9/21/2010

1

Microbial Fuel Cells

Iman Rusmana

Department of BiologyBogor Agricultural University

Oil and Fossil Fuels• Existing (coal, oil shale) and new potential energy

Carbon-based alternatives (methane hydrates, coal togas) pose continued environmental challenges.

• A reduction in CO2 emissions is the main driver forrenewable (CO2-neutral) energy production.

9/21/2010

Energy Utilization in the USA

• US energy use:

• US electricity generation:• 5% used for W&WW:

97 quad

13 quad0.6 quad

• Energy needed for H2 for transportation-Via electrolysis:Using new biomass process:

12 quad1.2 quad

97 quad [quadrillion BTUs]= 28,400 terawatt hours

Energy production: Needs to become more diverse and

CO2 neutral

• Solar

• Wind• Biomass

• Combustion: electricity

• Conventional biotechnology: ethanol,methane , other value products

• Novel biotechnological approaches: electricityand hydrogen

2

9/21/2010

3

Energy can be recovered in many forms viabiotechnological approaches

• Methane• Value: $0.43/kg-CH4

• Elevated temperatures required for bioreactors• Need very long hydraulic detention times (big reactors)

• Hydrogen• Value: $6 /kg, 2.2 × heat value of methane• Produced-low yield from fermentation from sugars• Produced from any biodegradable organic matter using the

BEAMR process

• Electricity• Directly generated using microbial fuel cells

Renewable Energy Production

• Electricity production using microbial fuelcells

• Overcoming the “fermentation barrier”:

high-yield H2 production from biomass

load

-

-

e

Fuel

O2

(wastes)

+

H

Oxidation H2O

products

Anode Cathode(CO )

2

Proton Exchange

membrane

Source: Liu et al., Environ. Sci. Technol., (2004)

bac

teri

a

9/21/2010

4

• Microbial fuel cell powered by organichousehold waste

• Produces 8x as much energy as similarfuel cells and no waste

• Estimated cost - $15• By 2005, NEC plans to

sell fuel cell- poweredcomputers

Bacteria-Driven Battery

“Gastronome”(2000)

9/21/2010

GASTROBOT (Gastro-Robot) – a robot with a stomachU. South Carolina 2000.

Uses an (MFC) system to convert carbohydrate fueldirectly to electrical power without combustion

Microbes from the bacteria(E. coli) decompose thecarbohydrates (in food),releasing electrons.MFC output keeps Ni-Cdbatteries charged, to runcontrol systems/motors.

“Chew-Chew” –meat-fueledGastrobot

The wagons contain a “stomach”, “lung”, gastric pump, “heart”pump, and a six cell MFC stack. Ti-plates, carbon electrodes,proton exchange membrane and a microbial biocatalyst, etc.

Material Requirements of a Biofuel cell

biocatalysts to convert chemical into electrical energy(One can use biocatalysts, enzymes or even whole cell organisms)

Substrates for oxidation: Methanol, organic acids, glucose (organic rawmaterials as abundant sources of energy)

Substrates for reduction at cathode: Molecular oxygen or H2O2

The extractable power of a fuel cell Pcell = Vcell × Icell

Kinetic limitations of the electron transfer processes, ohmic resistancesand concentration gradients cause irreversible losses in the voltage ()

Vcell = Eox – Efuel - Where Eox – Efuel denotes the difference in the formal potentials of theoxidizer and fuel compounds.

Cell current controlled by electrode size, transport rates acrossMembrane.

BUT most redox enzymes do not transfer electrons directly.Therefore, one uses electron mediators (relays).

5

9/21/2010

6

Approach I: Fuel products (say hydrogen gas) are produced byfermentation of raw materials in the biocatalytic microbialreactor (BIOREACTOR) and transported to a biofuel cell.

The bioreactor is not directly integrated with the electrochemical part,

allowing H2/O2 fuel cells to be conjugated with it.

Approach II: Microbiological fermentation can proceed inthe anodic compartment itself.

It is a true biofuel cell!(not a combination of a bioreactor and a conventional fuel cell).

