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Chemical characterization of starch and starch: lignin films using micro-attenuated total reflectance Fourier Transform infrared spectroscopy (micro-ATR FTIR).


Biodegradable polymeric films are a possible alternative to the synthetic plastics so common in modern life. Most synthetic plastics are known to degrade at incredibly slow rates and are a known contributor to landfill mass. As an alternative to these synthetic plastics, research in the last decade or so has attempted to develop biodegradable polymer films from natural sources, such as starch, pectin, cellulose, and lignin [2,3,4].

In addition to the biodegradable nature of these films, they also offer important benefits to researchers requiring homogenous, smooth biological substrates for study of decomposition patterns in microbes and fungi. These materials represent analogous sub-sets of the more complex chemistry of plant leaves and stems, allowing more detailed investigation of selective enzyme activities on these chosen substrates [5,6]. In our studies, we required such a substrate to investigate the relative enzymatic activities of various fungi on lignin containing substrates. It was important that the substrate was a flat film that could be analyzed using both atomic force microscopy (AFM) and micro-attenuated total reflectance Fourier transform infrared spectroscopy (micro-ATR FTIR).

Fourier transform infrared (FTIR) spectroscopy involves the absorption of infrared radiation by the sample resulting in molecular vibrations (i.e. stretching or bending of infrared active covalent bonds in the mid-IR region of 4000 and 400 [cm.sup.-1]). Each type of molecular vibration absorbs IR radiation at a specific spectral wavelength thereby providing qualitative and quantitative chemical information about the sample. The complex pattern of peaks produced by a sample, its IR spectrum, can then be analyzed to obtain compositional information. Peaks can be correlated with bonds (e.g. C-H stretch vs. O-H stretch), while the fingerprint region (~1500 - 400 [cm.sup.-1]) exhibits a unique signature including many overlapping vibrations from various parts of each distinct molecule in the sample. FTIR-ATR (attenuated total reflectance) microscopy utilizes a microscope in conjunction with a traditional FTIR bench. This enables the infrared spectrum to be obtained from a specific point located under the microscope approximately 10 x 10 microns in size. Spectral information from the surface of the sample is obtained by the ATR sampling method; the sample must be in good contact with the internal reflectance element, generally Ge.

Atomic force microscopy (AFM) is a physical microscopy technique that can be used for measuring surface topography, adhesion, and elasticity. This is accomplished by deflecting a laser off a scanning probe that rapidly taps the surface of the sample and onto a photodiode that detects the surface characteristics [7]. Atomic force microscopy measures these characteristics at the |xm and nm scales with limited height variability. It is this limitation in height that requires the use of flat films as the substrate for the fungal decomposition.

Our proposed study was to evaluate the extent of influence of enzymes produced by an individual fungal hypha growing across defined substrates using a combination of ATR-FTIR and AFM at a resolution of Lim to nm away from the hyphal surface. However, after reproducing starch: lignin films according to the process of Vengal & Srikumar [1] it was found that lignin was not present in the starch: lignin films. Further research indicates that the process of preparing the lignin as a dissolved solute in sodium hydroxide (NaOH) causes alkaline hydrolysis of the molecule. The resulting film, therefore, had no lignin present, as is supported by our ATR-FTIR spectra.


It is imperative that the films produced via the method of Vengal & Srikumar [1] are composed of the materials published, as other researchers depend upon this accuracy for their continuing studies.


The methods of Vengal & Srikumar [1] were followed in preparation of starch: lignin and starch-only films, with exception of the lignin source. Vengal & Srikumar extracted their own lignin from wood, whereas we obtained lignin from Sigma Aldrich. Starch: lignin films were 90% starch, 10% lignin, whereas the starch-only films were 100% starch. Tapioca starch was used to produce the starch solution. The starch: lignin polymer films (90:10) were cast from a solution of 4g of tapioca starch with 75 ml of water at 100[degrees] C until the starch was dissolved. Glycerol (5 drops) was added to the mixture (as a plasticizer) and 10 ml of lignin solution (3g / 75 ml NaOH). The resulting solution was cast on plastic cover slips and allowed to cure. Starch only films were cast in the same manner excluding the addition of the lignin/ NaOH solution.

Following curing, the films were analyzed on an Agilent (formerly Bio-Rad) FTS 6000 infrared spectrophotometer with an attached UMA 500 microscope with a germanium ATR crystal. 256 scans were averaged at a resolution of 4 [cm.sup.-1]? Background was air? As spectra were compared to each other, no ATR correction was applied? Spectra were normalized? Spectra were obtained from 3 replicates of the 90:10, starch: lignin films and 3 replicates of the starch only films. Spectra of pure lignin were also obtained by pressing lignin into a pellet and contacting the pressed lignin pellet against the ATR crystal. The pellet was prepared by grinding lignin in a mortar and pestle then loading a small amount into a KBr pellet press to produce a solid pellet of lignin.


