The litter decomposition process depends on the litter chemical composition, especially the ratio between more labile compounds, cellulose, and the recalcitrant lignin and waxes. Their determination is crucial to predict the process, though lignin measurement presents some limitations due to drawbacks of the different methods. Thermal analysis has been successfully applied to several organic materials in order to obtain quali-quantitative information of the chemical structure of the sample. In this work TG-DTA was used in a short-term litter decomposition study of two broadleaf forest stands of contrasting ages, and the results were compared to those obtained with a chemical method (Klason’s method) commonly used to quantify cellulose and lignin. TG-DTA was applied to the litter and to the cell walls (CW) extracted from the litter, whose cellulose and lignin content was determined using the Klason’s method. When applied to litter, thermal analysis showed a weak correlation with the Klason’s method, though it allowed the detection of the dynamics of waxes, that increased during the decomposition and could influence the later stages of the process. Contrastingly, a good correlation between cellulose and lignin determined with the two methods was found when TG-DTA was applied to the CW. In this case TG-DTA, according to NMR data, also highlighted the changes in the CW chemical structure compared with that of the litters, in particular the loss of waxes and the decreased thermostability of aromatic components. Moreover, a new concept of quality of the decomposing litter, based on the balance between the energy stored in the litter and the energy needed to release it obtained by thermal analysis, was recently introduced. Samples of the old forest litter had an initial energetic balance more favorable than those collected in the young stand. At the end of the period, the decrease in litter quality was greater in the young than in the old forest samples, due to the combined effect of the higher degradation of thermolabile substances and the accumulation of more thermostable components. Thermal analysis seems to have a good potential in litter decomposition studies, as it can link structural and energetic changes during the process.
In recent years several methods have been suggested to evaluate the chemical composition of litter and its change during the decomposition process. When environmental factors, such as temperature, redox potential and water availability are the same, the litter decay depends on its chemical composition, in particular the nutrients content and the ratio between labile and recalcitrant compounds. These characteristics, together with the physical structure of decomposing litter define the litter quality that is a quantitative measure of the litter biodegradability by soil microflora. As decomposition proceeds the more labile substances are generally consumed first and faster than the recalcitrant ones, such as lignin and waxes, that tend to accumulate in the remaining litter, which quality consequently decreases. However, evidences have been reported in broad-leaf species that also recalcitrant components such as lignin can undergo microbial degradation since the beginning of decomposition, and decreases in lignin content up to 60% of the initial amount can be measured in the first six months of decomposition (
Many indexes of litter quality were suggested as predictors of decomposition rate (
For most of these indexes, a crucial point is the measurement of the lignin content, for which several methods have been developed over the last years. Some of them are non-destructive spectroscopic methods that exploit the chemical properties of lignin to absorb radiation in discrete regions of the electromagnetic spectrum. They are based on different spectroscopic technologies, such as infrared and diffuse-reflectance Fourier transform (DRIFT) spectroscopy (
Chemical methods have also been frequently used in litter decomposition studies to estimate lignin content (
Recently, thermal analyses were applied to understand modifications in chemical structures taking place during litter decomposition (
In this study we evaluate the potential of thermal analysis technique in determining structural changes during litter decomposition of two broadleaf forests differing in their age. A short-term decomposition experiment was carried out to evaluate whether even small quality changes of litters were detectable with this technique. To this purpose, TG-DTA data were compared to the cellulose and lignin dynamics measured by chemical method that requires, as a first step, the extraction of CW. The thermal behavior of CW was therefore investigated. Moreover, the energetic approach introduced by
The present study was carried out over a 9-month period, from April to December 2003, in two adjacent woodlands characterized by a similar species composition, but with different ages. The older stand was a 280-year old hardwood forest, while the younger was a 30-year old plantation established on former agricultural land. In both forests, oak (
Litter decomposition was studied using the litter-bag technique, nylon bags (20 × 20 cm) of 1 mm mesh size were used (
The CW was extracted according to the method of
Cellulose was determined using a 72% sulfuric acid digestion. Residual tissue from the digest was considered as lignin, and the mass lost during the digestion was considered as cellulose (
Elemental analyses were carried out on both whole litter samples and extracted CW. Carbon and N content were measured with an elemental analyser (CHNS-O mod. EA 1110 Thermo-Fisher) using acetanilide as a standard.
