J. Cent. South Univ. Technol. (2009) 16: 0581−0587 DOI: 10.1007/s11771−009−0097−0
Characterization of tricalcium phosphate solubilization by Stenotrophomonas maltophilia YC isolated from phosphate mines XIAO Chun-qiao(肖春桥)1, 2, CHI Ru-an(池汝安)2, HE Huan(何 环)1, ZHANG Wen-xue(张文学)3 (1. Key Laboratory of Biometallurgy of Ministry of Education, Central South University, Changsha 410083, China; 2. Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, Wuhan Institute of Technology, Wuhan 430073, China; 3. Yunnan Phosphate Chemical Group Co. Ltd, Jinning 650600, China) Abstract: The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tricalcium phosphate (TCP) as sole phosphorus (P) source. The strain YC is identified as Stenotrophomonas maltophilia (S. maltophilia) based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis. The results show that the strain S. maltophilia YC can solubilize TCP and release soluble P in NBRIP growth medium. A positive correlation between concentration of soluble P and population of the isolate and a negative correlation between concentration of soluble P and pH in the culture medium are observed from statistical analysis results. Moreover, gluconic acid is detected in the culture medium by HPLC analysis. It indicates that the isolate can release gluconic acid during the solubilizing experiment, which causes acidification of the culture medium and then TCP solubilization. S. maltophilia YC has a maximal TCP solubilizing capability when using maltose as carbon source and ammonium nitrate as nitrogen source, respectively, in NBRIP growth medium. Key words: tricalcium phosphate (TCP); Stenotrophomonas maltophilia (S. maltophilia); phosphate mines; phosphorus (P); gluconic acid
1 Introduction Phosphorus (P) is an essential macronutrient for plants [1]. However, a large portion of P applied to soil in the form of P fertilizers is rapidly immobilized soon after application and becomes unavailable to plants [2]. Microorganisms play a central role in the natural P cycle, and recently, they were receiving great attention as inoculants to solubilize the insoluble phosphates and transform them to soluble P [3−6]. These microorganisms are involved in a range of processes that affect the transformation of soil P and are thus an integral part of the soil P cycle. Considering this factor, many microorganisms have been isolated from different soils, and used to solubilize insoluble phosphates [7−9]. However, in fact, these microorganisms reported account for only a small percentage of the total microbial population, and few of them present a high potential to solubilize insoluble
phosphates, which seriously restrain the application of this microbially-based technique. Moreover, almost all of them were isolated from field soil, and seldom studies concerning the isolation of native phosphate solubilizing microorganisms had been conducted from phosphate mines. It is, therefore, widely accepted that a further isolation of phosphate solubilizing microorganisms from soil, especially from phosphate mines is important and necessary. It is generally accepted that phosphate solubilizing microorganisms render insoluble phosphates into soluble P through the process of acidification, chelation and exchange reactions [10]. In this process, the exudation of organic acids by microorganisms plays a significant role in solubilizing insoluble phosphates, and the following pH reduction in the culture medium is also thought to be responsible for the solubilization of insoluble phosphates [11]. Moreover, it was reported that a probable reason for phosphate solubilization, except for organic acids production, is the extrusion of H+ accompanying NH4+
Foundation item: Project(2004CB619201) supported by the Major State Basic Research and Development Program of China; Project(Z200515002) supported by the Key Project Foundation of the Education Department of Hubei Province, China; Project(GCP200801) supported by the Open Research Fund of Key Laboratory for Green Chemical Process of Ministry of Education, China; Project(Q200811) supported by the Youths Science Foundation of Wuhan Institute of Technology, China Received date: 2008−10−06; Accepted date: 2008−12−22 Corresponding author: CHI Ru-an, Professor, PhD; Tel: +86−27−87194500; E-mail:
[email protected]
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assimilation [12]. However, the mechanism of phosphate solubilization by microorganisms has not been still clear yet. Phosphate solubilization is not a simple phenomenon and may be determined by many factors, such as different species, nutritional status and growth condition of microorganisms. Therefore, it needs further studies to understand the solubilization mechanisms of different insoluble phosphates used by different phosphate solubilizing microorganisms under different environment conditions. In this work, a phosphate solubilizing bacterium of strain YC, which was isolated from phosphate mines (Hubei, China), was characterized and identified. Tricalcium phosphate (TCP) solubilizing characteristics of the isolate were also investigated. Moreover, the effects of concentration of gluconic acid on TCP solubilization in vitro and different carbon and nitrogen sources on the TCP solubilizing capability of the strain were also examined.
