Chapter 1 Introduction and Literature Review




НазваниеChapter 1 Introduction and Literature Review
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Figure 3.3.5.1: 1H–15N HR-MAS NMR HSQC spectra of A) initial microbial biomass (0 h), biomass degraded under ambient conditions for B) 6 and C) 14 weeks, and that degraded under UV irradiation for D) 6 and E) 14 weeks.


3.3.6 13C NMR analysis of microbial leachate

Figure 3.3.6.1 shows the 1-D 13C HR-MS NMR spectra of microbial leachates degraded under ambient and UV conditions. General assignments, consistent with those reported in the literature, can be made as to the major components present (Karl et al., 2007; Solomon et al., 2007). Major components are applicable to all 13C NMR spectra and include, 1) carbonyl-C in carboxylic acid, amide groups and ester bonds, 2) aromatic-C and olefinic-C possibly from unsaturated lipids, phospholipids or aromatic amino acids, 3) a mixture of carbohydrates, amino acids and lipids including the anomeric carbon and units adjacent to esters, and may be associated with bacterial cell wall, and 4) aliphatic components, primarily medium- to short-chained or branched terminal CH3, CH2, and tertiary- or quaternary-C.


After 6 weeks degradation under ambient conditions, the microbial leachate was dominated by aliphatic-C species (region 4; ~38% of total carbon integral of major biochemical pools), decreasing relatively by approximately 19% after 14 weeks. Simultaneously, there a relative increase (~18%) in carbohydrates (region 3) which was the dominant C species (34.22%) in the spectrum after 14 weeks degradation (Figure 3.3.6.1). These results are somewhat surprising and contradict previous findings (Knicker et al., 1996) which suggest that the predominant reactions occurring during prolonged degradation are degradation and removal of carbohydrates. However, considering that up to one half of the signals resonating in region 4 may be proteinaceous in origin, the loss of aliphatic-C is likely due to the degradation of labile proteinaceous material resonating in this region (Kögel-Knabner, 2002). Moreover, it is likely that lipids were lost to consumption by bacteria as a source of energy and/or hydrolysis by lipases contained in bacterial cells growing in the leachate (Yoshimura et al., 2009). The quantitative differences between the lipid composition of the degraded microbial biomass and microbial leachates are clearly observed in Chapter 5.


In contrast, under UV-induced decomposition, the microbial leachate was dominated by signals from carbohydrate species (36.5%) after 6 weeks degradation (Figure 3.3.6.1 C), increasing relatively to other components by approximately 18% after 14 weeks. The 13C HSQC NMR spectra in Figure 3.3.7.1 provided complementary and conclusive evidence to support the conclusion that the UV degraded samples were dominated by carbohydrates (cross-peaks in regions 4 and 5). We postulate that the relative concentration of polysaccharide in the degraded sample is due to the accumulation of water soluble material leached from the degraded biomass. It should also be noted that a moderate peak Figure 3.3.6.1 near 102 ppm was not attenuated, and would suggest that no carbohydrate was lost through oxidation as is usually the case (Wershaw et al., 1996). Qualitatively, it is difficult to distinguish a pattern of degradation within the aliphatic chemical shift region (region 4) of the 1-D HR-MAS 13C NMR spectra of the UV irradiated microbial leachates Figure 3.3.6.1 C and D. However, according to integrals of our 13C NMR spectra, aliphatic-C was largely unchanged over the course of the degradation process and accounted for approximately 31% of the total C atoms in each UV irradiated sample. These results have significant implications for C sequestration and stabilization in the subsoil and should be the focus of more detailed investigations.




Figure 3.3.6.1: 1D 13C NMR spectra of microbial leachates degraded under ambient conditions for A) 6 and B) 14 weeks, and that degraded under UV irradiation for C) 6 and D) 14 weeks. Numbers and brackets represent general assignments as follows: 1) C=O of esters, acids and/or amides, 2) olefinic and/or aromatic C=C, 3) C-O and/or C-N bonds and 4) CH2 groups in polymethylenic chain. The regions highlighted should only be used as a reference as to the predominant species in each area. Similar contributions from other species are present in some regions. More details of the general regions are given in the text.