Hydrogen gas is producedbiologically, but it is oxidizedelectrochemically in presence ofbiological components undermilder conditions (thanconventional Fuel cells) asdictated by the biological system

9/21/2010

7

Microbial Fuel Cell(Schematic)

Metabolizing reactions in anode chamber are run anaerobically.An oxidation-reduction mediator diverts electrons from the transportchain. The MEDIATOR enters the outer cell lipid membranes andplasma wall, gathers electrons, shuttles them to the anode.

Why do we need Artificial Electron Relays (mediators) ?

Reductive species produced by metabolic processes areisolated inside the intracellular bacterial space fromthe external world by a microbial membrane.

Hence, direct electron transfer from the microbial cellsto an anode surface is hardly possible!

Low-molecular weight redox-species (mediators) assist shuttlingof electrons between the intracellular bacterial space and anelectrode. To be efficient

a) Oxidized state of a mediator should easily penetrate thebacterial membrane;

b) Its redox-potential should be positive enough to provide fastelectron transfer from the metabolite; and

c) Its reduced state should easily escape from the cell through thebacterial membrane.

9/21/2010

Redox reactions in an MFC

A cell-permeable mediator,in its oxidised form interceptsa part of NADH (Nicotinamide

Adenine Dinucleotide) within themicrobial cell and oxidizes itto NAD+.

Reduced form of mediator is cell-permeable and diffuses to theanode where it is electro-catalytically re-oxidized.

Cell metabolism produces protons in the anodic chamber,which migrate through a selective membrane to the

cathodic chamber, are consumed by ferricyanide [Fe3-(CN)6]and incoming electrons, reducing it to ferrocyanide.

Which Bacteria & Algae are used to produce hydrogenin bioreactors under anaerobic conditions in fuel cells?

Escherichia coli, Enterobacter aerogenes,Clostridium acetobutylicum, Clostridium perfringens etc.

The process is most effective when glucose is fermented in thepresence of Clostridium butyricum (35 µmol h-1 H2 by 1g of

microorganism at 37 C).

This conversion of carbohydrate is done by a multienzymesystem:

Glucose + 2NAD+ -----Multienzyme Embden–Meyerhof pathway2Pyruvate + 2NADH

Pyruvate + Ferredoxinox -----Pyruvate–ferredoxin oxidoreductaseAcetyl-CoA + CO2 + Ferredoxinred

NADH + Ferredoxinox ----NADH-ferredoxin oxidoreductaseNAD+ + Ferredoxinred

+

8

9/21/2010

How do electrons reach the electrode?

Early evidence was that bacteriaproduced their own mediators

- Pseudomonas spp. Produce mediatorssuch as pyocyanin (Rabaey et al. 2004)

- Recent data suggests that Shewanellaspp. use other methods…

Carrier (oxidized)

e Carrier (reduced)

Bacterium Fe (III)

e

e

Mediators produced by Pseudomonasspp. have distinct colors.(Photo provided by Korneel Rabaey, GhentUniversity, Belgium; 2005).

e

Assembly of a simple MFCfrom a kit1.Oxidizing reagent

for cathodechamber. e.g.ferricyanide

anion [Fe(CN)6]3–

from K3Fe(CN)6

10cm3 (0.02 M).

2. Dried Baker’sYeast;

3. Methylene bluesolution 5cm3

(10mM);4. Glucose solution

5cm3 (1M).

Problems:1. Ferricyanide does not consume liberated H+ ions (which lower pH levels).2. Capacity of ferricyanide to collect electrons gets quickly exhausted.

9

9/21/2010

10

To overcome this, ferricyanide can be replaced with an efficient oxygen(air) cathode which would utilize a half-reaction of:

6O2 + 24H+ + 24e– 12H2O, thereby consuming the H+ ions.

Which microorganisms work best?

P. vulgaris and E. coli bacteria are extremely industrious.

A monosaccharide (glucose) MFC utilizing P. vulgaris has shownyields of 50%–65%, while E. coli has been reported at 70%–80%.

