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About OMICS International Conferences

About OMICS Group

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Mumbai.

Workshop:Metabolomic Research

using Bio-electrochemical Systems

John M. Pisciotta

April 27, 2015

Metabolomics and System Biology

Philadelphia ,PA

Workshop Overview

• Types of BESs & their Set up

• Reactor Configurations

• Electrode Types & Materials

• HPLC analysis of cell metabolites

• Bioelectrochemical techniques (CA, LSV)

• Cell & Metabolite Sampling

• Unique Opportunities for Metabolomics

• Considerations

• Summary / Demos Units

Bioelectrochemical Systems: Types

• Microbial Fuel Cells (MFCs) exo-electrogenic bacteria on anode breakdown chemicals and donate useful electrons through a circuit. (Air cathode) = Electricity

• Microbial Electrolysis Cells: exo-electrogenic bacteria on anode breakdown chemicals and donate useful electrons through a circuit (Anaerobic cathode + voltage boost = Hydrogen Gas)

• Microbial Electrosynthetic Cells: use electrotrophicbacteria on the MEC cathode to accept electrons to store input electrical energy as chemical bonds.

• Enzymatic BESs: use redox active enzymes attached to electrodes to carry out specific reactions.

BES History

• 1911: M.C. Potter, at the University of Durham discovered that bacteria could produce direct electric current.

• 1934: Cohen develops 35 V microbial ½ cell.

• 1999: BH Kim discovered microbes do not require external mediators to generate current.

• 2014: hundreds of new BES article every year!

O2 = Terminal Electron Acceptor (TEA) in Aerobic Systems

Aerobic respiration allows full use

of the energy in food/fuel, it is little wonder

This mode of existence predominates today

Aerobic Electron Transport Chain

BES = Bioelectrochemically

Controlled Combustion

• Iron reducing bacteria

Luef et al., 2013. ISME Journal

MFC electron flow

Understanding the

Engineering

Microbes consume organic waste and transfer high

potential electrons through an electrical device

positioned between microbes and the system‟s

cathode TEA (O2).

Not just any Microbe can donate

Electrons to MFC anode:

-EXOELECTROGENS

(ex. Geobacter sp.)

Electron Transfer Mechanisms

from exo-electrogen to anode

1)Direct electron transfer: Geobacter sulfurreducens

2)Soluble Mediators: Shewanella oneidensis

Metabolomic methods can help ID mediatorsImage Source

Seminar only

Other BESs use Enzymes:Metabolomic methods can determine product formation rates

Zebda et al., 2011. Nature Communications

Some BESs use Enzymes:

-Difficulty in purifying enzymes

-Difficulty in adhering to electrode

-Difficulty in sustaining enzyme activity (just catalysts)

-Can not self repair or regenerate.

-Can not spread

-Can not be genetically programmed Zebda et al., 2011.

Nature Communications

BES Configurations

• Single Chamber

Simple & inexpensive

Amenable to High throughput screening

• Dual Chamber

Complex & Expensive

Separation of Anolyte from Catholyte (PEM)

Preferred for metabolomic studies

Single Chamber MFC

Resistor

Air Cathode

Reference

Wire

Sediment

Brush anode

AEROBIC

ANAEROBIC

Potentiostat

a b

Counter electrodeWorking electrode

Reference

Headspace PEM

Ohm‟s law used to

Calculate current

I = V/R

Single Chamber Customization:

Spec Tube MFC

Screw cap clamps wire between Cathode’s inner Pt surface andGlass top of test tube

Paint-on Anode insulator Prevents short circuit, createsWater tight seal w/Cathode PTFE layer

Spec Tube MFC

0

0.1

0.2

0.3

1

53

10

5

15

7

20

9

26

1

31

3

36

5

41

7

46

9

52

1

57

3

62

5

67

7

72

9

78

1

83

3

88

5

93

7

98

9

10

41

10

93

11

45

11

97

12

49

13

01

13

53

Negative

WW Tube

Curr

ent

(mA

)

Time (days)

Electrogenic Activity(Current determined by V drop across resistor)

0 7 14 21 30

Days

Dual Chamber “H-type”

ReactorResistor

Air Cathode

Reference

Wire

Sediment

Brush anode

AEROBIC

ANAEROBIC

Potentiostat

a b

Counter electrodeWorking electrode

Reference

Headspace PEM

Potentiostat

• Electronic hardware required to control a

three electrode cell and run most

electroanalytical experiments.

