Is it possible to determine formation processes of organic deposits through polysaccharides? Pilot study from the prehistoric site in Brandýs nad Labem,Czech Republic

Abstract

This paper deals with a possible interpretation value of biochemical methods in comparison with the classic tools of geoarchaeology for the evaluation of formation processes. Organic rich layers from the archaeological site Brandýs nad Labem-Vrábí were tested with the aim to determine the origin of several different types of soil organic material by analyzing the content of different sugars. The studied soil body showed signs of cultural layer, redeposited soils, and in situ developed soil. The analysis of different sugars was highlighted: soil samples taken from these layers were analyzed to assess the ratios of mannose + galactose to arabinose + xylose, and of rhamnose + fucose to arabinose + xylose, content of C_org and different nitrates, as well as different rates of absorbance. The results show that the interpretation values of polysaccharides evaluation didn’t bring significant results itself, but in combination with classical tools of geoarchaeology may bring interpretable and new results.

Keywords

C content geoarchaeology humic substances N content soil biogeochemistry sugars the Early Iron Age the Final Bronze Age

Introduction

There are two possible ways of organic matter decomposing in soil body. One of them is caused by macrobiota and the second one by the microbiota action. To measure the content of organic matter and to distinguish the state of decomposition and the factors affecting the decomposition is a question of different methodological approaches (Babel, 1975; Weaver et al., 1994). The origin of soil organic material (Diochon et al., 2013) can be accessed via analysis of the content of different sugars, as soils with a higher content of plant remains show a higher content of polysaccharides and saccharides derived from cellulose or starch (Gupta and Sowden, 1964) and also a relatively higher amount of the pentoses – AX – arabinose and xylose (Oades, 1967). In substances of microbial origin, the make-up is more complex: the primary storage polysaccharide in fungi and bacteria is glycogen which is analogous to the starch stored by plants. Structurally, it is similar to amylopectin, but its chains are more extensively branched: each branch is about six to seven glucose units in length and the segments between branching points are formed of about three glucose units. Therefore, soils with a higher content of microbial metabolism products (Child, 1995) are typified by the presence of microbial polysaccharides. Compared with plant polysaccharides, a hydrolysis of microbial polysaccharides results in a more diverse mixture of monosaccharides (Rumpel and Dignac, 2006). Microbial polysaccharides are found in the form of extracellular polysaccharides, cell wall polysaccharides, or intercellular polysaccharides. It is obvious that there must be a difference between the remains of dominant bacteria and the remains of fungi, typically with detectable levels of chitin. Bacterial polysaccharides are mostly linked to proteins and lipids, and therefore, they are found in the form of glycoproteins and glycolipids. Their hydrolysis leads to formation of glucose, glucuronic acid, glucosamine, inositol, and various deoxyhexoses, but first to mannose and galactose (MG), and rhamnose and fucose (RF). Because of the work of Cheshire (1977), we are aware that stability of polysaccharides in soil is not related to their chemical composition but to their unavailability which is primarily caused by (1) inaccessibility within undecomposed biological residues and (2) insolubility resulting from adsorption on clay. Furthermore, we know that this can be caused by inhibition of enzymatic hydrolysis because of complexing by both metals and humic substances. The methodological differences between different extractions of the carbohydrates, by hot-water extraction and those extracted by acid hydrolysis, were described by Angers et al. (1993) and Ball et al. (1996).

From the biochemical point of view, the study was focused on the interrelationship between soil carbohydrate composition and soil physics which led to understanding of both the origin and stability of soil saccharides, and soil organic matter as a whole. Mobility of organic polymers; their aliphatic, cyclic, and aromatic nature; and the isotopic composition of soil organic matter reflecting human influence on vegetation cover have been scrutinized long and eagerly in the mirror of archaeological records (Hjulström and Isaksson, 2009). Second, the study dealt with the evaluation of quantity and biodegradability of soil organic matter by gas chromatographic analysis. The specific composition of soil saccharides of plant (Chao et al., 2007) and of microbial (Rakhuba et al., 2009) origin causes differences in their decomposition – understanding these differences would allow us to detect the specific features of the archaeological records (memory of soil from the aspect of archaeology).