2 Experimental 2.1 Isolation of strain YC Soil samples for strain isolation were obtained from phosphate mines (Hubei, China), which contain almost 70% of rock phosphate, 20% of clay and 10% of silt. 10 g soil sample was added to 100 mL sterilized water and mixed on the magnetic blender for 20 min to separate microorganisms from the soil. The serially diluted soil solution was planted on National Botanical Research Institute’s phosphate (NBRIP) growth agar [13] (glucose, 10.0 g; (NH4)2SO4, 0.15 g; KCl, 0.2 g; MgCl2·6H2O, 0.5 g; MgSO4·7H2O, 0.25 g; agar, 20.0 g; distilled water, 1 L) containing 5.0 g TCP as sole P source for selectively screening of microorganisms that have TCP solubilizing capability. After 3 d of incubation at 30 ℃, isolate colonies with clear zones were further purified by replanting on the above culture medium supplemented with TCP. One purified bacterium from the preliminary screening was selected based on the concentration of soluble P in the culture medium and was again assayed for TCP solubilization. The isolate was designated as YC. 2.2 Identification of strain YC Physiological and biochemical characteristics of the isolate were determined according to the methods described in Ref.[14]. The Gram reaction was performed according to standard procedures. The 16S rRNA gene was amplified and sequenced according to the methods described in Ref.[15]. The sequences of 16S rRNA gene were first analyzed using the BLAST searching program at the National Center for Biotechnology Information
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(NCBI) website: http://www.ncbi.nlm.nih.gov/BLAST/. Related sequences were preliminarily aligned with the default setting of Clustal X (2.0) [16]. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 4 [17]. 2.3 TCP solubilization experiment by isolate Solubilization experiment was carried out in flasks with 50 mL of NBRIP growth medium (without agar) using 0.1 g TCP as sole P source. The initial pH of the culture medium was adjusted to be 7.0. Cell suspensions of the isolate were counted by a haemocytometer to adjust the count to approximately 1.0×107 mL−1. Each flask was inoculated with the cell suspensions at 10% (volume fraction). Flasks were shaken under 140 r/min at 30 ℃ for 7 d. Autoclaved, uninoculated NBRIP growth medium served as control. During the solubilizing experiment, certain volume of fresh NBRIP growth medium was added to each flask so that the volume of culture medium was maintained at 50 mL. Each flask with 50 mL culture medium was taken daily for examination for 7 d. Culture media were first filtered through filter paper and then through 0.22 µm Millipore filter to collect cells of the isolate. Cells were then resuspended in autoclaved deionized water and counted. The filtrate was centrifuged at 10 000 r/min for 20 min, and the supernatant was assessed for the concentrations of soluble P, organic acid released and pH. 2.4 Effect of concentration of gluconic acid on TCP solubilization in vitro Certain volume of gluconic acid was added to six flasks containing 50 mL NBRIP growth medium, and the concentrations of gluconic acid were 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 mmol/L, respectively. 0.1 g TCP was added to the flasks respectively to study the effect of concentration of gluconic acid on TCP solubilization. Each flask was shaken under 140 r/min at 30 ℃ for 7 d. Flask containing 50 mL NBRIP growth medium but without gluconic acid served as control. 2.5 Effects of carbon and nitrogen sources on TCP solubilization The effect of different carbon sources on TCP solubilization by the isolate was observed by replacement of glucose with six carbon sources (fructose, xylose, maltose, sucrose, mannose and lactose, respectively) and added to NBRIP growth medium before inoculation. The nitrogen sources were evaluated similarly by replacing ammonium sulphate with six nitrogen sources viz. ammonium chloride, ammonium nitrate, calcium nitrate, potassium nitrate, sodium nitrate and peptone, respectively. The procedure of solubilizing experiment was similar to that in Section 2.3.