An electrical yield close to the theoretical maximum has even

been demonstrated using a disaccharide (sucrose C12H22O11)substrate, although it is metabolized slower with a lower electrontransfer rate than when using glucose.

9/21/2010

Dyes function as effective mediators when they are rapidlyreduced by microorganisms, or have sufficient negative potentials

Thionine serves as a mediator of electron transport from Proteus Vulgarisand from E. coli.

phenoxazines (brilliant cresyl blue, gallocyanine, resorufin)phenazines (phenazine ethosulfate, safranine),phenothiazines (alizarine brilliant blue, N,N-dimethyl-disulfonated

thionine, methylene blue, phenothiazine, toluidine blue), and2,6-dichlorophenolindophenol, 2-hydroxy-1,4-naphthoquinonebenzylviologen, are organic dyes

that work with the following bacteria

Alcaligenes eutrophus, Anacystis nidulans, Azotobacter chroococcum,Bacillus subtilis, Clostridium butyricum, Escherichia coli, Proteus vulgaris,Pseudomonas aeruginosa, Pseudomonas putida, and StaphylococcusAureus, using glucose and succinate as substrates.

The dyes: phenoxazine, phenothiazine, phenazine, indophenol, thioninebipyridilium derivatives, and 2-hydroxy-1,4-naphthoquinone maintainhigh cell voltage output when current is drawn from the biofuel cell.

Bacteria used in biofuel cells when membrane-penetratingElectron Transfer Mediators (dyes) are applied

Alcaligenes eutrophus

Proteusvulgaris

E. Coli (image width 9.5 m)

Bacillus subtilis

Anacystis nidulans 200 nm

Pseudomonas putida

Pseudomonasaeruginosa

11

Streptococcuslactis

12

9/21/2010

Electrical wiring of MFCs to theanode using mediators.(Co-immobilization of the microbial cellsand the mediator at the anode surface)

(A) A diffusional mediator shuttlingbetween the microbial suspension andanode surface is co-valently bonded.

‘1’ is the organic dye Neutral red.

(B) A diffusional mediator shuttlingbetween the anode and microbial cellscovalently linked (amide bond) to theelectrode. ‘2’ is Thionine.

(C) No diffusional mediator. Microbialcells functionalized with mediators’The mediator ‘3’ i.e. TCNQ adsorbedon the surface of the microbial cell.

MFCs using electron relays for coupling of intracellular electrontransfer processes with electrochemical reactions at anodes

Microorganism

Pseudomonasmethanica

Escherichia coli

Proteus vulgarisBacillus subtilisEscherichia coli

Proteus vulgaris

NutritionalSubtrate

CH4

Glucose

Glucose

Sucrose

Mediator

1-Naphthol-2- sulfonate indo2,6-dichloro-phenol

Methylene blue

Thionine

Thionine

Lactobacillus plantarum Glucose Fe(III)EDTA

Escherichia coli Acetate Neutral Red

9/21/2010

The redox cofactors Nicotinamide Adenine Dinucleotide (NAD+) andNicotinamide Adenine Dinucleotide Phosphate (NADP+) playimportant roles in biological electron transport, and in activating thebiocatalytic functions of dehydrogenases – the major redox enzymes.

(NAD+) (NADP+)

J. Nießen and Fritz Scholz Angew. Chem. Int. Ed. 2003, 42, 2880 – 2883

Use of NAD(P)+-dependent enzymes (e.g. lactate dehydrogenase; alcoholdehydrogenase; glucose dehydrogenase) in biofuel cells allows the use oflactate, alcohols and glucose as fuels. Biocatalytic oxidation of thesesubstrates requires efficient electrochemical regeneration of NAD(P)+-cofactors in the anodic compartment.

Current Density output from Microbial Fuel Cells is Low!e.g.

1. Dissolved artificial redox mediators penetrate the bacterial cells,shuttle electrons from internal metabolites to anode. Currentdensities : 5–20 Acm-2

2. Metal-reducing bacteria (e.g. Shewanella putrefaciens) having specialcytochromes bound to their outer membrane, pass electrons directlyto anode. Current densities : maximum of 16 Acm-2

3. An MFC based on the hydrogen evolution by immobilized cells ofClostridium butyricum yielded short circuit currents of 120 Acm-2

by using lactate as the substrate.