The objective of this paper is to demonstrate how the analyses of polysaccharides, especially the mannose + galactose/arabinose + xylose (MG/AX), may shed light on the understanding of the formation processes and the origin of organic matter. Suitable locality for such a study was found in the area with intensive Holocene erosion (Roberts, 2014), resulting in the accumulation of rich organic deposits where there were both physicochemical and biological reasons for its persistence (Schmidt et al., 2011). The area was also lately occupied by humans and therefore there occurred within these organic rich deposits of Holocene age the archaeological objects containing organic rich bands. We suppose that natural accumulation of organic deposits of uncertain Holocene age resulted mainly from redeposited soils by colluviation, that is, the material influenced mainly by the microbial decomposition while the intentionally deposited archaeological deposits (floors, surfaces of abandoned objects) are represented either by the redeposited soils as well as by the plant material decomposed by macrobiota. When plant material is only partly decomposed, these might be used for such evaluation, for example, the method of micromorphology (Novák et al., 2012), but if the decomposition is intensive, then there is a need for additional methodology.

The main aims of this paper are therefore to (1) evaluate biochemically the decomposed organic matter within organic rich sediments of different age and (2) compare the applicability of suggested methodology together with the classical geoarchaeological tools for the identification of formation processes, that is, to distinguish the natural colluviation with polysaccharides of microbe-dominated origin from the in situ soils or archaeological deposits with organic matter of plant dominated origins.

Materials and methods

Description of the site and soil profile

The study site Brandýs nad Labem–Vrábí, (50°10′29.l33″N, 14°39′09.57″E) is situated in the area between the Elbe River valley in Brandýs nad Labem and the southeast edge of the Prague plateau (Figure 1). The geological background of the site is composed of loess overlying both Cenomanian sandstones and locally also marls and spongolites (Chlupáč et al., 2011). The area was, during the glacial period, intensively affected by frost action resulting into the development of a number of frost edges as well as cryogenic structures which form in some areas of polygonal soils (Figure 1). Subsequently, a presence of these structures caused surface instability which resulted in the system of erosional depressions lately infilled by organic rich sandy loams. The erosion of the area is more or less continuous since the beginning of Holocene till today. Modern age and Medieval erosion is reflected in eroded archaeological structures (Turek and Turková, 2012; Turek et al., 2013). Also, redeposited archaeological material of prehistorical age was possible to track in infillings of the natural depressions. Recent soils are represented by zonal Chernozems (IUSS Working Group WRB, 2006), which is typical soil type for loess substrate in this area and Fluvisols connected with the alluvial zone of Elbe River (Kozák et al., 2009).

Figure 1.

The general layout of the vicinity of the study area Brandýs nad Labem–Vrábí in the Czech republic.

Archaeological excavation in the study area reports the findings of different structures (pits, sunken houses, and graves) belonging to the Funnel Beaker Culture and Bell Beaker Culture of the Chalcolithic period, overlaid by the structures of Early Bronze Age Únětice, Bylany and Štítary Cultures (Turek and Turková, 2012; Turek et al., 2013). Because of the fact that the infillings of natural depressions are markedly large and contain organic rich deposits, there were sometimes issues with distinguishing the upper or lower limits of archaeological structures.

The soil section discussed in this paper (Figure 2) is composed of two different features. The infilling of natural depression (right lower part of Figure 2) is composed of regularly alternating layers of darker humic loam bands and lighter loess material. The darker humic bands represent layers where a reduction in the organic matter decomposition rate was present. There is visible inclination of bands in a west-east direction at an angle of 30°–40° with one organic rich layer marked as B-1. According to the macroscopic observation, it is quite clear that the material was redeposited into the former depression by colluviation. Lately, the archaeological feature discussed in this paper was partially cut into the former infilling of erosion feature, partly into the loess background. The infilling of archaeological feature is composed of macroscopically different set of layers. There was an observed 10 cm-thin organic rich layer (homogeneous loam of dark brown color with abrupt transition above and undulated surface) at the bottom of the sunken feature. This layer marked as B-4 contained ceramic shreds archaeologically dated into the Final Bronze Age or the Early Iron Age (Štítary and Bylany Cultures) and macroscopically was evaluated as the in situ cultural layer. Approximately 50 cm-thick brown layer of loam containing rip-up clasts of lighter loam (B-5) was recorded above the layer B-4. The presence of the rip-up clasts suggests colluviation as the most probable formation process in the development of the infillings of archaeological objects (Lisa et al., 2015). The transition into the layer above is gradual, which suggests that the layer above went through in situ pedogenetical process. The layer B-6 (Figure 2) is composed of approximately 60 cm-thick dark brown homogeneous loam as the evidence of humified material that was protected from decay. This layer seems to be continuous and also outside the archaeological feature and the transition above is abrupt. The uppermost layer marked as B-7 is brown loam continual outside the archaeological feature. The abrupt transition is because of the plowing, so the origin of the layers B-6 and B-7 is generally identical. The above described organic rich layers B-1, B-4, B-5, and B-7 were sampled for laboratory analyses. Samples were collected from each of the layers – sampling was carried out by randomly selecting four individual samples of each of the layers, then mixing them together (for each of the layer) and storing them at 4°C.

