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Microbial Biosensors-Intelligent Tools in Hydrocarbon Bioremediation

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Microbial Biosensors-Intelligent Tools in Hydrocarbon Bioremediation Powered By Docstoc
					Microbial Biosensors-Intelligent Tools in
     Hydrocarbon Bioremediation



               Graeme I. Paton

          University of Aberdeen and

              Remedios Limited
      Oil pollution and Remediation
4 Oil pollution
   • many localised hotspots
4 Bioremediation is a successful method for dealing with
  oil contamination
4 Current methods of assessing success are usually
  chemically based
    Trade-off between relevance and
                simplicity


                      Ease of
Increasing            use       Decreasing
ecological                      understanding
relevance and                   and ease of
complexity                      performing
                                assays




                complexity       Paton et al., SUM 2005
         Tests chosen cover three levels
                  of complexity


Biosensors (single           Ease of
species)                     use


Biolog (rapid
culturable method)


PLFA profiles (based
on entire community)
                       complexity      Bundy et al., SBB 2004
                     Constitutive

Bacterial




            Increasing Toxicity

  Yeast
        Induction/degradation


                    Sensor unexposed (-H-C) and
                    exposed (+H-C) to induction
                    target - sole carbon source


- H-C       + H-C



                    Same plates photographed in
                    darkness.
                    Carbon-Source
                   Utilisation Profiles
4 Biolog microtitre plates can be used for
  rapid and easy analysis
   • contain 95 different carbon sources
   • inoculate aqueous extract of soil community
   • output analysed by multivariate


4 Results are dominated by effects of
  culturable, fast-growing bacteria
4 Trendy in the early mid 90s but falling
  out of favour
           PLFA community analysis
4 Phospholipids are extracted directly from soils:

                       extraction

                       fractionation

                       derivatization

                       identification

4 Reports on entire microbial community
4 Slower to perform than Biolog profiles
                     Experimental design
A:       Three soil types treated with +/- diesel oil
     • ST1: sandy loam, pH 5.9
     • ST2: sandy loam, pH 5.2
     • ST3: loamy sand, pH 6.2


B:       One soil treated with three different oils
     • paraffin (light refined oil)
     • diesel (medium refined oil)
     • motor oil (heavy refined oil)

4 Soils adjusted to 55% WHC
4 C:N:P ratio of 100:12:1
4 Replicates destructively sampled at 0, 4, 9, and 15 weeks
                     Disappearance of hydrocarbons: % loss

                   125                                            125
                                    A                                              B
Diesel loss (% )




                   100                                            100




                                                  Oil loss (% )
                    75                                             75
                                                                                            MO
                    50                                             50
                                                                                       D
                    25                                             25
                                                                                   P
                     0                                              0
                         0      5       10   15                         0      5           10    15
                             Tim e (w eeks)                                 Tim e (w eeks)
                                  Constitutive biosensor results
                                (toxicity): response to diesel, ST1
Luminescence (% of control)




                              100

                               75                                       0
                                                                        3
                               50                                       9
                                                                        15
                               25

                                0

                                     E. coli    P. putida    P. fluor
                              Specific biosensor results: response
                                          to diesel, ST1
Luminescence (% of control)


                              300
                                                                     0
                              250
                                                                     3
                              200                                    9
                                                                     15
                              150

                              100

                               50

                                0

                                     OCT         NAP         IPB
                 Biosensor Results
4 Specific biosensors do not respond strongly to refined oils
  (do for key analytes).
4 Metabolic biosensors show an initial increase in toxicity
  associated with bioremediation
4 Optimal: sensitive catabolic (octane biosensor) + relevant,
  sensitive metabolic (P. putida).
                    Canonical variate analysis of
                  Biolog data: three soils +/- diesel
       8
                                                     ST1

       3
                                                     ST2
                                                     ST3
CV 2




       -2

                                                    cont..
       -7                                           +oil

   -12
            -12         -7      -2   CV 1   3   8
                        CVA analysis of Biolog data: ST1,
                         three different oil treatments
             11


             6
CV 2 (17%)




             1

                                                          control
             -4
                                                           diesel
                                                         paraffin
             -9
                                                        motor oil
        -14
                  -15             0   CV 1 (76%)   15       30
                    Biolog Results
4 Incubation effect is larger than exposure effect.

