Monitoring transformation of two tropical lignocellulosics and their lignins after residence in Benin soils

Chemical composition of the G. arborea and S. latifolius RCWs after 0 (RCW0), 6 (RCW6), 12 (RCW12) and 18 (RCW18) months of soil incubation

As presented in Fig. 1, the chemical constituents of studied samples varied significantly as shown by polynomial contrasts (linear and quadratic) with the progress of RCW decomposition in soils for G. arborea and S. latifolius. Total extractive contents decreased by more than 60% (compared to the initial content) for the two species after 18 months of soil incubation. This result is consistent with those described in other published reports, which indicated that extractive contents progressively decreased during wood decomposition^34,35. Extractives, given that they are “free molecules,” most notably phenolic products, are the first to be released during decomposition. In both species, total carbohydrate contents also were initially much higher than total lignin contents (Fig. 1), which is consistent with what has often been reported in the literature, including our own work^29,36,37,38.

As RCW decomposition progressed in soils, total lignin contents increased linearly, whereas total carbohydrate contents decreased with the same trend. For G. arborea, total carbohydrate loss occurred rapidly during the first 6 months of decomposition, in contrast to S. latifolius, for which the greatest carbohydrate loss was determined after 12 months. During organic matter decomposition, rapid initial losses of matter often can be attributed to leaching^36,39. However, the rapid loss of water-insoluble components, such as polymer carbohydrates (polysaccharides), during the first 6 to 12 months of decomposition, suggests more rapid microbial catabolism of carbohydrates from RCW. The latter phenomenon seems to be much more pronounced for G. arborea than in S. latifolius. In addition to this response, several studies have reported that carbohydrates are preferentially degraded by brown-rot fungi (e.g., Gloeophyllum spp.) with a concomitant increase in relative lignin content with wood mass loss^{21,40,41,42,43}. The decline in carbohydrate content could also be attributed to slow chemical autohydrolysis during decomposition in the soils⁴³. Indeed, lignin is known to be recalcitrant to degradation relative to other wood components. Only a limited range of microorganisms, primarily white-rot fungi (Basidiomycotina), are reported to be capable of extensively depolymerizing the lignin structures^21,41,44.

Ash content of the RCW increased with linear (p < 0.0001) and quadratic (p < 0.0001) trends as the RCW decomposition proceeded, reaching up to four and two times the initial ash content of RCW of G. arborea and S. latifolius, respectively. As indicated in our previous work², the initial ash contents of study RCW were comparable to those reported in the literature. The significant increase of ash contents after 18 months of RCW decomposition suggests the potential contribution of microbial activity to mineralization, leading to release of minerals favourable for soil fertility and crop nutrition.

Remaining mass, elemental analysis and degradability indices of RCW of G. arborea and S. latifolius during soil incubation

Figure 2 summarizes the data on remaining mass (Fig. 2A), elemental composition and degradability indices (Fig. 2B) and mineral content (Fig. 2C) of the study RCW. RCW mass losses decelerated over time for S. latifolius and G. arborea and had linear trend after 6, 12 and 18 months in the soils (Fig. 2A). The total mass loss of RCW throughout the decomposition period was higher for G. arborea (80%) than for S. latifolius (70%). The percentage of maximum mass loss was recorded between months 12 and 18 for S. latifolius RCW and between months 0 and 6 for G. arborea RCW. This difference in mass loss between the two species could be associated with the high amount of labile C in the carbohydrate and extractive components of G. arborea RCWand high quantities of recalcitrant C that was associated with the lignin of S. latifolius RCW, as reported in our previous work². The C content for both species showed a relative decrease in time, while the O contents generally exhibited a slight increase, which was probably related to oxidative microbial transformation.