But recentlyCurrent Outputs Boosted by an order of magnitude! U. Schroder,

Using PANI modified Pt electrode immersed in anaerobic culture ofEscherichia coli K12. CV response different for different stages offermentation: a) sterile medium; b) exponential bacterial growth; and c)stationary phase of bacterial growth.

13

9/21/2010

MFC Reactors

• Aqueous-cathode MFCs:– Salt Bridge proton exchange

system

– Membrane (Nafion)

• Direct air cathodes MFCs:– Single Chamber system for

wastewater

– Flat plate system

– Small batch system foroptimizing electricitygeneration

0.25 yr-- 0.3 mW/m2

0.5 yr-- 2 mW/m2

1 yr-- 45 mW/m2

1.5 yr-- 500 mW/m2

Current-- 1500 mW/m2

Detail of CellOpen circuit potential:

895 mV.Steady state 30 mA undershort circuit conditions.Max. currents measured:

150 mAMax. power output: 9 mW.

Operates a ventilator drivenby 0.4 V motor, continuously

Operation costs low.

Anaerobically growing suspension of E.coli K12 in glucose in the Reactor.Bacterial Medium pumped thru anode compartment.Anode is woven graphite cloth, platinized, And PANI-modified.

Cathode is unmodified woven graphite. Catholyte (50 mm ferricyanidesolution in a phosphate buffer).Nafion proton conducting membrane separates anode and cathodecompartments.

14

9/21/2010

Electricity from Direct Oxidation of Glucose in Mediatorless MFCSwadesh Choudhury and Derek Lovely, U.Mass, Amherst,Nature Biotechnol. 21 (Oct. 2003) 1229

The prototype produced 0.5 V, enough to power a tinylamp. A cup of sugar could power a 60-W light bulb for 17hours.But, the generation of electrons by bacteria is too slow topower commercial applications. Increasing the contactsurface of electrode (making it porous) may bring in morebacteria in contact.

15

The device doesn’t need toxic/expensive mediators because thebacteria Rhodoferax ferrireducens attach to the electrode’s surface andtransfer electrons directly to it.

The microbe(isolated frommarine sedimentsin Va., USA)metabolizesGlucose/sugarsinto CO2

producing e’s.

80% efficiencyfor convertingsugar intoelectricity

Graphite electrode is immersed into asolution containing glucose and thebacteria.The microbe R. ferrireducens is an"iron breather” – microorganismswhich transfer electrons to ironcompounds. It can also transferelectrons to metal-like graphite.

R. ferrireducens can feed on organicmatter (sugar), and harvest electrons.

Rhodoferax ferrireducenson electrode

9/21/2010

16

Harnessing microbially generated power on the seafloor

Seafloor has sediments meters thick containing 0.1-10% oxidizableorganic carbon by weight – an immense source of energy reserve.Energy density of such sediments assuming 2% organic carboncontent and complete oxidization is 6.1x104 J/L (17 W h/L),

– a remarkable value considering the sediment volumefor the Gulf of Mexico alone is 6.3x1014 liters.

Microorganisms use a bit of this energy reserve (limited bythe oxidant supply of the overlying seawater), and thus create avoltage drop as large as 0.8 V within the top few mm’s to cm’s ofsediment surfaces.

This voltage gradient across the water-sediment interface inmarine environments can be exploited by a fuel cell consisting ofan anode embedded in marine sediment and a cathode inoverlying seawater to generate electrical power in situ.

MFCs could power devices located at the bottom of the ocean,where the bacteria would feed on sugar-containing sediments.

Geobacters in Boston Harbor sediments colonizeelectrodes placed in the mud to power a timer

Geobacters have novel electron transfer capabilities, useful forbioremediation and for harvesting electricity from organic waste.

