World J Microbiol Biotechnol DOI 10.1007/s11274-015-1827-0
ORIGINAL PAPER
Isolation and the interaction between a mineral-weathering Rhizobium tropici Q34 and silicate minerals Rong Rong Wang • Qi Wang • Lin Yan He Gang Qiu • Xia Fang Sheng
•
Received: 31 August 2014 / Accepted: 17 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract The purposes of this study were to isolate and evaluate the interaction between mineral-weathering bacteria and silicate minerals (feldspar and biotite). A mineralweathering bacterium was isolated from weathered rocks and identified as Rhizobium tropici Q34 based on 16S rRNA gene sequence analysis. Si and K concentrations were increased by 1.3- to 4.0-fold and 1.1- to 1.7-fold in the live bacterium-inoculated cultures compared with the controls respectively. Significant increases in the productions of tartaric and succinic acids and extracellular polysaccharides by strain Q34 were observed in cultures with minerals. Furthermore, significantly more tartaric acid and polysaccharide productions by strain Q34 were obtained in the presence of feldspar, while better growth and more citric acid production of strain Q34 were observed in the presence of biotite. Mineral dissolution experiments showed that the organic acids and polysaccharides produced by strain Q34 were also capable of promoting the release of Si and K from the minerals. The results showed that the growth and metabolite production of strain Q34 were enhanced in the presence of the minerals and different mineral exerted distinct impacts on the growth and metabolite production. The bio-weathering process is probably a synergistic action of organic acids and extracellular polysaccharides produced by the bacterium. Keywords Rhizobium tropici Q34 Interaction Metabolite Silicate mineral Mineral dissolution
R. R. Wang Q. Wang L. Y. He G. Qiu X. F. Sheng (&) Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China e-mail: [email protected]
Introduction Silicate minerals such as feldspar and biotite are the most common minerals in the Earth’s crust and are a source of inorganic nutrients in soils (Uroz et al. 2007). Potassium (K) and silicon (Si) are the main elements in feldspar and biotite and are the essential soil nutrients that perform a multitude of important biological functions to maintain plant growth, development, yield, and biotic and abiotic resistance (Ma 2004; Read et al. 2006). However, plants cannot directly utilize mineral K and Si unless they are released by weathering or dissolved in soil water. It is urgent to search for effective approaches to use the mineral K and Si in silicate minerals. Furthermore, silicate mineral weathering also plays important roles in the soil formation, maintenance of soil fertility, and environmental change through removal of carbon dioxide from the atmosphere (Barker et al. 1998; Shirokova et al. 2012). Mineral-associated microbial communities can accelerate mineral weathering (Uroz et al. 2009). Previous studies only focused on the effects of bacteria on the mineral weathering and the mechanisms involved in the bio-weathering processes (Hutchens et al. 2006; Sheng et al. 2008; Uroz et al. 2011). As a consequence, the interaction between minerals and mineral-weathering bacteria has not been investigated in detail. Bacteria have been reported to play a pivotal role in silicate mineral weathering (Lapanje et al. 2012). Bacteria that produce organic or inorganic acids, extracellular polymers and siderophores could significantly increase the release of Si, Fe and Al from silicate minerals (Balogh-Brunstad et al. 2008; Balland et al. 2010; Garcia et al. 2013). A detailed understanding of how bacteria affect mineral dissolution rates is essential to quantify mineral weathering rates and the effects of weathering on element availability and cycling.
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Rhizobia are soil bacteria that fix nitrogen after becoming established inside root nodules of legumes. It is well established that in addition to symbiotic association with legumes, a rather large population of non-symbiotic rhizobia are found in the bulk soil, the rhizospheres (Berge et al. 2009), wastewater and oil-contaminated soil (Yan et al. 2010; Zhang et al. 2012). Besides the capacity of nitrogen fixing, rhizobia species can also promote the growth of plants and weather silicate minerals (Zhao et al. 2013). The objectives of this study were to isolate mineralweathering bacteria from weathered rock surfaces and to evaluate the impacts of feldspar and biotite on the growth and metabolite productions of one mineral-weathering Rhizobium tropici Q34. The effects of strain Q34 on the mineral weathering and the mechanisms involved were characterized.
Materials and methods Isolation of mineral-weathering bacteria The solid K-limited medium (KLM) [1 % sucrose, 0.1 % (NH4)2SO4, 0.05 % Na2HPO4, 0.05 % MgSO4, 0.01 % NaCl, 0.05 % yeast extract, 0.1 % feldspar powders (75–150 lm), 2 % agar in distilled water, pH 7.2] was used to isolate mineral-weathering bacteria (Zhao et al. 2013). The weathered rocks were aseptically collected from the Longshan (Nanjing, China) (31°470 N, 118°450 E). A 10 mL volume of sterile distilled water was added to 2 g of weathered rocks and shaken at 150 rpm for 40 min to allow bacteria to detach from rock surfaces. The suspension was then allowed to stand for about 30 min before 0.1 mL of supernatant was spread over solid KLM. The plates were incubated for 4 days at 28 °C. Twenty-two bacterial isolates were randomly selected and further purified on the same medium. Mineral weathering ability of the isolates The liquid KLM was used to test the feldspar dissolution ability of the 22 bacterial isolates. The mineral weathering experiment was performed according to the method of Zhao et al. (2013). Because Si is a structural element in feldspar, Si release to solution was used as an indicator of mineral dissolution. The flasks were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. Controls with feldspar, but no bacterial cells, to monitor the range of abiotic dissolution were treated in the same manner. Si concentration was determined using the inductively coupled plasma-atomic emission spectrometry (ICP-AES, Optimal 2100 DV, Perkin Elmer) methods.
