- Email: [email protected]
Effects of low-molecular-weight organic ligands and phosphate on DNA adsorption by soil colloids and minerals
Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
Effects of low-molecular-weight organic ligands and phosphate on DNA adsorption by soil colloids and minerals Peng Cai, Qiaoyun Huang ∗ , Jun Zhu, Daihua Jiang, Xueyong Zhou, Xingmin Rong, Wei Liang Key Laboratory of Agricultural Resources and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agriculture University, Wuhan 430070, China Received 27 April 2006; received in revised form 19 July 2006; accepted 20 July 2006 Available online 27 July 2006
Abstract Adsorption of DNA on montmorillonite, kaolinite, goethite and soil clays from an Alfisol in the presence of citrate, tartrate and phosphate was studied. A marked decrease in DNA adsorption was observed on montmorillonite and kaolinite with increasing anion concentrations from 0 to 5 mM. However, the amount of DNA adsorbed by montmorillonite and kaolinite was enhanced when ligand concentration was higher than 5 mM. In the system of soil colloids and goethite, with the increase of anion concentrations, a steady decrease was found and the ability of ligands in depressing DNA adsorption followed the sequence: phosphate > citrate > tartrate. Compared to H2 O2 -treated clays (inorganic clays), a sharp decrease in DNA adsorption was observed on goethite and organo-mineral complexes (organic clays) with increasing ligand concentrations. The results suggest that the influence of anions on DNA adsorption varies with the type and concentration of anion as well as the surface properties of soil components. Introduction of DNA into the system before the addition of ligands had the greatest amount of DNA adsorption on soil colloids and goethite. Organic and inorganic ligands promoted DNA adsorption on montmorillonite and kaolinite when ligands were introduced into the system before the addition of DNA. The results obtained in this study have important implications for the understanding of the persistence and fate of DNA in soil environments especially rhizosphere soil where various organic and inorganic ligands are active. © 2006 Elsevier B.V. All rights reserved. Keywords: Soil colloid; DNA; Organic ligand; Phosphate; Adsorption
1. Introduction DNA is the genetic material of various organisms. In soil environments, DNA molecules are liberated by excretion from microorganisms, plants and animals or by lysis of dying cells [1]. A proportion of the extracellular DNA molecules in soil is soluble in the aqueous phase and a proportion is physically and chemically adsorbed to soil active particles [2,3]. The adsorbed DNA molecules are partially protected against degradation by nucleases and retain the capacity to transform competent bacterial cells [4–7]. As a result, the adsorption of DNA on soil components plays a vital role in controlling the ultimate fate of extracellular DNA in soils. DNA molecules can be adsorbed on soil colloids and minerals through electrostatic interaction, non-electrostatic forces
∗
Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail address: [email protected] (Q. Huang).
0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.07.013
such as hydrophobic ones, ligand exchange, hydrogen bonding and van der Waals forces [8,9]. An increase in the concentration of cations and/or a decrease in the pH favored DNA adsorption on soil colloids and clay minerals [6,9,10]. The amount of supercoiled plasmid DNA adsorbed by sand was slightly less than that of linearized or open circular plasmid DNA [11]. The lower molecular mass DNA adsorbed and bound by Ca-montmorillonite and Ca-kaolinite showed higher adsorption capacity than the higher molecular mass DNA [12]. Organic matter played an inhibitory role in DNA adsorption on permanentcharge soil colloids. Montmorillonite clay dominates the amount of DNA adsorption and kaolinite probably plays a key role in the extent of DNA binding in permanent-charge soil [9]. Low-molecular-weight (LMW) organic acids secreted from plant roots and produced through the decomposition of organic material residues occur widely in soils, especially in the immediate zone of the soil–root interface [13,14]. The concentration of LMW organic acids in rhizosphere could be as high as 10–20 mmol L−1 [13]. Nutrients (phosphate, sulfate) and LMW
54
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
Table 1 Selected properties of soil colloids and minerals studied Soil colloid or mineral
O.M. (g kg−1 )
SESA (m2 g−1 )
PZC
CEC (cmol kg−1 )
Clay mineral composition
Organic clay Inorganic clay Goethite Montmorillonite Kaolinite
71.0 9.6 – – –
64.4 77.1 83.0 73.6 22.9
2.1 3.3 8.7 2.5 3.6
57.6 47.9 0 90.2 7.1
Hydromica (55%), vermiculite (30%), kaolinite (15%) Hydromica (55%), vermiculite (30%), kaolinite (15%) Goethite Montmorillonite Kaolinite
Clay mineral was determined by X-ray diffraction analysis. Organic matter (O.M.), point of zero charge (PZC), cation-exchange capacity (CEC) and specific external surface area (SESA) were analyzed by K2 Cr2 O7 digestion, Mehlich, NH4 AcO method [32] and N2 adsorption method (Beijing Analytical Instrument Company), respectively.