Figure 2.

The photo of the studied section. The right lower part of the area belongs to the older infilling as the archaeological feature is cut into it. White ground control point (crosses) are used to create a photogrammetric plan. The top layers are associated with today’s agricultural activities, sampling place B-7. Below these layers is the archaeologically affected layer that partially overlaps the archaeological artifact in the lower left (sampling place B-5 and B-4). In the area of the artifact are diagonally alternating layers of dark brown with ocher. These lower layers (B-1) extend outside the archaeologically explored area.

Laboratory analyses

Bulk samples were taken from each lithological divided layer and proceeded by the set of following methods. Laboratory methods were aimed at determining the ratios of polysaccharides in soil, namely, MG/AX and rhamnose + fucose/arabinose + xylose (RF/AX). Polysaccharides were determined according to Amelung et al. (1996). This method allows us to determine the origin of the prevailing sugars by hot-water extraction of carbohydrates with subsequent assessments of the MG/AX and RF/AX ratios (Oades, 1984). After purification, the polysaccharides were uniquely identified by retention data and their EI mass spectra. There are both the GC-MS chromatograms of 1µg standards with the detailed parameters for all the polysaccharides determined, and the GC-MS chromatograms of 1µg of each sugar standard and one of the sample analyzed as the example (Figure 3).

Figure 3.

(a) GC-MS chromatogram of 1 µg standards and (b) GC-MS chromatogram of 1 µg of each sugar standard (upper graph) and sample no. 4 (lower graph). After purification, sugars in the individual samples were uniquely identified by retention data and their EI mass spectra.

An amount of 50 mg of samples B-1, B-4, B-5, and B-7 was hydrolyzed with 2M TFA at 105°C for 2 h and the hydrolyzate was evaporated to dryness. The residue was dissolved in re-distilled water, cleaned up on a column with XAD-7 sorbent, and then passed through Dowex W X8 (200–400 mesh). After being evaporated to dryness, the saccharides from water eluates were oximated and silylated and subsequently analyzed using the GC-MS method; electron ionization, 70 eV; helium carrier gas, constant flow 1.1 mL/min^–1; injector temperature 240°C; injection volume 1 µL split; ion source temperature 220°C; and GC-MS interface temperature 250°C. The standards of the studied sugars were processed through columns of both sorbents in the same way as the soil samples, for development of three-point calibration curves with ranges of 20, 500, and 1000 µg/g (n = 2). The organic matter distinctive character was shown by the humus quality coefficient Q4/6 (Chen et al., 1977). The quality of humification products following decomposition and translocation of organic matter was subject to color quotient (Q4/6), calculated from the equation Q4/6 =E465/E665, where E465 and E665 are the extinctions (Na₄P₂O₇ extract) at wavelengths 465 and 665 nm (Hofman et al., 2014; Welte, 1956). In addition, samples B-1, B-4, B-5, and B-7 were subjected to other routine laboratory analyses: oxidizable carbon (C_ox) was determined spectrophotometrically after an oxidation by H₂SO₄ + K₂Cr₂O₇ (International Organization for Standardization (ISO) 14235:1998, 1998), total N content by Kjeldahl method using titanium dioxide as catalyst according to ISO 11261:1996 (1996), nitrate nitrogen content by the UV spectrophometry (Zbíral et al., 2011), and ammonium nitrogen content spectrophotometrically after an extraction with potassium chloride solution (ISO 14256:2003, 2003).

Results

The highest values of measured polysaccharides were recorded within layers B-7 and B-4. In case of B-7 layer, the values of single polysaccharides are more than twice higher than in the case of layers B-1 and B-5. Comparing results of MG/AX and RF/AX, the given results seem to be fairly homogeneous. The lowest ratio of MG/AX was detected in the case of the layer B-1, while the highest was in the case of the layer B-7. The lowest ratio of MG/AX as well as RF/AX was detected in the layer B-1, while the highest was detected in the layer B-7 (Table 1).

Table 1.

