Determination of total polysaccharides in soils affected by forest fires E. Atanasova and I. Atanassova* Department of Agroecology, Department of Soil Chemistry, Nikola Poushkarov Institute of Soil Science, Sofia, Bulgaria 7 Shosse Bankya, Sofia 1080, Bulgaria
Abstract Cinnamonic forest soils (Luvisols) on the ridge and the hillside of Lyulin mountain subject to a fire in the late summer of 2007, a bogged soil at the footslope and a control (unburnt) soil on the opposite site of the hill were studied. For the first time a modified procedure (Lowe, 1993) for spectrophotometric determination of polysaccharides in Bulgarian soils was developed. Total sugars of < 2 mm and < 250 μm soil fractions were analysed through extraction with c. H2SO4 with subsequent spectrophotometric determination..We found predominant accumulation of total polysaccharides in the finer fraction (< 250 μ) of the burnt soils on the ridge, the upper and the middle parts of the hill compared with the control soil. For the coarser (< 2 mm) soil fractions the opposite trend was observed, i.e. lower contents were found in the burnt soils than in the control soil. The results show that the finer soil fraction is more indicative for the changes in the polysaccharides as part of soil organic matter (both unhumified and humified) occurring in the soils after fire. Key words: total polysaccharides, soil, acid hydrolysis, fire Corresponding author e-mail:
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Introduction Extraction and accurate determination of soil carbohydrates is essential to assess the role of carbohydrates in stabilizing soil aggregates (Piccolo, 1996). Various colorimetric methods have been used over the years to determine soil total carbohydrates. The isolation and quantitative measurement of total soil carbohydrates has been reported by many authors. Soil poly- and oligosaccharides are usually acidhydrolyzed first to corresponding monosaccharides and then assayed either as reducing sugars using an alkaline ferricyanide procedure or with the aid of various sugar developers (Cheshire et al.,1984). Soil aggregation was studied in relation to microbial processes and to organic matter. It’s widely accepted, that soil carbohydrates improve aggregate stability and for this reason there is a need for simple and rapid method for measuring carbohydrates. Partial hydrolysis with mild sulfuric acid conditions (0.25-0.5M H2SO4) are frequently used for determination of soil saccharides (Murayama, 1984). Other extractants such as water, EDTA and urea have been used with limited success (Cheshire, 1977). Various methods including spectrophotometric and gas and liquid chromatography have been utilized for determination of soil saccharides (Martens and Frankenberger, 1991). The objectives of our study are to: (i) analyse total soil polysaccharides in soils affected by forest fire having occurred in the autumn of 2007 and (ii) evaluate the effect of fire on the total hydrolysable carbohydrates, which include the free sugars generated by the combustion of plant material and in soil organic matter in the burnt soils and those included in the organic matter unaffected by fire in the unburnt soils.
This study will further elucidate the role of soil polysaccharides in stabilising soil aggregates. To our knowledge it is the first study to measure total polyssacharides in Bulgarian soils by means of the spectrometric modified method of Lowe (1993). Materials and Methods The study sites were cinnamonic forest soils (Luvisols) on the ridge and the hillside, a bogged soil at the footslope (Gleysol) and a control (unburned) soil on the opposite site of the hill of Lyulin mountain subject to a fire in the late summer of 2007 (FAO classification, Teoharov, 2004). For the investigations, the surface soil (06 cm) and lower horizons at five soil sites were studied: on the ridge (sample 601), the upper and the lower parts of the hill (samples 602 and 603), as well as at the foot of the mountain (Sample 604). A control soil (sample 606) was sampled on the opposite side of the hill, not affected by fire. Sampling took place in the spring of 2008 after eliminating the litter layer. The vegetation was represented by black pine (Pinus nigra) mostly affected by fire. At the foot of the hill due to bogging and waterlogging, the predominant vegetation is bullrush (Typha angustifolia). Other vegetation species are represented by meadow fescue (Festuca Pratensis). The morphological and climatological characteristics of the area were presented in Atanassova et al., (2009) and Atanassova & Teoharov, (2010). Total organic carbon increased in all the burnt soils, due to partially combusted organic remnants (Atanassova et al., 2009). The procedure of Lowe (1993) with modifications was adopted. Soil samples were dried at room temperature and homogenized. Before analysis samples were sieved (2mm and 0.25mm). 1 g soil samples (in triplicate) were weighed into 250ml Erlenmayer flasks and 4 ml 12M H2SO4 was added. The flasks were covered with the watch glass and were left to equilibrate for 2 hours. Distilled water (92 ml) was added to achieve (0.5 M H2SO4). The flasks were covered with watch glass and left onto a sand bath for 2 h., then cooled, filtered into 250 ml volumetric flasks and the residue was thoroughly washed. Standard curve was prepared as follows: 1 ml of each standard was pipetted into separate cuvettes. To each cuvette 1ml phenol solution (5%w/v) was added, followed by addition of 5ml conc. H2SO4. After standing for 10 min cuvettes were placed in a water bath at 25-300C for 25 min. Reading of absorbance was done at 490 nm on a spectrophotometer UV/VIS Unicam. Zero absorbance was set with reagent blank prepared using 1ml distilled water in place of a standard. Analysis of sample hydrolysates was carried out according to the same procedure as for the standards. Calibration curve was prepared and regression line calculated. Polysaccharide concentration was determined from preparation of standard curve, and percentage polysaccharide content calculated. Results were recorded as percent total polysaccharide (glucose equivalent).