organic ligands can be adsorbed by soil colloids and minerals and resulted in changes in surface properties of soil components [15,16]. Therefore, various organic and inorganic ligands such as citric and tartaric acids and phosphate, which are relatively abundant in soil, may exert significant effects on the adsorption of DNA by soil components. Pietramellara et al. [12] reported that DNA adsorption on Ca-montmorillonite and Ca-kaolinite was reduced markedly in the presence of sodium metaphosphate, indicating a strong competition between phosphate groups and DNA molecules on the mineral surface. In our previous study, it was found that a percentage of 20–40% of adsorbed DNA on soil colloids from an Alfisol was still released by sodium phosphate buffer after extensive washes with Tris buffer and NaCl [9]. However, little information is available regarding the effect of organic ligands on DNA adsorption. The aim of the present work was to investigate and compare the effects of selected LMW organic acids and phosphate on the adsorption of DNA on soil clays, montmorillonite, kaolinite and goethite which are common minerals in soils. 2. Materials and methods 2.1. DNA Salmon sperm DNA was bought from Sigma Chemical Co., St. Louis, MO. 2.2. Soil colloids and minerals A clay loamy Brown Soil (Alfisol) was sampled from the 0–17 cm layer of a forest land in Tianwai village, Taishan, Shandong province, China. Pertinent characteristics of the soil include: water pH 6.3, organic matter 42.4 g kg−1 and cationexchange capacity of 15.3 cmol kg−1 . After removal of organic residue the soil was rinsed in deionized distilled water (ddH2 O) and dispersed by adding 0.01 M NaOH solution dropwise together with sonication. The <2 m clay fraction of the soil was separated by sedimentation. The two treatments applied to clays were organic matter left on the sample (organic clay) and organic matter removed from the sample by H2 O2 (inorganic clay). After flocculation by the addition of CaCl2 solution, the colloidal suspension was washed to be free of Cl− ions by ddH2 O and ethanol, and then air-dried. Some properties of the clay fractions are listed in Table 1.
Fig. 1. Adsorption of DNA on soil colloids and minerals in the presence of increasing concentration of organic acids and phosphate.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
Kaolinite and montmorillonite were prepared according to Cai et al. [9]. Goethite was synthesized as described previously by Huang et al. [17]. All the soil colloids and minerals prepared were ground to pass a 100 mesh sieve. 2.3. DNA adsorption experiment In a centrifuge tube, 2.1 ml of organic and inorganic ligands and 0.4 ml of 10 mM Tris buffer (pH 7.0) containing 200 g DNA were mixed to yield final ligand concentrations ranging from 0 to 80 mM for tartrate and citrate or 0–200 mM for phosphate. After that, 10 mg of soil colloid or mineral was added to the tube. The mixture was gently shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. DNA in the supernatant was determined directly at 260 nm spectrophotometrically. The amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that remaining in the supernatant. The experiment was also designed to investigate the influence of the addition order of ligands and DNA on the adsorption of DNA on soil colloids and minerals. (1) Introduction of DNA after LMW organic anions or phosphate adsorption. Ten mil-
55
ligrams of soil colloid or mineral and 2.1 ml anionic solution containing 5–50 mM of citrate or tartrate or 5–100 mM of phosphate were added into the centrifuge tube and shaken for 1 h at 25 ◦ C. Four hundred microlitres of 10 mM Tris buffer (pH 7.0) containing 200 g DNA was added for another 1 h. The mixture was centrifuged and the content of DNA in the supernatant was analyzed. (2) Introduction of LMW organic acids or phosphate after DNA adsorption. Four hundred microlitres of 10 mM Tris buffer (pH 7.0) containing 200 g DNA and 10 mg soil colloid or mineral was mixed and shaken for 1 h at 25 ◦ C. After the period 2.1 ml of anionic solution containing 5–50 mM of citrate or tartrate or 5–100 mM of phosphate was then added. The suspension was shaken for an additional 1 h and DNA in the supernatant was analyzed. 3. Results and discussion 3.1. Effect of ligand concentrations on DNA adsorption Fig. 1 shows the adsorption of DNA by soil colloids and minerals in the presence of organic acids and phosphate. The amount of DNA adsorbed by montmorillonite and kaolinite decreased
Fig. 2. Adsorption of DNA on soil colloids and minerals as affected by the addition order of citrate and DNA: (䊉) DNA/citrate; () citrate/DNA; () citrate + DNA.