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Strain Q34 that showed the highest enhancing effect on feldspar dissolution was selected for the interaction between the strain and silicate minerals. Mineral weathering experiments of strain Q34 The liquid KLM was used to test feldspar and biotite dissolution ability of strain Q34. To prepare the inoculum, strain Q34 was initially grown in sterilized (autoclaved at 121 °C for 30 min) sucrose-salts medium (SSM) [1 % sucrose, 0.1 % (NH4)2SO4, 0.001 % NaCl, 0.05 % MgSO47H2O, 0.2 % K2HPO4, 0.05 % yeast extract, 0.001 % CaCO3, pH 7.2] (Zhao et al. 2013) at 28 °C for 18 h on a rotary shaker at 150 rpm, and harvested by centrifugation at 5000 rpm for 10 min. Inoculum was washed two times in sterile distilled water, and cell pellets were then resuspended in sterile distilled water to a final concentration of 108 cells mL-1 before the dissolution experiment was started. Triplicate 250 mL polycarbonate Erlenmeyer culture flasks containing 50 mL of above KLM and 0.25 g feldspar or biotite powders were sterilized at 121 °C for 30 min and then inoculated with 2.5 mL of a bacterial suspension (Inoculum). A dead bacterial (autoclaved at 121 °C for 30 min) inoculated sample was prepared as a control. The flasks were incubated at 28 °C on a rotary shaker at 150 rpm. The dissolution of feldspar and biotite and the cell numbers and pH of the cultures were measured at 0, 1, 2, 4, 7, 10, 15, and 20 days after inoculation. Samples for chemical releasing measures were filtered through a 0.22 lm Millipore filter; 20 mL of the filtrate from each flask were centrifuged at 10,000 rpm for 10 min and divided into four aliquots. One aliquot was used for pH determination and the other aliquots were acidified with HNO3 (final concentration 2 % v/v). Si and K concentrations were determined using ICP-AES methods. Cell numbers of the cultures were estimated by plate counts using SSM described above. The concentrations of organic acids in the medium were determined using the HPLC analytical protocol previously described (Sheng et al. 2008). The isolation and purification of extracellular polysaccharides were performed according to the methods of Corzo et al. (1994) and van Casterena et al. (1998). Protein and nucleic acid contamination of polysaccharide preparations was monitored by recording the absorption spectrum of polysaccharide solutions (0.5 mg mL-1) in water between 190 and 350 nm. Mineral dissolution of the metabolites produced by strain Q34 Solutions were prepared using sterile deionized water and the sterilized silicate minerals. Dominant organic acids produced by strain Q34 in the presence of the minerals (HPLC analysis showed that the tartaric and succinic acids were dominant components in the metabolites) and
World J Microbiol Biotechnol
polysaccharides (protein and nucleic acid were not detected in the prepared polysaccharide) were used for the mineral dissolution. Mineral dissolution experiments were treated as follows: treated with (1) sterile deionized water (control); or (2) organic acids (mixture of tartaric and succinic acids, free of Si and K; or (3) pure polysaccharides or (4) mixture of organic acids and polysaccharides. The contents of organic acids and polysaccharides employed were identical to the maximum contents of organic acids and polysaccharides (Table 1). The flasks were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. The concentrations of Si and K in the solutions were determined by ICP-AES. Extraction of DNA from strain Q34, PCR amplification, and sequencing analysis The extraction of DNA, PCR amplification, and sequencing analysis of the strain were performed according to the method of Zhao et al. (2013). The nucleotide sequence determined in this study has been deposited in the NCBI database under accession number EU685810. Statistical analyses One-way analysis of variance (ANOVA) and the Fisher’s Least Significant Difference test (Fisher’s LSD) (p \ 0.05) were used to compare the means for pH and cell counts of each culture and to compare the means for Si and K released from the feldspar or biotite by live strain Q34 or the bacterial metabolites (organic acids and extracellular polysaccharides) with dead bacterial or water controls. The statistical analyses were carried out using SAS 8.2 (Statistical Analysis System, USA).
Results Isolation of mineral-weathering bacteria In this study, the obtained 22 bacterial strains isolated from the surfaces of the weathered rocks had the ability
to significantly release Si from feldspar compared with the control, among which strain Q34 was found to be the best ability to release Si from feldspar. Strain Q34 was identified as R. tropici based on the 16S rRNA gene sequence analysis (99 % 16S rRNA gene sequence similarity). Time courses of release of Si and K from the minerals by strain Q34 The time courses of Si and K release in the presence of feldspar and biotite are shown in Fig. 1. In the presence of the live strain Q34, Si release from feldspar was fluctuated (Fig. 1a). The water-soluble Si significantly (p \ 0.05) increased after 1 day of incubation. The highest Si release from feldspar was observed at the 7 days of incubation. After that, the content of water-soluble Si significantly declined between 7 and 15 days of incubation, followed by it’s a rising after 15 days of incubation. However, Si release from biotite continuously increased during the culturing time, reaching a maximum Si release at the end of data collection (Fig. 1a). We found that inoculation with the live strain Q34 increased the water-soluble Si release from feldspar and biotite by 48–284 and 27–179 %, respectively, compared to the dead bacteria inoculated controls. Similar to the Si release from the minerals with the presence of the strain, K release from feldspar was fluctuated (Fig. 1b). The water-soluble K significantly (p \ 0.05) increased after 1 day of incubation. The highest K release from feldspar was observed at the 4 days of incubation. After that, the concentration of K? significantly declined on 7 days of incubation, followed by it’s a rising after 7 days of incubation (p \ 0.05). However, K release from biotite continuously increased during the experimental period, reaching a maximum K release at 20 days of incubation (Fig. 1b). We found that inoculation with the live strain Q34 increased the watersoluble K release from feldspar and biotite by 8.5–66 and 9.3–39 %, respectively, compared to the dead bacteria inoculated controls.
