October 5, 2015, 6th

World Congress on Biotechnology, New Delhi, India

Dr. Karen Trchounian,

PhD in Biophysics and Biotechnology

Deputy Director of Scientific-Research Institute of Biology

YEREVAN STATE UNIVERSITY, 0025 YEREVAN, ARMENIA

k.trchounian@ysu.am

Application of mixture of carbon sources to enhance H2 production by Escherichia

coli

Biohydrogen as an alternative energy source of future

Molecular hydrogen (H2) produced by bacterial biomass is a 100% ecologically clean, renewable fuel that burns efficiently and generates no toxic byproducts (Momirlan & Veziroglu (2005) Int. J. Hydrogen Energy, 33, 795-802, Hallenbeck et al. (2012) Bioresour. Technol, 110, 1–9 Trchounian and Trchounian (2015) Appl. Energy, 156, 174-184).

As it is well known oil and gas are not renewable energy sources and H2 can replace existing fuel and gas (DOE 2004 Hydrogen Energy Program Report).

H2 is very effective energy carrier; it is ~3 time more effective than fuel and gas

Over the worldEuropean Union Hydrogen Highway

The European Union hydrogen highway network is at present a loose affiliation of H2 refueling stations

developed by various countries. Leading the charge is Germany who has the most hydrogen refueling

stations.

Austria 2

Belgium 1

Copenhagen 1

Czech Republic 1

Denmark 14

Finland 2

France 5

Germany 41

Greece 2

Greenland 1

Iceland 2

Italy 21

Luxembourg 1

Norway 10

Portugal 1

Spain 4

Sweden 5

Switzerland 2

The Netherlands 4

Turkey 3

United Kingdom 20

http://www.hydrogencarsnow.com/eu-hydrogen-highway.htm

Over the worldNowadays in United States,

Japan, United Kingdom, Netherlands, Germany, India etc. already H2 filling stations are exploited and different cars, buses and other motor

vehicles are working on hydrogen.

http://www.hydrogencarsnow.com/eu-hydrogen-highway.htm

Over the worldDifferent bacteria are used to produce H2 either via dark fermentation or photofermentation from organic agricultural and industrial wastes (Ueno et al. (2007) Environ. Sci. Technol., 2007, 41 (4), 1413–1419; O-Thong et al. (2008) Int. J. Hydrogen Energy, 33, 1204–1214; Keskin et al. (2011) Bioresour. Technol. 102, 8557–8568; Gabrielyan & Trchounian (2012), Biomass and Bioenergy, 36, 333-338).

Maeda et al. (2008) Microb. Biotechnol. 1, 30-39

constructed E. coli strain which produces ~141 fold more H2 than wild type.

From economic side nowadays 1 liter of H2

costs 2-4$.

~65 million tonnes/yr and yearly

increase in 10-15%

Biohydrogen as an alternative energy source of future

H2 is produced chemically and biologically:

Disadvantages of chemical production

High temperature (heating)

Limited yield not renewable

Short-term technology

High Energy Demand

Biological method of H2 production is possible through special

enzymes named hydrogenases catalyzing the simple redox

reaction

2H+

+2e- H2 (Trchounian et al. (2012) Crit. Rev. Biochem. Mol. Biol. 47,

236-249)

Advantages of biological production

low temperature (without heating)

Renewable

Long-term technology

Possibility of further improvement of technology for cheap H2

production

Glycerol fermentation by E. coli

RECENT DISCOVERYDharmadi et al. (2006) Biotechnol. Bioeng. 94, 821-829) have shown

that E. coli can ferment glycerol in acidic conditions (pH 6.3). We have shown first time that glycerol can be fermented also at pH

7.5 which has many interesting applications in biology and medicine

(Trchounian, Trchounian (2009) Int. J. Hydrogen Energy 34, 8839-8845; Trchounian et al. (2011) Ibid 36, 4323-4331).

SIGNIFICANCEglycerol is very cheap carbon source (crude glycerol costs 5-15

cents/lb). glycerol has higher reduced state, compared to other carbon

sources such as glucose, which promises significant increase in the product yield of different chemicals such as succinate, ethanol, H2, etc.

Mixed acid fermentation and H2 production by E. coli

Mixed-acid fermentation in E. coli. Formation of lactic, formic, acetic, succinic acids and the other end-

products (highlighted with yellow) of fermentation of glucose or glycerol as well as further oxidation of formate to CO2 and H2 are shown. On the ways

from phosphoenolpyruvate to pyruvate or from acetyl phosphate to acetate ATP is synthesized on the level of

substrate phosphorylation (Trchounian et al. (2012) Crit. Rev. Biochem. Mol. Biol. 47:

236-249,Trchounian & Sawers (2014) IUBMB Life, 66, 1-7.

