Bioluminescent method using Photobacterium leiognathi strain AK-MIE for rapid screening of heavy metals
Anthropogenic activities have contributed to the release of harmful toxicants into the environment which will eventually end up in sediments and soils which are the foundation of food web. Application of instrumental analysis solely is time consuming, costly, and only limited to the concentration...
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/89933/1/FP%202020%209%20ir.pdf |
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| Summary: | Anthropogenic activities have contributed to the release of harmful toxicants into
the environment which will eventually end up in sediments and soils which are
the foundation of food web. Application of instrumental analysis solely is time
consuming, costly, and only limited to the concentration. Incorporating a rapid,
sensitive, and cheap bioluminescent based bioassay could indicate the toxicity
level and reduce analysis time and cost. However, commercially available
system uses a test organism with an optimum temperature of 15ºC-25ºC that
required an expensive thermostat. In this study, we aim to isolate bioluminescent
bacteria with broad range of growth temperature to assess the toxicity of
environmental samples. A new bioluminescent bacterium strain was isolated
from squid (Loligo duvauceli) and identified as Photobacterium leiognathi strain
AK-MIE (AKS01) using 16S rRNA phylogeny analysis. The bacterial culture was
grown in a luminescent broth media with different concentrations (0%-0.6%) of
peptone, yeast extract, and salt, and a wide range of pH (5.5-8) and
temperatures (4ºC-50ºC). Analysis through OFAT showed the best
concentrations were at 0.2% of peptone, 0.3% NaCl, and 0.4% yeast extract, at
pH 7.5 and temperatures ranging from 22ºC to 33ºC. The optimisation of medium
composition was carried out using Response Surface Methodology (RSM) and
Artificial Neural Network (ANN) with four parameters employed (NaCl, peptone,
yeast extract, and pH). The predicted optimum conditions by RSM (2.76% NaCl,
2.28% peptone, 0.34% yeast extract, and pH 6.83) and ANN (2.21% NaCl,
2.27% peptone, 0.39% yeast extract, and pH 6.94) produced 541,211.80 RLU
and 541,986.20 RLU respectively. The coefficient of determination value (R2) for
RSM (0.9440) and ANN (0.9934) indicate a high correlation between the
experimental and predicted values with ANN showing a superior data fitting
capability. The strain was tested with different concentrations of toxicants that
showed inhibitive effects between the range of 0.001 to 100 mg/L for toxic
metals and 0.01 to 1000 mg/L for both pesticides and xenobiotics. The toxicity effects were determined by measuring the luminescence after 15-30 minutes of
incubation period. The results based on IC50 values at 30 minutes exposure time
showed that the bacterium was most sensitive to mercury (0.00978 mg L-1)
followed by cadmium (0.5288 mg L-1), copper (0.8117 mg L-1), silver (1.109 mg
L-1), lead (10.71 mg L-1), chromium (36.17 mg L-1), zinc (72.83 mg L-1), and nickel
(97.85 mg L-1). The high values of R2 (>0.95) indicate a good relationship
between the toxicity of heavy metals and the bioluminescence inhibition. There
was no marked bioluminescence inhibition for pesticides and xenobiotics.
Twelve out of 20 sediment samples collected from different factories in Bangi
and Seri Kembangan showed positive toxic responses with more than 30%
luminescence inhibition. The highest luminescence inhibition recorded was from
sample 2 (61.43%). The validation method carried out using Inductively Coupled
Plasma-Mass Spectrometer (ICP-MS) proved the presence of mercury, copper,
cadmium, lead, and silver. Ten sediment samples collected from the Tekala
River as a control showed no toxic responses with minimal to none luminescence
inhibition. |
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