56
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
with increasing tartrate, citrate and phosphate concentrations from 0 to 5 mM, whereas an increase for DNA adsorption was found in the range of 5–80 mM tartrate and citrate and 5–200 mM phosphate. A steady decrease in DNA adsorption was observed on soil colloids and goethite with the increase of organic ligand and phosphate concentrations. The inhibition of DNA adsorption by anions was mainly due to the competition of tartrate, citrate and phosphate with DNA for adsorption sites of soil clays and minerals surfaces. This agrees with the commonly observed competition of these high affinity organic and inorganic ligands with the adsorption of inorganic oxyanions [18–22] and enzyme [16,23] as well as chromosomal and plasmid DNA molecules [12]. The promotive effect of high concentrations of ligands on the adsorption of DNA on kaolinite and montmorillonite could likely be explained by dissolution reactions in the presence of organic ligands which create new sites on the surfaces of clay minerals and the formation of precipitates (Al-phosphate) which may facilitate DNA adsorption. LMW organic acids could accelerate aluminum dissolution from kaolinite, feldspar and aluminum oxides through proton-promoted and ligand enhanced
effects [24–26]. The soluble Al3+ forming bridges between phosphate groups of DNA and the negatively charged sites of minerals may be also responsible for the higher DNA adsorption on kaolinite and montmorillonite at higher ligand concentrations. The above finding suggests that montmorillonite and kaolinite play a more important role in the adsorption of DNA molecules in rhizosphere soil where higher concentrations of various ligands are present. 3.2. Effect of ligand types on DNA adsorption As shown in Fig. 1, the examined organic ligands and phosphate present different inhibiting effects on DNA adsorption by soil colloids and minerals. In order to evaluate the depressing ability of organic ligands and phosphate, the percentage efficiency of the ligand was calculated according to the following expression: efficiency of the ligand (%) = 1 − (DNA adsorbed in the presence of ligand/DNA adsorbed when applied alone). The efficiency of three ligands was similar for decreasing DNA adsorption on montmorillonite and kaolinite at lower anion concentrations. In organic clay system, with the increase of
Fig. 3. Adsorption of DNA on soil colloids and minerals as affected by the addition order of tartrate and DNA: (䊉) DNA/tartrate; () tartrate/DNA; () tartrate + DNA.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
anion concentration from 5 to 50 mM, the efficiency of ligands increased from 40 to 89, 33 to 56, and 37 to 53% for phosphate, citrate and tartrate, respectively. The efficiency of anions for reducing DNA adsorption on inorganic clay was 16–29% for phosphate, 12–22% for citrate and 8–18% for tartrate. In the system of goethite, the efficiency of phosphate increased from 41 to 77% and that for citrate was from 25 to 55% and that for tartrate was from 6 to 14%. The above results indicate that the ability of ligands in depressing DNA adsorption by soil colloids and goethite was in the order of phosphate > citrate > tartrate. In goethite system, the higher inhibiting efficiency of phosphate suggests that there are large amounts of adsorptive sites on goethite for ligand exchange and these sites are common for phosphate and DNA molecules. The values of point of zero charge (PZC) of organic clay and inorganic clay were 2.1 and 3.3, respectively, and the cation-exchange capacity (CEC) value of organic clay was higher than that of inorganic clay (Table 1). The isoelectric point of DNA is about 5.0 [27]. DNA molecules are negatively charged due to phosphate groups when pH is above the isoelectric point [28]. Therefore, there is a stronger electro-
57
static repulsion force between the negatively charged DNA and organic clay at pH 7.0. It was reported by Xu et al. [16] that the presence of organic anions led to an increase in negative charges and a decrease in positive charges of soils. Therefore, the higher inhibition of DNA adsorption by ligands on organic clay than that on inorganic clay may be ascribed to the greater amount of negative charges on organic clay surfaces than those on inorganic clay with the increase of ligand concentrations which result in stronger electrostatic repulsion forces between DNA and organic clay. The greater capacity of phosphate than citrate and tartrate to prevent DNA adsorption on soil clays and goethite could be due to more adsorptive sites occupied by phosphate on adsorbents which are common for DNA molecules. Franchi et al. [29] also suggested that DNA bases and sugars do not participate directly in the binding of polynucleotides on montmorillonite and kaolinite, indicating that the negatively charged phosphate group of DNA was the best candidate for interactions with clay surfaces. The adsorption affinity and the competitiveness of organic anions were related to the stability constant (an equilibrium con-
Fig. 4. Adsorption of DNA on soil colloids and minerals as affected by the addition order of phosphate and DNA: (䊉) DNA/phosphate; () phosphate/DNA; () phosphate + DNA.
58
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
stant that express the propensity of a substance to form from its component parts, Ks ) of some Al-organic complexes. The larger the stability constant, the stronger the organic anion adsorption adsorbed [19,30]. Therefore, the higher inhibition capacity of citrate than tartrate was probably related to the greater log Ks value of citrate (7.98) than tartrate (5.61) [31]. According to Huang et al. [17], the steric hindrance of ligand may play an important role in competing with enzyme molecules on soil colloids and minerals. Therefore, the larger surface coverage of citrate on soil components may also account for the higher inhibition capacity of citrate than tartrate. 3.3. Effect of the order of anion addition on DNA adsorption As shown in Figs. 2–4, the adsorption of DNA on soil colloids and minerals was investigated in three cases, i.e., ligands were introduced into the soil colloids and minerals systems simultaneously with DNA (L + DNA) and before the addition of DNA (L/DNA) and after the addition of DNA (DNA/L). Fig. 2 shows that DNA/citrate had the greatest amount of DNA adsorption on organic clay, inorganic clay and goethite. There were no obvious differences on DNA adsorption by organic and inorganic clays between the treatments citrate/DNA and DNA + citrate, while citrate/DNA had the least amount of DNA adsorption in the system of goethite. The amount of DNA adsorbed by kaolinite and montmorillonite was the greatest when citrate was introduced before the addition of DNA, and similar effects were found for the other two treatments. Compared with the addition order of citrate and DNA, similar trends were observed for DNA adsorption on soil colloids and minerals as affected by the addition order of tartrate and DNA (Fig. 3). As shown in Fig. 4, in the systems of organic clay, inorganic clay and goethite systems, DNA/phosphate exhibited the greatest amount of DNA adsorption, and equal amount of DNA adsorption was found between phosphate/DNA and DNA + phosphate. On the contrary, greater amount of DNA was adsorbed by kaolinite with the treatment of phosphate/DNA than the other treatments. DNA adsorbed by montmorillonite for the three treatments followed the order of phosphate/DNA > DNA/phosphate > DNA + phosphate. According to the above results, we can conclude that in soil colloids and goethite systems, DNA/L usually had the greatest amount of DNA adsorption, and L/DNA showed the least amount of DNA adsorption on goethite especially when citrate and tartrate were added, suggesting that organic ligands and phosphate have strong competitions with DNA molecules for the adsorptive sites on soil colloids and goethite. However, as for montmorillonite and kaolinite, the amount of DNA adsorbed was greater in L/DNA as compared to DNA/L and DNA + L in the presence of organic acids and phosphate. The possible explanation for the phenomenon could be that the adsorbed ligands or the soluble Al may act as bridges between mineral surfaces and DNA molecules, which enhances DNA adsorption. These results indicate that the sequence of ligands and DNA release into soil systems have different influences on the adsorption of DNA on surfaces of various soil colloidal particles.