GLUCOSE

Phosphoenolpyruvate

Pyruvate

Fructose-1,6-diphosphate

ATP

ADP + Pi

Dihydroxyacetone

phosphate

Glyceraldehyde-3-phosphate

1,3-bisphoshpogycerate

NAD+

+ Pi

NADH + H

ADP + Pi

ATP

Phosphoenolpyruvate

CO2

ADP + Pi

ATP

OxaloacetateMalate

2H

Pyruvate

GLYCEROL

4H

Lactate

2H

Fumarate

Succinate

2H

Acetyl -CoA Formate 2H + CO2

Acetyl phosphate H2

ADP + Pi

ATP

Acetate

Acetaldehyde

Ethanol

2H

GLYCEROL

(!) Insufficient knowledge on H2 metabolism.

Lactate formation during glycerol fermentation

at pH 7.5 is absent.

(Cintolesi et al. (2011) Biotechnol. Bioeng. 109, 187-198;

Poladyan et al. (2013) Curr. Microbiol. 66, 49-55)

Hydrogenases and formate hydrogen lyases in E. coli

Schematic representation of the localization and arrangement of hydrogenases and formate

hydrogen lyases (Trchounian et al. (2012) Crit Rev Biochem. Mol. Biol. 47, 236-249)

H2 uptaking and oxidizing hydrogenase 1 (Hya) and hydrogenase 2 (Hyb):

(Forzi and Sawers (2007) Biometals 20, 565-578)

H2 producing formate hydrogen lyase 1, formed by Fdh-H and Hyd-3 (Hyc) as proposed by

Sauter et al. (1992) Mol. Microbiol. 6, 1523-1532), and formate hydrogen lyase 2, formed by Fdh-H and

Hyd-4 (Hyf) as proposed by Andrews et al. (1997) Microbiology 143, 3633-3647).

Formate is electron donor for Fdh-H and H+

is terminal acceptor for electron. H+

translocation (dotted arrows) is suggested (Andrews et al. (1997) Microbiology 143, 3633-3647).

membrane cytoplasm

HyaC

H2 2e- HyaA

HyaB

2H+ Quinone Pool

HybC

HybO H2 2e-

HybB

2H+ HybA

HycB FdhF SeMo HycC Fe-S CO2+H+

2e-

HycD HycF Fe-S HCOO-

HycE

HycG 2e- 2H+ Ni H2

HyfA FdhF SeMo CO2+H+ HyfC Fe-S 2e-

HyfH HCOO- HyfE Fe-S HyfI

Fe-S HyfG 2e- 2H+ HyfBDF Ni

2H+ H2

H+

translocation

?

Hyd-1

Hyd-2

FHL-1

FHL-2

Glycerol fermentation and redox potential decrease by E. coli at slightly alkaline pH

In the E. coli wild type suspension upon addition of glycerol, the redox potential

(Eh), determined by a platinum (Pt) electrode, shift

down from the positive values to strong negative ones

(up to ~-650 mV) was observed, pH 7.5.

In the other mutants Eh was decreased too but in a

different manner.(Trchounian, Trchounian (2009) Int.

J. Hydrogen Energy 34, 8839-8845).

-800

-700

-600

-500

-400

-300

-200

-100

0

100

200

300

400

0 1 2 3 4 5

t (min)

Eh (

mV

)

w ild type

hyaB hybC

fhlA

hyaB

hybC

Glycerol fermentation, hydrogenases and H2 production by E. coli at slightly alkaline pH

The results show that under glycerol fermentation by E. coli at neutral and alkaline pH

Hyd-2 mostly and Hyd-1 partially are involved in H2 production by bacteria; no relation with FHL activity is observed;

Hyd-2 and Hyd-1 are reversible depending on fermentation substrate.

(Trchounian, Trchounian (2009) Int. J. Hydrogen Energy 34, 8839-8845).

(!) This is absolutely novel finding although under glycerol fermentation at acidic pH FHL complex is required for H2 production

These results confirm data reported by Sawers with coworkers (J. Bacteriol. 164 (1985) 1324-1331) that under glucose fermentation FHL

activity includes neither Hyd-1 nor Hyd-2. The latter activity determination under glycerol fermentation at a low Eh seems to be in

accordance with data about low Eh-dependent activity of Hyd-2 (Laurinavichene et al. (2002) Arch. Microbiol. 178, 437-442;

Laurinavichene, Tsygankov (2001) FEMS Microbiol. Lett. 202, 121-124).