First geobacter, known as Geobacter metallireducens, (strain GS-15) discovered in 1987 was found to oxidize organic compounds to

CO2 with iron oxides as the electron acceptor.

i.e. Geobacter metallireducens gains its energy by using iron oxidesin the same way that humans use oxygen. It may also explaingeological phenomena, such as the massive accumulation ofmagnetite in ancient iron formations.

17

and oxygen as oxidant

9/21/2010

Geobatteries powering a calculator

Geobacter colonizing a graphite electrode surface

Fuel cells convert chemical energy directly to electricalenergy. Reduced fuel is oxidized at anode – transferringelectrons to an acceptor molecule, e.g., oxygen, at cathode.

Fuel cell with hydrogen gas as fuel

Oxygen gas when passed overcathode surface gets reduced,combines with H+ ions(produced electrochemically atanode) arriving at cathode thruthe membrane, to form water.

One needs:An electrolyte medium;Catalysts;

(to enhance rate of reaction),Ion-exchange membrane;

(to separate the cathode and

anode compartments).

18

9/21/2010

Examples of microbial-based biofuel cells utilizing fermentationproducts for their oxidation at anodes

Microorganism

Clostridiumbutyricum

Clostridiumbutyricum

Clostridiumbutyricum

Enterobacteraerogenes

Desulfovibriodesulfuricans

Nutritional

substrate

Waste water

Molasses

Lactate

Glucose

Dextrose

FermentationProduct

H2

H2

H2

H2

H2S

Biofuel cell

voltage

0.62 V(at 1 )

0.66 V(at 1 )

0.6 V (oc)d

1.04 V (oc)

2.8 V (oc)

Biofuel cell currentor current density

0.8 A(at 2.2 V)

---

120 A cm-2

(sc)e

60 A cm-2

(sc)

1A

Anodec

Pt-blackened Ni,165 cm2

(5 anodes in series)

Pt-blackened Ni,85 cm2

Pt-black,50 cm2

Pt-blackened stainlesssteel, 25 cm2

Graphite, Co(OH)2

impregnated (3 anodesin series)

9/21/2010

19

Integrated microbial biofuel cells producing electrochemicallyactive metabolites in the anodic compartment of biofuel cells

Microbial cells producing H2 during fermentation immobilizeddirectly in the anodic compartment of a H2/O2 fuel cell

A rolled Pt-electrode was introduced into a suspension ofClostridium butyricum microorganisms. Fermentation conducted

directly at the electrode, supplying anode with H2 fuel.Byproducts of the fermentation process (hydrogen, 0.60 mol; formicacid, 0.20mol; acetic acid, 0.60mol; lactic acid, 0.15 mol) could alsobe utilized as fuel. For example, pyruvate produced can be oxidized:

Pyruvate ----Pyruvate–formate lyaseFormate

Metabolically produced formate is directly oxidized at the anodewhen the fermentation solution passes the anode compartment

HCOO- CO2 + H+ + 2e- (to anode)

The biofuel cell that included ca. 0.4 g of wet microbial cells (0.1 gof dry material) yielded the outputs Vcell = 0.4V and Icell = 0.6mA.

NAD+-dependent dehydrogenases oxidize CH3OH to CO2; diaphorase (D)catalyzes the oxidation of NADH to NAD+ using benzylviologen, BV2+

(N,N'-dibenzyl-4,4-bipyridinium as the electron acceptor.BV.+ is oxidized to BV2+ at a graphite anode and thus, releases electrons for the

reduction of dioxygen at a platinum cathode. The cell provided Voc = 0.8 V anda maximum power density of ca. 0.68 mW cm–2 at 0.49 V.

Methanol/dioxygen biofuel cell, based on enzymes (producing NADH uponbiocatalytic oxidation of primary substrate) and diaphorase (electricallycontacted via an electron relay and providing bioelectrocatalytic oxidationof the NADH to NAD+

Enzymes:ADH: alcohol dehydrogenaseAlDH: aldehyde dehydrogenaseFDH: formate dehydrogenase

9/21/2010

20

Viability of Robots working on MFCs

Main source of energy in plants is carbohydrates in the form of sugarsand starches. Foliage most accessible to robots such as spinach, turnipgreens, cabbage, broccoli, lettuce, mushroom, celery and asparagusmay contain about 4% carbohydrate by weight.