Table 1 Impacts of minerals on the productions of organic acids and polysaccharides by strain Q34 after 10 days of incubation in liquid cultures Contents of organic acid (mg L-1) Oxalic acid
Tartaric acid
Contents of polysaccharide (g L-1) Malic acid
Citric acid
Succinic acid
No mineral
31 ± 0.1a*
533 ± 57c
148 ± 7a
BDL
131 ± 16b
4.7 ± 0.4c
Feldspar
40 ± 7.6a
1431 ± 39a
169 ± 17a
51 ± 3b
212 ± 41a
6.7 ± 1.2a
Biotite
35 ± 11a
659 ± 27b
151 ± 5a
71 ± 3a
216 ± 42a
5.5 ± 0.2b
Average ± standard deviation from three separate experiments BDL below the detection limit * Means followed by the same letter within a column are not significantly different (p [ 0.05) according to Tukey’s test
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Feldspar (live bacterium)
45
Feldspar (dead bacterium) *
*
Biotite (live bacterium)
40
Biotite (dead bacterium)
*
35
*
30
*
*
*
25 20
*
*
8
6000
*
*
15 10
*
7
5000
*
6 4000
*
Cell count (feldspar)
5
* *
Cell count (biotite)
*
Cell count (no mineral)
3000
4
pH (feldspar) pH (biotite)
3
*
pH (no mineral)
2000
pH in solution
50
Cell count in solution (× 10 6 cfu mL-1)
Si in solution (mg SiO 2 L-1)
a
2 1000
1
5 0
0
0
1
2
4
7
10
15
20
0 0
1
2
4
7
10
15
20
Time (day)
Time (day)
b
70
Feldspar (live bacterium) Feldspar (dead bacterium)
60
*
Biotite (live bacterium)
*
Biotite (dead bacterium)
K in solution (mg K L -1)
Fig. 2 The changes of cell counts and pH in the bacterium-inoculated medium added with feldspar or biotite minerals during 20 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a mineral treated cell count value significantly greater than the no mineral treated control value (p \ 0.05)
*
50
* *
*
*
* * *
40
30
20
10
0
0
1
2
4
7
10
15
20
Time (day)
Fig. 1 Influence of Rhizobium tropici Q34 on Si (a) and K (b) releases in the medium added with feldspar or biotite minerals during 20 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a live bacterium treated value significantly greater than the dead bacterium treated control value (p \ 0.05)
Effects of mineral on the growth and metabolic productions of strain Q34 During the first 4 days (feldspar) or 2 days (biotite) of incubation, the bacterium was in lag phase. The cell numbers in each culture did not increase. After 4 days (feldspar) or 2 days (biotite) of incubation, the cell numbers rapidly increased, from initial 5.00 9 106 cfu mL-1 to approximately 4–5 9 109 cfu mL-1 at 10 days of incubation. After 10 days of incubation, the cell counts in the culture gradually decreased in the presence of feldspar or biotite (Fig. 2). Furthermore, the cell counts in the
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mineral-containing microcosm were significantly (p \ 0.05) higher than those in the mineral free culture (p \ 0.05). More cell counts were obtained in the microcosm with biotite than in that with feldspar. There was significant (p \ 0.05) change in the pH values (5.55–6.68) of cultures during the mineral weathering process. In the presence and absence of feldspar and biotite, the productions of organic acids and extracellular polysaccharides by strain Q34 are shown in Table 1. There were no significant (p \ 0.05) differences in the production of oxalic and malic acids when the bacterium was cultured with or without minerals. However, the production of tartaric and succinic acids was significantly changed when minerals were supplied. In the presence of the minerals, the concentrations of tartaric and succinic acids in the cultures were increased by 24–168 and 62–65 % respectively compared with those in cultures without minerals (Table 1). Notably, in the presence of feldspar and biotite, strain Q34 could produce 51–71 mg L-1 of citric acid, while the production of citric acid was below the detection limit in the bacterial culture without any minerals (Table 1). In addition, strain Q34 could produce significantly (p \ 0.05) more tartaric acid in the presence of feldspar than in the presence of biotite, while strain Q34 produced significantly (p \ 0.05) more citric acid in the presence of biotite than in the presence of feldspar. No significant (p [ 0.05) differences in the productions of oxalic, malic, and succinic acids by the strain were observed between the feldspar and biotite supplemented microcosms. The addition of feldspar and biotite in the media also significantly (p \ 0.05) promoted the production of extracellular polysaccharides by strain Q34. The content of extracellular polysaccharides was increased by 17–43 % in
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the presence of the minerals compared with that of extracellular polysaccharides in the absence of the minerals. Furthermore, the strain could produce significantly (p \ 0.05) more extracellular polysaccharides in the presence of feldspar than in the presence of biotite. Effects of metabolites on mineral dissolution The metabolites produced by strain Q34 could significantly (p \ 0.05) promote the releases of Si and K from the minerals (Fig. 3). Si releases from the minerals were increased by 45–83, 73–98, and 113–133 % in the presence of organic acids, polysaccharides, and the mixture of organic acids and polysaccharides, respectively; while the K releases from the minerals were increased by 27, 28–53, and 92–114 % in the presence of organic acids, polysaccharides, and the mixture of organic acids and polysaccharides, respectively. Mixture of organic acids and polysaccharides possess a significantly higher Si and K release promotion ability than organic acids or polysaccharides alone. Si releases from the minerals were increased by 28–47 and 18–23 % in the presence of the mixture of organic acids and polysaccharides compared with the organic acid and polysaccharide treatments respectively (Fig. 3). K releases from the minerals were increased by 51–68 and 25–67 % in the presence of the mixture of organic acids and polysaccharides compared with the organic acid and polysaccharide treatments respectively.
