An improved method for extracting cellulose from fossil wood and its paleoclimatic implications

Abstract

The stable carbon isotope composition of cellulose (δ¹³C_cell) in fossil wood is valuable for reconstructing past climatic and ecological changes, on seasonal to decadal timescales. However, extracting cellulose from fossil wood is challenging, leading to a lack of δ¹³C_cell data over deep time; moreover, there is a debate about whether the stable carbon isotope composition of whole wood (δ¹³C_wood) can reliably reflect past paleoclimatic or palaeoecological conditions. Here, we present an improved method for extracting cellulose from fossil wood. We initially used conventional methods to extract cellulose from a fossil wood sample a drill core from the Yuncheng Basin, near the Chinese Loess Plateau; however, we were unsuccessful. Subsequently, we successfully extracted cellulose and recovered 94% of the cellulose after modifying the conventional procedure. This involved increasing the reaction time during lignin removal, reducing the concentration of NaOH solution during hemicellulose removal, and employing multiple centrifugation steps for sample separation instead of a single step. We examined the relationship between δ¹³C_cell and δ¹³C_wood values (n = 136), and the results revealed a positive correlation between them (R² = 0.51, P < 0.001). This indicates that δ¹³C_wood is a dependable proxy for qualitative paleoclimatic reconstruction. However, the apparent enrichment factor ε between δ¹³C_cell and δ¹³C_wood values varied between samples, highlighting the need for caution when using records of δ¹³C_wood for quantitative paleoenvironmental reconstruction.

Graphical abstract

Keywords

fossil wood / stable carbon isotopes / tree rings / cellulose extraction / Paleoclimatic reconstruction

1 Introduction

The stable carbon isotope composition (δ¹³C) of fossil wood is a valuable proxy for reconstructing paleoclimatic and palaeoecological conditions on seasonal to decadal timescales in deep time (Schubert et al., 2017; Vornlocher et al., 2021). Wood primarily consists of cellulose, lignin, hemicellulose, and lipids, each with distinct carbon isotope compositions and climatic and ecological signatures (Wilson and Grinsted, 1977; Loader et al., 2003). In modern tree-ring studies, most researchers have focused on cellulose (δ¹³C_cell) rather than on whole wood (δ¹³C_wood), as the carbon isotopic signals of whole wood are a mixture of different wood components and can be influenced by varying proportions of non-structural elements unrelated to climatic factors, complicating their interpretation. For fossil wood studies, δ¹³C_cell records are particularly important, because its cellulose isotopic composition is more stable on long timescales and it is less affected by diagenetic processes, enabling δ¹³Ccell values to record the climatic conditions at the time the wood was formed (Macko et al., 1991; Leavitt and Danzer, 1993; Tao and Liu, 1995; Macfarlane et al., 1999; Loader et al., 2003; Rani et al., 2023). Therefore, extracting cellulose is crucial for stable isotope studies of fossil wood.

While methods for extracting cellulose from modern wood are well established, techniques for fossil wood extraction are underexplored. Green (1963) established the Jayme-Wise method, which uses an acidified sodium chlorite solution to remove lignin. Brendel et al. (2000) developed a rapid extraction technique for cellulose, involving treatment with a mixed solution of acetic acid and nitric acid. However, each method has limitations and cannot be universally applied to all fossil wood samples (Hook et al., 2015; Kagawa et al., 2015). These methodological constraints have hindered the collection of δ¹³C_cell data in deep time (Lukens et al., 2019), leading to an ongoing debate about whether δ¹³C_wood values can reliably reflect past climatic and ecological conditions. For instance, Lukens et al. (2019) found a strong linear correlation between δ¹³C_wood and δ¹³C_cell, indicating that δ¹³C_wood could be used as a valid proxy for paleoclimatic and paleoecological reconstruction. However, other researchers have preferred to use records of δ¹³C_cell, because of the possibility that molecular variability across tree rings could influence δ¹³C_wood values (Bechtel et al., 2003; Ma et al., 2003; Pawelczyk et al., 2004).

In 2019, we obtained a well-preserved fossil wood specimen from deep within a drill core from the Yuncheng Basin in China. This specimen provided an excellent opportunity to explore different cellulose extraction methods for fossil wood, and to determine the relationship between the stable carbon isotope composition of whole wood and its cellulose components.