High Resolution Magic Angle Spinning (HR-MAS) NMR spectra were recorded with a Bruker FT-NMR Avance 400 Spectrometer at 298K using 8kHz spinning rate. Nominal frequencies were 400.13 MHz for 1H and 100.61 MHz for 13C. An internal lock on the deuterium of DMSO-
All the samples were investigated by applying different pulse sequences, in order to obtain 1D 1H NMR spectra. Carr-Purcell-Meiboom-Gill (CPMG) sequence combined with a pre-saturation pulse for water suppression allowed the best results. Since integrated areas of the same spectral region may significantly vary according to the pulse sequence applied for acquisition, only integrated areas derived from CPMG for semi-quantitative discussion were used.
Additional information should be derived from carbon resonances. However, the weak sensitivity of 13C nuclei and its long relaxation delays give poor signal-to-noise ratio, despite very long acquisition times that may exclude extremely high time-consuming 1D 13C NMR, in favor of 2D 1H 13C correlated NMR experiments, which exploit the power of “inverse detection” and pulsed-field gradients.
2D homonuclear shift correlation (H, H-COSY) spectra was obtained using the Bruker pulse sequence “cosygpqf” implying gradient pulses for selection with second flip angle being 90°.
The phase sensitive 2D 1H, 13C HSQC was performed via the double INEPT transfer using Echo/Antiecho-TPPI gradient selection, and decoupling during acquisition was performed using TRIM pulses in INEPT transfer with multiplicity editing during the selection step (
Simultaneous thermogravimetric (TG) and differential thermal (DTA) analyses were performed on all samples to record continuous weight losses during sample heating and the corresponding energy changes as endothermic or exothermic reactions. The beginning and the end of mass loss for each reaction, was calculated by DTG (the first derivative of TG curve, which is the rate at which the mass of decomposing samples changes with respect to its changing temperature). DTG curve allows converting the TG trace into well defined peaks.
Two high purity standards indium and aluminum (both 99.99%) were used to calibrate TG-DTA for both temperature (°C) and enthalpy (J g-1 -
Changes in litter quality during decomposition were evaluated applying the energetic balance concept reported by
Data were analyzed by means of ANOVA using the STATGRAPHICS® software package (Statpoint Technologies, USA) using the stand age and the decomposition time as independent variables, and their interaction was also evaluated at α=0.05.
ANCOVA was used to compare slopes and intercepts of the linear regression between EXO1 mass loss (TG-DTA data) and cellulose content, and between EXO2 mass loss (TG-DTA data) and lignin content.
The litter mass (% of the original biomass) at the end of the study did not differ between the two stands analyzed, though a significant loss was observed just after 3 months in the young forest (
Throughout the experiment, the C content of the CW was about 6% less than the C content of the young forest litter and 3% less than the C content of the old forest litter (
The evaluation of phase sensitive 1H, 13C HSQC experiments of old forest and young forest litters (
After 9 months of degradation the differences observed between the two litters at time zero were dramatically reduced. For both forest litters, the decomposition is reflected in the broadening of resonance peaks, which is indicative of biopolymer functionalization and/or oxidation, resulting in a heterogeneous mixture of compounds (
The CW extraction considerably decreased the content of aliphatic structures (3-5 regions), especially in cuticles and waxes, while the aromatic signals, related to lignins, resulted more evident due to an increased signal-to-noise ratio. When compared to litters, CW NMR spectra (
Phase sensitive 1H, 13C HSQC experiments of CW support the results from 1D 1H spectra, showing (
The differential thermal analysis (DTA) of litter samples from both forests during the whole decomposition period was characterized by the presence of a small endothermic peak related to the sample dehydration and three exothermic peaks (
Based on TG data, the OM content of litters at the beginning of the experiment was on average 79.2% in the young litter and 80.7% in the old forest litter. After 9 months of decomposition, the OM content decreased from the initial amount by 24% and 14% for the young and the old forest litters, respectively.