4. Conclusions The presence of high concentration (higher than 5 mM) of ligands promoted DNA adsorption on montmorillonite and kaolinite especially when ligands were added before DNA molecules. As for soil colloids and goethite, phosphate showed the most significant inhibition on DNA adsorption, and the ability of citrate in depressing DNA adsorption was higher than that of tartrate. The presence of humic substances in colloidal particles increased the inhibition of organic and inorganic ligands for DNA adsorption. The rhizosphere is a favorable habitat for acid-producing bacteria which usually excrete many biomolecules including DNA. The presence of root exudates at the soil–plant interface as well as inorganic fertilizer (phosphate, sulfate) may strongly influence the adsorption of DNA on soil colloids, and even affect the transformation ability of free and bound DNA in soil ecosystems. The mechanisms for the competitive and promotive effects of various organic and inorganic ligands on the adsorption of DNA on soil components deserve further attention. Acknowledgement The authors are grateful to the National Natural Science Foundation of China for the financial support of the research (Project no. 40271064). References [1] A. Ogram, G.S. Sayler, D. Gustin, R.J. Lewis, Environ. Sci. Technol. 22 (1988) 982. [2] M. Khanna, G. Stotzky, Appl. Environ. Microbiol. 58 (1992) 1930. [3] E. Gallori, M. Bazzicalupo, D.L. Canto, R. Fani, P. Nannipieri, C. Vettori, G. Stotzky, FEMS Microbiol. Ecol. 15 (1994) 119. [4] S.A.E. Blum, M.G. Lorenz, W. Wackernagel, Syst. Appl. Microbiol. 20 (1997) 513. [5] C. Crecchio, G. Stotzky, Soil Biol. Biochem. 30 (1998) 1061. [6] G. Stotzky, J. Environ. Qual. 29 (2000) 691. [7] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Pedosphere 15 (2005) 16. [8] C.A. Haynes, W. Norde, Colloids Surf. B 2 (1994) 517. [9] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Soil Biol. Biochem. 38 (2006) 471. [10] M. Khanna, M. Yoder, L. Calamai, G. Stotzky, J. Soil Sci. 3 (1998) 1. [11] G. Romanowski, M.G. Lorenz, W. Wackernagel, Appl. Environ. Microbiol. 57 (1991) 1057. [12] G. Pietramellara, M. Franchi, E. Gallori, P. Nannipieri, Biol. Fertil. Soils 33 (2001) 402. [13] D.L. Jone, Plant Soil 205 (1998) 25. [14] B.W. Stobel, Geoderma 99 (2001) 169. [15] R.K. Xu, C.B. Li, G.L. Ji, J. Colloid Interface Sci. 277 (2004) 243. [16] R.K. Xu, A.Z. Zhao, G.L. Ji, J. Colloid Interface Sci. 264 (2003) 322. [17] Q.Y. Huang, Z.H. Zhao, W.L. Chen, Chemosphere 52 (2003) 571. [18] A. Violante, L. Gianfreda, Soil Sci. Soc. Am. J. 57 (1993) 1235. [19] J.J. Dynes, P.M. Huang, Soil Sci. Soc. Am. J. 61 (1997) 772. [20] J.S. Geelhoed, T. Hiemstra, W.H. Van Riemsdijk, Environ. Sci. Technol. 32 (1998) 2119. [21] F. Liu, J.Z. He, C. Colombo, A. Violante, Soil Sci. 1643 (1999) 180. [22] H.Q. Hu, J.Z. He, X.Y. Li, F. Liu, Environ. Int. 26 (2001) 353. [23] A. Naidja, P.M. Huang, J. Mol. Catal. A: Chem. 106 (1995) 255. [24] D.B. Word, P.V. Brady, Clay Miner. 46 (1998) 453. [25] R.E. Blake, L.M. Walter, Geochimica 63 (1999) 2043.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59 [26] J.Y. Li, R.K. Xu, S.C. Xiao, G.L. Ji, J. Colloid Interface Sci. 284 (2005) 393. [27] D.O. Jordan, The physical properties of nucleic acids, in: E. Chargaff, J.N. Davidson (Eds.), The Nucleic Acids, Academic Press, New York, 1955. [28] E. Paget, P. Simonet, FEMS Microbiol. Ecol. 15 (1994) 109.
59
[29] M. Franchi, J.P. Ferris, E. Gallori, Origins Life Evol. B 33 (2003) 1. [30] H. Wijinjia, C.P. Schulthess, Soil Sci. Soc. Am. J. 64 (2000) 898. [31] T.R. Fox, N.B. Comerford, W.W. Mcfec, Soil Sci. Soc. Am. J. 54 (1990) 1763. [32] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1985.