An interesting observation is that a dominant peak characteristic of polymethylenic [(CH2)n] C in long-chained aliphatic structures (Kögel-Knabner, 1997, 2002) that was observed (near 32 ppm) in the 13C NMR spectra of the ambient and UV degraded biomass (Figure 3.3.1.1) was significantly attenuated in the 13C NMR spectra of all microbial leachates, as were other aliphatic signals. The relative reduction in concentration of aliphatic-C species observed in the leachates may be interpreted as the preferential adsorption of aliphatic components to soil minerals as they leached through the soil (Figure 3.3.6.1). Convincing evidence of the preferential adsorption of aliphatic-C to clay minerals is presented in Chapter 4. Moreover, this suggestion is strongly supported by the findings of Kaal et al. (2005) who attributed the limited detection of tannins in soil leachates to their adsorption to soil minerals. Additionally, the hydrophobic nature of lipids may have influenced the amounts of aliphatic material leached and accumulated.


Signal 2 (aromatic and olefinic-C) in Figure 3.3.6.1 showed little variation in concentration in of the ambient and the UV degraded microbial leachates accounting for roughly 21% of the total C in all samples over the degradation period. However, signal 1 (carboxyl/amide-C) was slightly more variable, accounting for 14 and 11% of the total C in the 13C NMR spectra of leachates degraded under ambient conditions for 6 and 14 weeks, respectively, and approximately 12 and 6% of the total C in UV degraded samples over the same period. The relative reduction in concentration of carboxyl/amide-C is most likely do to biological and photochemical degradation of proteinaceous materials and labile fatty acids in the samples. Figure 3.3.7.1 also serves to complement these data by spreading the signals over two dimensions to circumvent overlapping signals.


3.3.7 13C HSQC NMR analysis of microbial leachate

The HSQC NMR spectra in Figure 3.3.7.1 support assignments made from the 1-D 13C NMR spectra and identifies a range of chemical constituents present, including 1) aromatic fragments, with possible resonances from aromatic amino acids, 2) double bonds and esters, possibly from lipids or phospholipids, 3) anomeric protons, 4) C-H bonds in carbohydrates, 6) α-protons/carbons in protein/peptides and 7) C-H bonds from various aliphatic structures including fatty acids and amino acids. More specific assignments are; 5) CH2 in carbohydrates, 8) aliphatic methyl (CH2)n in lipids, and 9) terminal CH3 or CH3 in methylated amino acid side chain residues. General and specific assignments have also been made using a full range of multidimensional NMR experiments as well as literature assignments (Simpson et al., 2007a).





Figure 3.3.7.1: 1H–13C HR-MAS HSQC spectra of A) microbial leachates degraded under ambient conditions for 6 weeks (black) and 14 weeks (green) and B) that degraded under UV irradiation for 6 (black) and 14 weeks (green). Basic assignments for the major structural groups are as follows: 1) aromatic fragments, 2) double bonds, 3) anomeric protons, 4) C-H bonds in carbohydrates, 6) α-protons/carbons in protein/peptides and 7) C-H bonds from various aliphatic structures including fatty acids and amino acids. More specific assignments are; 5) CH2 in carbohydrates, 8) aliphatic methyl (CH2)n in lipids, and 9) terminal CH3 or CH3 in methylated amino acid side chain residues and P; N-acetyl peptidoglycan.


It has been demonstrated that compounds containing double bonds are highly reactive (Goni and Hedges, 1990) and are known to degrade readily due to the presence of these labile allylic centres (Rontani et al., 2009). However, signals resonating from double bonds (region 2) showed no obvious pattern of decomposition under ambient degradation Figure 3.3.7.1A. These results are not surprising, as it has also been demonstrated that potentially labile bonds (Largeau et al., 1986) and double bonds in algenan (Simpson et al., 2003) persist in the environment for long periods of time (Deshmukh et al., 2003), and may be protected by biopolymers that shield the labile linkers in an aliphatic chain (Kelleher et al., 2006). This stabilization suggests that these double bonds may be present in aliphatic chains as indicated in Chapter 5 (Section 5.3.3). In contrast, signals assigned to double bonds were depleted in the UV degraded microbial leachate after 14 weeks (Section 5.3.4). The significant loss of these structures from the UV irradiated samples may be due to photochemical degradation of protective biopolymer into biologically oxidizable or volatile structures, as has been shown for marine DOM (Kieber et al., 1989; Mopper et al., 1991).


