Atomic force microscopic (AFM) images of humic acids show discrete, globular particles, where particles of the order of magnitude 100 to 300 nm dominate the image fields; the humic acids had been grown to a steady state at pH 5.0. The AFM data are consistent with our previously reported small-angle neutron scattering (SANS) study done under similar conditions. In further agreement, the cluster-cluster interactions shown in our previous SANS study may have their counterparts in closely interacting particles appearing as twin particles in the AFM images

Ikai, A.

Osterberg, R.

English

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Scanning Microscopy Volume 10 Number 4 Article 3 10-31-1996 Atomic Force Microscopy of Humic Acids A. Ikai Tokyo Institute of Technology R. Osterberg Swedish University of Agricultural Sciences Follow this and additional works at: https://digitalcommons.usu.edu/microscopy  Part of the Biology Commons Recommended Citation Ikai, A. and Osterberg, R. (1996) "Atomic Force Microscopy of Humic Acids," Scanning Microscopy: Vol. 10 : No. 4 , Article 3. Available at: https://digitalcommons.usu.edu/microscopy/vol10/iss4/3 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Scanning Microscopy Vol. 10, No. 4, 1996 (Pages 947-951) 0891-7035/96$5.00+ .25 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA ATOMIC FORCE MICROSCOPY OF HUMIC ACIDS A. Ikai and R. Osterberg 1•• Laboratory of Biodynamics, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama, Japan 1Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden (Received for publication July 5, 1995 and in revis ed form October 31, 1996) Abstract Atomic force microscopic (AFM) images of humic acids show discrete , globular particl es, where particles of the order of magnitude 100 to 300 nm dominate the image fields; the humic acids had been grown to a steady state at pH 5.0. The AFM data are consistent with our previously reported small-angle neutron scatter-ing (SANS) study done under similar conditions . In fur-ther agreement, the cluster-cluster interactions shown in our previous SANS study may have their counterparts in closely interacting particles appearing as twin particles in the AFM images . Key Words: Atomic force microscopy, hurnic acids, particl es, size, cluster-clu ster interaction . • Address for correspondence : Ragnar Osterberg Department of Chemistry, Swedish University of Agricultural Sciences Box 7015, S-750 07 Uppsala, Sweden Telephone number : 48-18-671544 FAX number: 48-18-673476 947 Introduction Humic acids, which form the important interphase between the biosphere and inorganic material, play a key role in the food chain by being mainly responsible for the release of nutrients to the plant kingdom {see, for example, the reviews by Kononova (1966) and Hayes et al. (1989b)}. Numerous attempts have been made to analyze the structure of humic acids but, so far , there is only a limited amount of knowledge about their size and shape (cf. Hayes et al., 1989a) as well as about their molecular structure (cf . Schulten and Schnitzer, 1993). Previously, we reported that small-angle neutron scat-tering (SANS) from humic acid solutions obey a power law, revealing that the particles in solution have a fractal structur e with a fractal dimension (ill of 2.35 ± 0.05. The particl e size varied within the range 100-320 nm, depending on the source and method of preparation (Osterberg et al., 1993, 1994, 1995; Osterberg and Mort ensen, 1992, 1994a). Titrations indicated that the aggregation pro cess of humic acids involves both protons and electrons (Osterberg et al., 1995; Wahlberg, per-sonal communi cation), although the detailed mechanism of aggregation is unknown. Current models such as Witt en-Sanders' diffusion limited aggregation ill = 2.5) and cluster-clu ster aggregation ill = 1. 78), apply only under certain conditions, for instance , at low temper-ature ~ 9°C (Osterberg et al., 1994). The humic acid system is kinetic in the sense that it is not at a thermo-dynamic equilibrium, and the "end" stage of the poly-merization process varies due to the source and method of preparation (Osterberg and Mortensen, 1994a). For example, humic acids prepared by a gentle method from brown soil yield a steady-state, where large particles remain in the solution even after storage for several months. In addition, we observe interactions of large clusters and gel formation (Osterberg and Mortensen, 1994a) . In an attempt to further analyze cluster size and cluster-cluster interactions, we have now studied humic acids with an independent method: atomic force micros-copy (AFM). This method has proven to be successful when applied to various