991). Ninety mililitre of 5 M KOH were added to a constantly stirred solution of 50 mL of 1 M Fe(NO3)3
9H2O in a polyethylene bottle. The solution was diluted immediately with deionized water to 1 L and incubated at 70 1C for at least 60 h. Then the supernatant was decanted and the precipitate centrifuged at 4000 rpm. The precipitate was dialyzed (Visking dialysis tubing 17/8, 49 mm diameter, Serva, Heidelberg, Germany) in several changes of deionized water for 24 h until the pH in the water no longer became alkaline. The goethite was oven dried at 40 1C and finely ground with mortar and pestle. The ground goethite was loaded with P according to Dye (1995). Forty-four milligram KH2PO4 and 2 g of goethite were added to 200 mL of deionized water. In preliminary experiments this soluble P/ goethite ratio was shown to maximize P adsorption to goethite. The phosphate/goethite suspension was mixed on a horizontal shaker for at least 24 h, dried at 40 1C and the residue ground with mortar and pestle. The concentration of phosphate loaded onto the goethite was calculated from the difference between P concentration in solution before and after shaking. One gram goethite per litre adsorbed 0.57 mM P which is equivalent to 1.77 g P kg 1 goethite. Of this P adsorbed to goethite 16.2% was soluble in the liquid medium at the beginning of the experiment.

ARTICLE IN PRESS Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown

Experimental design The P saturated goethite was used at a concentration of 0.7 g L 1 medium which is equivalent to the goethite concentration found in many European soils (Schachtschabel et al., 1989). This resulted in a soluble P concentration of 2.0 mg P L 1or 0.07 mM P. To provide approximately the same amount of soluble P, the low soluble P concentration was 0.1 mM KH2PO4 (low soluble P), which resulted in a soluble P concentration of 3.1 mg P L 1. The high soluble P concentration was 1.0 mM KH2PO4 (high soluble P). This concentration is much higher than the P concentration in the soil solution but it was used in order to ensure adequate P supply even at high cell growth rates.

Description of isolates Two strains were used in this study. P. fluorescens Pf-5 was isolated from the rhizosphere of Gossypium spp. (Loper and Lindow, 1994). Previous in vitro tests had shown that P. fluorescens Pf-5 could solubilize Ca-P. Pseudomonads are common soil bacteria and many have plant growth promoting effects. The second strain used in this study, Gordonia sp., an actinomycete, was isolated from the rhizosphere of Theobroma grandiflorum (Wild. ex Spreng.) Schum on a fieldsite on the terra firme near Manaus, Brazil (Marino, 2000). The soil at the field site is a xanthic ferralsol, has a pH of 4 and contains high concentrations of Fe and Al. Gordonia sp. was selected due to its high capacity to grow and take up P with Al-P or Fe-P as sole P source (Marino, 2000).

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soluble P at two concentrations (0.1 mM (low soluble P) or 1.0 mM P (high soluble P) as KH2PO4) or with P adsorbed to the iron oxide/hydroxide goethite (Goe-P, with a goethite concentration of 0.07 g 100 mL 1) (three replicates). Non-inoculated flasks served as controls (two replicates). All media were autoclaved before inoculation. Despite identical pre-culture conditions the population density of Gordonia sp. was lower than that of P. fluorescens. The initial culturable cell density of Gordonia sp. was log 1.2 cfu L 1, while it was log 3.7 cfu L 1 for P. fluorescens.

pH The pH of the media was adjusted to 6.5 before autoclaving and measured at the beginning and the end of the experiment.

Determination of culturable cells The number of colony forming units (cfu) was determined immediately after inoculation and on days 3, 6 and 10 by plating an appropriate dilution of the liquid media on solid Muromcev medium with 1.0 mM P as KH2PO4 (composition see above with 10 g L 1 agar) and on nutrient agar (LUFCO medium, Difco Laboratories, Detroit, USA). Only data on Muromcev medium are presented because cfu numbers did not differ from those on nutrient agar.