More interestingly, the N content of RCW increased substantially, reaching 2.6 and 1.9 times the initial content for G. arborea and S. latifolius, respectively. Progressive N-enrichment of RCW indicates that its application could improve the release and availability of N in soils, which is fundamental to maintaining soil fertility and important for plant nutrition. These results are in agreement with published reports, which also confirmed an increase of N content in decomposing biomass in soils^29,45,46. This could be explained, at least in part, by an immobilization stage involving microorganisms, the enzymes that they secrete, and the protein cores of the former which contain nitrogen⁴⁵. Pei and coworkers demonstrated that N released during decomposition can be immobilized by microorganisms, stabilized in the soil organo-mineral particles or absorbed to sustain crop productivity^38,46,47.

C/N ratios are often used as degradability indices to explain the decomposition patterns of lignocellulosic materials under study. The initial RCW had a C/N ratio between 82.76:1 and 94.92:1 and, theoretically, would result in strong nitrogen immobilization. The C/N ratio of RCW progressively decreased throughout the 18 months of the study, achieving a value of almost one-third (around 33.32 ± 0.2) and half (40.65 ± 0.23) of the initial ratios that have determined respectively for G. arborea and S. latifolius RCW. There is a positive correlation (0.94, P < 0.001) between C/N ratio and the remaining mass; the C/N ratio of RCW at 18 months is slightly higher than 25:1, indicating N-immobilization, but to a lesser degree than in the initial stages of decay. For both species, there was a negative correlation between C/N ratio and N content (− 0.95, P < 0.001), indicating that C/N ratio was a key factor affecting N enrichment as reported by Pei et al.⁴⁶.

Mineral contents of RCW (Fig. 2C) varied in various linear and quadratic trends with the RCW decomposition progress in soils for both species. Phosphorus and potassium contents significantly decreased, while calcium and manganese contents increased in RCW after 18 months of decomposition in soils for both G. arborea and S. latifolius RCW. The increase in manganese content could be related to the activity of white rot fungi since their enzymatic system comprises normally manganese-dependent lignin peroxidase^41,48. Magnesium content decreased with RCW decay in G. arborea, while the opposite result was observed for S. latifolius. This relative variation in mineral contents reflects different dynamics of their release during RCW mineralization in soils, which is intended to improve nutrient status of the soils.

Analytical pyrolysis Py-GC/MS of the RCW after residence in soils

Thermally assisted Hydrolysis and Methylation was conducted to follow changes in the composition of RCW from G. arborea and S. latifolius as these materials decomposed in the soil. Pyrograms of RCW from G. arborea and S. latifolius are shown in Figure S1 (supporting data). The compounds that are reported in Table 1 revealed that the syringyl guaiacyl (S + G) sum corresponding to lignins increased, while the sum of the compounds corresponding to carbohydrate content decreased. Several papers revealed that pyrolysis technique is less effective in detecting carbohydrate units in respect to the lignin component^49,50. This is probably the result of pyrolytic rearrangement of polysaccharides in complex matrices combined with the derivatizing agent (TMAH) which prevent chemical access and are believed to negatively interfere in the diagnostic release of carbohydrates derivatives and polysaccharides components. However, these trends of decreasing carbohydrate content are also indicated by CH/L ratio (carbohydrate/lignin ratio), confirming the results of global chemical analyses that are presented in Fig. 1. The main guaiacyl (G) moieties that were released by RCW samples from G. arborea have been identified as peaks 10R, 16R and 22R (Table 1), while the main syringyl (S) moieties of the same samples were identified as peaks 25R and 26R. Results showed that G. arborea samples contain GS-type lignin derivatives, dominated by S units, as confirmed by their lignin analyses presented below and reported in our previous study². The S/G ratio of G. arborea samples increased slightly with RCW decomposition in soils, except for RCW18. This result could be explained by the fact that lignins from G. arborea are S-dominant. However, the moieties consisted of S units that are known to cleave more rapidly. Due to the easier cleavage of the ether bond by which the S-units are preferentially linked, the S unit content remained high. The very high initial S/G in G. arborea samples could perhaps explain the slight increase of the S/G ratio in the RCW samples even after 18 months incubation in soils. As for the RCW samples from S. latifolius, the main G moieties were identified (Table 1) in addition to those that were identified in G. arborea samples. The major compounds based on S units are the same as those identified in G. arborea samples, but with a lower relative abundance. The relative abundance of lignin derivatives units (S, G) (Table 2) showed that RCW from S. latifolius has GS-type lignin that is dominated by G units. The trend is linear (p < 0.0001) and, there is an indication of decrease in the S/G ratio, indicating greater recalcitrance of G-lignin. The lower initial value of the S/G ratio in S. latifolius compared to G. arborea is consistent with the results of other analytical methods (higher Klason lignin + acid solubles, higher C content) as has been reported in previous studies^{28,30,31,51,52,53}.

