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Microbial Fuel Cell

Rhodoferax ferrireducens

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Scientific American News 
September 08, 2003

Bacterial Battery Converts Sugar into Electricity  
  
 Image: ENVIRONMENTAL BIOTECHNOLOGY CENTER/UMASS  
    
A tiny bacterium recovered from sediment may power batteries of the future. In a report published today in Nature Biotechnology, researchers describe a primitive microbial fuel cell that can convert simple sugars into electricity with 81 percent efficiency. Unlike previous attempts to manufacture such batteries, the novel design does not require unstable intermediaries to shuttle electrons and thus holds promise for producing energy from sugar-containing waste materials. 
Swades K. Chaudhuri and Derek R. Lovley of the University of Massachusetts used Rhodoferax ferrireducens, a bacterium first isolated from sediments collected from an aquifer in Virginia, for their bacterial battery. When the researchers exposed R. ferrireducens to a solution of glucose in a chamber containing a graphite electrode they found that when the bacterium fed on the sugar, it transferred electrons directly to the electrode, producing a current. In addition, the sugar-fed R. ferrireducens continued to grow, resulting in stable, long-term power production. The scientists also tested the bacterium's ability to convert other sugars, including fructose, sucrose and xylose (present in wood and straw), and found it to be equally efficient. 
 
The new findings should help scientists harness the abundant energy currently stored in waste from agricultural, municipal and industrial sources. The prototype fuel cells have such desirable features as the ability to recharge and minimum loss of energy while idling. Perhaps one day electronics will be sold with the caveat "bacteria not included." --Sarah Graham

Discovery of Rhodoferax Ferrireducens

Microbial Fuel Cells 
 
 http://powermin.nic.in/information_center/pdf/BARC-1.ppt.

J.V. Yakhmi

Technical Physics & Prototype Engineering Division,

Bhabha Atomic Research Centre

Advanced fuel cells for �Hydrogen Economy�

When do we need Fuel Cells?

when small,  light power sources are needed that can sustain

over long periods, in remote locations (space and exploration).

Biofuel cells copy nature�s solutions to energy

generation!  They consume renewable BIOMASS!

Microbes (bacteria) use substrates (fuel) from renewable sources,

convert them into benign by-products & generate electricity.

The fuel can even be taken from a living organism ! (e.g.  Glucose

from the blood stream). Implantable devices (e.g. pacemakers,

drug delivery devices) could take advantage! � to draw power as

long as the subject lives.

Large  scale production of power possible from renewable sources:

� sugars found in waste material (in sewage) or

� sugars in carbohydrate-rich crops (corn).

What is BIOMASS?

Certain organisms use sunlight (Photosynthesis) in the visible range

(400�800 nm) to synthesize (lipids, proteins, carbohydrates)

Only about 0.12% of solar radiation energy falling on

earth is converted into chemically bonded energy in

the form of biomass (from plants):

~ 170 Gt per year, with energy content of ~3,000 EJ (Exa Joule)

i.e. 3x1021J, which is ten times of mankind�s annual needs!

Generally, microorganisms (e.g. algae and bacteria)

alone can�t convert biomass into electrical energy, but

do so using enzymes, such as the oxidoreductases,

which catalyze  electron transfer for the redox

reactions involved in fuel cells.

Microbial fuel cell powered by organic household waste 
Produces 8x as much energy as similar fuel cells and no waste 
Estimated cost - $15 
By 2005, NEC plans to 
    sell fuel cell- powered

computers

Bacteria-Driven Battery

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

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.

GASTROBOT (Gastro-Robot) � a robot with a stomach

U. South Carolina 2000.

�Gastronome�  (2000)

�Chew-Chew� �meat-fueled

Gastrobot

Microbes from the bacteria (E. coli) decompose the carbohydrates (in food), releasing electrons.

MFC output keeps Ni-Cd batteries charged, to run  control systems/motors.

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 raw

materials 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 resistances and

concentration gradients cause irreversible losses in the voltage ( )

Vcell = Eox � Efuel -

Where Eox � Efuel denotes the difference in the formal potentials of the

oxidizer and fuel compounds.

Cell current controlled by electrode size, transport rates across Membrane.

BUT most redox enzymes do not transfer electrons directly.

Therefore, one uses electron mediators (relays).

Approach I:  Fuel products (say hydrogen gas) are produced by

fermentation of raw materials in the biocatalytic microbial

reactor (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 in

the anodic compartment itself.