Effects of low-molecular-weight organic ligands and phosphate on DNA adsorption by soil colloids and minerals Peng Cai, Qiaoyun Huang ∗ , Jun Zhu, Daihua Jiang, Xueyong Zhou, Xingmin Rong, Wei Liang Key Laboratory of Agricultural Resources and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agriculture University, Wuhan 430070, China Received 27 April 2006; received in revised form 19 July 2006; accepted 20 July 2006 Available online 27 July 2006
Abstract Adsorption of DNA on montmorillonite, kaolinite, goethite and soil clays from an Alfisol in the presence of citrate, tartrate and phosphate was studied. A marked decrease in DNA adsorption was observed on montmorillonite and kaolinite with increasing anion concentrations from 0 to 5 mM. However, the amount of DNA adsorbed by montmorillonite and kaolinite was enhanced when ligand concentration was higher than 5 mM. In the system of soil colloids and goethite, with the increase of anion concentrations, a steady decrease was found and the ability of ligands in depressing DNA adsorption followed the sequence: phosphate > citrate > tartrate. Compared to H2 O2 -treated clays (inorganic clays), a sharp decrease in DNA adsorption was observed on goethite and organo-mineral complexes (organic clays) with increasing ligand concentrations. The results suggest that the influence of anions on DNA adsorption varies with the type and concentration of anion as well as the surface properties of soil components. Introduction of DNA into the system before the addition of ligands had the greatest amount of DNA adsorption on soil colloids and goethite. Organic and inorganic ligands promoted DNA adsorption on montmorillonite and kaolinite when ligands were introduced into the system before the addition of DNA. The results obtained in this study have important implications for the understanding of the persistence and fate of DNA in soil environments especially rhizosphere soil where various organic and inorganic ligands are active. © 2006 Elsevier B.V. All rights reserved. Keywords: Soil colloid; DNA; Organic ligand; Phosphate; Adsorption
1. Introduction DNA is the genetic material of various organisms. In soil environments, DNA molecules are liberated by excretion from microorganisms, plants and animals or by lysis of dying cells [1]. A proportion of the extracellular DNA molecules in soil is soluble in the aqueous phase and a proportion is physically and chemically adsorbed to soil active particles [2,3]. The adsorbed DNA molecules are partially protected against degradation by nucleases and retain the capacity to transform competent bacterial cells [4–7]. As a result, the adsorption of DNA on soil components plays a vital role in controlling the ultimate fate of extracellular DNA in soils. DNA molecules can be adsorbed on soil colloids and minerals through electrostatic interaction, non-electrostatic forces
∗
Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670. E-mail address: [email protected] (Q. Huang).
0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.07.013
such as hydrophobic ones, ligand exchange, hydrogen bonding and van der Waals forces [8,9]. An increase in the concentration of cations and/or a decrease in the pH favored DNA adsorption on soil colloids and clay minerals [6,9,10]. The amount of supercoiled plasmid DNA adsorbed by sand was slightly less than that of linearized or open circular plasmid DNA [11]. The lower molecular mass DNA adsorbed and bound by Ca-montmorillonite and Ca-kaolinite showed higher adsorption capacity than the higher molecular mass DNA [12]. Organic matter played an inhibitory role in DNA adsorption on permanentcharge soil colloids. Montmorillonite clay dominates the amount of DNA adsorption and kaolinite probably plays a key role in the extent of DNA binding in permanent-charge soil [9]. Low-molecular-weight (LMW) organic acids secreted from plant roots and produced through the decomposition of organic material residues occur widely in soils, especially in the immediate zone of the soil–root interface [13,14]. The concentration of LMW organic acids in rhizosphere could be as high as 10–20 mmol L−1 [13]. Nutrients (phosphate, sulfate) and LMW
54
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
Table 1 Selected properties of soil colloids and minerals studied Soil colloid or mineral
O.M. (g kg−1 )
SESA (m2 g−1 )
PZC
CEC (cmol kg−1 )
Clay mineral composition
Organic clay Inorganic clay Goethite Montmorillonite Kaolinite
71.0 9.6 – – –
64.4 77.1 83.0 73.6 22.9
2.1 3.3 8.7 2.5 3.6
57.6 47.9 0 90.2 7.1
Hydromica (55%), vermiculite (30%), kaolinite (15%) Hydromica (55%), vermiculite (30%), kaolinite (15%) Goethite Montmorillonite Kaolinite
Clay mineral was determined by X-ray diffraction analysis. Organic matter (O.M.), point of zero charge (PZC), cation-exchange capacity (CEC) and specific external surface area (SESA) were analyzed by K2 Cr2 O7 digestion, Mehlich, NH4 AcO method [32] and N2 adsorption method (Beijing Analytical Instrument Company), respectively.