At the beginning of the experiment, EXO1 accounted for about 58% of litter biomass in both litters (
The third most-recalcitrant peak (EXO3) was the smallest in terms of mass loss, comprising about 3% in the young and 3.6% in the old forest litter. Unlike EXO1 and EXO2, EXO3 constantly increased during decomposition until the end of the experiment, accounting for 4.2% in the young forest litter, with an increase by 40% of the initial amount. In the old stand litter, a higher increase of 58% of the initial EXO3 content was observed, and the final value accounted for 5.7% of residual litter biomass (
The extraction procedure modified the thermal behavior of the CW as compared with that of the untreated litters (
The OM content of the CW was lower than that of the litters, with a decrease of 6.2% and 5.7% in the young and old forest, respectively. At the end of the study a decrease in OM of 25% of the initial content in the young forest and 18% in the old one was observed.
The dynamics of the two exothermic reactions in the CW is shown in
As mentioned earlier, the oxidative degradation of cellulose takes place in EXO1 and that of lignin in EXO2, but in the complex structure of intact litters many others substances are lost together with these litter constituents. The presence of hemicelluloses and cellulose in the first peak, and tannins and polyphenolics other than lignin in the second peak, may lead to an overestimation of both litter components. As an effect of the extraction procedure, the CW has a structure simpler than that of the litter, since at least some of the substances interfering with the thermal measurement of cellulose and lignin have been eliminated. Therefore, TG-DTA applied to CW should be more accurate in the estimate of the cellulose and lignin content through EXO1 and EXO2 mass loss.
Taking into account the previous assumption and considering the litter, the estimates of cellulose and lignin content obtained with the chemical method and those obtained as mass loss of EXO1 and EXO2 showed a weak correlation for both forest litters (
When the regression lines between EXO1 mass loss and cellulose content were compared by ANCOVA, no difference in slope values was found between stands (young
At time zero, the two litters were characterized by a similar total energy content, but the energetic cost to recover such stored energy, as measured by the T50 for DTA, was higher in the young than the old forest litter (
During decomposition, the greater loss of C observed in the young forest litter could be related to the larger abundance of branched aliphatic chains and low molecular weight substances (such as amino acids), as revealed by the NMR spectra. In fact, these compounds are mainly responsible for the first phase of litter decomposition, since they are readily available to microorganisms and highly susceptible to mineralization and quick conversion to CO2 (
The loss of C (
The cellulose and lignin content in the litter estimated by the mass loss associated with EXO1 and EXO2 showed a weak correlation with their contents measured with the chemical method (
The differences between the thermal behavior of litter and CW samples confirm that the extraction procedure affect the structure of the samples. The main effects were a shift of EXO1 and EXO2 peaks towards lower temperatures and the lack of the third exothermic peak related to the most recalcitrant organic compounds present in the litters. The shift of 30-40 °C that involved EXO1 highlighted a weakening of the cellulose structure. As reported by
During decomposition, the quality of the decomposing litter, as a measure of its biodegradability by the soil microbial community, is expected to decrease as more recalcitrant substances tend to accumulate. The energetic approach recently introduced by
In contrast with our results,
The use of thermal analysis allowed tracing shifts in litter chemical structure and quality during a short decomposition period, giving qualitative and quantitative information on the main classes of organic compounds involved in the decomposition process. Thermal analysis highlighted changes in the litter structure and complexity, including components removed by the extraction procedure such as cuticles and waxes as showed by NMR data. Such more recalcitrant substances tend to accumulate and could influence the decomposition rate at later stages of the process. The good correlation between cellulose and lignin content obtained by chemical method and TG data of CW suggests that thermal analysis may be a useful tool to directly measure these litter components without further steps. Furthermore, the thermal analysis carried out has highlighted a better energetic balance of the old than the young forest litter, allowing to assess their quality based on their energetic balance.
Thermal analysis is a powerful technique with potential interest in litter decomposition studies as it provides both structural and energetic information on litter changes occurring during the decomposition process. However, further studies are needed to better understand the actual relationship between the energetic balance and the microbial growth and activity, particularly with respect to the maturation of the entire litter and potential addition of micro and macrofaunal organic constituents to the litter.
Changes of cellulose and lignin content in the leaf litter of young and old forest stands during the decomposition period. Different capital letters indicate significant differences among sampling times, asterisks denote significant differences between sites (P ≤ 0.05, NS = not significant).