Glucose and glycerol fermentation and hydrogenase activity by E. coli at different pHs

7.5 6.5 5.50

0.5

1

1.5

2

2.5

3

3.5

4

wt

selC

hyaB

hybC

hyaB hybC

hyaB hybC selC

pH

Hyd

roge

nase

spe

cifi

c ac

tivi

ty, U

/mg

prot

ein-

1

Hyd-activity of E. coli wild type and different mutants grown at different pH on peptone medium supplemented with glucose (A) or glycerol (B)

at different pH. The results for single, double and triple mutants with defects in Hyd-1 and Hyd-2 and formate dehydrogenases are shown.

(Trchounian et al. (2012) Cell Biochem. Biophys. 62, 433-439)

Hyd-activity measured was H2-dependent reduction of benzyl viologen.

7.5 6.5 5.50

0.5

1

1.5

2

2.5

3

3.5

wt

selC

hyaB

hybC

hyaB hybC

hyaB hybC selC

pH

Hy

dro

gena

se s

pec

ific

act

ivit

y,

U/m

g

pro

tein

-1

A B

Glucose and glycerol fermentation and hydrogenase activity by E. coli at different pHs

Identification of active Hyd-1 and Hyd-2 by activity staining after

native-PAGE. Crude extracts derived from E. coli wild type

and different mutants grown on glucose (A-C) or glycerol (C-F) at different pH were analyzed. The locations of Hyd-1 and Hyd-2 in the gels are shown on the

right of each panel. Where 1’ is signified this indicates a rapidly migrating form

of Hyd-1 and where 2’ is shown, this signifies a more rapidly migrating form of Hyd-2. The asterisk near the top of

each gel designates a Hyd-independent activity band. To simplify the

nomenclature of the strains used and which are listed above and below the

panels, the wild type (Wt, BW25113) and mutant strains were given the following

phenotypic designations: D1 (hyaB, JW0955); D2 (hybC, JW2962); D3 (fhlA,

JW2701); D4 (hyfG, JW2472); D(1+2) (hyaB + hybC, MW1000); DF (hypF,

DHP-F2).(Trchounian et al. (2012) Cell Biochem.

Biophys. 62, 433-439)

Hyd-4

Hyd-3

Hyd-1

Hyd-2

2H+

+2e- H2

VH2 = {V(Hyd-3) – {V(Hyd-1) + V(Hyd-2) + V(Hyd-4)}

H2 2H+

+2e-

GLYCEROL

pH 5.5

GLUCOSEHyd-4

Hyd-3

Hyd-1

Hyd-2

2H+

+2e- H2

VH2 = {V(Hyd-2) +V(Hyd-1)} – {V(Hyd-3) + V(Hyd-4)}

H2 2H+

+2e-

GLYCEROL

pH 7.5

Different H2 producing and H2 uptaking Hyd-enzymes expressed by E. coli under glycerol fermentation at pH 7.5 or pH 5.5. VH2 is H2

producing rate by whole cells; V(Hyd) is H2 producing or H2 oxidizing rate by appropriate Hyd-enzyme. Arrows are for direction of

enzyme operation to produce and/or to oxidize H2. The mode for Hyd-enzymes functioning at pH 5.5 upon glucose fermentation is

similar with that under glycerol fermentation.

(Trchounian et al. (2011) Int. J. Hydrogen Energy 36, 4323-4331)

Interaction between hydrogen and proton cycles at neutral and slightly alkaline pH

Fig. 1.

[pH]out 7.5

In

Out

Hyd-1

b H2

n H+ m H+

Hyd-2

FHL-2

Hyd-3

F0F1

H2->2H++2e-

a H2

2H++2e-

->H2

H2

H+

m H+

H+

nH+ K+

K+

H+

H2

nH+

FORMATE

2H+

Hyd-4

TrkA

CO2

F0F1

FdhF 2e-

2e- HycB

H+

According to the model, for a transfer of energy from F0F1 reducing

equivalents (2(H++e-) are required. They can be donated from formate through Fdh-H and via HycB. The subsequent

transfer of 2H through F0F1 to TrkA implies that dithiol on TrkA can perform

the role of some "intermediator", because the future liberation of 2H and

restoration of disulfide may lead to energy release, used for the work of

counter-gradient K+ uptake. 2H can then be employed for evolution of H2 by Hyd-

4.

This model is proposed for slightly alkaline or neutral pH

(Trchounian (2004) Biochem. Biophys. Res. Commun. 315: 1051-1057; Trchounian et al. (2012) Crit.

Rev. Biochem. Mol. Biol. 47:236-249) Trchounian & Sawers (2014) IUBMB Life, 66, 1-7

Questions:What is the F0F1-activity depending on

pH? How is a model for acidic pH?

