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Four different geochemical fractions of
the sediment were used to determine the metal content in each fraction,
measured using selective sequential extraction method. The four fractions
tested included the exchangeable and carbonate bound fraction, iron and
manganese oxide fraction, organic and sulfide fraction, and the residual
fraction from the solid. The results from this test was used as the before
experiments to be compared to the metal partitions of the sediment samples
after bioremediation.

In the biostimulation experiments where the
sediment samples were bio-remediated under eight different conditions, the
independent variables were the absence or presence of sodium acetate, lactose,
and/or inorganic compounds in different combinations. The dependent variables in
this part of the experiment are both the change in metal partitioning observed
in the different fractions and the change in bacterial density in the fractions.

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The control fractions did not have any substrates added. All experimental and
control conditions were incubated at 20ºC for 60 days, where small aliquots were
collected after 30 days and 60 days and analyzed for metal partitioning and
bacterial density. The experimental control used was very effective because it
contained no modification of added substrates and can be used to compare the change
in metal partitioning and bacterial density. There were significant increases
in metal partitioning in the exchangeable fraction for all metals except for
Cr. Metal partitioning of Zn and Cd decreased in the oxidizable fraction. Pb
and As displayed a decrease of partitioning in the residual phase. For the most
part, the change in Zn partitioning across the experimental conditions remained
the same despite the different conditions. Cd and Pb were more mobilized in the
presence of inorganic compounds. For As, the increase in the exchangeable
fraction was facilitated by the addition of acetate and lactose. Under all
conditions, bacterial density increased.

Bacterial diversity was then analyzed by Automated
Ribosomal Intergenic Spacer Analysis (ARISA). The DNA extracted from the
sediments were amplified using bacterial primers 16S-1392F and 23S-125R, and
analyzed using PCR. Negative controls for contamination include the PCR mixture
without the primers, while positive controls contained the DNA of Escherichia coli. Analysis by ARISA
determined a decrease in the amount of bacterial ribotypes after 30 days of
incubation and an increase in bacterial ribotypes after 60 days of incubation.

A positive correlation was seen between bacterial ribotype richness and the
addition of inorganic compounds. Bacterial assemblage by phyla was determined
using 454 sequencing of bacterial 16S rRNA genes of the sediment samples
collected before and after incubation. PCR was used again to track
contamination content in the samples. Sequence analysis was run by the MOTHUR
pipeline and Operating Taxonomic Units (OTU) were generated in order to
classify groups of present microorganisms in the sediment samples. Results showed
that with the addition of inorganic compounds, the number of sulfate-reducing
bacteria in the genus Desulfobacteraceae and
Desulfobulbaceae decreased while the
number of bacteria in the Flavobacteriaceae
increased when compared to the controls because the sulfate-reducing bacteria
are unable to thrive off of the inorganic compounds.

Overall, these experiments concluded that
bio-treatments of contaminated sediment samples increased mobility of Zn, Pb,
Cd, and As. Along with increase in mobility, an increase in bacterial density
was seen in the experimental conditions after incubation. The control also
displayed bacterial growth, indicating that materials present in the
contaminated sediment were degraded and used up for bacterial metabolism. Experimental
conditions in which organic compounds were added showed an increase in the
sulfate-reducing bacteria in the genus Desulfobacteraceae
and Desulfobulbaceae and a
decrease in bacteria in the genus Flavobacteriaceae.

Conditions with inorganic compounds added showed the opposite results. The
increase in sulfate-reducing bacteria indicates their ability to metabolize the
organic compounds in the contaminants and release byproducts that are harmless
to the environment. The results of this study suggest that the partitioning of
metals depends on both organic matter decomposition and sulfate reduction
processes facilitated by microbes. From the results of this study, the authors
suggest further exploration on the contributions of bacterial assemblages on the
solubilization of metals (1).


About the Microorganisms

is a family that
comprises of 21 genera, typically characterized by their 16S rRNA gene sequence
phylogeny. These are motile Gram negative bacteria found in freshwater,
brackish water, marine, and haloalkaline environments. Their shapes range from
cocci to vibrioand rods. The bacteria in this family are strictly anaerobic,
with some capable of fermentative metabolism. They can oxidize organic
substrates to carbon dioxide through the C1 pathway, except for the genus Desulfobacter, which use a modified
TCA-cycle. Desulfobacteraceae is
known for its sulfate-reducing abilities under mesophilic or psychrophilic
conditions. They are capable of this because their mode of action uses sulfate
and thiosulfate as the electron acceptor, and uses reduced sulfur compounds as
a carbon source (4). It was found that members of this family and other
sulfate-reducing microorganisms were identified as contributors of carbon
cycling and climate change in the wetlands (5).

Desulfobulbaceae consists of seven genera, also characterized by their 16S rRNA gene sequence phylogeny. These are motile, Gram negative,
and strictly anaerobic bacteria, with possible fermemtative metabolism
capabilities. Most species are mesophilic and psychrophilic, but can also be
obligate alkaliphilic. They are best known for their sulfur-reducing
capabilities, and are found in marine, brackish, and freshwater habitats. They
have similar characteristics to members of the Desulfobacteraceae
family (6). One
study about this family showed that some members of this family are mercury
methylators and are capable of breaking down inorganic divalent mercury in
marine environments, which is a beneficial process because mercury
contamination poses a dangerous health risk to marine life and humans (7).

Flavobacteriaceae is a large family consisting of 90 genera,
found in marine, freshwater, and soil habitats. They are Gram negative
bacteria, and most are aerobic respiratory metabolizers. What differentiates
them from other families is that they use menaquinone of type 6 as the major
respiratory quinone. Most members of their family are able to secrete proteins outside
the outer membrane via the Por secretion system.


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