Discussion Although diverse bacteria have been reported to be able to weather silicate minerals (Uroz et al. 2011; Dopson et al. Si and K in solution (mg SiO 2 or K L-1)
80 70
Si (feldspar) K (feldspar)
Si (biotite) K (biotite)
*
*
60 *
50
* *
*
40 *
30
*
*
*
* *
20 10 0
Water (control)
Organic acid
Polysaccharide
Organic acid + polysaccharide
Fig. 3 Influence of bacterial metabolites on Si and K releases in the solution added with feldspar and biotite during 7 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a metabolite treated value significantly greater than the control value (p \ 0.05)
2009; Ward et al. 2013), little is known about the function of Rhizobium in silicate mineral weathering. Moreover, no study on the interaction between silicate mineral and mineral-weathering R. tropici Q34 has been reported yet. Mineral dissolution experiment showed that R. tropici Q34 could be very effective in enhancing feldspar and biotite dissolution (Fig. 1). The enhancement of Si and K release observed in the biotic systems agreed with the findings of other studies (Hutchens et al. 2006; Sheng et al. 2008; Zhao et al. 2013). Furthermore, in the presence of R. tropici Q34, dynamics of Si and K released from feldspar followed a different pattern from those of Si and K released from biotite (Fig. 1). However, the growth of R. tropici Q34 with feldspar followed a similar pattern with that with biotite (Fig. 1). The fluctuation in concentrations of Si and K released from feldspar with live R. tropici Q34 may be caused by the re-precipitation and re-dissolution of secondary minerals (Delvasto et al. 2006). Welch et al. (1999) showed that secondary phase for mineral formation occurred via dissolution of the primary mineral, complexation of cations by polysaccharides, and adsorption of other elements (Si) to the cation-polysaccharide complex. In the mineral dissolution process, the addition of silicate minerals exerted a significant impact on the growth and metabolism of strain Q34 (Table 1; Fig. 2). Furthermore, different mineral exerted distinct impacts on the growth and metabolite production of the strain. The limited nutrients from the silicate minerals may be contributory factors for the stimulation of the growth and metabolism of strain Q34. Rogers and Bennett (2004) showed that limiting nutrients from silicates were responsible for the mineral stimulation of subsurface microorganisms. The production of organic acids and extracellular polysaccharides by strain Q34 were significantly promoted in the presence of feldspar and biotite (Table 1). Balland et al. (2010) suggested that mineral could exert a control on the release of organic acids. Notably, different bacteria produced different kinds of metabolites during the silicate mineral weathering process. For instance, our previous study showed that gluconic acid and acetic acids were the main organic acids produced by Bacillus globisporus Q12 in the silicate mineral weathering process (Sheng et al. 2008). However, our isolate R. tropici Q34 produced tartaric and succinic acids as the main organic acids during the silicate mineral weathering process (Table 1). Furthermore, our previous study found that a mineral-solubilizing Rhizobium sp. Q32 only produced extracellular polysaccharides in the mineral weathering process (Zhao et al. 2013), however, our isolate R. tropici Q34 could produce both organic acids and extracellular polysaccharides in the mineral weathering process. This study analyzed the productions of organic acids and extracellular polysaccharides by strain Q34 in the absence and presence of silicates, whereas in the study of
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Zhao et al. (2013), the production of extracellular polysaccharides by strain Q32 was analyzed only in the presence of silicate minerals. In addition, the effect of the metabolites (organic acids, polysaccharides and the mixture of organic acids and polysaccharides) produced by R. tropici Q34 on the mineral dissolution was evaluated in this study, however, the effect of the polysaccharides produced by Rhizobium sp. Q32 on the mineral dissolution was not evaluated in the study of Zhao et al. (2013). It was shown that bacteria increase the rate of silicate mineral dissolution by producing proton, hydroxyl, metabolic products (organic acids, extracellular polymeric substances and siderophore) or oxidation or reduction of metals in the mineral (Welch et al. 2002). In this study, we performed the abiotic mineral dissolution experiment and found that the organic acids and extracellular polysaccharides produced by strain Q34 could be effective in enhancing Si and K releases from feldspar and biotite and the mixture of organic acids and polysaccharides were more effective in enhancing Si and K releases than single component (Fig. 3). Additionally, the growth of R. tropici Q34 leaded to limited pH changes (6.68–5.55) (Fig. 2). Our results suggested that the increasing Si and K release from feldspar and biotite may be caused by ligand (organic acids and polysaccharides)-promoted dissolution (White and Brantley 1995). This study showed the interaction between a mineralweathering R. tropici Q34 and silicate minerals. The results showed the effect of strain Q34 on the K and Si release from the minerals, revealed the bio-weathering mechanism of silicate minerals and highlighted the potential of the mineral-weathering R. tropici Q34 as a biofertilizer for plant nutrition. Further studies linking biogeochemistry to ecology and genetics are needed to fully understand the role of R. tropici Q34 in mineral weathering in the field environments. Acknowledgments Support was provided by the Chinese National Natural Science Foundation (41071173, 41473075).
References Balland C, Poszwa A, Leyval C, Mustin C (2010) Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity. Geochim Cosmochim Acta 74:5478–5493 Balogh-Brunstad Z, Keller CK, Dickinson JT, Stevens F, Li CY, Bormann BT (2008) Biotite weathering and nutrient uptake by ectomycorrhizal fungus, Suillus tomentosus, in liquid-culture experiments. Geochim Cosmochim Acta 72:2601–2618 Barker WW, Welch SA, Chu S, Banfield JF (1998) Experimental observations of the effects of bacteria on aluminosilicate weathering. Am Mineral 83:1551–1563 Berge O, Lodhi A, Brandelet G, Santaella C, Roncato MA, Christen R, Heulin T, Achouak W (2009) Rhizobium alamii sp. nov., an