Results and Discussion We observed the following trends given in Figure1. Increased contents of total polysaccharides were found in the fine textured (< 250 μ) soil. This concerns especially the 601, 602 and 603 soils on the higher parts of the hill. In contrast, in the control soil 606 lower contents of polysaccharides were found. The reason must be due to the predominant accumulation in soils 601, 602 and 603 of fine particulate organic matter which includes unhumified plant residues added to the soil during the incomplete combustion in the soils under study. This corresponds to the higher content of total organic carbon of soil 601 (Atanassova et al., 2009).
For the coarser (< 2 mm) soil fraction the opposite trend was observed, i.e. lower contents were found in the burnt soils 601 and 602 than in the control soil 606. The total polysaccharides exhibit higher contents in the finer fraction of the burnt soils (601- 603) than in the unburnt 604 and control 606 soils. 400 350 0,25 mm
300
2 mm
mg/g
250 200 150 100 50 0 601
602
603
604
606
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Figure 1. Variation of sugar contents in the experimental soils.
The results show that the finer soil fraction is more indicative for the changes in the polysaccharides as part of soil organic matter (both unhumified and humified) occurring in the soils after fire. Levoglucosan (1,6-anhydro-β-D-glucopyranose), a well-known cellulose combustion product (Shafizadeh, 1984) was not detected in the burnt samples in the study by Atanassova & Teoharov (2010). Most probably this biomarker was degraded in the forest soil environment with low temperatures and frequent rainfall in the following wet months after the occurrence of fire. Conclusion We found predominant accumulation of total polysaccharides in the finer fraction (< 250 μ) of the burnt soils on the ridge, the upper and the middle parts of the hill of Lyulin mountain affected by fire in the autumn of 2007 compared with the control (unburnt) soil. For the coarser (< 2 mm) soil fractions lower contents were found in the burnt soils than in the control soil. The results show that the composition of the finer soil fraction is more indicative for the changes in the polysaccharides as part of soil organic matter (both unhumified and humified) occurring in the soils after fire. References Atanassova, I., M. Teoharov and P. Ivanov. 2009. Characterisation of a fire affected catena sequence from Lyulin Mountain, Bulgaria. Bulgarian Journal of Agricultural Science, 15, 334-341. Atanassova, I. and M. Teoharov. 2010. Variation in lipid abundance and composition in a fire affected hillside from Lyulin mountain, Bulgaria. Agricultural Science and Tehnology Journal. 2 (3), 153-159.
Cheshire, M.V. 1977. Origin and stability of soil polysaccharides, Journal of Soil Science, 28, 1-10. Cheshire, M.V., Sparling G. P., C.M. Mundie. 1984. Influence of soil type, crop and air drying on residual carbohydrate content and aggregate stability after treatment with periodate and tetraorate. Plant and Soil, 76, 339-347. Lowe, L. E. 1993. Soil Sampling and Methods of Analysis, Eds. Martin Carter, Lewis Publishers, chapter 36, 823 Martens, D.A., W.T. Frankenberger Jr 1991. Determination of saccharides in biological materials by high performance anion-exchange chromatography. Journal of chromatography. 546, 297-309. Murayama, S. 1984. Changes in the monosaccharide composition during the decomposition of straus under field conditions. Soil Science and Plant Nutrition 30, 367-381. Piccolo, A. 1996. Humus and soil conservation, In: A. Piccolo (ed.), Humic Substances in Terresrtrial Ecosystems, Elsevier, Amsterdam, Netherlands, pp. 225264. Teoharov, M., 2004, FAO classification,