It is a true biofuel cell!

(not a combination of a bioreactor and a conventional fuel cell).

Hydrogen gas is produced

biologically, but it is oxidized

electrochemically in presence of

biological components under

milder conditions (than

conventional Fuel cells) as

dictated by the biological system

Microbial Fuel Cell

(Schematic)

Metabolizing reactions in anode chamber are run anaerobically.

An oxidation-reduction mediator diverts electrons from the transport

chain. The MEDIATOR enters the outer cell lipid membranes and

plasma wall, gathers electrons, shuttles them to the anode.

(Nafion)

Why do we need Artificial Electron Relays (mediators) ?

Reductive species produced by metabolic processes are

isolated inside the intracellular bacterial space from

the external world by a microbial membrane.

Hence, direct electron transfer from the microbial cells

to an anode surface is hardly possible!

Low-molecular weight redox-species (mediators) assist shuttling

of electrons between the intracellular bacterial space and an

electrode. To be efficient

a) Oxidized state of a mediator should easily penetrate the

bacterial membrane;

b) Its redox-potential should be positive enough to provide fast

electron transfer from the metabolite; and

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

bacterial membrane.

Reduced form of mediator is cell-permeable and diffuses to the

anode 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.

A cell-permeable mediator,

in its oxidised form intercepts

a part of NADH (Nicotinamide

Adenine Dinucleotide) within the

microbial cell and oxidizes it

to NAD+.

Redox reactions in an MFC

Which Bacteria & Algae are used to produce hydrogen

in 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 the

presence of Clostridium butyricum (35 �mol h-1 H2 by 1g of

microorganism at 37 C).

This conversion of carbohydrate is done by a multienzyme system:

Glucose + 2NAD+     -----Multienzyme Embden�Meyerhof pathway

2Pyruvate + 2NADH

Pyruvate + Ferredoxinox   -----Pyruvate�ferredoxin oxidoreductase

Acetyl-CoA + CO2 + Ferredoxinred

NADH + Ferredoxinox  ----NADH-ferredoxin oxidoreductase

NAD+ + Ferredoxinred

Ferredoxinred + 2H+   ----Hydrogenase Ferredoxinox + H2

Assembly of a simple MFC from a kit

1.Oxidizing reagent

for cathode

chamber. e.g.

ferricyanide

anion [Fe(CN)6]3�

from K3Fe(CN)6

10cm3 (0.02 M).

2. Dried Baker�s

Yeast;

3. Methylene blue

solution 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.

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 shown

coulombic yields of 50%�65%, while the more voracious 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 electron

transfer rate than when using glucose.

Dyes function as effective mediators when they are rapidly

reduced by microorganisms, or have sufficient negative potentials

Thionine serves as a mediator of electron transport from Proteus Vulgaris

and 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), and

2,6-dichlorophenolindophenol, 2-hydroxy-1,4-naphthoquinone

benzylviologen, 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 Staphylococcus

Aureus,   using glucose and succinate as substrates.

The dyes:  phenoxazine, phenothiazine, phenazine, indophenol, thionine

bipyridilium derivatives, and 2-hydroxy-1,4-naphthoquinone maintain

high cell voltage output when current is drawn from the biofuel cell.

Bacteria used in biofuel cells  when membrane-penetrating

Electron Transfer Mediators (dyes) are applied

Alcaligenes eutrophus

E. Coli (image width 9.5 m)

Anacystis nidulans 200 nm

Proteus

vulgaris

Bacillus subtilis

Pseudomonas putida

Pseudomonas

aeruginosa

Streptococcus

lactis

Electrical wiring of MFCs to the

anode using mediators.

(Co-immobilization of the microbial cells

and the mediator at the anode surface)

(A) A diffusional mediator shuttling

between the microbial suspension and

anode surface is co-valently bonded.

�1� is the organic dye Neutral red.

(B) A diffusional mediator shuttling

between the anode and microbial cells

covalently linked (amide bond) to the

electrode. �2� is Thionine.

cells functionalized with mediators

�The mediator �3� i.e. TCNQ adsorbed

on the surface of the microbial cell.

