RNA-based sequencing techniques are powerful tools that have the potential to identify functional traits of organisms and provide insight to their function in different ecosystems. Even though these techniques have been successfully used for some decades in the study of isolated species, its use in highly biodiverse environments as soil is still a challenge. The work in this PhD-thesis is contributing to fill in this gap, by exploring the use of RNA based approaches (16S rRNA gene transcript amplicon sequencing and Metatranscriptomics) to study different disturbed soil systems. The overall goal is to provide insights into the ecological role of soil microbes living in a community and its capabilities to cope with short- and long-term stresses.
In the introduction, the problem of using RNA based approaches in soil ecology is presented in parallel with the importance of soil microbes for the ecosystem functions and human societies. Furthermore, the effects of heavy metal pollution and climate change on soil microbial communities are considered and the general concepts of resistance and adaptation of microbial communities are further discussed. At last, a general discussion of the main findings and future research directions is presented. This PhD-thesis resulted in four draft-manuscripts where RNA sequencing techniques were used to answer different research questions related to the response of soil microorganisms to different types of stress:
MANUSCRIPT 1 explores the effect of soil sieving to the structure and gene expression pattern of the transcriptionally active soil microbial communities. In this study, sieving proved to be an efficient homogenization method, maintaining levels of richness and evenness overtime. However, it also promoted a significant change in the structure of the transcriptionally active community, corresponding to alterations in the dynamic relationship between the oligo- and copiotrophic fractions of the soil microbial community. While in the non-sieved controls the community was in equilibrium with oligotrophs and copiotrophs cohabiting in the different niches originated by the natural soil compartmentalization, in samples homogenized by sieving, the macro-structure of soil was disrupted and the better contact with nutrients, water and other bacteria promoted the fast-growing opportunistic copiotrophs in detriment of the slow-growing specialist oligotrophs.
From these findings, it became clear that sieving soil for RNA-based studies need careful consideration. While the efficient homogenization is a valuable feature to be considered in model laboratory experiments (e.g. microcosms), the potential enrichment of opportunistic strategists coupled to the high sensitivity of RNA-based techniques may impact the results and conclusions of studies addressing the functioning of natural soil ecosystems. Taking these pros and cons of sieving into consideration, a working frame was designed to study the effects of different disturbances to the transcriptionally active soil microbial communities, where microcosms and field sampling were applied.
Sieved agriculture soil microcosms were used in MANUSCRIPT 2 to test the effects of unpredictable temperature increase on the structure of soil bacterial communities, by using different doses of microwaving. Bacterial groups with different tolerance towards microwaving-heat were detected and corresponded to traits conserved at high taxonomical level. Moreover, using the detected tolerance ranges, it was possible to point nitrification as “at risk” in systems exposed to rapid heat stress, even though some functional redundancy may have occurred for other nitrogen cycle related functions. Potential bioindicators for nitrogen fixation status, soil nitrification capacity and heat tolerance were also indicated.
The following two manuscripts are addressing the soil microbial capacity to cope with longterm stresses by studying the effects of a century of copper exposure to the structure and gene expression patters of soil bacterial communities using in situ soil sampling.
MANUSCRIPT 3 shows copper as the main driving force for the observed decrease in richness and evenness of the transcriptionally active soil bacterial communities in the studied soils. Furthermore, this study shows a differentiation of the soil microbial communities according to copper contamination level, revealing an increasing proportion of transcriptionally active Acidobacteria and Nitrospira with increasing levels of contamination. These two genera also presented potential use as bioindicators of copper contamination status and copper resistance.
Knowing that a century of copper exposure resulted in structurally different transcriptionally active communities, MANUSCRIPT 4 addresses the potential legacy burden that this restructuration had in the soil microbial response to seasonal fluctuations. This study points the existence of functional redundancy for broad functions related with nutrition and cold protection, which fluctuate according to season and regardless of copper contamination level. However, specific functions involved in resistance to drought were reduced or lost in the contaminated areas. Overall, this report reinforces the importance of microbial biodiversity for the maintenance of functional redundancy and the consequent capacity to resist to changes in the environment.