INTRODUCTION

Microbial diversity studies are important in order to understand the microbial

ecology in the soil. Winogradsky columns incubated in sunlight allow us to
recreate phototrophic environments and oxygen/anoxygenic gradients, which
will determine the growth of different groups of bacteria. Different depths in the
column will correspond to different environments and therefore will promote the
growth of different organisms. This is an excellent tool to determine the diversity
of major bacterial communities present in a soil sample (Zavarin 2006).
Phototrophic Bacteria are a group of bacteria that use sunlight as their
only energy source in a process called photosynthesis. We can distinguish two
groups of phototrophic bacteria: the Anoxygenic Phototrophic Bacteria (divided
into aerobic and anaerobic Anoxygenic Phototrophic Bacteria), and oxygenic
phototrophic bacteria (Pelczar et al. 2003).
The Anoxygenic Phototrophic Bacteria contain a specific photosynthetic
pigment called bacteriochlorophyl and do not produce oxygen during
photosynthesis. This group is constituted by two subgroups of bacteria: the
Aerobic Anoxygenic Phototrophic Bacteria, which are obligate aerobes that can
be found in aquatic environments, and the anaerobic Anoxygenic Phototrophic
Bacteria, which are anaerobic organisms that only grow phototrophically under
anaerobic conditions. We can characterise two types of Anoxygenic Anaerobic
phototrophic organisms: the purple phototrophic bacteria (Purple-sulphur
Bacteria, photolitotrophs (H
2
S) or Purple Non-sulphur Bacteria,
photoorganotrophs), which are pigmented with bacteriochlorophyll A or B and
various carotenoids giving them purple colours, and the Green Phototrophic
Bacteria (Green-sulphur Bacteria, photolitotrophs (H
2
S) or Green Non-sulphur
Bacteria, photoorganotrophs), which contain bacteriochlorophyll C or D an little
amounts of A (Blankenship et al. 1996).
In the other hand, the Oxygenic Phototrophic Bacteria contain chlorophyll as
photosynthetic pigment and produce oxygen during photosynthesis. Belonging
to this group of organisms we find the Cyanobacteria. Cyanobacteria or blue-
green algae are an aquatic and photosynthetic group of nitrogen fixing
organisms that can be found in a wide variety of environments. They get their
colour form the bluish pigment phycocyanin, used to capture light for
photosynthesis (Nianzhi et al. 2003)
Finally, the Prochlorophytes are a group of photosynthetic prokaryotic
organisms that belong to the phytoplankton group Picoplankton. This group of
prokaryotic organisms morphologically resemble Cyanobacteria but they also
contain chlorophyll B in addition to A and a unique photosynthetic pigment call
divinyl-chlorophyll. They can be found in nutrient-poor waters (Lewin 2002).
In the last years, cultivation-independent molecular methods to
investigate bacteria diversity in natural environments have been commonly
applied (Neufeld and Mohn 2006). Employing the 16S rRNA gene as a maker
has been widely applied for phylogenetic studies (Weidner et al. 1996) as it is
highly conserved between different species of bacteria and archaea giving
information about the evolutionary process (Coenye and Vandamme 2003).
In addition, the analysis of photosynthetic gene sequences of pufM genes,
allows determining de diversity of phototrophic bacteria present in soil
environments. The pufM gene encodes the M subunit of a pigment binding
protein in the photosynthetic reaction center (Achenbach et al. 2001). This gene
is part of the puf operon, universally distributed among purple phototrophic
bacteria and also in Chloroflexus species (Heck et al. 2000). In anaerobic
anoxygenic phototrophic bacteria, this operon is repressed by oxygen, whereas
in aerobic anoxygenic phototrophs it is repressed by high light intensities
(Nishimura et al. 1996, Masuda et al. 2000).
The main objective of the present study was to study the diversity and
ecology of bacteria communities growing in a Vancouver Island soil sample
incubated under sunlight conditions in a Winogradsky column, with special
emphasis in phototrophic bacteria diversity, and also to better understand the
evolution process of these organisms. To assess the diversity and ecology of
these organisms, molecular biology and microbiological techniques such as
DNA extraction, PCR, colony hybridization, DNA sequencing and bioinformatics
analysis were employed. We hypothesize the presence of anaerobic
phototrophic organisms in anoxygenic environments in the deeper levels of the
column and aerobic organisms in the liquid and shallow depths with higher
oxygen levels.