biological specimens, such as A. lkai and R. Osterberg cell surfaces (Bonfiglio et al., 1992), DNA (Yang et al., 1992) and bacteriophages (lkai et al., 1993); for a review, see Hansma and Hoh (1995). Here, we report direct observation of humic acid particles using the AFM method; the results are in good agreement with our earlier SANS studies (Osterberg and Mortensen, 1992, 1994a,b; Osterberg et al., 1995). Materials and Methods Hwnic acids Humic acids were prepared from Al horizon, brown soil taken from a meadow outside Uppsala, Sweden (cf. Osterberg et al., 1994). The soil was extracted with 0.05 M pyrophosphate buffer pH 7.0 (Buffer A) in an argon atmosphere at 30 ± 0.5°C. After centrifugation, the supernatant was treated with cold 6 M HCl on an ice bath until the humic acid precipitated. The precipitate was recovered via centrifugation at 4°C and then dis-solved in Buffer A. After adjusting the resulting solu-tion to pH 7 and separating a minor precipitate via cen-trifugation, the procedure involving precipitation with HCl followed by dissolution in Buffer A was repeated thrice. The final humic acid sample obtained was stored in Buffer A at -80°C; elementary analysis gave a C/N molar ratio of 11.5. The elementary analysis of C was also used for the determination of the humic acid con-centration and then the concentration of C in dry humus was set to 50% (cf. Lindqvist, 1982). In order to remove metal ions and to minimize the degree of polymerization, the sample ofhumic acids was dialyzed first against ethylenediaminetetra -acetate at pH 7 .0 and then against bidistilled water. Finally, the sample was dialyzed against an acetate buffer of pH 5.0 with 0.1 M ionic strength (NaCl) . Before subjecting the sample to AFM, it was diluted 1: 1000 with bidistilled water. Atomic force microscopy A Nanoscope III (Digital Instruments, Santa Bar-bara, CA) was used in Tapping (Digital Instruments registered) AFM mode and a 3700 scanning probe microscope (Seiko Instruments Co., Tokyo, Japan) in contact AFM mode was used in air at 20 to 23 °C with a relative humidity of 40 to 50 % . Pyramidal shaped commercial tips made of silicon nitride were used. About 5 µ.l of diluted humic acid solution (x 1/1000) was placed on a freshly cleaved graphite surface, then dried under reduced pressure and finally subjected to AFM measurements within 60 minutes. Dilution was repeated when the particles of the dried sample were found to be too crowded for the visual inspection of AFM images. 948 Results Figure 1 shows a stepwise magnification of the diluted sample from 1 to 500 in area size illustrating humic acid particles. Please note that, even at low mag-nification, discrete particles are clearly shown, although the particles tend to adhere to each other. The sizes of the humic acid particles observed by AFM show a sig-nificant distribution, although particles of the order of magnitude 100 to 300 nm dominate the image fields. It should be noted that the particles sizes shown by Figures 1 and 2 are those observed after changes have occurred due to evaporation and deformation. It is the high water content of the particles (according to the fractal dimen-sion 2.35: > 90 volume% water), which results in an instability that leads to the deformation of the particles shown in Figures 1B and 1 C, and to the relatively small height shown in Figure ID. Also, the moving of the AFM tip over the particles may increase the flattening and deformation. As a result, the dimensions of the particles, except the height, have probably been some-what smaller in solution. We also believe that most of the particles have had an essentially spherical shape in solution, which is common for fractal particles formed via random growth processes. This is probably also true for the single particle displayed in Figure 1D, which has the dimensions 230 nm x 260 nm x 50 nm. Using SANS data recorded from similar samples of humic acids with a fractal dimension of 2. 35 ( cf. Osterberg and Mortensen 1992, 1994a; Osterberg et al., 1995), we may estimate the order of magnitude for the molecular mass of this particle to be 5,000 kDa. Then it is assumed that in solution a corresponding spherical parti-cle exists and that its volume is essentially the same as that of the particle in Figure ID. The calculation was done using the procedure and parameters described in Osterberg et al. (1993). As shown by Figure 2, AFM reveals the interaction of large clusters. Of particular interest is the group of three particles in Figure 2A, where two of the particles appear to be involved in cluster-cluster interactions with clusters of about the same size. One of these "twin" particles is displayed