Water-soluble P concentration Water-soluble P was determined according to Murphy and Riley (1962).

Analysis of organic acids Culture of microorganisms Gordonia sp. and P. fluorescens were precultured in liquid Muromcev medium ((g L 1) glucose 10, L-asparagine 1, K2SO4 0.2, MgSO4
7H2O 0.4, NaH2SO4 0.14) with 1.0 mM P as KH2PO4 (Muromcev, 1958; Deubel et al., 2000). This medium was chosen because it does not contain Ca, thus poorly soluble Ca-P, which would decrease P availability are not formed. Additionally it is free of ill-defined compounds such as yeast extract or peptone, which could potentially be a source of organic acids. The flasks were placed on an orbital shaker at 80 rpm and incubated at room temperature (22 1C). After 6 days of pre-culture with repeated transfer every 2 days to maintain an exponentially growing culture, an aliquot was transferred to 100 mL Muromcev medium with

To determine amount and composition of organic acids, a 5 mL aliquot of the medium was removed on days 3, 6 and 10 and frozen immediately. Samples were lyophilized (a I-6-Lyophilisationsystem, Christ, Osterode, Germany) and purified with cation/anion exchange resin (Chromabond SA/SAX, Macherey & Nagel, Du ¨ren, Germany). This purification was necessary to remove salts, which interfere with the organic acid detection by HPLC. Preliminary tests had shown that the recovery rate of organic acids with this method was 90–95%. Despite the removal of a large amount of salts, a residual salt peak remained and its elution time coincided with that of oxalate which therefore could not be detected. The resins were preconditioned in deionized water and methanol (1:1) and then washed with deionized water before use. The

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thawed media samples were dissolved in 2 mL 10 mmol L 1 H2SO4 and incubated with 0.3 g cation exchange resin for 1 h on an orbital shaker (80 rpm) at room temperature. Samples were centrifuged for 5 min at 4000 rpm and the supernatant incubated for another hour under the same conditions with the anion exchange resin (0.6 g). The purified supernatant was filtered through a 0.45 mm HPLC-membrane filter (Carl Roth, Karlsruhe, Germany) into HPLC-vials and stored at 20 1C until analysis. Isocratic separation of the organic acids was carried out on an AMINEX Ion Exclusion HPX-87 H column (BIO-RAD, Mu ¨nchen, Germany) at a column temperature of 38 1C. Eluent was 5 mmol L 1 H2SO4 at a rate of 0.6 mL min 1. Organic acids were detected at 210 nm (variable wavelength monitor, Knauer, Berlin, Germany) and integrated (Hewlett Packard, HP 3390 A integrator). Peaks were identified against a set of standard peaks obtained from known organic acids (Supelco, Taufkirchen, Germany). All samples and standards were stored at 20 1C and thawed immediately before analysis. The initial concentration of organic acids in the medium was determined before inoculation. They may have derived from impurities of the chemicals used or from microorganisms in the water that were killed during autoclaving. These values were subtracted from data measured in the inoculated treatments.

Statistical analyses Values were compared analysis of variance using general treatment structure with no blocking. Mean values were compared by least significant difference (GenStat 5. edition, Rothamsted Experimental Station 2000).

Results pH After autoclaving, the pH had decreased from 6.5 to 5.9 to 6.2 (Table 1). Both isolates strongly affected the pH of the medium (Table 1). Gordonia sp. increased the pH by 1.1–1.7 units to pH 7.0 with Goe-P and pH 7.8 with the soluble phosphate sources. For Gordonia sp., the final pH was significantly lower with Goe-P than with the soluble P sources. P. fluorescens lowered the pH in the media by 1.4–2.4 units with no significant differences between the treatments. The pH of the uninoculated control media did not change over time (data not shown).

