Correlation between bacterial decay and chemical changes in waterlogged archaeological wood analysed by light microscopy and Py-GC/MS

3.2.1 Pyrolysis products

Analytical pyrolysis coupled with GC-MS was applied to the archaeological waterlogged wood samples (“EB-non”, “EB-partly” and “EB-heavy”) and the reference sample. The pyrograms for the reference and the waterlogged pole samples with different degree of decay showed both holocellulose (cellulose and hemicelluloses) and lignin pyrolysis products with high abundances (Figure 2). Pyrolysis profiles of the waterlogged pole showed the same pyrolysis products as for the reference, but in different relative amounts. Since the same analytical conditions were used for all the samples, the different relative abundances obtained were the result of the changes undergone by the material at the molecular level, which influenced the relative yields of the pyrolytic processes (Tamburini et al. 2017). In fact, the differences in the relative abundances of pyrolysis products between the waterlogged samples (Figure 2), indicating that molecular changes in the lignocellulosic cell wall material is related to the gradient of decay in the waterlogged wood (Tamburini et al. 2017; Zoia et al. 2017). A total of 64 pyrolysis products from the reference material were identified and assigned to either holocellulose (H) or lignin (L). Table 1 lists the identified pyrolysis products of sound reference P. sylvestris together with the assigned category of holocellulose as furans (H-f), pyrans (H-p), cyclopentenones (H-c), hydroxybenzenes (H-h) and anhydrosugars (H-a) and lignin as monomers (L-m), short chain (L-s), long chain (L-l), carbonyl (L-c), demethylated (L-d) and acids (L-a).

Figure 2:

Pyrolysis profiles obtained for sound Pinus sylvestris (reference) and for the waterlogged P. sylvestris samples of the outer heavily decayed xylem (EB-heavy), the partly decayed xylem (EB-partly), and the inner morphological intact xylem (EB-non). Numbers refer to pyrolysis product peak listed in Table 1.

3.2.2 Holocellulose/lignin ratios

The ratio H/L is generally measured by gravimetric analysis and used for internal comparison of a data set (Babiński et al. 2019; Giachi et al. 2003; Tamburini et al. 2015). Previous studies have shown that the pyrolytic H/L ratio also provides a reliable and comparable estimation (Łucejko et al. 2012). For this reason, the pyrolytic holocellulose versus lignin ratio (H/L) was calculated as the ratio between the sum of the peak areas of holocellulose pyrolysis products (ΣH) and the lignin pyrolysis products (ΣL) (Table 2). This index is recognised as a powerful parameter for determining the extent of degradation of waterlogged wood, which is usually characterized by the loss of polysaccharides, therefore an decrease in H/L is observed when compared with sound undegraded wood (Huang et al. 2013; Łucejko et al. 2015; Łucejko et al. 2012). This semi-quantitative calculations highlighted that the waterlogged sample “EB-non” had undergone a loss of holocellulose of about 6% (H/L 2.2 ± 0.2), confirming its very good state of preservation, whereas the loss of holocellulose for “EB-partly” and “EB-heavy” was much higher as expected, leading to low or very low H/L ratios (1.0 ± 0.2 and 0.6 ± 0.1, respectively).

The lower value of the H/L ratio of waterlogged but morphological sound wood (“EB-non”, H/L 2.2 ± 0.2) compared to the reference sound wood (H/L 2.7 ± 0.1) shows that abiotic degradation of the holocellulose fraction most likely occurred. This indicates presence of chemical alteration in the lignocellulosic matrix not accompanied by morphologically altered structure detectable with light microscopy. The results obtained for “EB-partly” and “EB-heavy” confirm that erosion bacteria decay leads to a considerable depletion of carbohydrates, and that the depletion of holocellulose occurred to a higher degree in the surface layer of the pole that was heavily decayed by erosion bacteria (EB-heavy) than in the partly decayed xylem closer to the pith, which contained a higher proportion of un-decayed cell wall material (EB-partly). This is in agreement with results reported in the literature on chemical alteration induced by erosion bacteria decay (Pedersen et al. 2013). The H/L ratio gives an overall picture of carbohydrate depletion and lignin conservation but does not provide any information on the specific chemical changes that occurred in the wood polymers. However, molecular information can be obtained by examining the relative amounts of categorised pyrolysis products in more detail.

3.2.3 Changes in the chemical composition of holocellulose

A limitation of the analytical pyrolysis method is that it does not permit differentiating the pyrolysis products of cellulose and hemicelluloses, as the thermal degradation process form the same pyrolysis products from the two types of polymers. This means that the method cannot reveal details of chemical changes observed specifically in cellulose and hemicelluloses but will refer to chemical changes occurring in the overall polysaccharide (holocellulose) fraction of the wood samples. Pyrolysis products formed during the thermal degradation of holocellulose were classified into five categories: cyclopentenones, furans, pyranones hydroxybenzenes and anhydrosugars, according to (Moldoveanu 1998a; Pouwels et al. 1989; Ramirez-Corredores 2013). The sum of the peak areas assigned to each category (Table 1) were expressed as a percentage of the total abundance of holocellulose pyrolysis products in each sample (Figure 3a).