in Figure 2B. It is shown, especially for the solid image (on the right hand side of Fig. 2B) that the two clusters penetrate into the inner structures of each other. Since these two particles ap-pear to be "squeezed" into each other rather than inter-acting with their outer surface, it seems likely that this interaction is not an artifact of evaporation but the result of interactions occurring in solution. Likewise, this solid image of Figure 2B reveals protrusions and inden-tations which are in agreement with the idea that the par-ticles grow via the interactions of relatively large parti-cles. However, caution should be exercised before any Figure 1. Atomic force microscopic images of humic acids (from a solutions of pH S diluted 1: 1000 with distilled water) dried on a graphite plate. Imaging areas: (A) 10 µm x 10 µm, (B) 4 µm x 4 µm, (C) 650 nm x 650 nm and (D) 448 nm x 448 nm. The linear "structures" seen at the lower left of Figure IA are artifacts which arose during the drying procedure. AFM of humic acids Figure 2. AFM images of humic acids showing cluster-cluster interactions. (A) Imaging area 1680 nm x 1680 nm. (B) Left. Image of single particle involved in cluster-cluster interaction on the area 608 nm x 608 nm. (B) Right. Solid height image of the particle on the left with the same magnification. 949 A. lkai and R. Osterberg final conclusions are drawn about solution cluster-cluster interactions based on a study in the dry state. Discussion The previous sections have furnished support for the idea that dilute solutions of humic acids of pH 5, and grown to a steady state, involve discrete clusters, where particles of the order of 100 to 300 nm dominate the image fields (Fig. 1). The present AFM images also yield an excellent resolution of the humic acid particles (Figs. 1 and 2). Similar images of discrete particles, but with less resolution, have recently been obtained by transmission electron microscopy (TEM) (Drozd, 1986; Osterberg and Drozd, unpublished results). It should be kept in mind that the soil source and the method of prep-aration might influence the particle size; for instance, harsher methods involving sodium hydroxide extractions appear to yield smaller particles (Osterberg and Morten-son, 1992; Osterberg et al., 1993). This might be one reason that early studies using TEM and scanning elec-tron microscopy under somewhat different conditions, showed smaller, less defined particles, 9-50 nm; how-ever, their images also showed aggregates and conglom-erates expanding into filaments or/and network (Chen and Schnitzer, 1976; 1989; Stevenson and Schnitzer, 1982; cf. Schnitzer and Kodama, 1975) . These latter structures do not seem to have their counterparts among solution studies using SANS (Osterberg and Mortenson , 1992, 1994a,b; Osterberg et al., 1995). The present AFM images, on the other hand, are in good agreement with these SANS studies: both AFM and SANS indicate that humic acid solutions grown to a steady state involve discrete globular particles. And, particles of the same order of magnitude as those observed by SANS (100-300 nm) appear to dominate the AFM image fields (Fig. 1), although some smaller particles also are shown. It should be noted that SANS strongly emphasizes the larger particles, since it yields the weight average squared. Furthermore, both methods indicate that clus-ter-cluster interactions are involved (see Fig. 2 and Osterberg and Mortenson, 1994a). Finally, from the resolution of the twin humic acid particle (see, e.g ., Fig. 2B) showing protrusion and indentations, it is tempting to speculate that the humic acid clusters grow as a result of cluster-cluster inter-actions involving an ever increasing cluster size. Acknowledgement Financial support from the Swedish Agricultural Sciences Research Council is gratefully acknowledged. 950 References Bonfiglio A, Parodi MT, Tonni GP (1995) Subcellu-lar details of early events of differentiation induced by retinoic acid in human neuroblastoma cells detected by atomic force microscope. Exp Cell Res 216: 73-79. Chen Y, Schnitzer M (1976) Scanning electron microscopy of a humic acid and a fulvic acid and its metal and clay complexes. Soil Sci Am J 40: 682-686. Chen Y, Schnitzer M (1989) Sizes and shapes of humic substances by electron microscopy. In: Humic Substances II, In Search of Structure. Hayes MHB, Maccarthy P, Malcolm RL, Swift R (eds). Wiley, Chicester. pp. 621-638. Drozd J (1986) Application of the transmission elec-tron microscopy in studies of humic acids in muck soils under varying moisture conditions. Roczn Gleb V.34: 55-62 . Hansma HG, Hoh JH (1995) Biomolecular imaging with the atomic force microscope. Ann Rev Biophys Biomol Struct 23: 115-139. Hayes MHB, Maccarthy P, Malcolm RL, Swift R (eds.) (1989a) Humic Substances II, In Search of Structure. Wiley, Chicester. pp. 15-253. Hayes MHB, Maccarthy P, Malcolm RL, Swift, R (1989b) The search for structure: Setting the scene. In: Humic Substances II, In Search of Structure. Hayes MHB, Maccarthy P, Malcolm RL, Swift R (eds.). Wiley, Chicester. pp. 3-31. lkai A, Yoshimura K, Arisaka F, Ritani A, Imai K (1993) Atomic force microscopy of bacteriophage T4 and its tube-baseplate complex. FEBS Lett 326: 39-41. Kononova MM (1966) Soil Organic Matter, Its Nature, Its Role in Soil Formation and Soil Fertility. Pergamon, Oxford. pp . 