Culturable cell density The cell density of Gordonia sp. and P. fluorescens increased significantly with time (Table 2). The two isolates differed in their growth pattern. The population density of Gordonia sp. increased significantly from log 1.2 (day 0) to log 4.9–5.3 cfu L 1 (day 10). In contrast, the population density of P. fluorescens increased from log 3.7 on day 0 to log 5.4–5.6 cfu L 1 on day 3 but then remained at this level until day 10. Thus, by the end of the experiment, the cell density of both isolates was similar. The cell density of both isolates was lower with Goe-P than the two soluble P sources on day 3. On day 6 the cell density of Gordonia sp. with Goe-P was still lower than in both soluble P treatments, but that of P. fluorescens grown with Goe-P was similar to that at low soluble P. By day 10, the cell density of Gordonia sp. was higher in Goe-P than with both soluble P treatments and the cell density of P. fluorescens was similar in

Table 1. pH of media amended with high and low concentrations of soluble P (1.0 and 0.1 mM) or P adsorbed to goethite (Goe-P) inoculated with Gordonia sp. or Pseudomonas fluorescens at the start and the end of incubation (day 10), as well as non-inoculated control. Means of three replicates P treatment

1.0 mM sol. P 0.1 mM sol. P Goe-P

Start

6.20 6.00 5.90

pH Control

Gordonia sp.

6.18 6.09 5.53

7.68 7.75 7.02

P. fluorescens 3.77 3.56 4.48

ANOVA Source of variation

lsd

P

P treatment Isolate P treatment  isolate

0.38 0.33 0.56

0.868 o 0.001 0.002

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Table 2. Density of culturable cells (log cfu L 1) in media amended with high and low concentrations of soluble P (1.0 mM and 0.1 mM) or P adsorbed to goethite (Goe-P) inoculated with Gordonia sp. or Pseudomonas fluorescens on day 3, 6 and 10. Means of three replicates P treatment

Isolate

Cell density (log cfu L 1) Day 3

1.0 mM sol. P 0.1 mM sol. P Goe-P

Gordonia sp. P. fluorescens Gordonia sp. P. fluorescens Gordonia sp. P. fluorescens

3.03 5.63 3.22 5.60 2.61 5.39

Day 6

Day 10

4.86 4.74 4.78 5.79 4.41 4.98

5.06 5.21 4.92 5.45 5.31 5.30

ANOVA Source of variation

lsd

P

Day P treatment Isolate Day  P treatment (data not shown) Day  isolate P treatment  isolate Day  P treatment  isolate

0.20 0.20 0.16 0.35 0.28 0.28 0.49

o 0.001 0.016 o 0.001 0.032 o 0.001 0.108 0.052

all P treatments. Throughout the experiment, no culturable cells were detected in the uninoculated controls.

Water-soluble P concentration in the media In the high soluble P treatment, Gordonia sp. decreased the P concentration in the medium slowly from 32.0 mg P mL 1 on day 0 to 29.6 mg P mL 1 on day 3 and 11.4 mg P mL 1 on day 10 (Fig. 1). P. fluorescens reduced the P concentration much faster; it decreased from 32.0 mg P mL 1 on day 0 to 7.2 mg P mL 1 on day 3 and to 6.1 mg P mL 1 at the end of the experiment. A similar difference between the two strains was found in the low soluble P treatment, where Gordonia sp. decreased the P concentration from 3.2 mg P mL 1 on day 0 to 0.5 mg P mL 1 on day 10 whereas P. fluorescens had lowered the P concentration to 0.2 mg P mL 1 already by day 3 and to 0.1 mg P mL 1 at the end of the experiment. Thus in both soluble phosphate treatments P. fluorescens took up P more quickly than Gordonia sp. P uptake in the soluble P treatments was highest in P. fluorescens during the first 3 days and decreased rapidly after that. In the high P treatment 4.43 mg P (log cfu) 1 were taken up by P. fluorescens from day 0 to day 3 while Gordonia sp. took up 0.71 mg P (log cfu) 1. On the other hand, P uptake by