The infrared spectra of the starch: lignin and the starchonly films are nearly identical, see Fig. 1. Both films generated spectra with peaks at 1457, 1151, 1079, 1019, 931, and 852 cm-1. Replicates indicate similarity between samples. Overlaying spectra from the three films (lower right) highlights similarities in peak presence between the starch and the starch: lignin films that contrasts from those of lignin. Table 1 shows the main spectral peaks in the lignin pellet and both the starch: lignin and starch only films. X's indicate the presence of a peak within the spectrum and blank boxes indicate the absence of a peak. Starch only and starch: lignin films share the same peaks, but contrast with lignin in all peaks, with exception of 1079. It can be seen that other than a peak at 1079 [cm.sup.-1], there are no common peaks between the lignin and starch: lignin film. The starch: lignin film has complete congruence of major peaks with the spectrum of starch alone.


The results outlined above and in figure 1 and table 1 indicate that during the preparation of the starch: lignin films, there appears to be a reaction resulting in the loss of lignin from the system. The strong similarities of the starch and starch: lignin spectra, as well as their overwhelming contrast to the lignin spectra clearly indicate that there is no lignin present in the resulting starch: lignin films. According to Miller et al. [8], when exposed to an alkaline solvent lignin depolymerizes. This coincides with the known process of alkaline degradation of polysaccharides as outlined in Kennedy & White [9]. The only spectral peak that is common between the lignin and starch: lignin film is that at 1079 [cm.sup.-1]. This peak at 1079 [cm.sup.-1] is common in many polysaccharides [10], so it would be expected to still be present in the NaOH degraded lignin.

As a result of this research, we suggest caution in the manufacture of mixed component films. During the manufacture of films it is likely that compositional changes may occur in one or more components that renders the end product significantly different from the intended composition. The original intent of use of films by Vengal & Srikumar [1] was different from ours and may not have been influenced as much by the actual chemical composition of the end product of film casting. However, if one is attempting to produce films of known composition of the identical chemistries of component parts, caution must be taken to ensure that the end product does indeed have the desired components. For example the films created by the Vengal & Srikumar [1] process are inappropriately named as starch: lignin films, when the films are more representative of starch alone.


[1.] J.C. Vengal and M. Srikumar, Processing and Study of Novel Lignin-Starch and Lignin-gelatin Biodegradable Polymeric Films, Trends in Biomaterials and Artificial Organs, 237-241 (2005).

[2.] L. Mariniello, P. Di Pierro, C. Esposito, A. Sorrentino, P. Masi, and R. Porta, Preparation and Mechanical Properties of Edible Pectin-Soy Flour Films Obtained in the Absence or Presence of Transglutaminase, Journal of Biotechnology, 191-198 (2003).

[3.] R. Singh, S. Singh, K.D. Trimukhe, K.V. Pandare, K.B. Bastawade, D.V. Gokhale, and A.J. Varma, Lignin-Carbohydrate Complexes from Sugarcane Bagasse: Preparation, Purification, and Characterization, Carbohydrate Polymers, 57-66 (2005).

[4.] D. Tapia-Blacido, P. J. Sobral, and F.C. Menegalli, Development and Characterization of Biofilms Based on Amaranth Flour (Amaranthus caudatus), Journal of Food Engineering, 215-223 (2005).

[5.] P.M. Latter and D.W.H. Walton, The Cotton Strip Assay for Cellulose Decomposition Studies in Soil: History of the Assay and Development, Cotton Strip Assay: An Index of Decomposition in Soils,7-10 (1988).

[6.] J.C. Went and F. De Jong, Decomposition of Cellulose in Soils, Antonie van Leeuwenhoek, 39-56 (1966).

[7.] V.J. Morris, A.R. Kirby, and A.P. Gunning, Atomic Force Microscopy for Biologists, 2nd edition (Imperial College Press, 2010) p 1-33.

[8.] J. E. Miller, L.R. Evans, J.E. Mudd, and K.A. Brown, "Batch Microreactor Studies of Lignin Depolymerization by Bases", Sandia National Laboratories, SAND2002-1318, (2002)

[9.] J.F. Kennedy and C.A. White, Bioactive Carbohydrates (Ellis-Horwood Publishers, 1983) p66-76.

[10.] H.H. Mantsch and D. Chapman, Infrared Spectroscopy of Biomolecules (John Wiley and Sons, Inc., Publications, 1996) p 207-210.

Jennifer Oberle-Kilic * (1), John Dighton (2), Georgia Arbuckle-Keil (3)

(1) School of Environmental and Biological Sciences, (2) Pinelands Field Station, (3) Camden Campus, Rutgers The State University of New Jersey, New Brunswick, NJ, USA

* Corresponding author: Jennifer Oberle-Kilic (

Received 15 December 2011; Accepted 28 March 2012; Available online 27 April 2012
Table 1: Comparison of main spectral peaks between lignin,
starch, and 90:10 starch: lignin films

Wavenumber       Lignin      Starch     Starch Lignin

1594                X
1512                X
1457                            X             X
1453                X
1368                X
1266                X
1214                X
1151                            X             X
1139                X
1130                X
1079                X           X             X
1030                X
1019                            X             X
931                             X             X
852                             X             X
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Title Annotation:Short Communication
Author:Oberle-Kilic, Jennifer; Dighton, John; Arbuckle-Keil, Georgia
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2012
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