Abstract
Subsurface sediments at depths below 40 cm are estimated to contain up to 50% of the
microbial biomass in the terrestrial ecosystem. Still, most microbial ecology studies have
focused on the top soil leaving the subsurface relatively unexplored. The role of
microorganisms in cycling of elements and in various other functions in the subsurface
highlights the importance of describing the microbial communities and the processes they are
involved in more detailed. In clayey till, biopores and tectonic fractures serve as preferential
flow paths, which have potentially created habitats different from those in the matrix sediments.
In some sediment profiles, water flows primarily through these flow paths carrying nutrients,
organic carbon and microorganisms. Hence, increased microbial abundance and activity could
be expected in flow paths pinpointing the need for studies of the microbial communities and
their ecology in order to close some of the gaps in our knowledge regarding the subsurface
environments.
This thesis aims at describing the microbial community structures in clayey till subsurface
sediments and preferential flow paths. To further understand the ecology of the
microorganisms, functional genes of interest were investigated quantitatively and semiquantitatively.
Additionally, active bacterial dispersal also known as motility was investigated
using a novel method for detection of motile bacterial communities from environmental
samples.
In Manuscript I, bacterial communities from a 6 m deep clayey till profile including both
matrix sediments and preferential flow paths were described using 16S rRNA amplicon
sequencing. Bacterial abundance was determined based on qPCR results from 16S rRNA
genes, while a high-throughput qPCR method was used to determine the abundance of genes
involved in cycling of nitrogen and sulphur. Bacterial abundance and diversity were higher in
preferential flow paths compared to matrix sediments at all depths. Identification of aerobic
taxa and plant material decomposers such as Nitrospirae, Acidobacteria and Planctomycetes
at greater depths in preferential flow paths compared with the matrix sediments, indicated an
impact of oxygen and plant derived organic carbon deeper in the flow paths. The abundances
of functional genes, like archaeal amoA, and bacterial nirK and dsrB likewise indicated a
transition from aerobic to anaerobic conditions at greater depths in the preferential flow paths.
The results illustrated that the preferential flow paths provide different living conditions to the
bacteria colonizing them, compared to matrix sediments.
To study the potential for active dispersal of bacterial communities in environmental samples,
a novel method, expanding on the porous surface model, was developed in Manuscript II. The
method was verified using two motile bacterial strains, a gliding Flavobacterium johnsoniae
and a flagellated Pseudomonas putida, and their non-motile mutants. The application of the
method was further tested on bacterial communities from lake and soil samples, revealing that
the dispersing communties were substantially less diverse than the total communities. Although
dispersal was retarded by low matric potential (-3.1 kPa) previously argued to be too dry for
flagellar motility, a subset of motile bacteria was recovered from the model at this matric
potential. 16S rRNA amplicon sequencing of the fastest dispersers showed that Pseudomonas
and Aeromonas strains dominated the dispersers from the soil and lake, respectively.
The method was used in Manuscript III to survey active dispersal potential from different
domains in a clayey till profile. The domains included plough layer, matrix sediments and
preferential flow paths down to 350 cm below ground surface. This study expanded the lower
boundary for active bacterial dispersal down to a matric potential of -8.4 kPa. The active
dispersing communities were much less diverse than the total communities, and comprised
primarily Pseudomonas, and for the plough layer, Rahnella as well. The most dominant, active
dispersers from the matrix sediments at 350 cm below ground surface belonged to the genus
Pantoea. Hydrologically connected domains shared an increased proportion of dispersing
amplicon sequence variants (ASVs) compared to nondispersing ASVs. The results suggest that
active dispersal is an important trait for colonization in the preferential flow paths.
In Manuscript IV, lifestyles of bacterial communities in preferential flow paths were studied
at different depths. Fluctuating input of water, oxygen, organic carbon and nutrients was
hypothesized to have resulted in adaptations to variable nutrient conditions and environmental
stresses, which could be deciphered through analysis of the metagenomes representing the
bacterial communities’ functional potential. We applied amplicon sequencing of the 16S rRNA
gene and shotgun metagenomics to characterize bacterial communities from seven different
domains from clayey till to a depth of 4 meter below ground surface. We found that
communities changed with depth for both bacteria and archaea along the preferential flow
paths. Analysis of metagenomics sequences showed that communities in biopores had higher
abundance of genes related to flagellar motility and aerobic vitamin B12 biosynthesis, than
communities in the surrounding matrix sediment. Additionally, the biopore microbial
communities had more functions related to protection against desiccation and oxygen stress
than the communities in the deeper fractures. Abundant gene clusters in the matrix sediment
communities adjacent to biopores indicated that microbes in this habitat rely on anaerobic
biosynthesis of vitamin B12. Furthermore, deeper matrix sediment communities had more
genes involved in biosynthesis of aromatic amino acids, indicating that interaction among
bacteria and external supply of these essential amino acids are limited. Our results suggest that
there is a higher abundance of flagellated bacteria in biopores and that biopore communities
are better adapted to resist environmental stress than microorganisms in the deeper fractures.