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exopolysaccharide-producing species isolated from legume and non-legume rhizospheres. Int J Syst Evol Microbiol 59:367–372 Corzo J, Leo´n-Barrios M, Hernando-Rico V, Gutierrez-Navarro AM (1994) Precipitation of metallic cations by the acidic exopolysaccharides from Bradyrhizobium japonicum and Bradyrhizobium (Chamaecytisus) strain BGA-1. Appl Environ Microbiol 60:4531–4536 Delvasto P, Valverde A, Ballester A, Igual JM, Mun˜oz JA, Gonza´lez F, Bla´zquez ML, Garcı´a C (2006) Characterization of brushite as a re-crystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biol Biochem 38:2645–2654 Dopson M, Lovgren L, Bostrom D (2009) Silicate mineral dissolution in the presence of acidophilic microorganisms: implications for heap bioleaching. Hydrometallurgy 96:288–293 Garcia B, Lemelle L, Rose-Koga E, Perriat P, Basset R, Gillet P, Albare`de F (2013) An experimental model approach of biologically-assisted silicate dissolution with olivine and Escherichia coli—impact on chemical weathering of mafic rocks and atmospheric CO2 drawdown. Appl Geochem 31:216–227 Hutchens E, Valsami-Jones E, Harouiya N, Chairat C, Oelkers E, McEldowney S (2006) An experimental investigation of the effect of Bacillus megaterium on apatite dissolution. Geomicrobiol J 23:177–182 Lapanje A, Wimmersberger C, Furrer G, Brunner I, Frey B (2012) Pattern of elemental release during the granite dissolution can be changed by aerobic heterotrophic bacterial strains isolated from Damma glacier (central Alps) deglaciated granite sand. Microb Ecol 63:865–882 Ma JF (2004) Role of silicon in enhancing the resistance of plants to biotic and abiotic stresses. Soil Sci Plant Nutr 50:11–18 Read JJ, Reddy KR, Jenkins JN (2006) Yield and fiber quality of upland cotton as influenced by nitrogen and potassium nutrition. Eur J Agron 24:282–290 Rogers JR, Bennett P (2004) Mineral stimulation of subsurface microorganisms: release of limiting nutrients from silicates. Chem Geol 203:91–108 Sheng XF, Zhao F, He LY, Qiu G, Chen L (2008) Isolation and characterization of silicate mineral-solubilizing Bacillus globisporus Q12 from the surfaces of weathered feldspar. Can J Microbiol 54:1064–1068 Shirokova LS, Be´ne´zeth P, Pokrovsky OS, Gerard E, Me´nez B, Alfredsson H (2012) Effect of the heterotrophic bacterium Pseudomonas reactans on olivine dissolution kinetics and implications for CO2 storage in basalts. Geochim Cosmochim Acta 80:30–50 Uroz S, Calvaruso C, Turpault MP, Pierrat JC, Mustin C, Frey-Klett P (2007) Effect of the mycorrhizosphere on the genotypic and metabolic diversity of the bacterial communities involved in mineral weathering in a forest soil. Appl Environ Microbiol 73:3019–3027 Uroz S, Calvaruso C, Turpault MP, Frey-Klett P (2009) Mineral weathering by bacteria: ecology, actors and mechanisms. Trends Microbiol 17:378–386 Uroz S, Oger P, Lepleux C, Collignon C, Frey-Klett P, Turpault MP (2011) Bacterial weathering and its contribution to nutrient cycling in temperate forest ecosystems. Res Microbiol 162:820–831 van Casterena WHM, Dijkemab HC, Scholsa A, Beldmana G, Voragen AGJ (1998) Characterisation and modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B40. Carbohydr Polym 37:123–130 Ward MB, Kapitulcˇinova´ D, Brown AP, Heard PJ, Cherns D, Cockell CS, Hallam KR, Ragnarsdo´ttir KV (2013) Investigating the role of microbes in mineral weathering: nanometre-scale characterisation of the cell-mineral interface using FIB and TEM. Micron 47:10–17
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ORIGINAL PAPER
Isolation and the interaction between a mineral-weathering Rhizobium tropici Q34 and silicate minerals Rong Rong Wang • Qi Wang • Lin Yan He Gang Qiu • Xia Fang Sheng
•
Received: 31 August 2014 / Accepted: 17 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract The purposes of this study were to isolate and evaluate the interaction between mineral-weathering bacteria and silicate minerals (feldspar and biotite). A mineralweathering bacterium was isolated from weathered rocks and identified as Rhizobium tropici Q34 based on 16S rRNA gene sequence analysis. Si and K concentrations were increased by 1.3- to 4.0-fold and 1.1- to 1.7-fold in the live bacterium-inoculated cultures compared with the controls respectively. Significant increases in the productions of tartaric and succinic acids and extracellular polysaccharides by strain Q34 were observed in cultures with minerals. Furthermore, significantly more tartaric acid and polysaccharide productions by strain Q34 were obtained in the presence of feldspar, while better growth and more citric acid production of strain Q34 were observed in the presence of biotite. Mineral dissolution experiments showed that the organic acids and polysaccharides produced by strain Q34 were also capable of promoting the release of Si and K from the minerals. The results showed that the growth and metabolite production of strain Q34 were enhanced in the presence of the minerals and different mineral exerted distinct impacts on the growth and metabolite production. The bio-weathering process is probably a synergistic action of organic acids and extracellular polysaccharides produced by the bacterium. Keywords Rhizobium tropici Q34 Interaction Metabolite Silicate mineral Mineral dissolution
R. R. Wang Q. Wang L. Y. He G. Qiu X. F. Sheng (&) Key Laboratory of Agricultural and Environmental Microbiology, Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China e-mail: [email protected]
Introduction Silicate minerals such as feldspar and biotite are the most common minerals in the Earth’s crust and are a source of inorganic nutrients in soils (Uroz et al. 2007). Potassium (K) and silicon (Si) are the main elements in feldspar and biotite and are the essential soil nutrients that perform a multitude of important biological functions to maintain plant growth, development, yield, and biotic and abiotic resistance (Ma 2004; Read et al. 2006). However, plants cannot directly utilize mineral K and Si unless they are released by weathering or dissolved in soil water. It is urgent to search for effective approaches to use the mineral K and Si in silicate minerals. Furthermore, silicate mineral weathering also plays important roles in the soil formation, maintenance of soil fertility, and environmental change through removal of carbon dioxide from the atmosphere (Barker et al. 1998; Shirokova et al. 2012). Mineral-associated microbial communities can accelerate mineral weathering (Uroz et al. 2009). Previous studies only focused on the effects of bacteria on the mineral weathering and the mechanisms involved in the bio-weathering processes (Hutchens et al. 2006; Sheng et al. 2008; Uroz et al. 2011). As a consequence, the interaction between minerals and mineral-weathering bacteria has not been investigated in detail. Bacteria have been reported to play a pivotal role in silicate mineral weathering (Lapanje et al. 2012). Bacteria that produce organic or inorganic acids, extracellular polymers and siderophores could significantly increase the release of Si, Fe and Al from silicate minerals (Balogh-Brunstad et al. 2008; Balland et al. 2010; Garcia et al. 2013). A detailed understanding of how bacteria affect mineral dissolution rates is essential to quantify mineral weathering rates and the effects of weathering on element availability and cycling.
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Rhizobia are soil bacteria that fix nitrogen after becoming established inside root nodules of legumes. It is well established that in addition to symbiotic association with legumes, a rather large population of non-symbiotic rhizobia are found in the bulk soil, the rhizospheres (Berge et al. 2009), wastewater and oil-contaminated soil (Yan et al. 2010; Zhang et al. 2012). Besides the capacity of nitrogen fixing, rhizobia species can also promote the growth of plants and weather silicate minerals (Zhao et al. 2013). The objectives of this study were to isolate mineralweathering bacteria from weathered rock surfaces and to evaluate the impacts of feldspar and biotite on the growth and metabolite productions of one mineral-weathering Rhizobium tropici Q34. The effects of strain Q34 on the mineral weathering and the mechanisms involved were characterized.