organic ligands can be adsorbed by soil colloids and minerals and resulted in changes in surface properties of soil components [15,16]. Therefore, various organic and inorganic ligands such as citric and tartaric acids and phosphate, which are relatively abundant in soil, may exert significant effects on the adsorption of DNA by soil components. Pietramellara et al. [12] reported that DNA adsorption on Ca-montmorillonite and Ca-kaolinite was reduced markedly in the presence of sodium metaphosphate, indicating a strong competition between phosphate groups and DNA molecules on the mineral surface. In our previous study, it was found that a percentage of 20–40% of adsorbed DNA on soil colloids from an Alfisol was still released by sodium phosphate buffer after extensive washes with Tris buffer and NaCl [9]. However, little information is available regarding the effect of organic ligands on DNA adsorption. The aim of the present work was to investigate and compare the effects of selected LMW organic acids and phosphate on the adsorption of DNA on soil clays, montmorillonite, kaolinite and goethite which are common minerals in soils. 2. Materials and methods 2.1. DNA Salmon sperm DNA was bought from Sigma Chemical Co., St. Louis, MO. 2.2. Soil colloids and minerals A clay loamy Brown Soil (Alfisol) was sampled from the 0–17 cm layer of a forest land in Tianwai village, Taishan, Shandong province, China. Pertinent characteristics of the soil include: water pH 6.3, organic matter 42.4 g kg−1 and cationexchange capacity of 15.3 cmol kg−1 . After removal of organic residue the soil was rinsed in deionized distilled water (ddH2 O) and dispersed by adding 0.01 M NaOH solution dropwise together with sonication. The <2 m clay fraction of the soil was separated by sedimentation. The two treatments applied to clays were organic matter left on the sample (organic clay) and organic matter removed from the sample by H2 O2 (inorganic clay). After flocculation by the addition of CaCl2 solution, the colloidal suspension was washed to be free of Cl− ions by ddH2 O and ethanol, and then air-dried. Some properties of the clay fractions are listed in Table 1.
Fig. 1. Adsorption of DNA on soil colloids and minerals in the presence of increasing concentration of organic acids and phosphate.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
Kaolinite and montmorillonite were prepared according to Cai et al. [9]. Goethite was synthesized as described previously by Huang et al. [17]. All the soil colloids and minerals prepared were ground to pass a 100 mesh sieve. 2.3. DNA adsorption experiment In a centrifuge tube, 2.1 ml of organic and inorganic ligands and 0.4 ml of 10 mM Tris buffer (pH 7.0) containing 200 g DNA were mixed to yield final ligand concentrations ranging from 0 to 80 mM for tartrate and citrate or 0–200 mM for phosphate. After that, 10 mg of soil colloid or mineral was added to the tube. The mixture was gently shaken at 25 ◦ C for 2 h and centrifuged at 20,000 × g for 20 min. DNA in the supernatant was determined directly at 260 nm spectrophotometrically. The amount of DNA adsorbed was calculated by the difference between the amount of DNA added and that remaining in the supernatant. The experiment was also designed to investigate the influence of the addition order of ligands and DNA on the adsorption of DNA on soil colloids and minerals. (1) Introduction of DNA after LMW organic anions or phosphate adsorption. Ten mil-
55
ligrams of soil colloid or mineral and 2.1 ml anionic solution containing 5–50 mM of citrate or tartrate or 5–100 mM of phosphate were added into the centrifuge tube and shaken for 1 h at 25 ◦ C. Four hundred microlitres of 10 mM Tris buffer (pH 7.0) containing 200 g DNA was added for another 1 h. The mixture was centrifuged and the content of DNA in the supernatant was analyzed. (2) Introduction of LMW organic acids or phosphate after DNA adsorption. Four hundred microlitres of 10 mM Tris buffer (pH 7.0) containing 200 g DNA and 10 mg soil colloid or mineral was mixed and shaken for 1 h at 25 ◦ C. After the period 2.1 ml of anionic solution containing 5–50 mM of citrate or tartrate or 5–100 mM of phosphate was then added. The suspension was shaken for an additional 1 h and DNA in the supernatant was analyzed. 3. Results and discussion 3.1. Effect of ligand concentrations on DNA adsorption Fig. 1 shows the adsorption of DNA by soil colloids and minerals in the presence of organic acids and phosphate. The amount of DNA adsorbed by montmorillonite and kaolinite decreased
Fig. 2. Adsorption of DNA on soil colloids and minerals as affected by the addition order of citrate and DNA: (䊉) DNA/citrate; () citrate/DNA; () citrate + DNA.