These results indicate that the higher amount of energy and organic carbon provided by water
flow in biopores comes with a higher cost to cope with the environmental stressors
microbial biomass in the terrestrial ecosystem. Still, most microbial ecology studies have
focused on the top soil leaving the subsurface relatively unexplored. The role of
microorganisms in cycling of elements and in various other functions in the subsurface
highlights the importance of describing the microbial communities and the processes they are
involved in more detailed. In clayey till, biopores and tectonic fractures serve as preferential
flow paths, which have potentially created habitats different from those in the matrix sediments.
In some sediment profiles, water flows primarily through these flow paths carrying nutrients,
organic carbon and microorganisms. Hence, increased microbial abundance and activity could
be expected in flow paths pinpointing the need for studies of the microbial communities and
their ecology in order to close some of the gaps in our knowledge regarding the subsurface
environments.
This thesis aims at describing the microbial community structures in clayey till subsurface
sediments and preferential flow paths. To further understand the ecology of the
microorganisms, functional genes of interest were investigated quantitatively and semiquantitatively.
Additionally, active bacterial dispersal also known as motility was investigated
using a novel method for detection of motile bacterial communities from environmental
samples.
In Manuscript I, bacterial communities from a 6 m deep clayey till profile including both
matrix sediments and preferential flow paths were described using 16S rRNA amplicon
sequencing. Bacterial abundance was determined based on qPCR results from 16S rRNA
genes, while a high-throughput qPCR method was used to determine the abundance of genes
involved in cycling of nitrogen and sulphur. Bacterial abundance and diversity were higher in
preferential flow paths compared to matrix sediments at all depths. Identification of aerobic
taxa and plant material decomposers such as Nitrospirae, Acidobacteria and Planctomycetes
at greater depths in preferential flow paths compared with the matrix sediments, indicated an
impact of oxygen and plant derived organic carbon deeper in the flow paths. The abundances
of functional genes, like archaeal amoA, and bacterial nirK and dsrB likewise indicated a
transition from aerobic to anaerobic conditions at greater depths in the preferential flow paths.
The results illustrated that the preferential flow paths provide different living conditions to the
bacteria colonizing them, compared to matrix sediments.
To study the potential for active dispersal of bacterial communities in environmental samples,
a novel method, expanding on the porous surface model, was developed in Manuscript II. The
method was verified using two motile bacterial strains, a gliding Flavobacterium johnsoniae
and a flagellated Pseudomonas putida, and their non-motile mutants. The application of the
method was further tested on bacterial communities from lake and soil samples, revealing that
the dispersing communties were substantially less diverse than the total communities. Although
dispersal was retarded by low matric potential (-3.1 kPa) previously argued to be too dry for
flagellar motility, a subset of motile bacteria was recovered from the model at this matric
potential. 16S rRNA amplicon sequencing of the fastest dispersers showed that Pseudomonas
and Aeromonas strains dominated the dispersers from the soil and lake, respectively.
The method was used in Manuscript III to survey active dispersal potential from different
domains in a clayey till profile. The domains included plough layer, matrix sediments and
preferential flow paths down to 350 cm below ground surface. This study expanded the lower
boundary for active bacterial dispersal down to a matric potential of -8.4 kPa. The active
dispersing communities were much less diverse than the total communities, and comprised
primarily Pseudomonas, and for the plough layer, Rahnella as well. The most dominant, active
dispersers from the matrix sediments at 350 cm below ground surface belonged to the genus
Pantoea. Hydrologically connected domains shared an increased proportion of dispersing
amplicon sequence variants (ASVs) compared to nondispersing ASVs. The results suggest that
active dispersal is an important trait for colonization in the preferential flow paths.
In Manuscript IV, lifestyles of bacterial communities in preferential flow paths were studied
at different depths. Fluctuating input of water, oxygen, organic carbon and nutrients was
hypothesized to have resulted in adaptations to variable nutrient conditions and environmental
stresses, which could be deciphered through analysis of the metagenomes representing the
bacterial communities’ functional potential. We applied amplicon sequencing of the 16S rRNA
gene and shotgun metagenomics to characterize bacterial communities from seven different
domains from clayey till to a depth of 4 meter below ground surface. We found that
communities changed with depth for both bacteria and archaea along the preferential flow
paths. Analysis of metagenomics sequences showed that communities in biopores had higher
abundance of genes related to flagellar motility and aerobic vitamin B12 biosynthesis, than
communities in the surrounding matrix sediment. Additionally, the biopore microbial
communities had more functions related to protection against desiccation and oxygen stress
than the communities in the deeper fractures. Abundant gene clusters in the matrix sediment
communities adjacent to biopores indicated that microbes in this habitat rely on anaerobic
biosynthesis of vitamin B12. Furthermore, deeper matrix sediment communities had more
genes involved in biosynthesis of aromatic amino acids, indicating that interaction among
bacteria and external supply of these essential amino acids are limited. Our results suggest that
there is a higher abundance of flagellated bacteria in biopores and that biopore communities
are better adapted to resist environmental stress than microorganisms in the deeper fractures.
These results indicate that the higher amount of energy and organic carbon provided by water
flow in biopores comes with a higher cost to cope with the environmental stressors
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2019 |