• Serves to maintain electric potential of the

working electrode at a constant level versus

a reference electrode by adjusting current at

an auxiliary electrode.

Electrodes & Electrode Materials

used on other Type of BESs

• Photosynthetic Microbial Fuel Cells (UMD / Baskakov)

• Dark Electrotrophic Systems

(Penn State / Logan)

• Experimental Considerations for optimizing

Metabolomic analysis using BESs

Electrogenic and Electrotrophic

Microbial Fuel Cells

CO2

Biofuel

e-

Electrogenic Activity of

Cyanobacteria

Idea: Convert sunlight to electricity using

photosynthetic organisms on an anode and

H2O as electron source.

(A living solar panel)

PI: Ilia Baskakov,

U. Maryland Center For Marine Biotechnology

Photosynthetic Microbial Fuel Cell

-Illuminated biofilm donates electrons to anode (B).

For details see references 5, 6.

Single Chamber Cyanobacterial Fuel Cell

Carbon paint anode / carbon cloth Pt cathode

Rapid Light-Dependent Rise in Voltage

Photosynthetic Biofilm

Increasing light boosts electrogenic

response (polyaniline coating boosts voltage)

Poly A

Carbon Paint

Zou, et al. 2010, Bioelectrochemistry

Untreated

Poly Pyrrole

Poly Aniline

Conductive Polymer

coated electrodes allow

Greater Power-Carbon Paint Base-

Photosynthetic Biofilm

Poly Pyrrole nanostructure also

affects –Power-

Fibrular Poly P

Poly P

Fibrular Granular

Zou, et al. 2010, Bioelectrochemistry

Likely through Decreasing Resistance

Untreated

Poly Pyrrole, granular

Poly Pyrrole, fibrularPhotosynthetic Biofilm

EIS Analysis

Electrochemical impedance

spectroscopy (EIS)

• Allows analysis of the internal resistance

of BESs, electrodes & materials, catalyst

coatings, biofilms and reactions on the

anodes and the cathodes.

• Requires potetiostat

Non-phototrophic bacteria: Sediminibactetium

(a2), Prosthecobacter (a3, a4) and Methylococcus (a7)

detected.

Species Composition of

Photosynthetic biofilm

Pisciotta et al., 2010, PLoS One

Scenedesmus microalgae growing on PMFC

carbon nanofiber electrodes

Zou et al., 2009

Electrogenic fingerprints.

Pisciotta et. al., 2011. AMB

What is the Biological Basis?

Red

Blue

DO Thin lineE.A. Thick line

Electrogenic activity peaks

under strong light,

Suggesting possible

function: photo-protection

Red

Blue

DO Thin lineE.A. Thick line

Can metabolite production

be influenced by

Bioelectrochemical Interfacing?

Metabolomic HPLC analysis

Photo-protective Carotenoids less prominent in electrically-connected (thin line)

versus Disconnected (thick line) PMFCs.

Pisciotta et. al., 2011. AMB

DARK

Electrotrophic BES.

CO2

Biofuel

e-

Electrochemical Methods to ID effective

e- uptake from Biocathodes

1) Linear Sweep Voltammetry (LSV)

- Short term electron uptake (i.e. reduced overpotential)

2) Cyclic Voltammetry (CV)

Reversibility of electron exchange

/ mediator characterization

3) Chronoamperometry (CA)

- Long term electron uptake by monitoring Negative Current over time

4) Metabolomic Identification of Product (ex. GC of gas phase)

Electrotrophic CO2 fixation

using Biocathodes

• Idea: Enrich and isolate bacteria able to accept

electrons from a cathode for reduction of CO2 into fuel.

the “electrotrophs”

Sources Tested:

• Chesapeake Bay Sediment

• Baltimore Harbor Sediment

-200 mV*Unpoised

MFC establishment

conditions

* -200 mV vs Ag/AgCl reference electrode (aka “Set”)

40

Sediment MFC anode to MEC

cathode by Inversion Method.