The content of selected polysaccharides in μg g^–1 of soil (software Excalibur). The ratio of mannose and galactose to arabinose and xylose (the MG/AX ratios), and rhamnose and fucose to arabinose and xylose (the RF/AX ratios).

Soil sample	Mannose_Galactose_I	Galactose_II	Arabinose_I Xylose_II	Arabinose_ II	Xylose_I	Rhamnose_Fucose_I	Fucose_II	MG/AX	RF/AX
Soil sample	µg g⁻¹ dry soil							MG/AX	RF/AX
B-1	309.67	397.37	245.09	513.92	101.79	65.62	25.98	0.82138	0.10641
B-4	529.10	707.47	479.97	669.53	178.03	121.87	71.38	0.93148	0.14557
B-5	337.83	438.50	235.92	416.73	119.39	58.43	33.21	1.00556	0.11869
B-7	955.22	999.09	605.36	893.15	380.52	212.67	119.76	1.04006	0.17692

The values of oxidizable carbon (C_ox) vary between 1.07% and 1.80%, and the values of total nitrogen content vary from 0.07% to 0.15%. However, the C:N ratios counted from these data show interesting differences. For example, the layer B-4 exceeds seven times more the values counted for the layer B-7 and three to four times more the values counted for the layers B-5 and B-1 (Table 2). The evident differences between layer B-7 and the rest of the studied layers were recorded by the measurements of nitrate nitrogen (twice higher content) and ammonium nitrogen (twice lower content) (Table 2).

Table 2.

Available forms of mineral nitrogen, oxidizable carbon, total nitrogen, and C:N ratio.

Soil sample	Nitrate nitrogen, NO₃⁻ (mg kg⁻¹)	Ammonium nitrogen, NH₄⁺ (mg kg⁻¹)	Oxidizable carbon content, C_ox (%)	Total nitrogen content, N_t (%)	C:N
B-1	5.80	0.69	1.369	0.084	16.3
B-4	6.32	0.71	4.998	0.070	71.4
B-5	5.23	0.67	2.574	0.099	26.0
B-7	11.16	0.3	1.445	0.146	9.9

The values of absorption quotients of humic substances (Orlov, 1995) are also somewhat homogenous, and it seems that the only exception (though not notably significant) is the slightly higher content of absorbance A465 and absorbance A665 in the case of sample B-7 and slightly higher value of the humic substances absorbance coefficient Q4/6 in the case of sample B-1 (Table 3).

Table 3.

Determination of the quality parameters of humus based on the ratio of humic and fulvic acids detected by the absorption quotients of the humic substances, Q4/6.

Soil sample	Absorbance A₄₆₅	Absorbance A₆₆₅	The humic substances absorbance coefficient Q4/6
B-1	0.296	0.069	4.29
B-4	0.273	0.067	4.07
B-5	0.26	0.064	4.06
B-7	0.362	0.089	4.07

Discussion

Biogeochemical evaluation of humic layers

The values for MG/AX between 0.5 and 1.2, and RF/AX between 0.01 and 0.2, indicate that the analyzed organic matter is mainly of plant origin (Hatakeyama and Hatakeyama, 2005; Meier and Reid, 1982), although with some detectable presence of sugars of microbial origin (Morris and Harding, 2009). It means that when the ratio MG/AX is lower than 1.2, the organic matter is mainly of plant origin; when the ratio RF/AX is lower than 0.2, the organic matter is mainly of plant origin as well (Haynes and Francis, 1993; Oades, 1984; Sumner, 2000). Equally, both MG/AX and RF/AX values are closer to the upper boundary of the scale. As we see in Table 1, none of the four analyzed horizons exceed these values; therefore, it can be concluded that horizons sampled have been developed in direct connection with the storage of plant material.

It seems that the ratio of MG/AX and RF/AX does not detect slight differences in measured samples and that the simple values of measured polysaccharides have higher interpretation values. The layers B-4 and B-7 were macroscopically interpreted as in situ layer with no signs of redeposition. Consequently, the higher values of polysaccharides in these layers comparing with two layers B-1 and B-5 show that measured high content of polysaccharides may play important role in the final interpretation, which however have to be supported by additional data. The higher values of particular polysaccharides at the layers B-4 and B-7 were likely being produced by no redeposition of the organic material (Walkington, 2010). Such values could reasonably reflect pedogenetically the non-influenced B4 layer and slowly forming B-7 layer (Kristiansen, 2001).

