Miniaturizing MICREDOX A microplatform for environmental monitoring applications to BOD and Toxicity

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Miniaturizing MICREDOX A micro-platform for
environmental monitoring - applications to BOD
and Toxicity
J Webber A Tizzard A Scott J Hay N Pasco
PO Box 133, Lincoln, Christchurch 8152, New
Zealand
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Outline

• Microbial sensors
• BOD5 - MICREDOX BOD
• Miniaturization
• BOD
• Toxicity

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Microbial biosensors

• use whole-cells
• environmental applications
• analyte type
• detect target chemical(s)
• aggregate e.g. BOD
• selective e.g. priority contaminant
• effect type
• report biological impact
• e.g. toxicity

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Biochemical oxygen demand (BOD5)

• Measures organic pollutants in water
• Composite substrate
• Standard (GGA 150 mg/L)
• Oxygen demand - surrogate for unknown substrate
  
• Is a biological oxidation process
• 5-day incubation (300 ml)

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MICREDOX

• Rapid mediated microbial-based assay
• Mediator acts as electron acceptor
• Mediator registers amount of bioconversion
• Rate adjusted by cell concentration
• Substrate (unknown) now rate-limiting

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Miniaturization of assay

• signal detection
• microelectrode amperometry
• separate working electrode compartment

• incubation volume
• 15 ml ? 1.5 ml ? 0.15ml
• sparging ? shaking
• signal (sparge) ? signal (shaker)
• signal (shaker) ? f(v)

r2 0.97
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MICREDOX miniaturization
signal detection by microelectrode
incubation in multi-well plate
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• StandardMICREDOX
• 16 ml
• incubation 12 hour
• Falcon tube
• reduced mediator
• seed 27 mg
• mediator/GGA50 1
• linear range 0-250 mg/l

• BOD5
• 300 ml
• incubation 5 day
• glass bottle
• O2 consumed
• seed 0.15 mg
• O2/GGA1 6.5
• linear range 2-8 mg/l

• Miniaturized MICREDOX
• 0.2 ml
• incubation12 hour
• 96-well plate
• reduced mediator
• seed 0.33 mg
• mediator/GGA50 1
• linear range 0-250 mg/l

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MICREDOX BOD

• GGA
• glucose/glutamic acid
• simple substrate
• 1º BOD5 standard
• unsuitable as rapid BOD standard
• BOD5 100 mg/l

• OECD
• Peptone/meat extract
• complex substrate
• 2º BOD5 standard
• recommended as rapid BOD standard
• BOD5 185 mg/l

Use response to GGA and OECD standards as screen
for suitable microbes
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StrategyMultivariate analysis
MICREDOX BOD
MICREDOX BOD BOD5 MICREDOX signal
(OECD/GGA) BOD5 (OECD/GGA) 1.85

• Microbial strain (4)
• Growth media composition (3)
• Growth media strength (2)
• Harvest time (2)
• 48 factor-level combinations (quadruplicate)
• Measure OECD and GGA standard responses at each
  level

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Main factor effects on MICREDOX BOD
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Main factor trends
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MICREDOX toxicity

• Measures effect on microorganism
• Known amount of substrate (GGA)
• Toxicant modifies signal

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Attenuation of signal by toxin (E.coli / GGA /
3,5-DCP)
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3,5-dichlorophenol
B.subtilis
E.coli
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EC50 3,5-dichlorophenol
1 Standard MICREDOX
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Conclusions

• MICREDOX is amenable to miniaturisation
• BOD, toxicity
• Miniaturization significantly boosts throughput
  of assay
• BOD
• quantitative signal (amount of biodegredation)
• simple substrates can dominate short term signal
• Toxicity
• relative signal
• EC50s consistent with literature data
• selection of measurement range and replication
  allow for estimation of
• confidence interval

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Thanks
LVL co-workers Griffith University Dr G
Lloyd-Jones, Dr M Noonan NZ Foundation for
Research Science Technology Waste Technology
Group
PO Box 133, Lincoln, Christchurch 8152, New
Zealand