47-256. Lindqvist L (1982) Charge-transfer interaction of humic acids with donor molecules in aqueous solutions. Swed J Agric Res 12: 105-109. Osterberg R, Mortensen KM (1992) Fractal dimen-sion of humic acids. Eur Biophys J 12: 163-167. Osterberg R, Mortensen KM (1994a) The growth of fractal humic acids : Cluster correlation and gel formation . Radiat Environ Biophys 33: 269-276. Osterberg R, Mortensen KM (1994b) Fractal geom-etry of humic acids. Temperature-dependent restruc-turing studied by small-angle neutron scattering. In: Humic Substances in the Global Environment and Implications on Human Health. Senesi N, Miano TM (eds.) . Elsevier, Amsterdam. pp. 127-132. Osterberg R, Lindqvist L, Mortensen KM (1993) Particle size of humic acids. Soil Sci Soc Am J 57: 283-285 . Osterberg R, Szajdak L, Mortensen KM (1994) Temperature-dependent restructuring of fractal humic AFM of humic acids acids: A proton-dependent process. Environ Int 20: 77-80. Osterberg R, Mortensen KM, Ikai A (1995) Direct observation of humic acid clusters, a non-equilibrium system with a fractal structure. Naturwissenschaften 82: 137-139. Schnitzer M, Kodama H (1975) An electron micro-scopic examination of fulvic acid. Geoderma 13: 279-287. Schulten HR, Schnitzer M (1993) A state of the art structural concept for humic substances. Naturwissen-schaften 80: 29-30. Stevenson IL, Schnitzer M (1982) Transmission electron microscopy of extracted fulvic and humic acids. Soil Sci 133: 179-185. Yang J, Takeyasu K, Shao Z (1992) Atomic force microscopy of DNA molecules. FEBS Lett 301: 173-176. Discussion with Reviewers H. Hansma: What exactly are humic acids? Authors: Humic acids constitute the major organic part of the soil, and aromatic carboxylic acids dominate their composition. Humic acids involve degradation products from the plant kingdom and to a certain extent from the animal kingdom. In the soil, these products undergo aromatization with release of oxygen, and there they are subjected to microbiological activity. Autocatalytic polymerization and aggregation occur in the presence of oxygen, iron(III) or manganese(IV). H. Hansma: What does it mean that titrations indicate that both protons and electron are involved in the aggregation process? Authors: We observe that protons and electrons are consumed in a certain pH range, where it is indicated that aggregation processes occur. The true meaning of this is not known, and we can only speculate that hydro-gen bonds and free radicals are involved. 951 H. Hansma: What is the meaning of particle size in a fractal structure? Authors: A particle has to be sufficiently large in order to yield a fractal structure, although particle size and fractal structure in general do not depend on each other. The pair correlation function which form the basis for the mathematical analysis of fractals is, of course, only valid up to the limit of the particle size. Its Fourier transform, which yields the scattering curve in the form of a power law (indicating scale invariance), should ex-tend over at least one magnitude in order to consider the particles in solution to be fractals. As an example, a fractal unit of 0.5 nm would need a particle which is larger than 5 nm in order to be considered fractal. H. Hansma: Why was graphite used as the substrate, since it is known to show artifacts in, for example, scanning tunneling microscopy (STM) of DNA, instead of mica? Authors: We are aware of the study by Clemmer and Beebe (1991). However, these artifacts are on a much smaller scale than the objects observed in this study. As far as we know graphite does not cause any serious problem when used as substrate for AFM. Additional Reference Clemmer CR, Beebe Jr TP (1991) Graphite: A mimic for DNA and other biomolecules in scanning tunneling microscopy studies. Science 251: 640-643. 

Atomic Force Microscopy of Humic Acids

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Atomic Force Microscopy of Humic Acids

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