Yield, purity, and elemental analysis of organosolv lignins extracted from RCW as degradation proceeded in soils

It has been demonstrated previously² that the organosolv process is well-suited to get access to high purity lignins from the RCW of G. arborea and S. latifolius, thereby increasing their suitability for studies of chemical structures. Table 3 presents the properties and elemental analysis of organosolv lignins that were isolated from G. arborea and S. latifolius RCW. The yield of organosolv lignin from the G. arborea RCW was greater than 15% and did not vary substantially with the progress of RCW decomposition within the experimental timeframe. However, the yield of organosolv lignin from S. latifolius RCW samples decreased with RCW incubation time, as reported in Table 3. For both species, lignin recovery decreased with RCW decomposition and this decrease is much greater for lignin of S. latifolius than G. arborea. Thermally assisted hydrolysis and methylation (THM) determined that more condensed structures accumulated in RCW lignins with increasing duration of incubation (Table 4), which could explain the difficulty of extracting greater proportion of lignins by the organosolv process. Nevertheless, the selected organosolv process seems appropriate, given that it yielded, from both species, organosolv lignins with purities higher than 95% (sum of Klason plus acid soluble lignin of the isolated lignins) and, therefore, suitable for structural studies. The elemental composition of the isolated organosolv lignins is presented in Table 3, revealing significant variation in C, H, O, and N contents. The presence of N in the lignins under study could be due to the potential formation of protein-lignin complexes during the organosolv pulping process⁵⁴. Yet, this could also be explained by nitrogen presence in the native lignins, as nitrogen content has been determined in organosolv lignins extracted following all litter bag retrievals (0, 6, 12, 18 months) (Table 3). These findings indicate that lignin-protein chemical bonds are strong and, therefore, difficult to remove through pulping^2,54. It should be noted that the N content of the lignins significantly increased as RCW decomposition progressed in soils. This was demonstrated for all RCW samples (Fig. 2), indicating the potential reactions between lignins and proteins from enzymes or microorganisms as the likely sites of nitrogen accumulation upon RCW degradation in soils.