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2.6 Analytical methods The concentration of soluble P in the culture medium was determined by using the method described in Ref.[18]. The population of the isolate was counted by a haemocytometer in a microscope. For the analysis of organic acids, 10 µL of filtrate was injected to HPLC (Agilent 1100), using C18 columns (Thermo Electron Corporation). The mobile phase consisted of a phosphate buffer (50 mmol/L KH2PO4, pH 2.0) and acetonitrile (2.0%, volume fraction). Organic acids were detected at 214 nm with a flow rate of 1.0 mL/min for 20 min. The pH of culture medium was recorded with a pH meter equipped with glass electrode. All experiments were performed in triplicate.
3 Results 3.1 Identification of isolated strain YC The isolated strain YC is an aerobic Gram-negative and rod-shaped bacterium that has more than three polar flagella. The details of physiological and biochemical characteristics are summarized in Table 1. A phylogenetic tree of the strain YC is constructed in Fig.1. Its GenBank accession number is EU564819. The 16S rRNA gene sequence of the strain YC shows the closest match to that of Stenotrophomonas maltophilia (S. maltophilia) with a homology of more than 99%. Based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis, the strain YC is identified as S. maltophilia. 3.2 Characteristics of TCP solubilization by S. maltophilia YC Changes of the concentration of soluble P released by S. maltophilia YC in the culture medium during 7 d after incubation are presented in Fig.2. The result shows that the maximum solubilization of TCP is found at the fifth day, in which the concentration of soluble P reaches 180.5 mg/L. The concentration of soluble P in the culture medium increases significantly from the first day to the
583 Table 1 Physiological and biochemical characteristics of S. maltophilia YC Test item
YC
Test item
YC
Motility
+
Oxidase
−
Gram staining
−
Hydrolysis of gelatin
+
Aerobic growth
+
Starch
−
Anaerobic growth
−
H2S formation
+
Optimum temperature/℃
30−35
Citrate utilization
+
Optimum pH
7.0−7.5
Substrate utilization Glucose
+
Nitrate reduction
+
Maltose
+
Methyl red test
−
Mannose
+
Indole production
+
Lactose
+
Lysine decarboxylase
+
Sucrose
+
DNase
+
Fructose
−
Note: “+” denotes positive result; “−” denotes negative result.
fifth day, after which it begins to decrease gradually. This fact may be attributed to the depletion of substrate, which limits both the production of organic acid and bacterial activity [19], and the re-immobilization of a portion of the soluble P. Fig.2 also shows that there is no significant change in the concentration of soluble P for the control, which only has a slightly increase up to 15.8 mg/L during 7 d. The results indicate that strain S. maltophilia YC plays a vital role in TCP solubilization and releasing of soluble P in the culture medium. Fig.3 shows that strain S. maltophilia YC has high growth rate in the culture medium added with TCP. The population of the isolate increases rapidly after inoculation and attains the highest of 5.49×108 mL−1 after 4 d. However, the population of the isolate begins to decrease gradually after 4 d. It may be due to the lack of essential nutrients such as carbon and nitrogen sources in the culture medium, which retards the growth of the isolate. From Figs.2 and 3, a positive correlation (r=0.83; p＜0.01) between the concentration of soluble P and the population of the isolate in the culture medium is also
Fig.1 16S rRNA-based phylogenetic relationship between strain YC and representatives of other related taxa (Numbers at nodes indicate levels of bootstrap support based on data for 1 000 replicates; values inferred greater than 50% are only presented; scale bar indicates 0.005 substitutions per nucleotide position)
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Fig.2 Concentration of soluble P in culture medium during 7 d of solubilizing experiment
Fig.3 Population of S. maltophilia YC in culture medium during 7 d of solubilizing experiment
observed. Changes of pH in the culture medium during the solubilizing experiment are presented in Fig.4. The pH slightly increases to 7.2 from the initial value of 7.0 at the first day, which may be due to the consumption of acid during the proton attack to TCP. However, after the first day, an obvious decrease of pH is observed, and its value drops to the lowest value of 4.3 at the fourth day. However, no decrease but a slight increase in pH is found in the culture medium over the last three days. Fig.4 also shows that the pH of the control has only a little increase from 7.0 to about 7.5 during the solubilizing experiment. The results show that the increase of the concentration of soluble P released by S. maltophilia YC is accompanied by a significant drop of pH in the culture medium (Figs.2 and 4). A strong negative correlation (r= −0.86; p＜0.01) between the concentration of soluble P and pH is observed from statistical analysis result. This is in accordance with some studies that also showed a negative correlation between soluble P released and pH in the phosphate solubilization [20].