1) LSV

2) CV

3) Current Uptake

4) Chemical Analysis

A) MFC anodes in anaerobic sediment

establish electrogenic biofilm.

B) MFC anode then inverted to form functional

MEC biocathode.

Potentiostat

Cathode

Reference

Wire

Brush anode

Sediment

AEROBIC

ANAEROBIC

A B

(Pisciotta. et al., 2012. AEM)

Linear Sweep Voltammetry (LSV) indicates

highest

short term electron uptake by harbor biocathode.<I> vs. Ewe

LSV 7_1_11_marine_sed_PBSnoACE_12.mpr LSV 7_1_11_marine_sed_PBSnoACE_13.mpr LSV 7_1_11_marine_sed_PBSnoACE_14.mpr LSV 7_1_11_marine_sed_PBSnoACE_15.mpr

LSV 7_1_11_marine_sed_PBSnoACE_16.mpr #

x-axis

0.20-0.2-0.4-0.6-0.8

<I>

/m

A

0

-0.5

-1

-1.5

-2

-2.5

-3

-3.5

-4

-0.9 -0.6 -0.3 0

Biocathode Potential (vs Ag/AgCl)

Cu

rren

t (m

A)

0

2

4

ControlBayBay SETHarborHarbor SET

Cu

rren

t (m

A)

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0 12 24 36 48 60 72 84 96 108

Control

Bay

Bay Set

Harbor

Harbor Set

20:80 (CO2:N2)50 mM PBS, 10 ppt NaCl No substrate

Time (Hours)

Chronoamperometery

Development of negative current

(-650 mV vs Ag/AgCl)

-125

-100

-75

-50

-25

0

25

Sterile

Bay

Bay Set

Harbor

Harbor Set

Coulo

mbs o

f C

harg

e

Time (days)

0 1 2 3 4 5 6 7

Sustained current uptake Vs. sterile control.

0.0

10.0

20.0

30.0

40.0

n650

n750

Metabolomic GC Analysis

CO2 depletion from cathode chamber headspace

after 1 week at -650 mV or -750 mV

CO

2%

N

2 I.S

.

All Reactors

Degassed w/ 20:80 CO2/N2

Prior to each weekly trial

Week 1, -550 mV(no changes detected)

Week 2, -650 mV

Week 3, -750 mV-27%

-52%

0

10

20

30

0 1 2 4 7 16

H2

CO2

CH4

Time (Days)

Headspace g

as

Biogas production and electron consumption.

0

Current

0

-100

-200

-300

-400

I (uA

)

46

Electrotrophic growth is apparent

Bay, -650 mV (note Bay’s lack of gas bubbles ) Harbor, -650 mV

Follow up objective1) Transfer to sterile MECs

-Is direct perpetuation

possible?

Task 2.2.3 – Milestone

47

Delayed Growth was Apparent

Mass Spectrometric Analysis of

Cell Metabolites

1) Targeted Analysis

2) Metabolite Profiling

3) Global Metabolomic Analysis

.

JHU29-B2-PROF-2 #13-61 RT: 0.22-1.02 AV: 49 NL: 2.26E7

T: + p Q3MS [ 50.00-1500.00]

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

421.42

365.51

631.48

453.40603.43

201.14337.45

253.34 855.82675.29

287.28766.61

174.20

839.73 1172.31740.64 989.89463.34 865.90 1228.291057.6379.95 1481.321396.79

MSG

MPG

(Li Adduct of Isoprene acetal)

D(1S/3P)G

DSG

IS

DPG?

