Download Abstract Dead plant biomass is a key pool of carbon in terrestrial

Abstract
Dead plant biomass is a key pool of carbon in terrestrial ecosystems. Its decomposition in soil
environments is thus an essential process of the carbon cycle. Fungi are considered to be the
primary decomposers in soil ecosystems because of their physiological adaptations and
enzymatic apparatus composed from highly effective oxidative and hydrolytic enzymes.
Many recent works show that in addition to fungi, bacteria may also play a significant role in
lignocellulose decomposition and among bacteria, the members of the phylum Actinobacteria
are often regarded to significantly contribute to cellulose and lignocellulose decomposition.
This thesis is focused on the evaluation of the role that fungi and Actinobacteria play
in dead plant biomass degradation. First, it explored mechanisms involved in degradation, in
particular the enzymatic breakdown of major lignocellulose components as cellulose,
hemicelluloses and lignin. Enzymatic apparatus of the saprotrophic fungus Fomes fomentarius
was explored both in vitro as well as in vivo. Several Actinobacteria were isolated from soil
and comparative experiments, investigating production of hydrolytic enzymes, were carried
out to track the transformation of polysaccharides and lignin by these strains.
To explain the roles of lignocellulose decomposers in complex environments like soil,
the community composition of fungi and bacteria, with special focus on Actinobacteria, was
investigated. The use of next generation sequencing (NGS) methods to cover this task
required implementation and design of appropriate tools for data handling, not available at the
time when the studies were conducted. This resulted in the development of the software
pipeline SEED. With the help of this tool, active degraders in forest soil were identified by the
comparison of RNA and DNA communities. Further, it was demonstrated that some microbial
taxa show high RNA/DNA ratio or were detected only in RNA pool and thus they are
underestimated or missing in studies based on DNA analysis. Results also confirmed the
importance of Actinobacteria showing that they belong to the most active bacterial groups
especially in soil organic horizon.
To provide an in-depth analysis of actinobacterial communities along the gradient of
heavy metal contamination, where they were expected to represent the most metabolically
active group, the method for a selective community composition analysis of the
Actinobacteria using 454 pyrosequencing was developed that allows community composition
at a high resolution. The study showed that diversity of Actinobacteria was unaffected by
heavy metal content, but the contamination changed the community composition
significantly. Results also confirmed that Actinobacteria thrive better than other bacteria in
contaminated soils and may thus serve as important degraders of lignocellulose.
Finally, to get the most accurate estimates of the relative abundance of fungal and
bacterial taxa obtained by NGS, methods improving these estimates were proposed. This part
of study investigated the impact of the variability of 16S copy numbers among the bacterial
genomes on the diversity and abundances of different taxonomical groups and steps to refine
the abundance estimates were suggested. In the case of fungal communities, an alternative
marker derived from the single-copy gene rpb2 was compared with the widely used multicopy ITS with the aim to reduce the problems of community analysis due to the intragenome
variability and multicopy nature of the latter marker. Broad taxonomic coverage, suitability
for taxonomic assignments and sufficient variation for the use in phylogenetic analyses were
confirmed for the newly proposed marker.