Materials and methods Isolation of mineral-weathering bacteria The solid K-limited medium (KLM) [1 % sucrose, 0.1 % (NH4)2SO4, 0.05 % Na2HPO4, 0.05 % MgSO4, 0.01 % NaCl, 0.05 % yeast extract, 0.1 % feldspar powders (75–150 lm), 2 % agar in distilled water, pH 7.2] was used to isolate mineral-weathering bacteria (Zhao et al. 2013). The weathered rocks were aseptically collected from the Longshan (Nanjing, China) (31°470 N, 118°450 E). A 10 mL volume of sterile distilled water was added to 2 g of weathered rocks and shaken at 150 rpm for 40 min to allow bacteria to detach from rock surfaces. The suspension was then allowed to stand for about 30 min before 0.1 mL of supernatant was spread over solid KLM. The plates were incubated for 4 days at 28 °C. Twenty-two bacterial isolates were randomly selected and further purified on the same medium. Mineral weathering ability of the isolates The liquid KLM was used to test the feldspar dissolution ability of the 22 bacterial isolates. The mineral weathering experiment was performed according to the method of Zhao et al. (2013). Because Si is a structural element in feldspar, Si release to solution was used as an indicator of mineral dissolution. The flasks were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. Controls with feldspar, but no bacterial cells, to monitor the range of abiotic dissolution were treated in the same manner. Si concentration was determined using the inductively coupled plasma-atomic emission spectrometry (ICP-AES, Optimal 2100 DV, Perkin Elmer) methods.
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Strain Q34 that showed the highest enhancing effect on feldspar dissolution was selected for the interaction between the strain and silicate minerals. Mineral weathering experiments of strain Q34 The liquid KLM was used to test feldspar and biotite dissolution ability of strain Q34. To prepare the inoculum, strain Q34 was initially grown in sterilized (autoclaved at 121 °C for 30 min) sucrose-salts medium (SSM) [1 % sucrose, 0.1 % (NH4)2SO4, 0.001 % NaCl, 0.05 % MgSO47H2O, 0.2 % K2HPO4, 0.05 % yeast extract, 0.001 % CaCO3, pH 7.2] (Zhao et al. 2013) at 28 °C for 18 h on a rotary shaker at 150 rpm, and harvested by centrifugation at 5000 rpm for 10 min. Inoculum was washed two times in sterile distilled water, and cell pellets were then resuspended in sterile distilled water to a final concentration of 108 cells mL-1 before the dissolution experiment was started. Triplicate 250 mL polycarbonate Erlenmeyer culture flasks containing 50 mL of above KLM and 0.25 g feldspar or biotite powders were sterilized at 121 °C for 30 min and then inoculated with 2.5 mL of a bacterial suspension (Inoculum). A dead bacterial (autoclaved at 121 °C for 30 min) inoculated sample was prepared as a control. The flasks were incubated at 28 °C on a rotary shaker at 150 rpm. The dissolution of feldspar and biotite and the cell numbers and pH of the cultures were measured at 0, 1, 2, 4, 7, 10, 15, and 20 days after inoculation. Samples for chemical releasing measures were filtered through a 0.22 lm Millipore filter; 20 mL of the filtrate from each flask were centrifuged at 10,000 rpm for 10 min and divided into four aliquots. One aliquot was used for pH determination and the other aliquots were acidified with HNO3 (final concentration 2 % v/v). Si and K concentrations were determined using ICP-AES methods. Cell numbers of the cultures were estimated by plate counts using SSM described above. The concentrations of organic acids in the medium were determined using the HPLC analytical protocol previously described (Sheng et al. 2008). The isolation and purification of extracellular polysaccharides were performed according to the methods of Corzo et al. (1994) and van Casterena et al. (1998). Protein and nucleic acid contamination of polysaccharide preparations was monitored by recording the absorption spectrum of polysaccharide solutions (0.5 mg mL-1) in water between 190 and 350 nm. Mineral dissolution of the metabolites produced by strain Q34 Solutions were prepared using sterile deionized water and the sterilized silicate minerals. Dominant organic acids produced by strain Q34 in the presence of the minerals (HPLC analysis showed that the tartaric and succinic acids were dominant components in the metabolites) and
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polysaccharides (protein and nucleic acid were not detected in the prepared polysaccharide) were used for the mineral dissolution. Mineral dissolution experiments were treated as follows: treated with (1) sterile deionized water (control); or (2) organic acids (mixture of tartaric and succinic acids, free of Si and K; or (3) pure polysaccharides or (4) mixture of organic acids and polysaccharides. The contents of organic acids and polysaccharides employed were identical to the maximum contents of organic acids and polysaccharides (Table 1). The flasks were incubated at 28 °C on a rotary shaker at 150 rpm for 7 days. The concentrations of Si and K in the solutions were determined by ICP-AES. Extraction of DNA from strain Q34, PCR amplification, and sequencing analysis The extraction of DNA, PCR amplification, and sequencing analysis of the strain were performed according to the method of Zhao et al. (2013). The nucleotide sequence determined in this study has been deposited in the NCBI database under accession number EU685810. Statistical analyses One-way analysis of variance (ANOVA) and the Fisher’s Least Significant Difference test (Fisher’s LSD) (p \ 0.05) were used to compare the means for pH and cell counts of each culture and to compare the means for Si and K released from the feldspar or biotite by live strain Q34 or the bacterial metabolites (organic acids and extracellular polysaccharides) with dead bacterial or water controls. The statistical analyses were carried out using SAS 8.2 (Statistical Analysis System, USA).