Py-GC/MS of RCW G. arborea and S. latifolius lignins

Organosolv lignins from G. arborea and S. latifolius were also analyzed by the Thermally assisted Hydrolysis and Methylation (THM), as was the case for the RCWs (Table 1). Figure 3 shows the chromatograms of pyrolytic products that were detected by THM for the four lignins of G. arborea and S. latifolius, respectively. The identified products and their relative abundances are listed in Table S1 (Supporting data). Based upon the figure and Table 4, the distribution of the pyrolytic products from the lignins showed linear and quadratic variation in their chemical structure as the RCW degradation progressed in soils. The main S-unit moieties that were present in the lignins were identified as peaks 15L, 16L, 18L, 22L, 25L, 26L, 30L, 37L (L refers to lignin). While the G-unit moieties were identified as 7L, 10L, 11L, 17L, 19L, 21L, 23L, and 35L. A comparison of products from initial and decomposed RCW lignins of both species showed that benzene, cis-1-(3,4,5-trimethoxyphenyl)-1-methoxyprop-1-ene (peak 30) increased in abundance relative to other pyrolysis products upon degradation. The relative abundance of main compounds that were released and the S/G ratio of lignins are summarized in Table 4. S-unit abundance increased, while those of G-units decreased slightly for lignin samples as RCW decomposed in soils. p-hydroxyphenyl (H)-unit abundance is very low (0.33–0.94%), indicating that lignins are G-S type as determined previously. This distribution of lignin derivatives is similar to that observed following THM of lignin from several hardwoods^28,31. Carbohydrate pyrolysis products such as anhydrosugars (1,6-anhydro-β–d-glucopyranose), pyrans and furans^28,52 were not detected (Fig. 3), confirming the purity of the organosolv lignin. This reults also confirms the results regarding the high purity of the lignins under study (low carbohydrate content) (Table 3). The validity of choosing the organosolv process was affirmed, given that it led to high purity lignin preparations^33,55. It shoud be noted that the recovery of lignin by organosolv process steadily decreased for lignins, which had been isolated from the RCW after 6, 12 and 18 months (Table 3). Nevertheless, the isolated lignins were determined to be of high purity, a compromise that must be made regardless of the method of lignin extraction that is chosen. The S/G ratio of the studied lignins steadily increased for the lignins isolated from RCW after long incubations in the soils, which could reflect the fact that a more easily soluble part of partially decomposed lignin has been recovered preferentially by the organosolv method that was applied with the same parameters throughout this study. This result could also indicate progressive depolymerization of lignin during decomposition, since it is well known that with increasing decomposition of woody biomass, the S/G ratio is usually increasing^15,21.

Polymer properties of lignins isolated from the RCW samples

Values of the number average (Mn), weight-average (Mw), and polydiversity index (PDI) are presented in Table 5. The molecular weight distribution curves are shown in Figure S2 (Supporting data). Mn values varied significantly with time. No significant difference for Mw and PDI was apparent over time or between species, but lignin that was isolated from RCW after 18 months of incubation in soil appears much less stable than that of the original material given that lower polydispersity is indicative of good physicochemical stability of lignin⁵⁶. Mw and PDI both vary depending upon the lignin isolation process, molecular weight distribution methods, and the type of plant material and its degradation state^2,57. In considering the Mw data of all samples as a function of their incubation time in soil, an increase (although not significant) was noted in the average molecular weight (Mw) of all lignin samples, from 1703 to 2028 g/mol for G. arborea and from 1692 to 2158 g/mol for S. latifolius. This response clearly indicates that lignin polymerization reactions with enzymes of microorganisms are taking place, as observed in the RCW analysis.

2D-HSQC NMR analysis of the organosolv lignins isolated from the RCW of G. arborea and S. latifolius

To obtain additional information regarding the progress of changes in the studied lignin structures after incubation in the soil, 2D-HSQC NMR analyses were performed based upon methods that have been described in previous studies^20,57,58,59. Figures 4 and 5 present the aliphatic oxygenated region (δ_C/δ_H 50–90/2.5–5.8) and the aromatic/unsaturated regions (δ_C/δ_H 90–155/6.0–8.0) of the 2D-HSQC NMR spectra of the organosolv lignins from the RCW of G. arborea and S. latifolius, respectively. The assignments of cross-signals that are related to the structural units and interunit bonds in the study lignins are listed in Table S.1 and are based upon data that have been taken from the literature^2,19,37,60. The aliphatic oxygenated region of the spectra (Figs. 4, 5, top panel) have yielded useful information about the types of interunit linkages in the lignins under study. The correlation peaks (δ_C/δ_H) from methoxy (MeO) and β-O-4′ aryl ethers (A) were the most prominent in the HSQC spectra of the lignins of both species. It should be noted that this result confirms that the major interunit moiety of lignins does survive the decomposition process (Table 6). It is remarkable that the β-O-4′ moiety (Fig. 6), which could be regarded as the hallmark of lignin identity, is preserved. HSQC spectra revealed the presence of other characteristic signals which correspond to C_α-H_α (71.8/4.83 ppm), C_β-H_β (83.9/4.28 ppm), and C_γ-H_γ (60.2/3.66 ppm) correlations for the β-O-4′ substructures (A), C_β-H_β (86.9/4.08 ppm) correlations in γ-acylated β-O-4′ linkage. These β-O-4′ substrcutures were less prominent in the lignins from RCW corresponding to longer residence in soils. In addition, other correlation peaks are visible in the spectra including signals for β-β′ resinol (B) in all lignin samples and β-5′ phenylcoumaran (C), which is only present in the organosolv lignin from the RCW of S. latifolius. The presence of the latter confirms the importance of guaiacyl units for this lignin; this component has been determined for the lignin sample that was isolated from the initial RCW (time zero).

