1.3 Results and Discussion
1.3.3 Identification of specific metabolic pathways
1.3.3.1 Specific pathways of individual members of the microbial community in soils
The investigated compound classes of microbial products are on the one hand spe-cific for a certain biosynthetic pathway within microbial cells. On the other hand, PLFA and the amino sugars are also biomarkers for individual microbial groups in soils. This enables the identification of specific pathways of individual members of the soil microbial community.
Amino sugars enable fungi and bacteria to be distinguished between (Engelking et al., 2007, Glaser et al., 2004): In study 7, bacterial muramic acid showed the higher dy-namics of 13C replacement of the cell walls (study 7, Figure 3). In addition the DI of indi-vidual C positions changed strongly from day 3 to day 10 for bacterial muramic acid but remained constant for fungal galactosamine (study 7, Figure 2). This reflects a more ac-tive maintenance metabolism in bacteria, which caused an increasing incorporation of glucose metabolization fragments within a period of 10 days. Therefore, this study proved for the first time that differences in the C turnover in the slow- and fast-cycling branch of soil food webs can be attributed to metabolic processes (Moore et al., 2005).
A more detailed fingerprint of the microbial community, especially for the prokary-otic groups, can be gained by individual PLFA (MooreKucera and Dick, 2008). Combining statistical grouping of the fatty acids with known fatty acid fingerprints enables functional
microbial groups to be distinguished in soil (studies 3, 6 and 8). The DI revealed for glu-tamate incorporation into PLFA that two functional microbial groups – gram-positive II and fungi – showed a specific C allocation: Glutamate C-2 was transferred from the citric acid cycle towards fatty acid synthesis. Special reactions (e.g. the glyoxylate bypass) are necessary to avoid the oxidation of glutamate C-2 (Caspi et al., 2012). None of the other microbial groups showed C-2 incorporation into PLFA (see study 3, Figure 4). Conse-quently, this was the first time that specific pathways of individual members of the soil microbial community could be traced in soils in parallel.
1.3.3.2 Pathways under various concentrations of LMWOS
C allocation within metabolic pathways is strongly affected by environmental condi-tions. The bioavailability of C sources, especially LMWOS, is one of the main factors con-trolling the state of the microbial community - from maintenance to growth (Blagodatskaya et al., 2007, Fischer et al., 2010b; Schneckenberger et al., 2008). In study 4, alanine transformations were investigated within an alanine concentration gradi-ent represgradi-enting the full range of concgradi-entrations in soil: from bare soil (0.5 µM) to hot spots (5 mM).
At low alanine concentration, uptake was described by Michaelis-Menten kinetics, whereas with increasing concentration, linear kinetics dominated microbial uptake (Jones and Hodge, 1999). This shift in uptake kinetics revealed that two mechanisms were re-sponsible for the microbial uptake: An unsaturable, unspecific uptake of intact alanine at hot-spot concentrations and specific, active uptake mechanisms at low alanine concen-trations. The DI of the non-extractable pool of microbial transformation products (contain-ing macromolecules as well as lipids) reflected additional shifts in the intensity of the alanine metabolism pathways (Figure S8).
A significantly increased incorporation of C-1 under lowest C availability may indi-cate C starvation pathways e.g. anapleurotic pathways. A convergence of the DI of C-2 and C-3 for highest alanine concentration is characteristic of many anabolic pathways for biomass formation under growth conditions (e.g. lipid biosynthesis from alanine for the formation of new cell membranes: see figures S5 and S7).
Fig. S8 Concentration-dependent position-specific transformation index DIi (N=6, ± SEM) of alanine C position incorporated into the not-extractable pool of micro-bial biomass compounds.
These and further results from study 4 indicate an altered C allocation into individ-ual pathways that is dependent on substrate availability: from anabolic pathways charac-teristic for C deficiency via maintenance metabolism towards pathways common for growing cells (Figure S9).
Fig. S9 General biochemical pathways of amino acids metabolization in soil as depend-ing on alanine availability. Line width represents the qualitatively estimated rela-tive shifts of alanine C between certain pathways dependent on the alanine concentration.
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
0.5 µM 5 µM 50 µM 500 µM 5000 µM
DIi
C-1 Alanine C-2 Alanine C-3 Alanine
1.3.3.3 Pathways of sorbed LMWOS
Sorption is one of the most likely processes causing the long-term stabilization of C in soils (Gonod et al., 2006, Duemig et al., 2012). Nevertheless, a part of the sorbed LMWOS remains bioavailable: Study 5 showed for alanine that at least 20-50% of the mineral-sorbed alanine was microbially metabolized. However, sorption reduced the availability and consequently affected transformation pathways (Jones and Edwards, 1998). In study 5, transformations of position-specifically labeled alanine, sorbed to five sorbents (two iron oxides with different crystalline structure: goethite and hematite; two clay minerals with 2:1 layers – smectite, and 1:1 layers – kaolinite; and active coal) were investigated. Goethite and active coal showed the highest amount of sorbed alanine (~45% of added alanine), and the lowest portion of the sorbed alanine C was microbially utilized (26 and 22%, respectively), whereas clay minerals showed lower sorption (10-26% of added alanine) and a higher portion that was microbially available (30-35%).