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

Microorganism  Nutritional  Mediator

Subtrate

Pseudomonas   CH4   1-Naphthol-2- sulfonate indo  
methanica     2,6-dichloro-phenol

Escherichia coli   Glucose  Methylene blue

Proteus vulgaris   Glucose  Thionine

Bacillus subtilis  
Escherichia coli

Proteus vulgaris   Sucrose   Thionine

Lactobacillus plantarum Glucose  Fe(III)EDTA

Escherichia coli   Acetate   Neutral Red

The redox cofactors Nicotinamide Adenine Dinucleotide (NAD+) and

Nicotinamide Adenine Dinucleotide Phosphate (NADP+) play

important roles in biological electron transport, and in activating  the

biocatalytic functions of dehydrogenases � the major redox enzymes.

(NAD+)

(NADP+)

Use of NAD(P)+-dependent enzymes (e.g. lactate dehydrogenase; alcohol

dehydrogenase; glucose dehydrogenase) in biofuel cells allows the use of

lactate, alcohols and glucose as fuels. Biocatalytic oxidation of these

substrates requires efficient electrochemical regeneration of NAD(P)+-

cofactors in the anodic compartment.

But recently

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

J. Nie�en and Fritz Scholz             Angew. Chem. Int. Ed. 2003, 42, 2880 � 2883

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. Current densities : 5�20 Acm-2

2. Metal-reducing bacteria (e.g. Shewanella putrefaciens) having special

cytochromes bound to their outer membrane, pass electrons directly to

anode.  Current densities : maximum of 16 Acm-2

3. An MFC based on the hydrogen evolution by immobilized cells of

Clostridium butyricum yielded short circuit currents of  120 Acm-2

by using lactate as the substrate.

Using PANI modified Pt electrode immersed in anaerobic culture of Escherichia coli

K12. CV response different for different stages of fermentation: a) sterile medium;

b) exponential bacterial growth; and c) stationary phase of bacterial growth.

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 ferricyanide solution in a phosphate buffer).

Nafion proton conducting membrane separates anode and cathode compartments.

Open circuit potential:

895 mV.

Steady state 30 mA under

short circuit conditions.

Max. currents measured:

150 mA

Max. power output:  9 mW.

Operates a ventilator driven

by 0.4 V motor, continuously

Operation costs low.

Detail of Cell

Redox-active biocompatible PANI layer banishes artificial

compounds from bacterial medium & oxidizes excreted metabolites.

PANI layer also acts as barrier allowing metabolic products like H2

(but blocking large molecules) to diffuse to the electrocatalytically

active electrode surface, preventing the poisoning of electrocatalyst.

Current increases as the bacteria grows exponentially. However,

it decreases when the bacterial growth reaches stationary phase.

because of reduction in the electrocatalytic activity of the working

electrode caused by microbial catabolic.

The polymer layer slows down the deactivation but cannot fully

prevent it.

The anode deactivation is reversed, and its long-time stability

ensured with a regenerative-potential program, performed in situ,

by regularly applying short oxidative-potential pulses to the anode.

During the potential pulses, chemisorbed species are stripped off

from The electrode surface, which reactivates the surface of the

metallic electrocatalyst. This prevents the rapid diminution of the

anodic currents and increases the current densities upto 1.5

mA cm-2, offering potential for microbial electricity generation.

Electricity from Direct Oxidation of Glucose in Mediatorless MFC

Swadesh Choudhury and Derek Lovely,  U.Mass, Amherst, Nature Biotechnol. 21 (Oct. 2003) 1229

The device doesn�t need toxic/expensive mediators because the

bacteria Rhodoferax ferrireducens attach to the electrode�s surface and

transfer electrons directly to it.

The microbe

(isolated from

marine sediments

in Va., USA)

metabolizes

Glucose/sugars

into CO2

producing e�s.

80% efficiency

for converting

sugar into

electricity

(vis-�-vis 10%

usually in MFCs.

The  prototype produced 0.5 V, enough to power a tiny lamp. A cup of sugar could power a 60-W light bulb for 17 hours.

But, the generation of electrons by bacteria is too slow to power commercial applications. Increasing the contact surface of electrode (making it porous) may bring in more bacteria in contact.

Graphite electrode is immersed into a solution containing glucose and the bacteria.

The microbe R. ferrireducens is an "iron breather� � microorganisms which transfer electrons to iron compounds. It can also transfer electrons to metal-like graphite.

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

Rhodoferax ferrireducens

on electrode

Harnessing microbially generated power on the seafloor

Seafloor has sediments meters thick containing 0.1-10% oxidizable

organic carbon by weight �  an immense source of energy reserve.