In the aromatic unsaturated region of the HSQC spectra (Figs. 4, 5, bottom panel), the main correlation peaks that were found correspond to the aromatic rings of different lignin units (S, G), and to p-hydroxycinnamates (p-coumarates, pCA). A strong characteristic signal of S and G units was identified by their correlation peaks at δ_C/δ_H 104.2/6.61, 110.7/6.98, 115.5/6.67, 119.9/6.91 ppm corresponding to S_2,6, G₂, G₅ and G₆, respectively. Also, the signal corresponding to Cα-oxidized S-units (S′_2,6) at δ_C/δ_H 107.4/7.28 ppm was only observed in lignins from G. arborea samples. All study lignins were determined to contain a signal of p-coumarate (pCA), which was confirmed by important correlation (δ_C/δ_H) at 115.7/6.69 ppm (pCA_3,5), while signals corresponding to p-coumarate in C₇–H₇ (pCA₇) (137.3/7.87 ppm) and pCA2,6 (124.7/7.44 ppm) were only found in the spectral data for lignins from S. latifolius.

The relative content of the main lignin substructures, the molar content of S and G units, as well as the molar S/G ratio of the study lignins was evaluated from the contour integration volume in the 2D HSQC spectra. The results are presented in Table 6^20,57,58,59. The semiquantitative results of the lignins under study demonstrate a predominance of β-O-4′ aryl ether linkages, which decreased from 63.34 to 37.18% for G. arborea and from 65.67 to 34.92% for S. latifolius as the duration of soil incubation increased. At the same time, the relative content of β-β′ linkages decreased from 9.83% to 2.51% in G. arborea and from 20.1% to 7.49% in S. latifolius, respectively. Furthermore, β-5′ substructure was only determined for S. latifolius samples and it decreased from 7.77 to 2.98% with increasing length of incubation.

The results indicate a progressive degradation of lignins with the increase in residence time of RCWs in soils, which seems to be more pronounced for lignin of G. arborea than that of S. latifolius (Table 6). Previous research has indicated that the cleavage of β-O-4′ linkages was the main mechanism of lignin depolymerization during wood decomposition^41,59. This supports our result suggesting that RCW decomposition in soils is linked to the decline in β-O-4′ aryl ether linkages; nevertheless, they remain important moieties in the study lignins. Moreover, the S/G ratio of lignin is considered to be a major indicator of lignin depolymerization by white-rot fungi in soils^15,21,41. The S/G ratios that were calculated increased from 1.68:1 to 2.07:1 and from 0.83:1 to 1.01:1 for RCW lignins that were isolated respectively from G. arborea and S. latifolius. Increased S/G ratios indicated that effective degradation of lignin was occurring during RCW decomposition, which agrees with thermochemolysis using tetramethylammonium hydroxide (TMAH) results. It is also important to note the incorporation of p-hydroxycinnamates in lignin of Sarcocephalus latifolius RCW, which agrees in part with results of the thermochemolysis using TMAH that are presented in Table 4.