Gordonia sp. was higher than that of P. fluorescens from day 6 to day 10. In the third treatment, Goe-P, the initial P concentration in the medium was 2.0 mg P mL 1 (Fig. 1). Gordonia sp. reduced the P concentration to 0.6 mg P mL 1 on day 3, whereas it was decreased to 0.1 mg P mL 1 by P. fluorescens. In the following days Gordonia sp. decreased the P concentration to 0.03 mg P mL 1, while P. fluorescens did not lower the P concentration any further. On days 3 and 6 P uptake from Goe-P was higher by Gordonia sp. (0.50 and 0.14, mg P (log cfu) 1 respectively) than with P. fluorescens (0.32 and 0.00 mg P (log cfu) 1, respectively). On average over all P treatments, P. fluorescens decreased the P concentration in the medium to a significantly lower level than Gordonia sp. In the uninoculated control media the initial P concentration did not change over time (data not shown).

Concentration and composition of organic acids in the media Irrespective of the P source, the organic acid concentration in the media with P. fluourescens was 3–10 times higher than in those with Gordonia sp. (Tables 3 and 4, Fig. 2). The difference in the total

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E. Hoberg et al. 40 1.0 M sol. P 30 20

lsd

10 0 0.1 M sol. P

mg PL-1

3 2

lsd 1 0 Goe-P

In Gordonia sp. the organic acid concentration of all three treatments was highest on day 3. It was 10.9 mmol L 1 with high soluble P and 0.8 and 0.6 mmol L 1 with low soluble P and Goe-P, respectively. This difference between the treatments was significant throughout the experiment. The concentration of organic acids in the media decreased during the experiment. On day 10 the total organic acid concentration had decreased to 5.9 mmol L 1 in the high soluble P treatment, whereas organic acids could no longer be detected in the other two treatments. In the culture media of Gordonia sp. five organic acids (citrate, formiate, lactate, malate, succinate) were detected in varying amounts (Tables 3 and 4). In all three treatments formiate was the dominant organic acid on day 3. Later it disappeared completely while succinate could still be found in the high P treatment on day 10.

3 Gordonia sp.

2

P. fluorescens 1

lsd

0 0

2

4

6 Days

8

10

Figure 1. Soluble P concentration (mg L 1) in media amended with high and low concentrations of soluble P (1.0 and 0.1 mM) or P adsorbed to goethite (Goe-P) inoculated with Gordonia sp. or Pseudomonas fluorescens on day 3, 6 and 10. Means of three replicates. Bar indicates least significant difference (lsd).

organic acid exudation was mainly due to high citrate exudation by P. fluorescens. In P. flourescens the total organic acid concentration in the media with Goe-P ranged from 33.4 mmol L 1 (day 3) to 54.0 mmol L 1 (day 10) and with high soluble P from 59.2 mmol L 1 (day 3) to 96.1 mmol L 1 (day 6). It increased significantly from day 3 to day 10. The total organic acid exudation of P. flourescens in both soluble P treatments was significantly higher than with Goe-P. Four different organic acids (Tables 3 and 4) were detected in the high and low soluble P treatments of P. fluorescens (citrate, lactate, malate, succinate), whereas only three (citrate, malate, succinate) were found in the Goe-P treatment. Irrespective of treatment and sampling time, citrate made up more than 90% of the organic acids detected in the media with P. fluorescens.

Discussion The two isolates showed distinct physiological differences, which has implications for their effect on P availability and their usefulness to enhance P uptake by plants and/or reduce Al toxicity. The actinomycete Gordonia sp., isolated from an acid tropical soil, increased the pH of the media. In acid soils this could decrease Al toxicity and increase P availability. However in alkaline soils, it may decrease P availability. P. fluorescens on the other hand, reduced the pH of the media and exuded large amounts of citrate. The pH decrease could increase P availability in alkaline soils, but may decrease P availability and increase Al toxicity in acid soils. However the high citrate exudation rate of P. flourescens could counteract the pH effect. Citrate can complex Al and Fe and displace phosphate anions from adsorption sites. Chelation of Al by citrate decreases its toxicity (Ryan et al., 1995). This ameliorating effect in soil may be limited, because Fe/Al-citrate/malate complexes are rapidly degraded by microorganisms (Jones et al., 1996). On the other hand citrate sorption can increase persistence against microbial decomposition (Jones and Edwards, 1998; Geelhoed et al., 1999) and continuous release of organic acids could compensate for degradation (Geelhoed et al., 1999). It should also be noted that the pH effect of the isolates may be less pronounced in soils with a high pH buffering capacity (Gyaneshwar et al., 2002). Hence, both isolates have the potential to increase P availability and decrease Al toxicity.