2 Materials and methods

2.1 Study area and samples

The Yuncheng Basin is in the south-western region of Shanxi Province, China, and is adjacent to the Chinese Loess Plateau. This region has a temperate continental monsoon climate, with an average annual temperature of 13.7°C and annual precipitation of ~550 mm. Most of the rainfall occurs between June and September.

The fossil wood analyzed in this study was collected at the depth of 279 m in a drill core (YC20, 35°02′N, 111°01′E, Fig. 1) from the Yuncheng Basin. The 7.5 cm diameter fossil wood specimen showed distinct boundaries between early and late wood after polishing (Fig. 2). Its age is estimated to be Middle Pleistocene, based on stratigraphic correlations with drill core P3 from a nearby region (Fig. 2), which is well-dated using paleomagnetism (Wang et al., 2002).

2.2 Cellulose extraction

We initially applied conventional extraction methods for fossil wood, designated Method A (Brendel et al., 2000) and Method B (Green, 1963). However, due to the inability of these methods to extract cellulose, we developed a series of improved procedures based on cellulose extraction methods for both modern tree rings and fossil wood (Green, 1963; Brendel et al., 2000; Evans and Schrag, 2004; Gaudinski et al., 2005; Anchukaitis et al., 2008; Liu et al., 2010; Xu et al., 2011; Hook et al., 2015; Kagawa et al., 2015; Ren et al., 2017). These methods are described below (Table 1).

Method A Lignin was removed with nitric acid and acetic acid, hemicellulose was removed with 17% NaOH solution, and lipids were removed with ethanol and acetone (Brendel et al., 2000).

Method A1 Modification of Method A. Hemicellulose was removed with 2% NaOH.

Method B This used the classical sodium chlorite processing method established by Green (Green, 1963), but with the extraction sequence of α-cellulose modified according to the improved extraction procedures developed by various workers (Gaudinski et al., 2005; Xu et al., 2011). These improved procedures involved the use of a solution of sodium chlorite and acetic acid added to remove lignin, after which 17% NaOH solution was added to remove hemicellulose, and then ethanol and acetone were added to remove lipids.

Method B1 Modification of Method B. Hemicellulose was removed with 2% NaOH.

The detailed steps (Fig. 3) are as follows.

1) Cleaning the sample. A scalpel was used to gently scrape away impurities adhering to the surface of the sample, which was then washed with deionized water and air-dried at room temperature.

2) Separating the sample. The samples were first cut into thin slices, separated under a microscope with a scalpel at high resolution, according to the annual tree rings, and placed in centrifuge tubes. Sample separation was performed on a glass plate, and to prevent cross-contamination, separated samples were placed in centrifuge tubes using sulfate paper and numbered. Finally, the scalpel was cleaned with anhydrous ethanol.

3) Bleaching with sodium hypochlorite to remove lignin. 1 g NaClO₂, 0.5 mL CH₃COOH, and 35 mL hot deionized water were added to the beaker. A pipette gun was then used to add the prepared solution to each centrifuge tube. The tubes were then placed on a heating plate at 70°C for 60 min, and after removal were separated by centrifugation, and the upper layer of liquid was carefully removed with a syringe. This step was repeated 4–7 times until the sample turned white. The tubes were then rinsed three times with deionized water, until the color (yellow) of the solution disappeared completely.

4) Removal of hemicellulose and the decomposition of lignin with NaOH. 2% NaOH was added to each tube and the tubes were placed on a hot plate at 80°C for 30 min. After removal from the hotplate the tubes were placed in a centrifuge. After centrifugation, the upper layer of clear liquid was carefully withdrawn using a syringe, and this step was repeated twice (1–3 reactions in total). The sample was then rinsed with deionized water and centrifuged, repeated four times.

5) Reduction of the alkalinity with dilute HCl. 1% HCl was added to each centrifuge tube, and after centrifugation the supernatant was withdrawn. The samples were cleaned using step (4) until the solution was neutral.

6) Removal of lipids with organic solvents. The cellulose was cleaned in a centrifuge tube with acetone and ethanol, separated by centrifugation, and the supernatant withdrawn. The α-cellulose in the tubes was dried in an oven at 50°C, or freeze-dried, and stored for subsequent stable isotope analysis.