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Fig.4 Changes of pH in culture medium during 7 d of solubilizing experiment
HPLC analysis detects different concentrations of gluconic acid (retention time 2.4 min) in the culture medium inoculated with S. maltophilia YC during the solubilizing experiment (Fig.5). The highest of 15.5 mmol/L gluconic acid is presented at the fourth day. Earlier or later than it, the concentration of gluconic acid decreases sharply, and the concentrations of gluconic acid are very lower and cannot be detected at the first day and after the fifth day. Gluconic acid is produced in the periplasm of many Gram-negative bacteria through a direct oxidation pathway of glucose by the quinoproteins glucose dehydrogenase (GDH) and gluconate dehydrogenase (GADH) [21]. Consequently, gluconic acid can diffuse freely outside the cells and decrease the pH in the culture medium, thus cause TCP solubilization and release soluble P in the culture medium.
Fig.5 Changes of concentration of gluconic acid in culture medium during 7 d of solubilizing experiment
3.3 Effect of concentration of gluconic acid on TCP solubilization in vitro The concentration of gluconic acid was detected in the culture medium during the TCP solubilizing
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experiment by S. maltophilia YC (Fig.5). Therefore, the TCP solubilizing capability of different concentrations of gluconic acid in vitro was further explored. Fig.6 shows that gluconic acid exhibits TCP solubilizing capability in the culture medium containing 0.1 g TCP. The TCP solubilizing capability of gluconic acid is positively related with gluconic acid concentration (lower than 20.0 mmol/L). It is noteworthy that as the concentration of gluconic acid is higher than 20.0 mmol/L, the concentration of soluble P in the culture medium begins to decrease (Fig.6). The results of Figs.5 and 6 show clearly that gluconic acid is an important factor for TCP solubilization, which chelates the cations bound to phosphate through its hydroxyl and carboxyl groups, thereby converting insoluble phosphates into soluble forms. This further confirms the positive action of gluconic acid released by phosphate solubilizing microorganisms on the TCP solubilization [22].
Fig.6 Concentration of soluble P solubilized by different concentrations of gluconic acid in culture medium
3.4 Effects of carbon and nitrogen sources on TCP solubilization by S. maltophilia YC Figs.7 and 8 show that strain S. maltophilia YC can solubilize TCP using different sources of carbon and nitrogen, respectively. The maximum concentration of soluble P (250.1 mg/L) is achieved with maltose as carbon source, followed by lactose, glucose, sucrose, mannose, xylose and fructose (Fig.7). Among nitrogen sources, the maximum concentration of soluble P of 178.0 mg/L is obtained with ammonium nitrate, followed by ammonium sulphate, ammonium chloride, sodium nitrate, potassium nitrate, calcium nitrate and peptone (Fig.8). This shows that the soluble P concentration in the culture medium using ammonium salts as nitrogen source is higher than that with other nitrogen sources, suggesting that the acidification of culture medium by H+ extrusion during NH4+ assimilation may be involved in the TCP solubilization. The result is in accordance with the report in Ref.[12].
