Microbial molybdate reduction to mo-blue by a cyanide-degrading bacterium
Molybdenum, an emerging pollutant, has recently being demonstrated to be toxic to spermatogenesis in several animal models. It is also very toxic to ruminants causing death at very low level (parts per million). Molybdenum is mined as a small byproduct of copper and gold mining in Malaysia. Molyb...
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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Online Access: | http://psasir.upm.edu.my/id/eprint/68504/1/FBSB%202018%202%20IR.pdf http://psasir.upm.edu.my/id/eprint/68504/ |
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| Summary: | Molybdenum, an emerging pollutant, has recently being demonstrated to be toxic to
spermatogenesis in several animal models. It is also very toxic to ruminants causing
death at very low level (parts per million). Molybdenum is mined as a small byproduct
of copper and gold mining in Malaysia. Molybdenum pollution is also found
at dumping sites of waste oil lubricant containing molybdenum disulfide, with levels
of up to hundreds of parts per million found. Molybdenum, in the soluble form,
molybdate can be reduced to molybdenum blue, a colloidal and relatively insoluble
product and this phenomenon forms the basis for bioremediation of molybdenum. This
research is therefore, aimed at screening, phylogenetic identification, characterization
and optimization of molybdenum-reducing activity of the best isolate via one-factorat-
a-time (OFAT) and response surface method (RSM); modelling the kinetics of
molybdenum reduction through primary and secondary models; finally purify and
characterize the molybdenum-reducing enzyme activity. One of the ten previously
isolated cyanide-degrading bacteria from gold mine soils in Malaysia exhibited a
novel molybdenum reduction to molybdenum-blue, with this best molybdate-reducer
further studied on a molybdate low phosphate minimal salts media supplemented with
glucose and ammonium sulfate as the carbon and nitrogen source, respectively. Strain
HMY3 via phylogenetic analyses reveals that the isolate belongs to Serratia genus.
Sucrose was the best carbon source supporting molybdate reduction in this strain and
was optimum at 20 g/L. Ammonium sulfate was the best source of nitrogen for strain
HMY3 and was optimal at 10 g/L. Strain HMY3 grew best at 35 oC and at pH 6.5.
Response Surface Method shows the best conditions for molybdenum reduction were
molybdate concentration between 55 and 57.5 mM, phosphate concentration of 3.95
mM, pH 7, sucrose concentration between 15 and 17.5 g/L and incubation time
between 48 and 60 h. Molybdenum reduction was inhibited by the heavy metals such
as copper, mercury, chromium and arsenic at concentrations higher than 1 ppm.
However, prolonged incubation succeeded in overcoming this inhibition. Analysis of
the reduction kinetics showed that molybdenum reduction over time can best be modelled using the modified Gompertz model while the reduction kinetics was best
modelled using the Luong model. Statistical analysis of these models has been carried
out and they exhibit low values for RMSE and AICc, highest adjusted R2 values, Ftest
and with Bias Factor and Accuracy Factor nearest to unity (1.0) over other models.
The calculated value for the Luong’s constants which are maximal reduction rate, half
saturation constant for maximal reduction, maximal concentration of substrate
tolerated and curve parameter that defines the steepness of the growth rate decline
from the maximum rate symbolized by qmax, Ks, Sm, and n were 0.06±0.1 hr-1,
47.95±10.12 mM, 69.63±0.8 mM and 0.69±0.11, respectively. The Luong model
clearly shows strong substrate inhibition to rate of reduction at high substrate (Mo)
concentration. The Mo-reducing enzyme has been purified using an ammonium
sulfate precipitation followed by gel-filtration chromatography. The results showed
that the best ammonium sulfate fraction giving the highest enzyme activity was
between 50 and 60% ammonium sulfate concentration. The molybdenum-reducing
enzyme was monomeric with an estimated mwt of 105 kDa. The enzyme was further
characterized, and the results show that the enzyme is most stable in Tris-buffer pH 7
containing 0.1 mM DTT, temperature 4 oC and not affected by metabolic inhibitors
and heavy metals (1 ppm). The enzyme attains optimum catalysis at pH 5.5,
temperature between 25 and 35 oC, substrate concentration (LPPM) 12 mM and
electron donor (NADH) concentration 5 mM. In conclusion, a novel cyanidedegrading
bacterium with a molybdenum reduction capacity has been isolated, and
statistical method using RSM has succeeded in optimizing reduction. Also, the
enzyme has been purified to homogeneity. The characteristic of this bacterium makes
it suitable for future bioremediation works in polluted soil containing cyanide and
molybdenum metal. |
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