Figure 3:

Percentage distribution of (a) holocellulose pyrolysis product grouped into categories, calculated in relation to the total amount of holocellulose in each sample; (b) anhydrosugars from the holocellulose pyrolysis products with three different degrees of sylilation calculated in relation to the total amount of anhydrosugars in each sample. The values are based on analyses of three replicas for each sample. Whiskers show the standard deviation of the replica measurements.

A comparison of the relative distributions of categorised holocellulose pyrolysis products shows difference between the reference pine and waterlogged samples, and as expected also within the waterlogged wood samples with different degree of bacterial decay (Figure 3a).

In particular, sample “EB-non” that is not attacked by bacteria and morphologically similar to the reference sound wood, presents a distribution profile of holocellulose pyrolysis products different from that of sound wood. This indicates that certain chemical changes in the carbohydrate polymers take place due to permanence in the waterlogged anoxic environment as abiotic decay in absence of bacterial activity. This is in agreement with the few available chemical analyses of morphological sound waterlogged wood performed in earlier studies (Borgin et al. 1975; Gelbrich et al. 2008; Pedersen et al. 2015).

A notable chemical difference relates to the categories of anhydrosugars and cyclopentenones. An increase in the relative abundances of anhydrosugars and decrease in cyclopentenones can be related to depolymerisation of polysaccharides (Tamburini et al. 2017). In this case the relative amount of anhydrosugars is lower in “EB-non” than in the reference. This indicates a lower amount of low molecular polysaccharides in “EB-non” than in the reference. This can be explained by microbial removal of easily accessible and degradable non-structural carbohydrates like starch stored in parenchyma cells and maybe also by removal of depolymerised structural carbohydrates from the wood structure due to abiotic physical and chemical processes in the waterlogged burial environment. After removal of small fragments, oligomers, and monomers the residual carbohydrates have a lower level of depolymerisation, resulting in a lower pyrolysis yield of anhydrosugars and higher of cyclopentenones.

Differences in the relative amount of cyclopentenones and anhydrosugars between the waterlogged wood samples is related to a higher relative amount of anhydrosugars (from 21 to 29%) and lower amount of cyclopentenones (from 46 to 40%) with increase in erosion bacteria decay (Figure 3a). Levoglucosan is the most abundant compound found in the group of anhydrosugars (Table 1), and its abundance can be related to the degree of polymerization of cellulose. Moldoveanu (1998b) reported that cellulose with a degree of polymerization (DP) of more than 200 generated about 20% levoglucosan during pyrolysis while cellulose with DP less than 200 formed 44% more levoglucosan. This confirms that erosion bacteria depolymerise the polysaccharides fraction of the cell wall.

The chemical differences in the pyrolytic profiles of the holocellulose fractions (Figure 3), are not pronounced compared to the distinctive anatomical changes in the material (Figure 1). This is probably due to the fact that erosion bacteria consume the polysaccharide fraction of the secondary cell wall without leaving residual sugars during and after decay as suggested by Pedersen et al. (2015). The cell wall is either decayed by erosion bacteria or sound; confirming that erosion bacteria has very close contact to the cell wall during decay and very effectively utilise all available sugars. The result thereby suggest that heavily decayed waterlogged wood contains intact holocellulose only in the morphologically sound compound middle lamella that is not degraded by erosion bacteria due to its high content of lignin.

The distribution profile of anhydrosugars with different degree of sylilation formed during pyrolysis and shown in Figure 3b. Three types of silylated anhydrosugars can be formed depending on the number of OH groups that have reacted with the silylating agent (HMDS) to create the corresponding trimethylsilyl ester: monosylilated, disilylated, or persilylated (three -OTMS groups) (Figure 3b). The degree of silylation of anhydrosugars gives an indication of the reactivity of the hydroxyl groups in terms of their hydrogen exchange with the TMS groups. The reaction is not quantitative due to steric hindrance, but the less the anhydrosugars are derivatised during pyrolysis, the tighter the holocellulose polymers are bound in the cell wall polymer network (Evershed 1993; Fabbri et al. 2003).