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Table 3. Organic acid concentrations (mmol L 1) in media amended with high and low concentrations of soluble P (1.0 mM and 0.1 mM) or P adsorbed to goethite (Goe-P) inoculated with Gordonia sp. or Pseudomonas fluorescens on day 3, 6 and 10. Means of three replicates P treatment

1.0 mM sol. P

0.1 mM sol. P

Goe-P

Day

3 6 10 3 6 10 3 6 10 3 6 10 3 6 10 3 6 10

Organic acid concentrations (mmol L 1)

Isolate

Gordonia sp.

P. fluorescens

Gordonia sp.

P. fluorescens

Gordonia sp.

P. fluorescens

Citrate

Formiate

Lactate

Malate

Succinate

0.2 0.3 0.3 43.5 80.1 71.8 0.2 0.1 — 51.3 75.2 82.0 — 0.2 — 33.3 65.2 52.9

5.4 — — — — — 0.5 — — — — — 0.6 0.5 — — — —

— 0.8 0.7 2.8 5.4 0.5 0.1 0.2 — 2.7 — — — — — — — —

1.6 1.9 — 6.2 3.8 4.8 — — — — — — — 0.3 — — — —

3.7 2.4 4.9 6.7 6.8 8.6 — — — — 0.3 0.8 — — — — 0.3 1.1

— Not detectable.

Table 4.

ANOVA results of organic acid concentrations in culture solutions (mmol L 1)

Source of variation

Citrate

Formiate

Lactate

Malate

Succinate

Sum

lsd

lsd

P

lsd

lsd

lsd

lsd

0.003 0.032 0.007 0.006 0.003 0.036 0.006

0.7 0.027 1.0 0.584 1.3 0.170 9.2 0.006 0.7 o0.001 1.0 o0.001 1.3 o0.001 9.2 0.003 0.5 o0.001 0.8 0.035 1.1 0.020 7.5 o0.001 1.1 0.005 1.7 0.755 2.3 0.598 16.0 0.910 0.9 0.024 1.4 0.845 1.9 0.438 13.1 0.001 0.9 0.001 1.4 0.010 1.9 0.078 13.1 0.048 1.6 0.009 2.5 0.984 3.2 0.855 22.6 0.777

However the decrease in water-soluble P in the media over time indicates that most of the mobilized P would be taken up by the microorganisms and may only be available to plants after cell death. When interpreting the results of the present study, the different growth patterns of the two organisms needs to be taken into account. The initial cell density of P. fluorescens was higher than in Gordonia sp., thus P. fluorescens reached the highest cell density already on day 3 of the experiment. The cell density of Gordonia sp. on the other hand increased until day 10. It should be

noted that the cell density of Gordonia sp. may have been underestimated because unlike singlecelled bacteria, actinomycetes can grow in mycelium-like structures which may not have been completely separated before plating. Nevertheless the more rapid decrease in solution P concentration in P. fluorescens is probably due to the higher initial cell density of P. fluorescens and not due to a higher uptake rate and/or storage capacity. In all three treatments cell density of P. fluorescens increased from day 0 to day 3 and P uptake was highest on day 3 (Fig. 1, Table 2). From day 3 to day 6 P uptake by P. fluorescens decreased

Day P treatment Isolate Day  P treatment Day  isolate P treatment  isolate Day  P treatment  isolate

P

8.4 0.001 0.7 8.4 0.067 0.7 6.9 o0.001 0.5 14.6 0.885 1.2 11.9 0.001 1.0 11.9 0.071 1.0 20.6 0.886 1.6

P

P

P

P

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Sum of organic acids (µmol L-1)

120 lsd

100

80 60

40

20

0 Day 3

6 10 Gord.