The energy content of carbohydrate is around 5 kcal/g (=21 kJ/g).This amounts to 0.82 kJ/ml for liquified vegetable matter, similar tothe energy density of a Lithium-ion battery. If converted into anelectrical form this would yield 5 kWh/kg for a pure monosaccharidesugar, or 0.2 kWh per liter of liquified vegetable matter.

Renewable Energy Production

• Electricity production using microbial fuelcells

• Overcoming the “fermentation barrier”:

high-yield H2 production from biomass

Bio

gas

(mL

)

9/21/2010

Current sources for H2

Production

Electrolysis

4%Coal

0 50 100 150 200 250

150

100

200

Molasses

Potato Starch

Glucose

Cellulose

SucroseLactate

50

0

18%

Natural Gas48%

Heavy oilsand naphtha

30%

Observation: H2 production results primarilyfrom sugars

300

Biogas:250

- 60% H2

- 40% CO2

Time (h)

Source: Logan, VanGinkel & Oh Environ. Sci. Technol. (2002)

21

2

2

H2 Yield increased

2

20

Source: Park, Hyun, Oh, Logan & Kim, Environ. Sci. Technol. (2005)

H2,

%

9/21/2010

22

9/21/2010

Observation: the “fermentation barrier”

Maximum: 12 mol--H2/mol--hexose

C6H12O6 + 2 H2O 4 H2

+ 2 C2H4O2 + 2 CO2

C6H12O6 2 H2 +C4H8O2 + 2 CO2

Maximum of 4 mol/mol(2 mol/mol in practice)

??????

How can we recover the remaining 8 to10 mol/mol?

Overcoming the “Fermentation Barrier”

• Bio-Electrochemically Assisted MicrobialReactor (BEAMR) [Process developed with Ion

Power, Inc.]:• Acetate: achieve 2.9 mol-H2/mol-acetate

(Maximum of 4 mol/mol)

• Couple fermentation + BEAMR process

→ 8 to 9 mol-H2/mole glucose• Not limited to glucose

23

CO

2 - - 2

Anode Cathode

+H

Cathode

2

Source: Liu, Grot and Logan, Environ. Sci. Technol. (2005)

24

••••

Anode potential= -300 mVCathode potential: 0 mV

Needed to make H2= 410 mV (theory)Circuit (300 mV) augmented with >110 mV= >410 mV

Anode: C2H4O2 + 2 H2O 2 CO2 + 8 e- + 8 H+

Cathode: 8 H+ + 8 e- 4 H2

9/21/2010

Essentials of the BEAMR Process

• Conventional MFC:• Anode potential= -300 mV• Cathode Potential= +200 mV (+804 mV theory)• Circuit working voltage= -(-300) + 200= 500 mV

Anode: C2H4O2 + 2 H2O 2 CO2 + 8 e- + 8 H+

Cathode: O2 + 4 H+ + 4 e- = 2 H2O

• BEAMR Process: No oxygen

(electron recovery)

2

80

-Overall:

70

2

60

H2 CE

Applied voltage (V)

Source: Liu, Grot and Logan, Environ. Sci. Technol. (2005)

1 -250

CD

0.8 -260

AP

(0.11 V theory)

0.6 -270

0 -300

0.2 0.4 0.6 0.8 1

Applied voltage (V)

Source: Liu, Grot and Logan, Environ. Sci. Technol. (2005)

Cur

rent

den

sity

(A

/m )

Rec

over

y (%

)

Ano

de p

oten

tial

(m

V)2

9/21/2010

25

9/21/2010

26

Observation 1: Industries currently “throw away” avaluable resource (land application)

Observation 2: Other countries are investingin development and scale-up of hydrogen

and alternative energy processes

Large-scale biohydrogenreactor being tested atHarbin University, China

(Director: Prof. Nanqi Ren)

9/21/2010

27