4 Soil types are still clearly defined even after 15 weeks of
  diesel contamination + biodegradation

4 No apparent differences between paraffin, diesel and
  motor oil
                       PCA analysis of PLFA data: three
                        different soil types +/- diesel
             6
                                                           ST1
             4
                                                           ST2
             2
PC 2 (19%)




                                                           ST3
             0

             -2                                           cont.
             -4                                           +oil
             -6

             -8
                  -6            -1
                                     PC 1 (21%)   4   9
                     PCA analysis of PLFA results:
                  ST1 + three different oil treatments
                                                       control
             8
                                                        diesel
             6                                        paraffin
                                                     motor oil
PC 2 (14%)




             4

             2

             0

             -2

             -4
                  -6                0   PC 1 (26%)        6
                      PLFA Results
4 Good separation between incubated diesel-spiked and
  control soils

4 No apparent differences between paraffin, diesel and
  motor oil

4 Less variability in controls than for Biolog results

4 Separated ST3 from ST1 and ST2
       ……….so what does this prove?

4 Hydrocarbon (mixed compounds) degradation is different
  from exotics (sole analytes)

4 Biotransformation not just mineralisation are important

4 Maintaining the population not just enrichment is important
                         Tool-kit
4 In addition to good solid chemical analysis- 3 ecologically
  based components
   • Community tests
      – Respiration
      – Most Probable Number (MPN)
   • Constraints to bioremediation
      – Metabolic biosensors
   • Induction of relevant contaminant degraders
      – Catabolic biosensors
                      Case Study
4 Small-scale spatial
  heterogeneity
   • Inter-tidal sediment
     impacted with 50,000
     L of diesel
   • Assessment of the
     Tool-kit
   • 64 sample locations
     in confined area
Total H-C
H-C Degraders (MPN)
Constitutive - E.coli HB101 (pUCD607)
Specific- E.coli DH5α (pGEc74, pJAMA7)
                       Case Study
4 Demonstrates significant variability of parameters within a
  small contaminated field area

4 Integration of microbial tools with chemical data can be
  applied to assess environmental impact of a H-C spill

4 Spatial presentation enables a focused remediative strategy
Importance of Scale
                     0.35
                                                                                                                                                      0.25
                     0.3               Microcosm Mesocosm Field




                                                                                                                  Respiration (mg carbon dioxide/g)
                                                                                                                                                                       MicrocosmMesocosmField
Hydrocarbon (mg/g)




                     0.25                                                                                                                             0.2

                     0.2
                                                                                                                                                      0.15
                     0.15
                                                                                                                                                      0.1
                     0.1

                     0.05
                                                                                                                                                      0.05
                       0
                            0   100   200   300    400                       500       600         700                                                  0
                                               Time                                                                                                         0    100     200   300   400   500   600   700
                                                                                                                                                                                 Time

                                                                            1400

                                                                            1200             Microcosm     Mesocosm                                    Field
                                                      MPN- relartiveunits




                                                                            1000

                                                                             800

                                                                             600                                                                        -

                                                                             400

                                                                             200

                                                                               0
                                                                                   0         200          400                         600                       800
                                                                                                         Time
                  Case Studies

Scotoil MGP                  Pesticide spill
Ferryhill                    Witton Chem
Total (various)              Nigg (various)
Shell (various)              Kuwait terminal
BP (various)                 Saudi Ramco
Corus (various)              Sheffield
C’hill Homes                 Dundee Oil
AHB                          Allied Bakers
Newcastle (various)          BTEX sites
Exeter (SEREBAR)             …..about 42 to date
Aldermaston (AWE)
         Resp                 [TPH]
BF =                 x                     x Inhibition
       (I x [TPH]           log (MPN)




            BF = bioremediation function
                    I = induction
             [TPH] =TPH concentration
            MPN = most probable number
                 Resp = respiration
                                      BF & Rate of Degradation

                   10000




                    1000
Rate (mg/kg/day)




                     100




                      10




                       1
                        1e+1   1e+2    1e+3    1e+4   1e+5   1e+6   1e+7   1e+8

                                                 BF
                                      BF & Rate of Degradation

                   10000




                    1000
Rate (mg/kg/day)




                     100




                      10




                       1
                        1e+1   1e+2    1e+3    1e+4   1e+5   1e+6   1e+7   1e+8

