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By Ji-Dong Gu
Laboratory of
Environmental Toxicology, Department of Ecology &
Biodiversity, The Swire Institute of Marine Science, The
University of Hong Kong, The People’s Republic of China
E-mail:
jdgu@hkucc.hku.hk)
Introduction
Aromatic compounds are either
homocyclic or heterocyclic aromatic nucleus. Actually two
thirds of known chemicals contains heterocyclic structures.
Examples of heterocyclic are pyridine, indoles and substituted
indoles and all have at least one carbon substituted by
nitrogen on their structures. Nitrogen (N) substituted
aromatics including indolics are common constituents of
petroleum, coal, dyestuffs and agricultural chemicals, and
they are known environmental pollutants found in aqueous waste
effluents associated with oil shale, coal mining and farming
and in groundwater as a result of industrial and agricultural
contamination (Dailey, 1981; Gu et al., 1992; 2001).
Marine sediments of Hong Kong are
classified as polluted due to the unrestricted discharge of
sewer, industrial and agricultural wastes into the coastal
environments between 60’s-80’s. Indole and 3-methylindole (skatole)
are degradation products during tryptophan metabolism and
their concentrations are particularly high in pig waste.
Contamination of groundwater and subsurface environments where
anoxic condition is predominant poses a serious hazard to
human health and affect the fate of these pollutants.
Information on the transformation and degradation of indolic
compounds are essential in evaluation of their environmental
toxicity and the impact to the environment. However, little
information is available on the transformation processes and
the microorganisms involved under anaerobic conditions (Kaiser
et al., 1996). Therefore, the objectives of this study were:
1) to enrich strictly anaerobic microorganisms capable of
degrading selected indolic compounds; 2) to elucidate the
degradation pathways; and 3) to immobilize the degradative
microorganisms on carrier surfaces.
Results and
Discussion
Anaerobic environments
Marine sediments
in Hong Kong are not only rich in organic including a wide
range of pollutants including polyaromatic hydrocarbons but
also heavy metals, the concentration of the latter can be as
high as 0.1% in selected location (Gu and Ma, unpublished
data). Under the polluted conditions anaerobic microbial
activities including methanogenesis (methane producing) and
sulfidogenesis (sulfide producing) have been observed to be
high. It is reasonable to speculate that anaerobic bacteria
capable of degrading specific organic pollutants have been
evolved or adapted to the polluted conditions, and they are
actively transforming organic compounds. Isolation of
microorganisms capable of degrading specific chemical is not
only scientifically interesting, but also offers biotechnology
option for treatment of wastes.
To enrich and
isolate anaerobic microorganisms, we followed the following
procedures. Initially, methanogenic and sulfidogenic
conditions were simulated by utilizing the Hungate anaerobic
technique and artificial culture media with Na2S as
a reducing agent. Sulfate salt was present in sulfidogenic
medium but omitted from methanogenic medium. Subsurface
sediment materials from Victoria Harbour of Hong Kong SAR were
sampled and used as an inoculum (source of microorganisms) to
enrich microorganisms capable of using indolic chemicals as
the sole source of carbon and energy. Serum bottles were used
and the indolic compounds were the only organic chemicals for
the selective microorganisms capable of metabolizing the
specific chemical. High Pressure Liquid Chromatography (HPLC)
was applied to detect the concentrations of chemicals in
aliquot and Gas Chromatography (GC) to measure the methane
produced in the headspace of serum bottle. During experiment,
samples of 1.0 mL were withdrawn periodically from serum
bottles and centrifuged before passing 0.2mm-pore-size-membrane
syringe filter (Gelman Science, An Arbor, Michigan). Filtrate
was analyzed on HPLC and concentration was obtained through
calibration using external standards.
Methanogenic condition
Our results showed
that indole (2,3-benzopyrrole) was degraded in serum bottles
amended with the marine sediment. Since the presence of
environmental organic made interpretation of complete
degradation difficult, enrichment transfer was implemented.
During the transferring processes, a fraction (20%) of the
initial content in an active serum bottle was transferred into
a fresh prepared serum bottle containing anaerobic medium,
then both chemical and gas produced were monitored. After 5
successive transfers, mineralization of indole was observed
repeatedly within 28 days by the enriched consortium of
anaerobic microorganisms to methane and carbon dioxide.