Based on the known fact that the C:N ratio value is around 10 in the biologically most active soils, around 30 in plant material, and around 60 in peat formation products (Gobat et al., 2004), it is possible to conclude that the analysis proved autochthonism of the soil processes on the study area surface. The bottom of archaeological feature represented by layer B-4 shows high C:N ratio corresponding to the decomposed plant material without any signs of long-term pedogenesis, that is, without clay-humus complexes with high-molecular organic compounds. The layer just below (B-1 sample) is a layer of accumulated material, transported from soil surface at a period of different climatic conditions compared with the more recent. As regards the deepest located, second cultural layer, its relation to pedogenesis and plant material accumulation is unclear. The possible interpretation of quite low C:N ratio may be because of the redeposition of material once affected by pedogenetic processes – the question is how such redeposition affects the nitrogen values (Coltrain and Ugan, 2011). This value is comparable also with layer B-5 where the redeposition of once pedogenetically influence material is also supposed.

Knowing that the mean values of soil nitrate nitrogen range between 3 and 6 mg kg⁻¹ (Rejsek et al., 2011), we can say that the rate of its highest formation in the highest located horizon fully corresponds to the recent character of nitrification with a high level of oxidation in that horizon, and to its high value of soil reaction (Subbarao et al., 2015) – typical for the region of Elbe development in the Bohemian Cretaceous Basin. The value of the ammonium nitrogen content on the soil surface stands out from its generally low values, fluctuating between 0.5 and 2 mg kg⁻¹ (Rejsek et al., 2011). This corresponds to the relation between the non-nitrified ammonization products and the higher level of nitrates (Glinski et al., 2007; Rosenkranz et al., 2012).

The similar values of the oxidizable carbon content found in the higher located layers did not allow us to form any definite conclusion. We could see the closeness of the two potential cultural layers (B-1 and B-4 samples). What is more, there is a possible link between the polysaccharides transferred by percolation (Malik et al., 1991) and accumulated in the B-4 sample, and the high content of organic matter in the surface horizon (Vranová et al., 2013). The results show that the youngest layer developed because of the input of substances from the higher located part of the soil body by precipitation water infiltration.

Applicability of the polysaccharides evaluation comparing with the classical methodology

The interpretation value of the infilling of archaeological objects is the subject of long-term investigation and a number of methods were tested to find out the environmental record. However, all types of deposits can be studied if longevity of them allows such investigations. Jia et al. (2008) and Brock et al. (2011) confirmed the longevity of survival of carbohydrates for peatlands, Rabbi et al. (2013) for soils, Amon and Benner (2003) for ocean waters, Hedges et al. (1994) for river waters, and Ogier et al. (2001) for lake waters. A relevant argument against determination of the origin of Holocene organic soil matter from deeper layers (Hansen and Møller, 1975) through biochemically assessed ratios of plant (arabinose and xylose) and microbial (mannose, galactose, rhamnose, and fucose) polysaccharides could be instability of saccharides in soil environment, often chemically aggressive. The study area is located in quite alkaline environment of loess which is typically a non-aggressive one. It is a question whether in such environment the organic substances become more concentrated, especially if they change their position within the soil body and form various organo-mineral complexes that either participate in colloidal bonds within their surrounding environment or get stabilized by physical-chemical forces (Rejsek et al., 2012) with smectite clay minerals.