                                                 BF
                   The “Algorithm”
4 Refined Algorithm is a valuable predictive tool

4 Reduces uncertainty

4 Widely applicable

4 Cost effective and competitive
A TCE (DNAPL) Plume
  AWE Aldermaston
Total concentration of CAHs in samples

                         Concentration in mg/l
Response of Pseudomonas fluorescens


                            % luminescense
                              Interpretox®

                                Charcoal                pH adjustment
Air sparging
             Removal of                       Removal
               volatile                    of non-volatile          Removal of
                                                                    adverse pH
               organic          (resuspension) organic
             compounds                       compounds

 high        low               high     low                  high   low




         Further sample
          manipulation
                                        Inorganic    pH constraint Further sample
                                                                    manipulation
                          non-VOC’s     constraint
 VOC’s
constraint                constraint
Response of Pseudomonas fluorescens


                            % luminescense
Response of Pseudomonas fluorescens to
           sparged samples


                               % luminescense
Response of Pseudomonas fluorescens to
       charcoal treated samples

                                % luminescense
Response of Escherichia coli DH5α (alkane
     induced) to untreated samples

                              Fold induction
Response of Escherichia coli DH5α (alkane
     induced) to sparged samples

                                Fold induction
Response of Pseudomonas putida TVA8 (TCE
 and toluene induced) to untreated samples


                                 Fold induction
Response of Escherichia coli DH5α (alkane
  induced) to charcoal treated samples

                                Fold induction
       Realisation of the Data Potential
4 Massive chemical and biological data sets

4 Often commercial contractors with no interest in
  interpretation

4 Does MNA take place (monitored natural attenuation)?

4 Seasonality
Summer
Winter
                 AWE Aldermaston

4 Multiple pollutant inputs

4 Focussed biosensor work- very successful

4 MNA needs other tools

4 Significance of sample strategy
Railway Yard Development
     Excavation area 2 – exposure of hydrocarbon
contaminated soils, confirmed by elevated FID readings
Protective Barrier Installation to Prevent
        Migration from Off Site
Conclusions 5
 Data Collection                                                 Material Being Excavated
                                                                                                                                                                   Assess against Risk
 Nutrient level
                                                                                                                                                                   Pass- then stockpile
 Moisture
                                                                                                                                                                   Fail- then biopile
 pH
 TPH
 MPN
                                                                             TPH Characterisation
 Biosensor
                                                                             On site FID                                        FID low levels
 CO2
                                                                             Off site analysis                                  Group material and send to AlControl
 O2

                                                                                                                                Phase material
                                                                                                                                Group material; do not add to biopiles
 End-point Criteria                               TPH levels
 Olfactory                                        If TPH between 0.05 and 10 g/kg
 Risk-based                                       Consider for biopiling                                                        Non-TPH
 Physical/ engineering                                                                                                          Group material and send to AlControl


                                                   Site Status                                                                  Excessive TPH                          Difficult Substances
                                                   Base of biopile area                                                         Levels exceeding 10 g/kg               Group and stockpile
                                                   Made ready as Page 6                                                                                                For decision later



                                                   Material Management
                                                   Data collection for characterisation




Nutrient Amendment             Moisture                           pH of soil                        TPH                                   MPN                       Biosensor
If trace levels are present,   Determine the water                Amend with lime or                Calibrate FID with AlControl/         A count of less than      Use MeOH and
use 100:10:1                   holding capacity of the soil       sulphur to reach pH of 6-         lab data and record for site          104 is too low and        water to assess
Select N source to suit pH     and maintain at levels as          7.5.                              characterisation                      augmentation is           bioavailability of co-
(urea, ammonium nitrate)       per manual.                        Use standard agricultural                                               requirted                 pollutant and TPH
Add before biopiling and mix                                      calculation but remember
well.                                                             CEC will be low




                                                                                                                                    Biopile Algorithm
                                                      Amend and Optimise before biopiling                                           Put derived data into equation (p29)
                                                                                                                                    and calculate decay



