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Location: Home > Research Areas > Soil Nutrient Cycling and Control Mechanisms
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Novel techniques for tracing microbial processes of soil carbon and nitrogen transformation - A chromatographic mass spectrometry technique for separating native

The transformation of soil carbon and nitrogen is driven primarily by microbial processes. Amino sugar analysis has become a powerful tool in characterizing microbial contributions to soil organic matter dynamics. However, the determination of amino sugars does not reveal any detailed information on the dynamic changes of old- and newly-synthesized amino sugars in the soil matrix. Such knowledge can only be gained by differentiating newly-synthesized microbial residues from native soil by isotope tracing techniques. For this purpose, a set of isotope-labeling-based gas chromatographic mass spectrometry techniques (GC/MS or LC/MS) were developed to separate native and newly-immobilized microbial residues. They are powerful tools for probing the origins and transformation of microbial residues at the molecular level and establishing a solid foundation for clarifying the temporal pattern of the renewal of soil carbon and nitrogen.

The main purpose of this approach is to quantify the intensity of both parent and heavy isotope-containing fragments. Since there is only a single N atom in one amino sugar molecule, the incorporation of 15N into amino sugars can be evaluated according to the fragment abundance ratio of mass [F+1] to F for soil samples with 15N amendments. However, 13C incorporation into amino sugars for the soil samples labeled with 13C should be estimated by the fragment abundance ratio of mass [F+n] to F, where n is the skeleton carbon number in the evaluated fragment ion. We used the fragment ratio in the original samples as the control. Both stable isotope ratios were expressed by the atom percentage excess (APE), which is calculated as:

 

where Re is the enriched ratio of [(F+1)/F] (for 15N enrichment) or [(F+n)/F] (for 13C enrichment) and Rc is the corresponding ratio obtained from original samples (control) analyzed on the same GC/MS run.

Our work has resulted in several papers published in Rapid Communications in Mass Spectrometry (2005) and Soil Biology & Biochemistry (2006), respectively.

Figure 21: Gas chromatograph mass spectrometry of soil amino sugars in CI- mode

A: original soil; B: incubated with 15N-labeled substrate; C: incubated with 13C-labeled substrate.

Identifying the transformation processes of amino acid enantiomers as microbial metabolites was essential to explore the fate, turnover and aging of soil nitrogen as important factors in the biogeochemical cycling. If this can be achieved by differentiating between newly-biosynthesized and inherent compounds in soil, then the isotope tracer method can be considered to be the most appropriate one. We therefore developed a gas chromatography/mass spectrometry (GC/MS) method to trace the incorporation of 15N or 13C isotopes into soil amino acid enantiomers after incubation with 15NH4+ + or U-13C-glucose substrates. The most significant fragments (F), as well as the related minor ions, were monitored by the full scan mode. The isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). The incorporation of 15NH4+ was evaluated according to the relative abundance increase of mass [F+1] to F for neutral and acidic amino acids; and [F+2] to F (mass 439) for lysine. The assessment of 13C enrichment in soil amino acids was more complicated than that of 15N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of mass [F+n] to F can indicate the direct conversion of added glucose to amino acids, but the total isotope incorporation from the added 13C can only be calculated according to all target isotope fragments. The abundance ratio increment summation from mass [Fa+1] through mass [Fa+T] represents the total incorporation of the added 13C, where Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule. This method is especially appropriate for the evaluation of high-abundance isotope enrichment in organic compounds compared to the GC/C/IRMS technique. In principle, this technique is also valid for normal amino acids in addition to enantiomers if stereoisomers are not the study target. Alternatively, differentiating between newly-biosynthesized and inherent amino acids can be achieved by using the liquid chromatography-mass spectrometry technique, which was developed after the previous method. These novel approaches will shed light on the biochemical mechanisms of microbial N and C transformation in terrestrial ecosystems.

 

Figure 22: The fragmentation pattern of L-alanine derivative in isotope-incubated soil samples.

(a) original sample; (b) incubated with 15N-labeled substrate; and (c) incubated with 13C-labeled substrate

Two types of techniques ¾ chromatography/mass spectrometry and chromatography/isotope ratio mass spectrometry ¾ are currently available for compound-specific isotope enrichment determination. According to the isotope quantification principle, the average isotope ratio in a molecule can be obtained for IRMS determination due to the combustion process. However, the isotope differentiation accomplished in our GC/MS techniques is based on ionization, and thus can be used for tracing the distribution of carbon and nitrogen in the molecular structure, which is especially powerful in investigating substrate metabolic pathways and the microbial transformation of soil carbon and nitrogen.

Amino sugars are one of the important microbial residue biomarkers, which are associated with the cycling of soil organic matter. However, little is known about their transformation kinetics in response to substrate availability, since living biomass only contributes a negligible amount to the total mass of amino sugars. By using the isotope tracing techniques, the newly-synthesized (labeled) amino sugars can be differentiated from the native portions in the soil matrix, making it possible to quantitatively evaluate the transformation pattern of amino sugars as well as to interpret the past and ongoing changes of microbial communities during the assimilation of extraneous 15N. Our study significantly improved the understanding of the turnover pattern of soil amino sugars and their roles in the microbial processes of soil C and N cycling. The determination of microbial biomarkers in combination with stable isotope tracer techniques can incorporate time effects within the investigation; thus the change from the instantaneous effect of the microbial biomarker to the soil organic matter dynamics can be achieved. This improvement was essential to assess the impact of microbial processes on carbon and nitrogen transformations and enhance our understanding of the role of microbial residues as biomarkers. Equally as important, the temporal pattern of the transformation and renewal of microbial-derived stable components can be inferred from the accumulated immobilization of extraneous carbon and nitrogen manipulated by microbial biomass utilization and necromass stabilization, the latter of which were recognized as “memory effect” in microbial processes. Thus stable microbial biomarkers have unique advantages in investigating microbial-driven carbon and nitrogen cycling.







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