Detailed procedures of transferring were described elsewhere (Gu
and Berry, 1991; 1992; Gu et al., 1992). At the same time,
our sterile controls showed a negligible loss of chemical
throughout the incubation period.
During degradation
of indole, at least two intermediates were observed on HPLC
chromatograms and they were further isolated, purified and
identified as oxindole and isatin (indole-2,3-dione) using a
combination of techniques including Thin-Layer Chromatography
(TCL), HPLC, UV-Visible spectrometry and Mass Spectrometry
(MS). The pathway of degradation follows two steps of
oxidation accomplished by hydroxylation and then
dehydrogenation at 2- and 3-positions sequentially prior to
the cleavage of the pyrrole ring between 2- and 3-positions.
It is interesting that degradation of indole by the enrichment
proceeds without an apparent lag phase indicating the
acclimation has successfully resulted in an effective
consortium capable of degradation. At the end of the
incubation, CH4 produced accounts for 86% of the
theoretical value using the following equation: C8H7N
+ 7H2O
¾®
4.5CH4 + 3.5 CO2 +NH3 and
substantial quantities of the added indole was mineralized.
It is expected that a fraction of the substrate is immobilized
in the biomass of microorganisms as biomass carbon.
No strong evidence
supports the degradation of methyl substituted indoles, namely
1-methylindole, 2-methylindole, and 3-methylindole
(3-methyl-1H-indole, skatole) under methanogenic conditions
using marine sediment taken from Victoria Harbour of Hong
Kong. Since substituting group on 1-, 2-, and 3-methyl
indoles may affect the activity of hydroxylation enzyme and
then inhibit the attack 2- and 3-positions, none of them
showed apparent degradation. However, it should be mentioned
that 3-methylindole (skatole) could not be mineralized under
methanogenic condition but was completely degraded under
sulfate-reducing condition in our recent study.
Sulfate-reducing conditions
Under
sulfate-reducing condition, sulfate serves as an electron
acceptor resulting in the production of H2S and at
the same time organic compound is oxidized serving as a source
of carbon. Sulfate-reducing bacteria from the marine
sediment showed degradative capability in transforming both
indole and 3-methylindole, none of the others including
1-methylindole and 2-methylindole was attacked. Previously
one bacterium capable of degrading indole was isolated and
identified as Desulfobacterium indolicum (Bak and
Widdel, 1986). Degradation of indole under sulfate-reducing
conditions followed a similar biochemical pathway as under
methanogenic condition, and both oxindole and isatin were also
identified as degradation intermediates. This illustrates the
universal occurring of hydroxylation enzymes in anaerobic
microorganisms. However, sulfate-reducing bacteria are more
competitive than methanogens in the marine environment due to
higher affinity for biologically produced molecular hydrogen
by the former. But both processes can take place
simultaneously in the marine sediment due to formation of
microniche and availability of hydrogen. Both degradation
process and the mechanism involved are currently under further
investigation. Our results suggest that N-heterocyclic
aromatic compounds can be degraded by selective anaerobic
microorganisms and the degradability is dictated by the
substituting groups and the position of substitution on
primarily the pyrrole ring of indolic compounds.
Surface immobilization
Since heterocyclic
aromatic compounds are generally more toxic than homocyclic
ones, treatment of wastewater or environments containing
heterocyclic aromatic compounds requires higher resistance to
the toxicity of chemical. In practice, one way to introduce
the microorganisms in bioremediation is by immobilization of
the microorganisms on surfaces of carrier materials as
biofilms because biofilms are more resistance to toxic
loading. We have successfully immobilized the consortium of
indole-degrading microorganisms on a carrier surface in a
bioreactor system.
In conclusion, our
results show substituted heterocyclic compounds may persist in
the environment, and the substituting group and the position
affect their fate and degradability. Both methanogenic and
sulfate-reducing bacteria possess similar biochemical pathways
in degrading indole, suggesting the ubiquitous of
hydroxylationa enzymes among anaerobic microorganisms. In
addition, it should also be point out that our bioreactor test
further provide evidence for treating pollutants in bioreactor
systems through immobilization of the degradative
microorganisms.
Acknowledgement
This research was
partial supported by CRCG grant of The University of Hong
Kong.
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