                                                                                                               Verify Algorithm
                                                                                                               Use microcosm to check algorithm
                                                                                                               prediction- max 2 weeks
                  Algorithm calculated                                                                     Defined Targets
                    to assess most                            Microcosm                                    Olfactory
                     suitable set-up                          Microcosm                                    Risk-based
                                                              experiment used to                           Physical/ engineering
                                                              verify algorithm



                       Constructed Biopile
                                                                                   TPH Measurements
                                                                                   Analysis carried out                                        YES   Continue monitoring
                                                                                   with calibrated FID
                                                                                                                   Levels change as
                                                                                                                   expected by
                                                                                   CO2 Measurements                algorithm
                  Specific Analysis            Routine Analysis
                                                                                   Gas Analysis
                                                                                                                                               NO    Plot data
                                                                                                                                                     To assess trends
                          Nutrient Amendment                                       O2 Measurements
                          Verify bioavailable status in KCl                        Gas Analysis                    WHC
                                                                                                                   Confirm the values are
                                                                                   Percent WHC                                                       Turning
                                                                                                                   between 60 and 80%
Biosensor                 Analysis                                                 Derived from                                                      If the system is air
Use MeOH                  Verify presence of toxic                                 moisture                                                          limited then turn
and water to              metabolites or co-pollutant                                                              pH
assess                                                                                                             Make sure 6-7.5 or
bioavailability                                                                    pH                                                                Moisture
                                                                                   In water                        amend: check buffering            Add/ remove as
of co-pollutant           Physical constraints
and TPH                                                                                                                                              required
                          Trial pits to make sure the
                          biopile is homogenised                                   Temperature                     Temperature
                                                                                   Using probe                     If the temp is too low
MPN                                                                                                                                                  pH
A count of                Assessment of Bioaugmentation                                                            (<10) then add steam; too         Trickle filter to adjust
less than 104             If the biopile was amended or                                                            high (>40) remove
is too low and            not,there should be a minimum                                                            covers.
                                                                                   None of the above
re-                       number of degraders present                              The cause of                                                      Temperature
augmentation                                                                       reduced degradation                                               Assess site weather
is requirted                                                                       rate cannot be                                                    and forecasts
                                                                                   related to one of the
                                                                                   above factors
Windrow - construction
Windrow - view to west
Construction of Windrow 2 - view to north west
                 Gantt Chart for Windrow Progress
             18/07 25/07 01/08 08/08 15/08 22/08 29/08 05/09 12/09 19/09 26/09 03/10 10/10 17/10 24/10 31/10 07/11 14/11 21/11 28/11



 Biopile 1
         2
         3

                                          t      t    t t     t t   t t   te te   t     t   t   t
Biopile 1A                          pH, N       A
                                         t       t    t   t   t t   t t   te te   t     t   t   t
        2A                          pH, N       A
                                                      t         t   t t   t t     t t       te te   t   t   t   t
        3A                                      pH, N         A
                                                      t        t    t t   t t     t t       te te   t   t   t   t
        4A                                      pH, N         A



                                          slippage               pH       adjust/ optimise pH
                                          impact of slippage     N        add nutrients
                                                                 t        turning
                                          sampling (week start) e         excavator for sampling
                                          sign off (week end)    A        Addition of inoculum
                                          back fill (week start)
Compaction of Material in Backfill Area
Ongoing and Future Research Strategies
4 Laboratory studies do little to help us prepare for the real
  environmental conditions
4 Need to link micro to meso to field!
4 …………and select the correct techniques
               General Conclusions

4 Bioremediation- being more widely used

4 End-point issues are important as is the re-use potential of
  the material

4 Priority for meaningful and easy to understand
  measurments
                  Acknowledgments
University of Aberdeen                Enterpris
Prof. Ken Killham                     Prof. David Read
Dr. Julian Dawson
Dr. Jake Bundy                        Remedios
Dr. CJ Patterson                      Diarmuid O’Sullivan
Dr. Chris Paynter                     Lucy Stuart
Mohammed Al-Alwadi                    Dave Mardlin
Dr. Saad Alamari                      Adam Ritchie
Dr. Jessica Bellarby
Dr. Leigh Cassidy                     Macaulay Institute
Liz Diplock                           Dr. Colin Campbell


                       Funding
                       BBSRC, NERC, EPSRC
                       SNIFFER, Enterpris, Remedios,
                       Commonwealth Universities

				
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