3

6 10 3 6 10 Pseud. Gord.

1.0 mM sol. P

3 6 10 Pseud.

0.1 mM sol. P

3

6 10 Gord.

3

6 10 Pseud.

Goethite P

Figure 2. Total concentration of organic acids in the media amended with high and low concentrations of soluble P (1.0 and 0.1 mM) or P adsorbed to goethite (Goethite P) inoculated with Gordonia sp. or Pseudomonas fluorescens on day 3, 6 and 10. Means of three replicates. Bar indicates least significant difference (lsd).

irrespective of the concentration of soluble P remaining in solution. This coincided with the entry into the stationary phase of growth. On the other hand, cell density of Gordonia sp. increased throughout the entire experiment. In the high and low soluble P treatment P uptake by Gordonia sp. was highest on day 6 while it was highest on day 3 in the Goe-P treatment. Although the soluble P concentration in the Goe-P treatment on day 6 was very low (0.03 mg P mL 1), Gordonia sp. continued to grow rapidly. This may be due to rapid turnover of P within the population with P released by dead cells rapidly been taken up by new cells. In soils, the inorganic P fraction is composed of poorly soluble salts of orthophosphates with Al, Fe and Ca and P adsorbed to Fe- and Al-oxides/ hydroxides (Gerke and Hermann, 1992). For goethite loaded with P, P solubility increases from pH 3 to pH 10 (Bar-Yosef, 1991). Organic acids, especially those with two or more carboxylic groups, can chelate Fe and other cations. Organic acid anions can also replace P adsorbed to iron oxides by anion exchange (Gerke, 1992). Both, chelation of Fe and exchange of P, can release P from goethite (Jones, 1998). For any organic anion, its desorption capacity is greatest at a pH that corresponds to its second pK value. At this degree of dissociation, the ratio of the stability of the Fe (or Al)-organic anion complex to the stability of the Fe (or Al)-P complex is highest. Fully dissociated organic acids on the other hand, are

largely ineffective in desorbing P (Bar-Yosef, 1991; Geelhoed et al., 1998). The differing strategies of the two isolates for P acquisition from goethite can be outlined as follows. The pH increase by Gordonia sp. would lead to a partial dissolution of goethite and release of adsorbed P. The dissolution of goethite may be further increased by the small amounts of malate exuded by Gordonia sp., because malate can reduce Fe(OH)3 (Jaureguri and Reisenauer, 1982). Due to the high pH, the majority of the organic acids will be fully dissociated and thus almost ineffective in desorbing P by anion exchange from goethite (Gerke, 1992; Geelhoed et al., 1998). Gordonia sp. therefore increased the P availability from goethite mainly by dissolution of goethite and not by desorption of P through organic acid exudation. In agreement with the results of the present study, Gordonia sp. grew well in liquid culture with Fe and Al-P as sole P source, increased the medium pH and only small amounts of organic acids were detectable in the medium (Marino, 2000). P. fluorescens decreased the pH of the media and exuded large amounts of citrate. This is in agreement with large pH decreases by Pseudomonas sp. found by Singh et al. (1984). Citrate sorption to iron oxides and thus P desorption are maximal at pH 4.3 (Bar-Yosef, 1991) which is only slightly lower than the pH measured in the goethite treatment with P. fluorescens (pH 4.5). At this pH citrate will effectively chelate Fe thus decreasing the number