Results Isolation of mineral-weathering bacteria In this study, the obtained 22 bacterial strains isolated from the surfaces of the weathered rocks had the ability
to significantly release Si from feldspar compared with the control, among which strain Q34 was found to be the best ability to release Si from feldspar. Strain Q34 was identified as R. tropici based on the 16S rRNA gene sequence analysis (99 % 16S rRNA gene sequence similarity). Time courses of release of Si and K from the minerals by strain Q34 The time courses of Si and K release in the presence of feldspar and biotite are shown in Fig. 1. In the presence of the live strain Q34, Si release from feldspar was fluctuated (Fig. 1a). The water-soluble Si significantly (p \ 0.05) increased after 1 day of incubation. The highest Si release from feldspar was observed at the 7 days of incubation. After that, the content of water-soluble Si significantly declined between 7 and 15 days of incubation, followed by it’s a rising after 15 days of incubation. However, Si release from biotite continuously increased during the culturing time, reaching a maximum Si release at the end of data collection (Fig. 1a). We found that inoculation with the live strain Q34 increased the water-soluble Si release from feldspar and biotite by 48–284 and 27–179 %, respectively, compared to the dead bacteria inoculated controls. Similar to the Si release from the minerals with the presence of the strain, K release from feldspar was fluctuated (Fig. 1b). The water-soluble K significantly (p \ 0.05) increased after 1 day of incubation. The highest K release from feldspar was observed at the 4 days of incubation. After that, the concentration of K? significantly declined on 7 days of incubation, followed by it’s a rising after 7 days of incubation (p \ 0.05). However, K release from biotite continuously increased during the experimental period, reaching a maximum K release at 20 days of incubation (Fig. 1b). We found that inoculation with the live strain Q34 increased the watersoluble K release from feldspar and biotite by 8.5–66 and 9.3–39 %, respectively, compared to the dead bacteria inoculated controls.
Table 1 Impacts of minerals on the productions of organic acids and polysaccharides by strain Q34 after 10 days of incubation in liquid cultures Contents of organic acid (mg L-1) Oxalic acid
Tartaric acid
Contents of polysaccharide (g L-1) Malic acid
Citric acid
Succinic acid
No mineral
31 ± 0.1a*
533 ± 57c
148 ± 7a
BDL
131 ± 16b
4.7 ± 0.4c
Feldspar
40 ± 7.6a
1431 ± 39a
169 ± 17a
51 ± 3b
212 ± 41a
6.7 ± 1.2a
Biotite
35 ± 11a
659 ± 27b
151 ± 5a
71 ± 3a
216 ± 42a
5.5 ± 0.2b
Average ± standard deviation from three separate experiments BDL below the detection limit * Means followed by the same letter within a column are not significantly different (p [ 0.05) according to Tukey’s test
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Feldspar (live bacterium)
45
Feldspar (dead bacterium) *
*
Biotite (live bacterium)
40
Biotite (dead bacterium)
*
35
*
30
*
*
*
25 20
*
*
8
6000
*
*
15 10
*
7
5000
*
6 4000
*
Cell count (feldspar)
5
* *
Cell count (biotite)
*
Cell count (no mineral)
3000
4
pH (feldspar) pH (biotite)
3
*
pH (no mineral)
2000
pH in solution
50
Cell count in solution (× 10 6 cfu mL-1)
Si in solution (mg SiO 2 L-1)
a
2 1000
1
5 0
0
0
1
2
4
7
10
15
20
0 0
1
2
4
7
10
15
20
Time (day)
Time (day)
b
70
Feldspar (live bacterium) Feldspar (dead bacterium)
60
*
Biotite (live bacterium)
*
Biotite (dead bacterium)
K in solution (mg K L -1)
Fig. 2 The changes of cell counts and pH in the bacterium-inoculated medium added with feldspar or biotite minerals during 20 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a mineral treated cell count value significantly greater than the no mineral treated control value (p \ 0.05)
*
50
* *
*
*
* * *
40
30
20
10
0
0
1
2
4
7
10
15
20
Time (day)
Fig. 1 Influence of Rhizobium tropici Q34 on Si (a) and K (b) releases in the medium added with feldspar or biotite minerals during 20 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a live bacterium treated value significantly greater than the dead bacterium treated control value (p \ 0.05)
Effects of mineral on the growth and metabolic productions of strain Q34 During the first 4 days (feldspar) or 2 days (biotite) of incubation, the bacterium was in lag phase. The cell numbers in each culture did not increase. After 4 days (feldspar) or 2 days (biotite) of incubation, the cell numbers rapidly increased, from initial 5.00 9 106 cfu mL-1 to approximately 4–5 9 109 cfu mL-1 at 10 days of incubation. After 10 days of incubation, the cell counts in the culture gradually decreased in the presence of feldspar or biotite (Fig. 2). Furthermore, the cell counts in the
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mineral-containing microcosm were significantly (p \ 0.05) higher than those in the mineral free culture (p \ 0.05). More cell counts were obtained in the microcosm with biotite than in that with feldspar. There was significant (p \ 0.05) change in the pH values (5.55–6.68) of cultures during the mineral weathering process. In the presence and absence of feldspar and biotite, the productions of organic acids and extracellular polysaccharides by strain Q34 are shown in Table 1. There were no significant (p \ 0.05) differences in the production of oxalic and malic acids when the bacterium was cultured with or without minerals. However, the production of tartaric and succinic acids was significantly changed when minerals were supplied. In the presence of the minerals, the concentrations of tartaric and succinic acids in the cultures were increased by 24–168 and 62–65 % respectively compared with those in cultures without minerals (Table 1). Notably, in the presence of feldspar and biotite, strain Q34 could produce 51–71 mg L-1 of citric acid, while the production of citric acid was below the detection limit in the bacterial culture without any minerals (Table 1). In addition, strain Q34 could produce significantly (p \ 0.05) more tartaric acid in the presence of feldspar than in the presence of biotite, while strain Q34 produced significantly (p \ 0.05) more citric acid in the presence of biotite than in the presence of feldspar. No significant (p [ 0.05) differences in the productions of oxalic, malic, and succinic acids by the strain were observed between the feldspar and biotite supplemented microcosms. The addition of feldspar and biotite in the media also significantly (p \ 0.05) promoted the production of extracellular polysaccharides by strain Q34. The content of extracellular polysaccharides was increased by 17–43 % in
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the presence of the minerals compared with that of extracellular polysaccharides in the absence of the minerals. Furthermore, the strain could produce significantly (p \ 0.05) more extracellular polysaccharides in the presence of feldspar than in the presence of biotite. Effects of metabolites on mineral dissolution The metabolites produced by strain Q34 could significantly (p \ 0.05) promote the releases of Si and K from the minerals (Fig. 3). Si releases from the minerals were increased by 45–83, 73–98, and 113–133 % in the presence of organic acids, polysaccharides, and the mixture of organic acids and polysaccharides, respectively; while the K releases from the minerals were increased by 27, 28–53, and 92–114 % in the presence of organic acids, polysaccharides, and the mixture of organic acids and polysaccharides, respectively. Mixture of organic acids and polysaccharides possess a significantly higher Si and K release promotion ability than organic acids or polysaccharides alone. Si releases from the minerals were increased by 28–47 and 18–23 % in the presence of the mixture of organic acids and polysaccharides compared with the organic acid and polysaccharide treatments respectively (Fig. 3). K releases from the minerals were increased by 51–68 and 25–67 % in the presence of the mixture of organic acids and polysaccharides compared with the organic acid and polysaccharide treatments respectively.