1H HRMAS NMR spectra of the litter from the young forest stand and their CW at the beginning (red line) and at the end (black line) of the decomposition period. (*): residual DMSO; (**): residual HOD.
1H HRMAS NMR spectra of the litter from the old forest stand and their CW at the beginning (red line) and at the end (black line) of the decomposition period. (*): residual DMSO; (**): residual HOD.
Phase sensitive 1H,13C HSQC spectra of young forest litter and its CW (red) and old forest litter and its CW (green) at the beginning of decomposition. Arrow indicates methoxyl groups of lignins, circles emphasize the main differences between the two forest stands. Squared box highlights main carbohydrates correlations.
DTA curves of litter and CW of the young and old forest stands at the beginning (a) and at the end (b) of the decomposition period.
EXO1, EXO2 and EXO3 mass loss of young and old forest litters during the decomposition period. Different capital letters indicate significant differences among sampling times, asterisks denote significant differences between sites (P ≤ 0.05).
EXO1 and EXO2 mass loss of CW extracted from young and old forest litters during the decomposition period. Different capital letters indicate significant differences among sampling times, asterisks denote significant differences between sites (P ≤ 0.05).
Relationship between OM content measured by thermal analysis (TG) and C content measured by elemental analysis (CHN) in litter and CW of young and old forest stands.
Relationship between cellulose and lignin content measured by thermal analysis (mass loss related to EXO1 and EXO2) and wet chemical method for young and old forest litters and their CW.
Quality change of litters of young (circles) and old (diamonds) stands during the decomposition period. T50 for DTA has been taken as an indicator of the energy needed to release the energy stored in litter. Filled symbols: initial decomposition stage. Empty symbols: final decomposition stage.
Mass remaining (% of the original) C and N content of the litter and relative CW from young and old forest stands during the decomposition period. Numbers within a column denoted by different capital letters are significantly different after Tukey’s test (P<0.05); numbers within each parameter followed by different lowercase letters denote statistically significant differences between Litter and CW values according to the Tukey test (P < 0.05); numbers within each parameter and type of plant tissue followed by a asterisk denote statistically significant differences between the two forests after Tukey’s test (P < 0.05). Values in brackets are standard errors (n = 3).
Months | Young Forest | Old Forest | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Massremaining | C (%) | N (%) | Mass remaining | C (%) | N (%) | |||||
Litter | CW | Litter | CW | Litter | CW | Litter | CW | |||
0 | - | 46.4 a A(0.14) | 39.2 b A(0.70) | 2.34 *a (0.12) | 1.89 b(0.01) | - | 46.1 a A(0.50) | 42.8 b A(0.38) | 1.76 * A(0.03) | 1.76(0.11) |
3 | 74.2 * A(2.03) | 40.5 a B(0.79) | 34.4 *b B(1.75) | 2.50 *a(0.07) | 2.10 b(0.06) | 89.04 * A(1.09) | 42.6 a B(1.16) | 39.2 *b B(1.67) | 2.20 * B(0.04) | 2.09(0.05) |
9 | 65.5 * B(4.49) | 35.8 *a C(1.87) | 29.9 *b C(2.61) | 2.59 *a(0.10) | 2.20 b(0.04) | 72.32 * B(5.86) | 38.3 *a C(0.77) | 35.2 *b C(0.29) | 2.10 * B(0.04) | 2.11(0.04) |
Comparison of intercept and slope of the linear regression between EXO1 and cellulose content and between EXO2 and lignin content. Different lowercase letters indicate statistically significant differences between sites according to the ANCOVA test (P< 0.05); different capital letters indicate statistically significant differences between tissues according to the ANCOVA test (P< 0.05).
Parameter | Tissue | Cellulose |
Lignin |
||
---|---|---|---|---|---|
Old Forest | Young Forest | Old Forest | Young Forest | ||
Intercept | Cw | 3.59 a B | -1.33 b B | -0.30 b A | 5.33 a A |
Litter | 8.39 a A | 3.93 a A | -0.23 a A | -11.15 b A | |
Slope | Cw | 0.88 a A | 0.84 a A | 0.80 a A | 0.73 a A |
Litter | 0.99 a A | 1.05 a A | 0.79 a A | 1.62 a A |