2.3 Evaluation of α-cellulose purity

We used Fourier transform infrared (FTIR) spectroscopy to assess the purity of the extracted cellulose. This was conducted using a Nicolet iS5 Fourier transform infrared spectrometer. The extracted α-cellulose was subjected to FTIR analysis and then compared with α-cellulose obtained from modern plant samples (Picea) and an international standard tree-ring sample. FTIR measurements were performed at the Analytical and Test Center, College of Chemistry and Chemical Engineering, Lanzhou University.

We utilized an Apreo S scanning electron microscope (SEM) for detailed observation of the α-cellulose samples. During the observation process, various working distances and magnifications were employed to enable precise visualization of the macrostructure of the cellulose. Before SEM analysis, both fossil wood α-cellulose and modern α-cellulose samples were gold coated to enhance conductivity. Additionally, energy dispersive spectroscopy (EDS) was employed to characterize the elemental composition of the fossil wood and extracts, as well as to detect any potential mineral contaminants. SEM and EDS were performed at the Electron Microscopy Center of Lanzhou University.

2.4 Stable carbon isotope analysis

Samples of 0.05 mg were enclosed in tin capsules and shaped into spheres or cubes. The samples were then converted to gas in a high-temperature conversion analyzer (Flash EA), and then the ¹²C/¹³C ratio was determined on a MAT-253 gas stable isotope mass spectrometer. The accuracy and reproducibility of the carbon isotope analysis were monitored via replicate analysis of standard graphite samples (16‰). The isotopic results for carbon are presented using the δ notation as per mil (‰) with respect to VPDB (Vienna Pee Dee Belemnite), with the δ¹³C analytical error (standard deviation) being < 0.05 ‰. δ¹³C was calculated using the following equation:

(1){\delta }^{13}\mathrm{C}=[(R_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}/R_{\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}})-1]\times 1000,

where R_sample and R_standard are the ¹³C/¹²C ratios in the sample and standard, respectively. The stable carbon isotopes of α-cellulose were determined in the Stable Isotope Laboratory of the Key Laboratory of Western China’s Environmental Systems, Lanzhou University, China.

3 Results

3.1 Comparison of the different extraction methods

We were unable to extract cellulose using either method A or method B. However, methods A1 and B1 successfully extracted cellulose with a high extraction rate when the NaOH concentration was reduced to 2%. Notably, the alpha-cellulose obtained using method A1 had a yellowish color compared to that extracted by method B1, suggesting that some component was not completely removed. Therefore, we suggest that extraction method B1 is the best choice for this type of sample.

3.2 Verification of α-cellulose

The results of Fourier transform infrared spectroscopy (FTIR) analysis showed that the extracted α-cellulose of the fossil wood had removed resin (1600 cm⁻¹), lignin (1500–1450 cm⁻¹), and non-cellulosic polysaccharide (1230–1180 cm⁻¹) (Anchukaitis et al., 2008). The FTIR of the α-cellulose extracted from the fossil wood, α-cellulose from modern plant samples (Picea), and the international standard tree ring α-cellulose were simultaneously analyzed and the results revealed a consistent trend. The transmittance range across all samples was uniform (Fig. 4). The transmittance of α-cellulose in the international standard tree ring was lower than that of the fossil wood and modern plant samples, most likely caused by the differences between the samples themselves and the different extraction methods. However, using the same method for extracting α-cellulose from the fossil wood and modern plant samples (Picea), we found that the infrared spectra of the α-cellulose were highly consistent. This indicates that the extracted α-cellulose is of high purity and the extraction method is effective.

SEM was employed to examine the structural characteristics of the α-cellulose extracted from fossil wood, and to compare it with the α-cellulose extracted from modern plant samples (Picea). The SEM images of the cellulose extracts revealed a fibrous structure, which is consistent with modern cellulose (Fig. 5). However, the α-cellulose extracted from fossil wood was noticeably degraded, with an overall fractured appearance, different from the three-dimensional structure of modern samples. The texture was relatively flat, and fine lines and folds were not clearly discernible. Cellulose chains exhibited varying degrees of folding and pitting, with some chains appearing twisted and entangled (Savard et al., 2012). This suggests structural alterations in the fossil wood cellulose compared to modern cellulose.