In the reference wood samples, the disilylated form prevails. The reference and “EB-non” samples are similar in the relative distribution of their derivatised anhydrosugars. This is in good agreement with the morphological similarity between the two material types. With increased erosion bacteria decay (“EB-partly” and “EB-heavy”), persilylated anhydrosugars become more abundant while disilylated anhydrosugars decrease. In “EB-partly” the amount of persilylated anhydrosugars are almost equal to the amount of disilylated anhydrosugars; in “EB-heavy” the amount of persilylated anhydrosugars are higher than the amount of disilylated anhydrosugars. This can either be explained by a partly unwinding of the cell wall polymer matrix during decay or by the difference in ultrastructure of un-decayed cell wall material left in the decayed wood material. The remaining holocellulose in “EB-heavy” is located in the primary cell wall of the compound middle lamella, whereas a high portion of the holocellulose in “EB-non” and “EB-partly” is located in the secondary cell walls. The cellulose in the primary cell wall is arranged in thin crossing layers embedded in pectin, whereas the cellulose and hemicelluloses of the secondary cell wall are arranged in a highly organised parallel structure (Abe et al. 2005). It is possible that the less ordered structure of the cellulose fibrils - or the higher amount of pectin present in the primary cell wall - has an effect in the difference in the relative distribution of silylated anhydrosugars.

3.2.4 Changes in chemical composition of lignin

Gymnosperms such as P. sylvestris contain lignin composed almost entirely of guaiacyl and small quantities of p-hydroxyphenyl units. The lignin pyrolysis products were divided into five categories according to their molecular structure (Huang et al. 2014; Kawamoto et al. 2008; Kotake et al. 2013; Kotake et al. 2014; Tamburini et al. 2015): Z- and E- coniferyl alcohols monomers, short side chain guaiacyl units, long side chain guaiacyl units, oxidised guaiacyl units with carbonyl and carboxyl functional groups, and demethylated guaiacyl units (Table 1). Variations between the relative amounts of these categories in the different types of wood material were observed (Figure 4a). This indicates chemical alterations in the lignin polymer.

Figure 4:

Percentage distribution of (a) lignin pyrolysis products grouped into categories; (b) selected individual lignin pyrolysis products. All values calculated in relation to the total amount of lignin in each sample and based on analyses of three replicas of each sample. Whiskers show the standard deviation of the replicas. TMS-trimethylsilyl group.

Chemical changes between sound reference wood and the morphologically sound waterlogged archaeological sample (EB-non) suggest abiotic decay as a consequence of the immersion in the waterlogged environment. The most pronounced difference in the lignin profile of these two samples is that the relative abundance of demethylated units (pyrolysis products) in the waterlogged wood is less than half of that of the sound wood (Figure 4a). Demethylation of lignin moieties in the waterlogged wood have been reported previously (Pedersen et al. 2013). This study shows that 200–300 years of burial in a waterlogged environment significantly affects the decrease of this type of functional group in the lignin polymer. The second distinct difference in relative amounts of pyrolysis products between reference sample and “EB-non” is the increase in the number of oxidised units, and the slight increase in short chain pyrolysis products (Figure 4a). These changes can suggest that the lignin polymer has undergone an alteration due to abiotic decay during the waterlogging period.

Chemical changes between the morphologically sound waterlogged archaeological sample (EB-non) and waterlogged wood decayed by erosion bacteria (“EB-partly” and “EB-heavy”) consist of relatively small changes in chemical composition of lignin compared to the great difference in the morphology. This indicates that the composition of the lignin polymer network is only slightly affected by the action of erosion bacteria. The chemical differences consist of a slight decrease in demethylated guaiacyl units, a decrease in coniferyl alcohols monomers, an increase in long side chain guaiacyl units, and an increase in oxidised guaiacyl units (Figure 4a). The decrease in the relative abundance of coniferyl alcohols (Z- and E- monomers) in the pyrolysis profile is directly related to the increase in the degree of erosion bacteria decay and is pronounced for both monomers, whereas the increase in long side chain units are most pronounced for vanillylpropanol and E-isoeugenol (Figure 4b). The changes observed in the lignin polymer indicate an alteration in the propanoid side chains, probably due to cleavage of the bonds and oxidation of some positions (Zoia et al. 2017) due to the erosion bacteria activity.

Oxidised guaiacyl units primarily stem from formation of guaiacyl units containing carbonyl and carboxyl functionalities in the alkyl side chain at the a-position, which are conjugated with the aromatic ring (Figure 4b) (Crestini et al. 2003; Tarabanko et al. 2017).

A slight depolymerisation, demethylation and oxidation of the lignin polymer are, by the design of this study, linked to erosion bacteria decay. This is a novel finding, although not surprising, as the erosion bacteria in some way need to rearrange the cell wall, and thereby the lignin polymer, to reach the “encapsulated” polymeric carbohydrates within the micro fibrils of the cell wall as these are their essential nutrient. This alteration of lignin was not reported in Raman imaging investigations (Pedersen et al. 2015). The oxidation induced by bacteria primarily results in the formation of guaiacyl units containing carbonyl functionalities in the alkyl side chain, vanillin, acetovanillone and coniferylaldehyde (Figure 4b). In particular, erosion bacteria oxidation leads to the preferential formation of aldehyde functional groups at the γ-position of the coniferyl alcohol monomers.