56
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
with increasing tartrate, citrate and phosphate concentrations from 0 to 5 mM, whereas an increase for DNA adsorption was found in the range of 5–80 mM tartrate and citrate and 5–200 mM phosphate. A steady decrease in DNA adsorption was observed on soil colloids and goethite with the increase of organic ligand and phosphate concentrations. The inhibition of DNA adsorption by anions was mainly due to the competition of tartrate, citrate and phosphate with DNA for adsorption sites of soil clays and minerals surfaces. This agrees with the commonly observed competition of these high affinity organic and inorganic ligands with the adsorption of inorganic oxyanions [18–22] and enzyme [16,23] as well as chromosomal and plasmid DNA molecules [12]. The promotive effect of high concentrations of ligands on the adsorption of DNA on kaolinite and montmorillonite could likely be explained by dissolution reactions in the presence of organic ligands which create new sites on the surfaces of clay minerals and the formation of precipitates (Al-phosphate) which may facilitate DNA adsorption. LMW organic acids could accelerate aluminum dissolution from kaolinite, feldspar and aluminum oxides through proton-promoted and ligand enhanced
effects [24–26]. The soluble Al3+ forming bridges between phosphate groups of DNA and the negatively charged sites of minerals may be also responsible for the higher DNA adsorption on kaolinite and montmorillonite at higher ligand concentrations. The above finding suggests that montmorillonite and kaolinite play a more important role in the adsorption of DNA molecules in rhizosphere soil where higher concentrations of various ligands are present. 3.2. Effect of ligand types on DNA adsorption As shown in Fig. 1, the examined organic ligands and phosphate present different inhibiting effects on DNA adsorption by soil colloids and minerals. In order to evaluate the depressing ability of organic ligands and phosphate, the percentage efficiency of the ligand was calculated according to the following expression: efficiency of the ligand (%) = 1 − (DNA adsorbed in the presence of ligand/DNA adsorbed when applied alone). The efficiency of three ligands was similar for decreasing DNA adsorption on montmorillonite and kaolinite at lower anion concentrations. In organic clay system, with the increase of
Fig. 3. Adsorption of DNA on soil colloids and minerals as affected by the addition order of tartrate and DNA: (䊉) DNA/tartrate; () tartrate/DNA; () tartrate + DNA.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
anion concentration from 5 to 50 mM, the efficiency of ligands increased from 40 to 89, 33 to 56, and 37 to 53% for phosphate, citrate and tartrate, respectively. The efficiency of anions for reducing DNA adsorption on inorganic clay was 16–29% for phosphate, 12–22% for citrate and 8–18% for tartrate. In the system of goethite, the efficiency of phosphate increased from 41 to 77% and that for citrate was from 25 to 55% and that for tartrate was from 6 to 14%. The above results indicate that the ability of ligands in depressing DNA adsorption by soil colloids and goethite was in the order of phosphate > citrate > tartrate. In goethite system, the higher inhibiting efficiency of phosphate suggests that there are large amounts of adsorptive sites on goethite for ligand exchange and these sites are common for phosphate and DNA molecules. The values of point of zero charge (PZC) of organic clay and inorganic clay were 2.1 and 3.3, respectively, and the cation-exchange capacity (CEC) value of organic clay was higher than that of inorganic clay (Table 1). The isoelectric point of DNA is about 5.0 [27]. DNA molecules are negatively charged due to phosphate groups when pH is above the isoelectric point [28]. Therefore, there is a stronger electro-
57
static repulsion force between the negatively charged DNA and organic clay at pH 7.0. It was reported by Xu et al. [16] that the presence of organic anions led to an increase in negative charges and a decrease in positive charges of soils. Therefore, the higher inhibition of DNA adsorption by ligands on organic clay than that on inorganic clay may be ascribed to the greater amount of negative charges on organic clay surfaces than those on inorganic clay with the increase of ligand concentrations which result in stronger electrostatic repulsion forces between DNA and organic clay. The greater capacity of phosphate than citrate and tartrate to prevent DNA adsorption on soil clays and goethite could be due to more adsorptive sites occupied by phosphate on adsorbents which are common for DNA molecules. Franchi et al. [29] also suggested that DNA bases and sugars do not participate directly in the binding of polynucleotides on montmorillonite and kaolinite, indicating that the negatively charged phosphate group of DNA was the best candidate for interactions with clay surfaces. The adsorption affinity and the competitiveness of organic anions were related to the stability constant (an equilibrium con-
Fig. 4. Adsorption of DNA on soil colloids and minerals as affected by the addition order of phosphate and DNA: (䊉) DNA/phosphate; () phosphate/DNA; () phosphate + DNA.
58
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59
stant that express the propensity of a substance to form from its component parts, Ks ) of some Al-organic complexes. The larger the stability constant, the stronger the organic anion adsorption adsorbed [19,30]. Therefore, the higher inhibition capacity of citrate than tartrate was probably related to the greater log Ks value of citrate (7.98) than tartrate (5.61) [31]. According to Huang et al. [17], the steric hindrance of ligand may play an important role in competing with enzyme molecules on soil colloids and minerals. Therefore, the larger surface coverage of citrate on soil components may also account for the higher inhibition capacity of citrate than tartrate. 3.3. Effect of the order of anion addition on DNA adsorption As shown in Figs. 2–4, the adsorption of DNA on soil colloids and minerals was investigated in three cases, i.e., ligands were introduced into the soil colloids and minerals systems simultaneously with DNA (L + DNA) and before the addition of DNA (L/DNA) and after the addition of DNA (DNA/L). Fig. 2 shows that DNA/citrate had the greatest amount of DNA adsorption on organic clay, inorganic clay and goethite. There were no obvious differences on DNA adsorption by organic and inorganic clays between the treatments citrate/DNA and DNA + citrate, while citrate/DNA had the least amount of DNA adsorption in the system of goethite. The amount of DNA adsorbed by kaolinite and montmorillonite was the greatest when citrate was introduced before the addition of DNA, and similar effects were found for the other two treatments. Compared with the addition order of citrate and DNA, similar trends were observed for DNA adsorption on soil colloids and minerals as affected by the addition order of tartrate and DNA (Fig. 3). As shown in Fig. 4, in the systems of organic clay, inorganic clay and goethite systems, DNA/phosphate exhibited the greatest amount of DNA adsorption, and equal amount of DNA adsorption was found between phosphate/DNA and DNA + phosphate. On the contrary, greater amount of DNA was adsorbed by kaolinite with the treatment of phosphate/DNA than the other treatments. DNA adsorbed by montmorillonite for the three treatments followed the order of phosphate/DNA > DNA/phosphate > DNA + phosphate. According to the above results, we can conclude that in soil colloids and goethite systems, DNA/L usually had the greatest amount of DNA adsorption, and L/DNA showed the least amount of DNA adsorption on goethite especially when citrate and tartrate were added, suggesting that organic ligands and phosphate have strong competitions with DNA molecules for the adsorptive sites on soil colloids and goethite. However, as for montmorillonite and kaolinite, the amount of DNA adsorbed was greater in L/DNA as compared to DNA/L and DNA + L in the presence of organic acids and phosphate. The possible explanation for the phenomenon could be that the adsorbed ligands or the soluble Al may act as bridges between mineral surfaces and DNA molecules, which enhances DNA adsorption. These results indicate that the sequence of ligands and DNA release into soil systems have different influences on the adsorption of DNA on surfaces of various soil colloidal particles.