PL

DiacylglyceridesMonoacylglycerides Triacylglycerides

Microbial Metabolomic (Lipidomic) Spectrum Profiling

Phospholipids

Note: DSG = 631

TSG = 897

.JHU29-B2-PROF-2-ms2-365 #4-85 RT: 0.05-0.97 AV: 74 NL: 7.51E5

F: + c Full ms2 365.50@-40.00 [ 50.00-450.00]

50 100 150 200 250 300 350 400 450

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

99.05

365.21

81.09

63.1398.04

71.10 251.13 291.26174.08159.12 209.94 237.26137.17109.13 305.06 333.34 347.41 386.89 398.69 443.29

Glycerol Backbone

Unfragmented

MSG C18:0Note loss of H2O (m/z 18)

From glycerol results in

81 and 63 m/z peaks

MS/MS Confirmation of MSG @ 365

MS/MS Identification vs Standard

Drugs Induce Distinct Metabolic Response Patterns

100

200

300

400

500

10 15 20 25 30 35 40 45 50

Time (min.)

100

200

300

400

500

10 15 20 25 30 35 40 45 50

m/z

Sta

rt

Time (min.)

Artemisinin vs. ControlChloroquine vs. Control

O

O

CH3

O

O

O

H

CH3

H

HH

CH3

Cl

N

NH

CH3

N

CH3

CH3

*Data From Two Independent Experiments

100

200

300

400

500

10 15 20 25 30 35 40 45 50

100

200

300

400

500

10 15 20 25 30 35 40 45 50Time (min.)

Pyrimethamine vs. Cont.

1 microM drug

3hr time

Isolated trophozoites

John Pisciotta,* Abhai Tripathi,* Joel Shuman,‡ Vladimir Shulaev‡ & David J.

Sullivan Jr*

Issues concerning

Metabolomic comparisons:

• Extraction method standardization

• Large library of metabolite standards needed

• Timing and degree of BES exposure

• Normalization of cell / enzyme number to

electrode surface area

- Hybrid Systems-Anode respiration to boost Photosynthetic

metabolite production

Enhanced waste to fuel conversion with a bioelectrochemically controlled

autotrophic bioreactor. Pisciotta et al., 2013. Blusens Industrial Report III

CO2CO2

Concluding Remarks:

• Metabolomic researchers interested in redox active

proteins can gain fresh insight by using BES systems.

• The MFC research community also has much to gain

from an improved understanding of Metabolomics.

• Stable Isotope studies may eventually help determine

how voltage influences electrically directed

metabolism in exo-electrogens and electrotrophs

54

Acknowledgments

• JHU Sullivan Lab: Metabolomic GC-MS/MS training using Malaria.

• UMD Baskakov Lab: Metabolomic analysis of pMFC using HPLC / PDA detector.

• PSU Logan Lab: Electochemical Biocathode analysis

• West Chester University: Hybrid Designs (MEC-PBR)

Central Dogma of Molecular

Biology, 1958

Francis Crick

1962 Nobel Prize

w. Crick and Wilkins

DNA Genomics

mRNA Transcriptomics

Protein Proteomics

Metabolites > Metabolomics

UV Stress >> Thiamine dimers >> Tyrosinase mRNA 2x >> Melanin 7xEller et al., Nature. 1994

Why Metabolomics?

• It sometimes provides best (or only)

insight into pathways involved in response

to a stress.A genetically hard-wired metabolic transcriptome in

Plasmodium falciparum fails to mount protective responses

to lethal antifolates.

Ganesan et al., 2008. PLoS Pathogens

Parent product ion MS/MS of synthetic PG

Q-1 1ppm PG scan m/z 110-990 (M –H) -

Sn1 16:0, Sn2 18:2

Q-3 product ion scan of m/z 747 scanned

m/z 110-990 Note 50X > sensitivity

SIM additional 5x > sensitivity ~ 250X

16s Clone Library Chesapeake Bay

24

22

10

10

3

3

3

3

19Mesorhizobium

Rhodococcus

Azospirillum

Gemmata obscuriglobus

Sphingopyxis alaskensis

Labrenzia aggregata

Endosymbiont of Tevnia

Marine actinobacterium

Remainder

(n = 97)

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