White particles were present on the surface of the polished fossil wood (Fig. 6(a)), manifested as white crystals under the microscope (Fig. 6(c)). These particles may have formed due to the prolonged burial of the fossil wood in Yuncheng Salt Lake, with mirabilite precipitating from the lake water and adhering to the wood surface. EDS analysis of the white particles revealed the presence of S, Fe, and a small amount of Mg. S and Fe were also detected in the whole wood samples, in addition to prominent peaks in C and O, indicating the presence of minerals within the wood composition. However, upon extraction, the α-cellulose was found to be devoid of these white particles, as confirmed by EDS analysis, showing strong peaks in C and O only, as illustrated in Fig. 7.

3.3 Stable carbon isotope results

The stable carbon isotope values of fossil wood α-cellulose (δ¹³C_cell) ranged from −22.22‰ to −25.18‰, with an average of −23.77‰. In comparison, the stable carbon isotope values of whole wood (δ¹³C_wood) ranged from −25.00‰ to −27.62‰, with an average of −26.36‰ (Table 2). Notably, the stable carbon isotopes of the whole wood value were lower than those of the α-cellulose. Orthogonal regression analysis conducted on fossil wood α-cellulose and whole wood samples yielded regression coefficients of determination (R²) of 0.51 (Fig. 8(a)), 0.54 for earlywood (Fig. 8(b)), and 0.49 for latewood (Fig. 8(c)), indicating a relatively strong linear correlation between δ¹³C_cell and δ¹³C_wood for the fossil wood samples. Additionally, the α-cellulose content of the fossil wood ranged from 0.2% to 3.5%, which was lower than that of other fossil wood samples reported in previous studies (Ren et al., 2017; Lukens et al., 2019; Ren et al., 2023). This discrepancy may be attributed to the old age of the samples and their prolonged preservation in a humid environment, leading to accelerated cellulose degradation and consequently a low cellulose content.

4 Discussion

4.1 Key procedures for extracting α-cellulose from fossil wood

The extraction of cellulose is crucial for stable isotope analysis. Fossil wood, undergoing extended geological burial processes (Rutherford et al., 2005), experiences differential degradation rates among its components (Benner et al., 1987; Spiker and Hatcher, 1987; Schleser et al., 1999; McCarroll and Loader, 2004). Cellulose and hemicelluloses tend to degrade more rapidly than lignin (Hedges et al., 1985; Schleser et al., 1999; van Bergen and Poole, 2002; Loader et al., 2003), presenting a significant challenge for α-cellulose extraction from fossil wood. In our experiments, we assessed both the Brendel method (Brendel et al., 2000) and the Jayme-Wise method (Green, 1963) for cellulose extraction. After numerous attempts, we found that the Brendel method was suboptimal for our sample. Modern tree ring α-cellulose is typically considered to be insoluble in a 17.5% NaOH solution; and our experiments showed that this concentration was not suitable for fossil wood, which is consistent with the findings of Hook et al. (2015). Due to the extreme age of fossil wood, hemicellulose degrades faster than α-cellulose (van Bergen and Poole, 2002; Gelbrich et al., 2008), and the structurally weaker hemicellulose is likely to have broken down into sugar monomers (Rissanen et al., 2014) and been slowly leached out of the material. Thus, the effect of hemicellulose on the δ¹³C results of fossil wood may be minor. To address potential residual hemicellulose, we tested different concentrations of NaOH, with reference to the concentrations in the experimental protocol of Hook et al. (2015) and found that a 2% NaOH solution was more suitable for our samples. Following NaOH treatment, alkaline residues may be present in the sample, requiring neutralization with a weak acid followed by washing with deionized water until the solution reaches neutrality to mitigate the effect of residual reagents during extraction.