Discussion Although diverse bacteria have been reported to be able to weather silicate minerals (Uroz et al. 2011; Dopson et al. Si and K in solution (mg SiO 2 or K L-1)
80 70
Si (feldspar) K (feldspar)
Si (biotite) K (biotite)
*
*
60 *
50
* *
*
40 *
30
*
*
*
* *
20 10 0
Water (control)
Organic acid
Polysaccharide
Organic acid + polysaccharide
Fig. 3 Influence of bacterial metabolites on Si and K releases in the solution added with feldspar and biotite during 7 days of incubation. Error bars are ±standard error (n = 3). An asterisk denotes a metabolite treated value significantly greater than the control value (p \ 0.05)
2009; Ward et al. 2013), little is known about the function of Rhizobium in silicate mineral weathering. Moreover, no study on the interaction between silicate mineral and mineral-weathering R. tropici Q34 has been reported yet. Mineral dissolution experiment showed that R. tropici Q34 could be very effective in enhancing feldspar and biotite dissolution (Fig. 1). The enhancement of Si and K release observed in the biotic systems agreed with the findings of other studies (Hutchens et al. 2006; Sheng et al. 2008; Zhao et al. 2013). Furthermore, in the presence of R. tropici Q34, dynamics of Si and K released from feldspar followed a different pattern from those of Si and K released from biotite (Fig. 1). However, the growth of R. tropici Q34 with feldspar followed a similar pattern with that with biotite (Fig. 1). The fluctuation in concentrations of Si and K released from feldspar with live R. tropici Q34 may be caused by the re-precipitation and re-dissolution of secondary minerals (Delvasto et al. 2006). Welch et al. (1999) showed that secondary phase for mineral formation occurred via dissolution of the primary mineral, complexation of cations by polysaccharides, and adsorption of other elements (Si) to the cation-polysaccharide complex. In the mineral dissolution process, the addition of silicate minerals exerted a significant impact on the growth and metabolism of strain Q34 (Table 1; Fig. 2). Furthermore, different mineral exerted distinct impacts on the growth and metabolite production of the strain. The limited nutrients from the silicate minerals may be contributory factors for the stimulation of the growth and metabolism of strain Q34. Rogers and Bennett (2004) showed that limiting nutrients from silicates were responsible for the mineral stimulation of subsurface microorganisms. The production of organic acids and extracellular polysaccharides by strain Q34 were significantly promoted in the presence of feldspar and biotite (Table 1). Balland et al. (2010) suggested that mineral could exert a control on the release of organic acids. Notably, different bacteria produced different kinds of metabolites during the silicate mineral weathering process. For instance, our previous study showed that gluconic acid and acetic acids were the main organic acids produced by Bacillus globisporus Q12 in the silicate mineral weathering process (Sheng et al. 2008). However, our isolate R. tropici Q34 produced tartaric and succinic acids as the main organic acids during the silicate mineral weathering process (Table 1). Furthermore, our previous study found that a mineral-solubilizing Rhizobium sp. Q32 only produced extracellular polysaccharides in the mineral weathering process (Zhao et al. 2013), however, our isolate R. tropici Q34 could produce both organic acids and extracellular polysaccharides in the mineral weathering process. This study analyzed the productions of organic acids and extracellular polysaccharides by strain Q34 in the absence and presence of silicates, whereas in the study of
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Zhao et al. (2013), the production of extracellular polysaccharides by strain Q32 was analyzed only in the presence of silicate minerals. In addition, the effect of the metabolites (organic acids, polysaccharides and the mixture of organic acids and polysaccharides) produced by R. tropici Q34 on the mineral dissolution was evaluated in this study, however, the effect of the polysaccharides produced by Rhizobium sp. Q32 on the mineral dissolution was not evaluated in the study of Zhao et al. (2013). It was shown that bacteria increase the rate of silicate mineral dissolution by producing proton, hydroxyl, metabolic products (organic acids, extracellular polymeric substances and siderophore) or oxidation or reduction of metals in the mineral (Welch et al. 2002). In this study, we performed the abiotic mineral dissolution experiment and found that the organic acids and extracellular polysaccharides produced by strain Q34 could be effective in enhancing Si and K releases from feldspar and biotite and the mixture of organic acids and polysaccharides were more effective in enhancing Si and K releases than single component (Fig. 3). Additionally, the growth of R. tropici Q34 leaded to limited pH changes (6.68–5.55) (Fig. 2). Our results suggested that the increasing Si and K release from feldspar and biotite may be caused by ligand (organic acids and polysaccharides)-promoted dissolution (White and Brantley 1995). This study showed the interaction between a mineralweathering R. tropici Q34 and silicate minerals. The results showed the effect of strain Q34 on the K and Si release from the minerals, revealed the bio-weathering mechanism of silicate minerals and highlighted the potential of the mineral-weathering R. tropici Q34 as a biofertilizer for plant nutrition. Further studies linking biogeochemistry to ecology and genetics are needed to fully understand the role of R. tropici Q34 in mineral weathering in the field environments. Acknowledgments Support was provided by the Chinese National Natural Science Foundation (41071173, 41473075).
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