Unfavorable environmental conditions: Consequences for microbial metabolism and C stabilization in soil
by Ezekiel Bore
Date of Examination:2017-11-14
Date of issue:2017-11-21
Advisor:Prof. Dr. Michaela Dippold
Referee:Prof. Dr. Sandra Spielvogel
Referee:Prof. Dr. Andreas von Tiedemann
Referee:Prof. Dr. Yakov Kuzyakov
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Abstract
English
Soil microorganisms are primary drivers of biogeochemical cycles, making them an important link between pedosphere and atmosphere. Soil microbes control the carbon (C) transfer from terrestrial ecosystem to the atmosphere via the decomposition of soil organic matter (SOM). Thus, soil microbes have the power to geo-engineer the climate through mineralization of C and nitrogen (N) compounds into greenhouse gases. However, soil microorganisms are frequently exposed to unfavorable conditions either naturally or anthropogenically. For every unfavorable condition, some microorganisms have been found to not only tolerate the conditions, but also often require the conditions to perform their functions. Therefore, understanding adaptation mechanisms enabling microorganisms to survive and function under the unfavorable conditions is essential predicting the response of nutrients and C cycle to diverse expressions of global change. Position-specific 13C and 14C labeling and compound-specific analysis were applied as the main methodological approach to study microbial adaptations to unfavorable conditions and mechanisms underlying C stabilization in soil. The unfavorable conditions were: 1) subzero temperatures, 2) respiration inhibition by toxicants, and 3) nutrient limitation induced by sorption in soil. Incorporation of 13C or 14C into CO2, bulk soil, microbial biomass and phospholipid fatty acids (PLFA) was quantified to identify adaptation mechanisms with the aid of metabolic tracing. Subzero temperatures induced a switch from pentose phosphate pathway (PPP) to glycolysis. 13C incorporated into microbial biomass was 3-fold higher at 5 than +5 °C, which points to a synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Even at 20 °C, less than 0.4% of 13C was recovered in dissolved organic C (DOC) after one day, demonstrating complete glucose uptake by microorganisms. Consequently, we attributed the 5-fold higher extra- than intracellular 13C to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. These mechanisms reflect the general response of microbial groups in soil. To understand the effect of freezing on individual microbial group in soil phospholipid fatty acid (PLFA) analysis was performed. Based on these results, a strong increase of mono-unsaturated fatty acids and corresponding 13C incorporation at -5 °C was attributed to 1) desaturation within existing fatty acid chains, and 2) de novo synthesis of PLFA. On contrary, 13C incorporation into short-chain branched fatty acids was dominant at -20 °C. This reflects adaptation of microbial membranes to subzero temperatures. A part from freezing, microbial activity can be hampared by toxicant exposure due to human activities. Despite exposure, CO2 is persistently released from soils. To determine the origin and understand the mechanism underlying such persistent CO2 release, soil microorganisms were exposed to sodium azide (NaN3) as model toxicant inhibiting respiration. Respiration inhibition prevented 13C incorporation into PLFA and decreased total CO2 release. However, 13C in CO2 increased by 12% compared to control soils due to an increased use of glucose for energy production. The allocation of glucose-derived carbon towards extracellular compounds, demonstrated by a 5-fold higher 13C recovery in bulk soil than in microbial biomass, suggests the synthesis of redox active substances for extracellular disposal of electrons to bypass inhibited electron transport chains within the cells. PLFA content doubled within 10 days of inhibition, demonstrating recovery of the microbial community. This growth was largely based on recycling of metabolically expensive biomass compounds, e.g., alkyl chains, from microbial necromass. The bypass of intracellular toxicity by extracellular electron transport permits the fast recovery of the microbial community. Toxicants are not the only limitation in soils. Soil is full of C; however, this C is not always avilabe because 70-100% is found in close association with organic and mineral fractions in soil. This means that soil microorganism suffer starvation induced by nutrient sorption in soil. To identify metabolic adaptations of soil microbes to nutrient limitation induced by sorption, we tracked transformation of sorbed alanine. Sorption of alanine decreased the initial mineralization peak by ≈80% compared to free alanine. Consequently, a 4-fold incorporation of 14C into microbial biomass was induced by sorption. Additionally, C-2 and C-3 of sorbed alanine remained in equal proportion in soil until day 3 contrary to free alanine in which significantly lower C-2 was incorporated than C-3. Even more vivid, an increased incorporation of C-2 into microbial biomass under sorption reveals a decrease of C flux through the citric acid cycle. Therefore, use of sorbed substrate shifts microbial metabolism towards a higher C use in anabolism, resulting in increased carbon use efficiency (CUE). In addition to changes in microbial metabolic activity, shift in microbial community structure to compensate for the loss of more sensitive populations also occurs under unfavorable conditions. The fungal/bacterial PLFA ratio revealed a shift towards bacteria at subzero temperatures and in presence of respiration inhibiting toxicants. However, susceptibility of fungal populations to toxicant was short-lived compared to subzero temperatures. This suggests that fungi are more resilient to toxicants than perturbations imposed by freezing. Changes in microbial metabolic activity and community structure due to changes in environmental conditions also influence ecosystem-level C, energy and nutrient flows, especially considering that C derived from labile compounds such as sugars have been shown to persist longer in soil than those from compounds of high recalcitrance such as lignin. Tracking glucose and ribose-derived C under long-term field conditions revealed that different mechanisms underlie their persistence. The persistence of glucose-derived C was mainly dominated by recycling. On contrary, stabilization in non-living SOM characterized the persistence of ribose-derived C. Therefore, even with the same class of labile compounds – the monosaccharides, the mechanisms responsible for C stabilization differ. Thus, stabilization of C in soil is largely influenced by metabolic transformation of the compound – affected not only by the compound class and its metabolic pathway, but also by the environmental conditions to which the microorganism is adapting its metabolism. Thus, for every condition investigated, some microorganisms have shown tolerance, implying that the limit of life on Earth is far from being well defined. Combining positions-specific labelling with compound-specific analysis revealed such mechanisms allowing microbes to survive and function under unfavorable conditions. Microorganisms induce a suite of not only metabolic and physiological changes, but also shifts in microbial community structure in response to unfavorable conditions. The changes in microbial metabolic activity influences C stabilization in soil. Knowledge on these adaptation strategies and their implications for C fluxes in crucial in predicting changes in C cycles induced by various phenomena of global change.
Keywords: Metabolic tracing, Position-specific labeling, unfavorable conditions, Resilience, Toxicants, Starvation, Freezing, Labile C