ARTICLE IN PRESS Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown of P sorption sites. The strategy of P. fluorescens therefore involves desorption of P from goethite and to a lesser extent dissolution of goethite. According to an in vitro study by Geelhoed et al. (1998) P mobilization from goethite was increased by citrate with the greatest mobilization observed at 10 3 M citrate. The mobilization decreased with decreasing citrate concentration but even 10 5 M citrate increased the concentration in the solution more than 10 fold compared to the water control. P. fluorescens released up to 60 mM citrate in the goethite treatment which corresponds to 6  10 5 M. Hence, citrate release by P. fluorescens is high enough to mobilize P from goethite. Geelhoed et al. (1998) did not use concentrations less than 10 5 M citrate, therefore the potential P release of the citrate release by Gordonia sp. (0.2  10 6 M) can not be estimated, but it is likely to be low. Both strategies appear to be effective because by the end of the experiment, the cell density of both isolates in the Goe-P treatment was higher or similar to that at high soluble P for Gordonia sp. or P. fluorescens, respectively. The capacity of microorganisms to utilize P adsorbed to goethite or Al precipitate was also shown by He and Zhu (1998) and Shang et al. (1996). He and Zhu (1998) concluded that the capacity of microorganisms to utilize adsorbed P is greater than that of plants. Despite the potential P mobilization from goethite either by organic acid release (P. fluorescens) or pH increase (Gordonia sp.), the P concentration in solution decreased with time (Fig. 1). We hypothesize that most of the mobilized P was taken up by the cells. Using the decrease in P concentration in the soluble P treatments, P uptake by actively growing cells can be estimated to range from 0.02 to 0.6 and 0.2 to 4.4 mg P log cfu 1 for low and high P, respectively. At the cell densities measured in the goethite treatment this amounts to 0.1–24 mg P L 1, indicating that the cells can take up substantial amounts of P. Ketogluconic acid has been shown to be very effective in Ca-P solubilization (Goldstein et al., 1993) and Banik and Dey (1983) found that among a range of organic acids produced, only exudation of ketogluconic acid is positively correlated with P solubilization from Al-P. However in our study, neither gluconic nor ketogluconic acid were detected in the media. The release of organic acids by the two isolates is likely to be higher than that measured in the solution. Organic acids are not only exuded but they are also easily available carbon sources for microorganisms (Jones and Darrah, 1994). Part of the exuded organic acids will therefore be miner-

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alized. Thus the data are net concentrations of the organic acids. In the presence of goethite the organic acid concentration may be further decreased by sorption of organic acids to goethite (Gerke, 1992; Geelhoed et al., 1998). Deubel et al. (2000) showed that the amount and composition of organic acids released by bacteria may be dependent on the type of sugars supplied and that sugar exudation by roofs is influenced by a range of factors such as plant species and nutrient availability. Thus the organic acid exudation observed in our study may be considerably influenced by the environmental conditions in the rhizosphere. Despite the ability to utilize poorly available adsorbed P and the potential to ameliorate Al toxicity of both rhizobacteria, the strategies observed in this study may not be effective in soil, because: (i) pH changes observed in vitro will be less pronounced in soils with high pH buffer capacity (Gyaneshwar et al., 2002), (ii) organic acids are rapidly mineralized by soil organisms (Jones and Darrah, 1994) and (iii) the microorganisms may only be able to carry out these strategies under the relatively nutrient-rich conditions in vitro but not in the soil where nutrient availability is often limiting microbial growth.

Conclusions The physiology of the two isolates in this study differed and both appear to be able to utilize poorly available adsorbed P. Based on their strategies, the isolates could potentially increase P availability to plants; however the P concentration measured in the solution remained low indicating that most of the liberated P is quickly taken up by growing cells. Besides the effect on the phosphate nutrition the potential of both isolates in ameliorating Al toxicity deserves more attention.

Acknowledgements This study was conducted within the SHIFT program (Studies on Human Impact on Forest and Floodplains in the Tropics, subproject (TP 4) ‘‘Recultivation of abandoned monoculture areas under special consideration of soil biological factors’’) supported by the German Federal Ministry of Education, Science, Research and Technology.

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