Energy density of such sediments assuming 2% organic carbon

content and complete oxidization is 6.1x104 J/L (17 W h/L),

� a remarkable value considering the sediment volume

for the Gulf of Mexico alone is 6.3x1014 liters.

Microorganisms use a bit of this energy reserve (limited by

the oxidant supply of the overlying seawater), and thus create a

voltage drop as large as 0.8 V within the top few mm�s to cm�s of

sediment surfaces.

This voltage gradient across the water-sediment interface in

marine environments can be exploited by a fuel cell consisting of

an anode embedded in marine sediment and a cathode in

overlying 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 colonize

electrodes placed in the mud to power a timer

Geobacters have novel electron transfer capabilities, useful for

bioremediation and for harvesting electricity from organic waste.

First geobacter, known as Geobacter metallireducens, (strain GS-

15) discovered in 1987 by Lovely�s group, 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 oxides

in the same way that humans use oxygen. It may also explain

geological phenomena, such as the massive accumulation of

magnetite in ancient iron formations.

Geobatteries powering a calculator

Geobacter colonizing a graphite electrode surface

Fuel cell with hydrogen gas as fuel

and oxygen as oxidant

Fuel cells convert chemical energy directly to electrical

energy. Reduced fuel is oxidized at anode � transferring

electrons to an acceptor molecule, e.g., oxygen, at cathode.

Oxygen gas when passed over

cathode surface gets reduced,

combines with H+ ions

(produced electrochemically at

anode) arriving at cathode thru

the 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).

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

Microorganism

Nutritional  
substrate

Fermentation  
Product

Biofuel cell  
voltage

Biofuel cell current  
or current density

Anodec

Clostridium  
butyricum

Waste water

0.62 V  
(at 1 W)

0.8 A  
(at 2.2 V)

Pt-blackened Ni,  
165 cm2  
(5 anodes in series)

Clostridium  
butyricum

Molasses

0.66 V  
(at 1 W)

---

Pt-blackened Ni,  
85 cm2

Clostridium  
butyricum

Lactate

0.6 V (oc)d

120 mA cm-2  
(sc)e

Pt-black,  
50 cm2

Enterobacter  
aerogenes

Glucose

1.04 V (oc)

60 mA cm-2  
(sc)

Pt-blackened stainless  
steel, 25 cm2

Desulfovibrio  
desulfuricans

Dextrose

H2S

2.8 V (oc)

1 A

Graphite, Co(OH)2  
impregnated (3 anodes  
in series)

Integrated microbial biofuel cells producing electrochemically

active metabolites in the anodic compartment of biofuel cells

Microbial cells producing H2 during fermentation immobilized

directly in the anodic compartment of a H2/O2 fuel cell

A rolled Pt-electrode was introduced into a suspension of

Clostridium butyricum microorganisms. Fermentation conducted

directly at the electrode, supplying anode with H2 fuel.

Byproducts of the fermentation process (hydrogen, 0.60 mol; formic

acid, 0.20mol; acetic acid, 0.60mol; lactic acid, 0.15 mol) could also

be utilized as fuel. For example, pyruvate produced can be oxidized:

Pyruvate  ----Pyruvate�formate lyase   Formate

Metabolically produced formate is directly oxidized at the anode

when 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 g

of 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 and

a maximum power density of ca. 0.68 mW cm�2 at 0.49 V.

Methanol/dioxygen biofuel cell, based on enzymes (producing NADH upon

biocatalytic oxidation of primary substrate) and diaphorase (electrically

contacted via an electron relay and providing bioelectrocatalytic oxidation

of the NADH to NAD+

Enzymes:

ADH: alcohol dehydrogenase

AlDH: aldehyde dehydrogenase

FDH: formate dehydrogenase

Viability of Robots working on MFCs

Main source of energy in plants is carbohydrates in the form of sugars

and starches. Foliage most accessible to robots such as spinach, turnip

greens, cabbage, broccoli, lettuce, mushroom, celery and asparagus

may 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 to

the energy density of a Lithium-ion battery. If converted into an

electrical form this would yield 5 kWh/kg for a pure monosaccharide

sugar, or 0.2 kWh per liter of liquified vegetable matter.

Challenges for assembling a working Gastrobot are to provide

facilities for Foraging (Food Location & Identification); Harvesting

(Food Gathering); Mastication (Chewing); Ingestion (Swallowing);

Digestion (Energy Extraction),and Defecation (Waste Removal).

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