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Fig.7 Concentration of soluble P in culture medium in presence of different carbon sources (Glu: glucose; Fru: fructose; Xyl: xylose; Mal: maltose; Suc: sucrose; Mao: mannose; Lac: lactose)
Fig.8 Concentration of soluble P in culture medium in presence of different nitrogen sources (AS: ammonium sulphate; AC: ammonium chloride; AN: ammonium nitrate; CN: calcium nitrate; PN: potassium nitrate; SN: sodium nitrate; PE: peptone)
4 Discussion Conventionally, insoluble phosphates are chemically processed by reacting with sulphuric acid or phosphoric acid into soluble P. However, this process increases P fertilizer cost, and has environmental implications. In view of environmental concerns and current developments in sustainability, research efforts are concentrated on the development of a technique that uses phosphate solubilizing microorganisms to solubilize insoluble phosphates [5, 23]. In this work, a phosphate solubilizing bacterium, which is identified as S. maltophilia, was isolated from phosphate mines (Hubei, China). Generally, the action of microorganisms leading to the solubilization of minerals is recognized as direct and indirect actions [24]. On one hand, for the direct action, microorganisms utilize minerals as their growth substrate,
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and the enzyme activities of the microorganisms during the growth cause the solubilization of minerals. On the other hand, for the indirect action, microorganisms produce some metabolites during the solubilizing periods, such as organic acids, which also solubilize minerals. It has been well established that, as a common strategy to release soluble P from insoluble phosphates, phosphate solubilizing microorganisms reduce the pH of the surroundings by the production of organic acids [8]. In this work, S. maltophilia YC presents high growth rate in NBRIP growth medium containing 0.1 g TCP, and a positive correlation between the concentration of soluble P and the population of the isolate in the culture medium is observed (Figs.2 and 3). The results show that the isolate can solubilize TCP as its growth substrate. It was reported that acid or alkaline phosphatases are produced in this process, thus also cause TCP solubilization [1]. This indicates that the direct action by S. maltophilia YC is one of the reasons for TCP solubilization. In addition, a decrease of pH is presented, and different concentrations of gluconic acid that cause TCP solubilization are detected in the culture medium inoculated with S. maltophilia YC during the solubilizing experiment (Figs.4 and 5). An inverse relationship between the concentration of soluble P and pH in the culture medium is observed (Figs.2 and 4). The results indicate that the indirect action by S. maltophilia YC is another reason for TCP solubilization. It further affirms that phosphate solubilization by microorganisms is involved with the production of organic acid released by microorganisms and followed by a decrease in the pH of the culture medium [25]. However, the mechanism of phosphate solubilization by microorganisms is also a subject of controversy today. Therefore, it needs further studies to understand the characteristics and mechanisms of phosphate solubilization by phosphate solubilizing microorganisms. Moreover, the role of phosphate solubilizing microorganisms on plant growth under field conditions is also important and necessary to be studied. It is expected that this report will prompt further screenings of phosphate solubilizing microorganisms so as to enhance agronomic value of soils and benefit crop growth.
5 Conclusions (1) A strain YC, which is isolated from phosphate mines (Hubei, China), is identified as S. maltophilia. It is an aerobic Gram-negative and rod-shaped bacterium. (2) The strain, S. maltophilia YC, could solubilize TCP and release soluble P in NBRIP growth medium containing 0.1 g TCP as sole P source. (3) The TCP solubilizing capability of S.
maltophilia YC is associated with an increase of its population and a decrease of the pH in NBRIP growth medium. A positive correlation (r=0.83; p ＜ 0.01) between the concentration of soluble P and the population of S. maltophilia YC in the culture medium and a negative correlation (r=−0.86; p＜0.01) between the concentration of soluble P and pH are observed from statistical analysis results. The result of different concentrations of gluconic acid on TCP solubilization in vitro indicates that gluconic acid is an important factor for TCP solubilization, which released by S. maltophilia YC during the TCP solubilizing experiment in vivo. (4) It is expected that this report will prompt further screenings of phosphate solubilizing microorganisms so as to enhance agronomic value of soils and benefit crop growth.
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