We also found that the number of centrifugation cycles and the reaction time were critical factors in cellulose extraction. A centrifuge is used to separate the waste liquids prior to each extraction in order to retain as much α-cellulose as possible; moreover, the samples are mixed during centrifugation to ensure homogeneity and minimize the loss of α-cellulose. Due to the longer burial time of fossil wood, a longer bleaching time is required to completely remove lignin and achieve a white sample compared to modern plants. During the delignification phase, the upper solution initially appeared reddish-brown in color which gradually turned to near-transparent yellow due to the formation of iron oxide, which is common in fossil wood samples (Richter et al., 2008). The incomplete removal of lignin can bias the isotopic signal, resulting in the δ¹³C values of whole wood and lignin being lighter than those of cellulose (Loader et al., 2003). Therefore, the bleaching time should be adjusted according to the burial time to maximize lignin removal while avoiding excessive cellulose loss and reducing the cellulose extraction rate. Furthermore, pre-treatment and the use of organic solvents should be considered. Due to the prolonged burial time, fossil wood often accumulates soil and other contaminants on its surface, some of which may be highly mineralized. It is therefore essential that samples are cleaned prior to chemical treatment to remove impurities. Treatment with organic solvent is time-consuming and toxic, and it has been suggested that it may not be necessary to remove resinous substances (Rinne et al., 2005). However, several studies have shown differences in isotopic values between samples with and without organic solvent extraction (Long, 2006; Au and Tardif, 2009). Therefore, to ensure the accuracy of isotope assays, we employed acetone and alcohol to remove resinous substances, ultimately obtaining α-cellulose with a high purity.

4.2 Comparison of δ¹³C values between whole wood and α-cellulose, and its paleoclimatic implications

Tree rings contain various substrates, and there are different opinions on which substrate should be used for δ¹³C analysis to better extract paleoclimatic information (Wilson and Grinsted, 1977; Borella et al., 1998; Loader et al., 2003; Cullen and Grierson, 2006). In stratigraphic sequences, fossil wood preserves basic woody structures such as cellulose and lignin formed during growth, and their stable isotope indices offer new research perspectives for investigating climate changes in deep time. However, there is a lack of stable isotope data for fossil wood due to preservation difficulties, low cellulose content (Ghavidel et al., 2020), and dating difficulties; hence, there is debate about which component of whole wood, or cellulose in fossil wood, better reflects paleoclimatic changes.

In our study, the R² value between whole wood (δ¹³C_wood) and α-cellulose (δ¹³C_cell) was 0.51 (Fig. 8(a)). This relationship is generally consistent with those reported by Lukens et al. (2019), which suggests that whole wood can be used for qualitative paleoclimatic reconstruction. Hence, if the extremely low cellulose content of fossil woods makes it difficult to meet the requirements of high-resolution paleoclimate reconstruction, this relatively high linear correlation shows that the use of whole wood rather than α-cellulose should be considered.

For quantitative paleoclimate reconstructions, the apparent enrichment factor (ε) between δ¹³C_wood and δ¹³C_cell is a critical parameter for determining the reliability of δ¹³C_wood records. Lukens et al. (2019) compiled 1210 paired δ¹³C_wood and δ¹³C_cell values of fossil wood and modern trees and found that the ε value and cellulose content were negatively correlated. Using data from the literature (Lukens et al., 2019) and our own data, we observed that the calculated ε values were negatively correlated with the cellulose content and very weakly correlated with the α-cellulose content. The lower the cellulose content the higher the ε value, and the higher the cellulose content the lower the ε value (Fig. 9). This suggests that the cellulose content may have an effect on δ¹³C_wood values, which further suggests that the use of whole wood carbon isotopes for paleoclimatic reconstruction may be biased when the cellulose content is low and thus α-cellulose extraction is required. We therefore recommend caution when using records of δ¹³C_wood for quantitative paleoclimatic reconstruction, and there is the need to consider the preservation state of the fossil wood and changes in the cellulose content.

5 Conclusions

Based on the analysis of fossil wood from the Yuncheng Basin, we have developed an improved method for α-cellulose extraction. We identified the reaction time, NaOH concentration, and the number of centrifugations as crucial factors influencing the efficiency of cellulose extraction. There was a high correlation between δ¹³C_wood and δ¹³C_cell, indicating that whole wood is a dependable proxy for α-cellulose to qualitatively determine paleoclimatic trends. However, the stable carbon isotope values of whole wood were lower than that of α-cellulose, and the ε values were negatively correlated with the cellulose content. This suggests that caution should be exercised when using records of δ¹³C_wood for quantitative paleoclimatic reconstruction.

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