3.3.8 1H and Diffusion Edited NMR analysis of microbial leachate

Figures 3.3.8.1 and 3.3.8.2 illustrate the conventional 1-D 1H HR-MS and the diffusion edited 1H NMR spectra of degraded microbial leachates dissolved in DMSO-d6, while the 1-D 1H spectrum of a standatd protein, bovine serum albumin is represented for comparison in Figure 3.3.8.2A. General peak designations represent the predominant species present and are consistent with those previously reported. The assignments can be defined as; contribution from amide signals in peptides and aromatic amino acid residues (regions 1 and 2) consistent with the presence of intact proteinaceous material. 3) Protons associated with double bonds (mono- and di-unsaturated acyl chains) and esters, possibly form lipids or phospholipids 4) predominantly signals from carbohydrates. Some signals from amino acids and proteins may also resonate in this region. 5) Signals from various substituted methylenes and methines β to a functionality in a hydrocarbon, as would be the case for a lipoprotein and lipid. Region 6) is consistent with aliphatic methylene (CH2)n carbons (likely from bacterial membrane) including that in aliphatic rings and chains and methyl groups bound to carbon (Lundberg et al., 2001: Kelleher et al., 2006; Karl et al., 2007). More specific assignments refer to i); terminal CH3 in methylated amino acid side chain residues, and are consistent with a side chain residue in the 1H NMR spectrum of bovine serum albumen (BSA), ii) contributions from N-acetyl group in peptidoglycans, and iii) novel peptides.





Figure 3.3.8.1: 1D 1H NMR spectra of microbial leachates degraded under ambient conditions for A) 6 and B) 14 weeks, and that degraded under UV irradiation for C) 6 and D) 14 weeks. Numbers and brackets represent general assignments as follows: 1) amides and amide signals in peptides 2) signals from aromatic rings including aromatic residues, some amide signals in peptides may also resonate in this area, 3) protons on α carbon in peptides, 4) protons in carbohydrates, protons α to an ester, ether, and hydroxyl in aliphatic chains will also resonate in this region, 5) signals from various substituted methylenes and methanes β to a functionality in hydrocarbons, signals from some amino acid side chains will also resonate here, 6) protons in aliphatic species. (i) CH3 in methylated amino acid side chain; (ii); peptidoglycan, (iii); novel peptides; u; unknown compound and *; resonance from natural silicate species.





Figure 3.3.8.2: 1-D 1H spectrum of a standatd protein, bovine serum albumin (A); diffusion edited NMR spectra of microbial leachates degraded under ambient conditions for B) 6 and C) 14 weeks, and that degraded under UV irradiation for D) 6 and E) 14 weeks. PG, peptidoglycan; Phe, phenylalanine and *; resonance from natural silicate species.


The samples investigated display largely similar spectral profiles and ratios of major biochemical components with a characteristic dominance from signals assigned to polymethylene [(CH2)n] C (~1.29 ppm) in aliphatic compounds indicating they are still preserved in rigid domains. Signals from carbohydrates also represent one of the dominant species indicating that they are preserved as large polymeric structures that could potentially be associated to bacterial cell wall (Lam et al., 2007). Resonances attributed to amide signals in protein/peptide and aromatic amino acids (regions 1 and 2) are partially unchanged by the diffusion gate, strongly suggesting that this material is of significant size and is likely to be intact polymeric protein structures that have survived the degradation process. These data are consistent with the findings of Kelleher et al. (2007) who reported the presence of intact polymeric protein structures in marine sediments using diffusion edited NMR.


This interpretation is further supported by the spectral similarity to the 1D 1H NMR spectrum of protein standard BSA, a characteristic resonance ascribed to CH3 in methylated amino acid side chains and the presence of α-protons from amino acids in Figure 3.3.7.1. Furthermore, substantial amounts of intact proteins were subsequently isolated from the samples investigated and separated on 1-D SDS-PAGE (Figure 6.3.1.3). Other signals previously identified in the conventional 1H NMR spectra of degraded microbial leachates (see annotation for Figure 3.3.8.1) also appear to be preserved in the rigid domain. We speculate that proteins were preserved as intact polymeric structures in the form of long-term protein-dissolved organic matter interactions or protein-mineral interactions that render them unavailable to bacterial and photolytic degradation (Aluwihare et al., 2005). Hydrophobic and hydrogen-bond interactions between proteinaceous material and non-protein components have been proposed as major forces of N stabilization (Nguyen and Harvey, 2003). Other possible mechanisms involved in the preservation of the major biochemical components in the degraded leachates were previously discussed.