The stronger the sorption by the individual sorbent, the lower the microbial utiliza-tion was (Jones and Hodge, 1999). The fate of individual molecule posiutiliza-tions reflected that, at least for the four mineral phases, alanine was processed by the classical bio-chemical pathways: deamination, decarboxylation of C-1 by pyruvate dehydrogenase and further oxidation of C-2 and C-3 in the citric acid cycle (Djikstra et al., 2011). However, the intensity of microbial pathways depended on the bioavailability of the sorbed sub-strate: the less alanine was accessible, the less was oxidized by catabolism and the more alanine C was used for anabolism, i.e. the formation of microbial biomass.
0 2 4 6 8
0 10 20 30 40 50 60 70 80
time (h) ratio C-1/C-2,3 in respired CO2 heamatite
goethite smectite kaolinite active coal
Fig. S10 Ratio of C-1 to (C-2+C-3)/2 respiration of alanine C for the 5 applied sorbents calculated from the fitted, position-specific oxidation rate.
The ratio of C-1 oxidation by pyruvate dehydrogenase versus oxidation of C-2 and C-3 in the citric-acid cycle (Figure S10) depended on the microbial availability of alanine:
High availability due to fast desorption of cation-exchangeable bound alanine caused an initial peak in C-1 oxidation by glycolysis for the two clay minerals and an abrupt shift to oxidation via the citric acid cycle. However, low microbial availability of alanine sorbed to iron minerals led to a parallel oxidation of all three positions by glycolysis and citric acid cycle (represented by the C-1/C-2,3 ratio in Figure S10). This slower oxidation rate was associated with an increase in C allocation towards anabolism (Djikstra et al., 2001) (Fig-ure S11).
Fig. S11 Metabolic pathways of alanine sorbed on clay minerals (smectite and kaolinite), iron oxides (hamatite and goethite) and active coal. Detailed explanations in text. Various colors show the pathways of C from individual positions of alanine.
Line width represents the qualitatively estimated relative shifts in the fate of alanine C positions between certain pathways dependent on the sorbent class.
Alanine sorbed to active coal showed a deviating behavior with preferential stabili-zation of C-3 and oxidation of C-1 and C-2 (study 5, Figure 3 and 4). This indicates that in addition to basic microbial mechanism, further stabilization and modified transformations of sorbed alanine occured, e.g. by exoenzymatic degradation (Lehmann et al., 2011).
Potential desorption processes and pathways are shown in Figure S11. In general, posi-tion-specific labeling revealed that the fate of amino acid C in soil is strongly affected by sorption: The stronger the sorption, the more metabolized LMWOS C is allocated into microbial biomass compounds (Figure S11).
1.3.3.4 Extra- versus intracellular transformation pathways
Besides intracellular microbial pathways, extracellular transformations may also play an important role in LMWOS transformations in soils, especially in microhabitats where intact cells have no access (von Luetzow et al., 2006). To distinguish extra- and intracellular transformation pathways, selective inhibition of cellular, energy-dependent pathways was performed in study 4 (Figure S12).
0.02 0.03 0.04 0.05
0 5 10 15 20 25 30 35 40
time (h)
bi ot ic A la -C re m ov al (µ m ol )
C-1 Alanine Microorganisms C-2 Alanine Microorganisms C-3 Alanine Microorganisms C-1 Alanine Exoenzymes C-2 Alanine Exoenzymes C-3 Alanine Exoenzymes
Fig. S12 Removal of alanine from soil solution by extra- and intracellular processes with-out inhibition (filled symbols, dashed line) and by extracellular transformation in respiration-inhibited treatments (open symbols, dotted line); Experimental points (means ± SEM, N=6) and fitted curves based on an exponential utilization model are presented.
This approach could prove the existence of extracellular transformation of LMWOS in soils: Alanine was decomposed by a stepwise extracellular oxidation starting from the
carboxylic group, presumably by rather unspecific exoenzymes (Hofrichter et al., 1998).
However, comparing the kinetics of extra- versus intracellular processes (Figure S12) revealed that cellular uptake of LMWOS always out-competed extracellular transforma-tions, which are quantitatively relevant only at very low alanine concentrations or in spe-cific microhabitats.