Methodologically, there is an established framework for interpreting the results of the carbohydrate analyses: the results discussed came from hot-water extraction followed by measurement of the ratios of MG and RF contents to AX contents where low MG/AX and RF/AX ratios indicate organic matter of plant origin (Haynes and Francis, 1993; Oades, 1984; Sumner, 2000). Meier and Reid (1982) and Hatakeyama and Hatakeyama (2005) presented data about polysaccharides of plant origin, and Morris and Harding (2009) data on some detectable presence of sugars of microbial origin. That approach is biologically important: even when we are aware that most plants are actually mycorrhizal, till now there are no hesitations in sound scientific literature about a routinely applicable methods for distinguishing polysaccharides of plant origin from the ones of microbial origin, that is, a soil organic matter of plant-derived origin is richer in pentose sugars such as arabinose and xylose, while a soil organic matter of microbe-dominated origins is richer in the above-mentioned microbial polysaccharides. Table 1 has its value based on assigning particular saccharides to their particular sources. Considering plant tissues of archaeological importance, hydrolyzed fiber of, on one side, oat and wheat and, on the other side, spruce, aspen, and birch was analyzed extensively by Rovio et al. (2008) confirming high amount of d-xylose; high amount of arabinose in gums of different plants was determined by Bonaduce et al. (2007). In addition, high amounts of both arabinose and xylose in beech and oak litter were confirmed by Sariyildiz and Anderson (2003). As regards microbial saccharides, higher amounts of mannose (Elloway et al., 2004; Ferreira et al., 2010; Geurtsen et al., 2009; Gruden-Movsesijan and Milosavljevic, 2006) and both galactose and mannose stated Moreira et al. (2003) and Chowdhury et al. (2011). In terms of human saccharides, irrespectively of the well-known fact that humans do not possess a functional gene for l-gulonolactone oxidase in ascorbic acid biosynthesis (Kim et al., 2012), l-rhamnose occurs in blood and urine (Fujii et al., 2001), N-acetyl-d-glucosamine in gastrointestinal tract (Kompella and Lee, 2001), and l-fucose in various human organs (Leck and Wiese, 2004). Because the timespan of the origin of the colluviated organic material was not of a goal for the research, the likely combination of potentially different sources of material was not a problem. The authors are aware of the sparse dataset, and the significance of the proposed approach is carefully evaluated. Positive results can be seen in answering the question at what point the microbial genesis of organic matter begins to outweigh the genesis linked to plant material accumulation.

The classical tools of geoarchaeological investigation, that is, the sedimentological evaluation followed by the archaeological soil organic chemistry (Bull et al., 1999; Hunt et al., 2015; Maghsoudi et al., 2014) link together proxies to establish interpretive frameworks which should be taken into account as the first ones because of the interrelationships between prehistoric human environment and alterations in soil bodies (Gerlach et al., 2006; Kuna et al., 2013; Leigh, 2001; Stinchcomb et al., 2013). Therefore, although the assessing ratios of MG to AX and of RF to AX did not bring significant results themselves, a combination of the biochemical analysis with macroscopic field evaluations and classical geoarchaeological methods can fruitfully bring well interpretable results.

The obtained information on the position of layers rich in organic matter could become a part of more extensive research on the prehistoric landscape (Ferme and Huerta, 2014; Matless, 2008) and its parameters, from the viewpoint of the settlement dynamics of the dated human occupation (Grayson and Millar, 2008). The first sedimentological evaluation of the study section corresponds with the results given by C and N analyses. The new analytical approaches didn’t show significant results in spite of the fact that there are signs of possible interpretations comparable with primary observations. For the future research, the organic material found in soil bodies (Evershed, 2008) should be subjected to genetic investigation (Eichmiller et al., 2014; Yang et al., 2014) combined with palynological research (Edwards, 1979; Montoya et al., 2011) determining the participating plant taxa, and with soil micromorphological studies (Kooistra and Kooistra, 2003; Lisa et al., 2014) with the aim of examining thin sections within the soil bodies studied.

Conclusion

This study demonstrates the limits of pedobiochemical approach to evaluating formation processes of the infillings of archaeological features. The authors propose to highlight the use of soil carbohydrates as a means of assessing the relative importance of plant versus microbial contributions to archaeological soil horizons but the given results are fairly homogeneous and hence do not support the macroscopic field evaluation. The ratios of MG/AX and RF/AX suggest the plant origin of organic matter in spite of the fact that the macroscopical evaluation as well as the interpretation of C:N ratio points to the different situation. However, the layers interpreted macroscopically as well as by the values of C and N as in situ cultural layer or in situ pedogenetically influenced layer show higher values of polysaccharides compared with those with evidently redeposited ones. It suggests that higher content of polysaccharides may play an important role in the final interpretation, which however has to be supported by additional data. Also, the second proposed biogeochemical approach, the detection of absorption quotients of humic substances, didn’t bring any well interpretable results. The only result (but not really significant for further interpretations) is the slightly higher content of absorbance A465 and absorbance A665 in case of layer influenced by recent pedogenesis and slightly higher value of the humic substances absorbance coefficient Q4/6 in case of the cultural layer.

Footnotes

Acknowledgements

The authors wish to thank RN Dr. Petr Šimek, CSc. from the Laboratory of Analytical Biochemistry and Metabolomics, Institute of Entomology, Biological Centre CAS, v.v.i. whose help made the successful completion of this research possible.

Funding

This study was supported by the Grant Agency CR project Proto-eneolithic enclosures in Bohemia, interpretation of their purpose and social meaning (Grant No. 15-02453S) and by internal program of Institute of Geology CAS RVO 67985831.

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