Contributions from microbial cell wall components (signal ii) observed in microbial leachates can be assigned to N-acetyl peptidoglycan (Figure 3.3.8.1) that is confirmed by cross-peaks also observed in HSQC spectra in Figure 3.3.7.1. Cross-peaks were identified by a comparative analysis with Advanced Chemistry Development (ACD) software and literature data on the basis of their characteristic chemical shifts. N-acetyl peptidoglycan comprises up to 90% by weight of gram-positive bacteria and is a key structural component in all microbial cell walls (Simpson et al., 2007a). Structurally, it comprises roughly equal amounts of amino sugars strands with peptide bridges (Hedges et al., 2001). However, it must be noted that it is not possible to accurately quantify contributions of peptide in the form of peptidoglycan in the sample due to spectral overlap. Using rough estimates based on deconvolution of spectral profiles, Simpson et al. (2007a) determined that the peptide contribution of peptidoglycan in humin was relatively small. The presence of peptidoglycan in the diffusion edited NMR spectra is of no surprise as it is resistant to many chemical and biological processes and has been found as a constituent of the most refractory components of SOM (Simpson et al., 2007b). It is likely that peptidoglycan also contributed to the intensity of the carbohydrate signals observed in the proton spectra of the samples investigated.


Another key observation is the presence of trace amounts of natural silicate species (Si) in the 1-D 1H HR-MS and the diffusion edited 1H NMR spectra of the degraded microbial leachates (Figures 3.3.8.1 and 3.3.8.2). This is particularly important since carbon sequestration in the ocean has been linked with the global cycling of silicon (Tréguer et al., 1995; Ragueneau et al., 2000). The conventional thought is that the primary source of continental silicon flux into the ocean is due to weathering in terrestrial biogeosystems (van Breemen and Buurman, 2002). However, silicon dynamics in terrestrial biogeosystems cannot be understood solely on the basis of mineral weathering (Sommer et al., 2006). The presence of silicate species in microbial leachates would therefore suggest that soil microorganisms are capable of accumulating stable amounts of silicon and may play a more vital role in silicon cycling than currently thought. If this is the case, then soil microbial biomass (or at least the heterothophic fraction) may play an even more significant role in the removal and sequestration of atmospheric CO2 in soil as inorganic bicarbonates. In addition, microbially accumulated silicon may further influence the hydrologic properties of soils and improve their capacity to stabilize the labile C pool and reduce atmospheric CO2 levels through the dissolution of CO2 in soil solution, ultimately producing carbonate which is then sequestered as inorganic C in soils. These findings further demonstrate shortfalls in our knowledge of microbial contributions to SOM and carbon sequestration and raise important new questions and highlight the need for greater research into the qualitative significance of silicon and its impact on the carbon biogeochemical cycle.


An even more significant discovery is the appearance of novel peptides (iii; ~7.4 ppm) in the 1D 1H HRMS NMR spectrum of the microbial leachate incubated under UV irradiation for 14 weeks (Figure 3.3.8.1D) that is confirmed by cross-peaks appearing in the 15N HSQC NMR spectrum in Figure 3.3.9.1 that cannot be found in any other sample. Also of interest is the appearance of an unknown compound in the same sample which appears to be preserved in the rigid domain (Figure 3.3.8.2E), indicating that it is macromolecular in nature. These findings demonstrate the potential for the photochemical transformation of organic material into novel photoproducts. The formation of novel photoproducts from DOM was previously reported (Kieber et al., 1989). It is also possible that these structures were formed after random condensation and polymerization reactions of degrading proteinaceous material (Nguyen and Harvey, 2003).

3.3.9 Nitrogen NMR analysis of microbial leachate

Figure 3.3.9.1 illustrates the 1H–15N HR-MAS NMR HSQC spectra of microbial leachates degraded under ambient and UV conditions for a period of 14 weeks. Considering the spectroscopic evidence, it emerges that amide-N functionalities, most likely protein/peptides represent the major form of the organic 15N in all samples. This would suggest that microbially derived amide-N is an integral part of the stable N pool of SOM. Although the major form of organic N in the 15N NMR spectra of the degraded samples is of a predominantly proteinaceous nature, the contribution of signals from heterocyclic nitrogen, such as purines/pyrimidine, indoles, imidazoles (including heterocyclic-N in histidine) and substituted pyrroles to the intensity of the resonance from amide-N (Knicker et al., 1996; Zang et al., 2001) cannot be discounted. Several studies of degraded algal residues have reported that signals from nitrogen in acetylated amino sugars, lactams, and carbazoles resonate between –145 and –220 ppm, but may expand to –270 ppm, incorporating the dominant resonance (–259 ppm) of the spectrum assigned to amide-N (Knicker et al., 1996; Kögel-Knabner, 1997). It is also noteworthy that the 1H–15N HR-MAS NMR HSQC spectra of the UV irradiated microbial varied slightly from that of the samples degraded under ambient conditions with the emergence of novel peptides in Figure 3.3.9.1D and new cross-peaks, possibly photoproducts unknown to us at this time. Or, alternatively they could be specific species synthesized from the labelled biomass due to UV-induced changes in the initial microbial community.




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