4. Conclusions The presence of high concentration (higher than 5 mM) of ligands promoted DNA adsorption on montmorillonite and kaolinite especially when ligands were added before DNA molecules. As for soil colloids and goethite, phosphate showed the most significant inhibition on DNA adsorption, and the ability of citrate in depressing DNA adsorption was higher than that of tartrate. The presence of humic substances in colloidal particles increased the inhibition of organic and inorganic ligands for DNA adsorption. The rhizosphere is a favorable habitat for acid-producing bacteria which usually excrete many biomolecules including DNA. The presence of root exudates at the soil–plant interface as well as inorganic fertilizer (phosphate, sulfate) may strongly influence the adsorption of DNA on soil colloids, and even affect the transformation ability of free and bound DNA in soil ecosystems. The mechanisms for the competitive and promotive effects of various organic and inorganic ligands on the adsorption of DNA on soil components deserve further attention. Acknowledgement The authors are grateful to the National Natural Science Foundation of China for the financial support of the research (Project no. 40271064). References [1] A. Ogram, G.S. Sayler, D. Gustin, R.J. Lewis, Environ. Sci. Technol. 22 (1988) 982. [2] M. Khanna, G. Stotzky, Appl. Environ. Microbiol. 58 (1992) 1930. [3] E. Gallori, M. Bazzicalupo, D.L. Canto, R. Fani, P. Nannipieri, C. Vettori, G. Stotzky, FEMS Microbiol. Ecol. 15 (1994) 119. [4] S.A.E. Blum, M.G. Lorenz, W. Wackernagel, Syst. Appl. Microbiol. 20 (1997) 513. [5] C. Crecchio, G. Stotzky, Soil Biol. Biochem. 30 (1998) 1061. [6] G. Stotzky, J. Environ. Qual. 29 (2000) 691. [7] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Pedosphere 15 (2005) 16. [8] C.A. Haynes, W. Norde, Colloids Surf. B 2 (1994) 517. [9] P. Cai, Q.Y. Huang, X.W. Zhang, H. Chen, Soil Biol. Biochem. 38 (2006) 471. [10] M. Khanna, M. Yoder, L. Calamai, G. Stotzky, J. Soil Sci. 3 (1998) 1. [11] G. Romanowski, M.G. Lorenz, W. Wackernagel, Appl. Environ. Microbiol. 57 (1991) 1057. [12] G. Pietramellara, M. Franchi, E. Gallori, P. Nannipieri, Biol. Fertil. Soils 33 (2001) 402. [13] D.L. Jone, Plant Soil 205 (1998) 25. [14] B.W. Stobel, Geoderma 99 (2001) 169. [15] R.K. Xu, C.B. Li, G.L. Ji, J. Colloid Interface Sci. 277 (2004) 243. [16] R.K. Xu, A.Z. Zhao, G.L. Ji, J. Colloid Interface Sci. 264 (2003) 322. [17] Q.Y. Huang, Z.H. Zhao, W.L. Chen, Chemosphere 52 (2003) 571. [18] A. Violante, L. Gianfreda, Soil Sci. Soc. Am. J. 57 (1993) 1235. [19] J.J. Dynes, P.M. Huang, Soil Sci. Soc. Am. J. 61 (1997) 772. [20] J.S. Geelhoed, T. Hiemstra, W.H. Van Riemsdijk, Environ. Sci. Technol. 32 (1998) 2119. [21] F. Liu, J.Z. He, C. Colombo, A. Violante, Soil Sci. 1643 (1999) 180. [22] H.Q. Hu, J.Z. He, X.Y. Li, F. Liu, Environ. Int. 26 (2001) 353. [23] A. Naidja, P.M. Huang, J. Mol. Catal. A: Chem. 106 (1995) 255. [24] D.B. Word, P.V. Brady, Clay Miner. 46 (1998) 453. [25] R.E. Blake, L.M. Walter, Geochimica 63 (1999) 2043.
P. Cai et al. / Colloids and Surfaces B: Biointerfaces 54 (2007) 53–59 [26] J.Y. Li, R.K. Xu, S.C. Xiao, G.L. Ji, J. Colloid Interface Sci. 284 (2005) 393. [27] D.O. Jordan, The physical properties of nucleic acids, in: E. Chargaff, J.N. Davidson (Eds.), The Nucleic Acids, Academic Press, New York, 1955. [28] E. Paget, P. Simonet, FEMS Microbiol. Ecol. 15 (1994) 109.
59
[29] M. Franchi, J.P. Ferris, E. Gallori, Origins Life Evol. B 33 (2003) 1. [30] H. Wijinjia, C.P. Schulthess, Soil Sci. Soc. Am. J. 64 (2000) 898. [31] T.R. Fox, N.B. Comerford, W.W. Mcfec, Soil Sci. Soc. Am. J. 54 (1990) 1763. [32] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1985.