Proton cycleH2 cycle

By decreasing glucose concentration from 0.2% to 0.05% H2 evolution was increased ~2 fold at pH 7.5 and ~3.5 fold at pH 6.5. Interestingly, at pH 5.5 the decrease of glucose concentration did

not enhance H2 production which was lowered ~1.6 fold. The decrease of glycerol concentration had no any affect on H2

formation either at slightly acidic or slightly alkaline pH. Only at pH 5.5 H2 production decreased ~1.6 fold.

0.2% glucose 0.1% glucose 0.05% glucose

0

2

4

6

8

10

12

14

16

18

pH 7.5

pH 6.5

pH 5.5

H2

pro

du

ctio

n r

ate

mV

E

h/m

in/m

g d

ry w

eigh

tH2 production during glucose or glycerol

fermentation at different pHs

1% glycerol 0.5% glycerol0

0.5

1

1.5

2

2.5

3

3.5

4

pH 7.5

pH 6.5

pH 5.5

H2

pro

du

ctio

n r

ate

mV

E

h/m

in/m

g d

ry w

eigh

t

H2 production during glucose or glycerol fermentation at different pHs

From these data it can be suggested that glucose has inhibitory effect on H2 producing activity of Hyd enzymes; this is in good conformity with glucose inhibitory effects on hyf operon expression (Self et al. 2004. J. Bacteriol. 186: 580-58).

At low pH high concentration of glucose did not inhibit H2 production due to that the other producing Hyd enzyme Hyd-3 is active and no inhibition of hyc operon expression is determined.

H2 production during glucose or glycerol fermentation at different pHs

pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.50.2% glucose 0.8% glucose

0

1

2

3

4

5

6

7

8MC4100

JRG3615

JRG3621

H2

prod

ctio

n ra

te E

h m

V/O

RP

/m

in/m

g dr

y w

eigh

t

pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.50.2% glucose 0.8% glucose

0

1

2

3

4

5

6MC4100

JRG3615

JRG3621

H2

prod

ucti

on r

ate

Eh

mV

/OR

P/m

in/m

g dr

y w

eigh

t First time it was shown that Hyd-4 activity depends on glucose concentration. Especially at pH 7.5 during fermentation of glucose

at 0.2% concentrations in hyfA-B and hyfB-R mutants H2 production is significantly lowered compared to the cells grown at 0.8%

glucose (Trchounian and Trchounian (2014) Int. J. Hydrogen Energy 39, 16914-16918).

H2 production during mixed carbon fermentation at

different pHs

During mixed carbon fermentation when

glycerol was supplemented wild type cells VH2 was the same as with glycerol only fermentation at pH 7.5 and

5.5. No any H2 gas was detected at pH 5.5 which was not observed when

cells were grown on glycerol only.

0

0.5

1

1.5

2

2.5

3

3.5

4

pH 7.5

pH 6.5

pH 5.5

H2

pro

du

cti

on

ra

te m

V E

h/m

in/m

g

dry

we

igh

t

Interestingly, at pH 5.5 no H2 gas was detected which might be that

glucose inhibits glycerol uptake enzymes which was shown for Klebsiella

pneumoniae (Sprenger et al. 1989. J. Gen. Microbiol. 135: 1255-1262).

H2 production during mixed carbon fermentation at different pHs

During mixed carbon (glucose+glycerol) sources

fermentation at pH 7.5 in the presence of 1% glycerol and 0.05% glucose, when glucose was supplemented into the

assays, H2 produced was ~2.5 fold higher compared to that

for the medium containing 1% glycerol and 0.2% glucose

Trchounian, K. et al., (2014). Int, J. Hydrogen Energy 39, 6419-6423.

2.5 ml/l glucose 1.25 ml/l glucose 10 ml/l glycerol + 2.5 ml/l glucose (glucose assay)

10 ml/l glycerol + 1.25 ml/l glucose (glucose assay)

0

2

4

6

8

10

12

14

16

18

pH 7.5

pH 6.5

pH 5.5

H2

pro

du

ctio

n r

ate

mV

Eh

/min

/mg

dry

wei

ght

At pH 7.5 mixture of 1% glycerol and 0.1% when supplementing glucose,

increased H2 production ~2.2 fold

At pH 6.5 H2 production ~1.7 fold

At pH 5.5 supplementation of glycerol into the medium increased H2

evolution

H2 production during glycerol and formate fermentation at different pHs

wt hyaB hybC hyaB hybC0

10

20

30

40

50

60

glycerol

formate

H2

pro

du

ctio

n, m

V E

h/m

in/m

g d

ry

wei

ght

wt hycE hyfG hyfG fhlA0

5

10

15

20

25

30

35

40

glycerol

formate

H2

pro

du

ctio

n, m

V E

h/m

in/m

g d

ry w

eigh

t

B

H2 production rate (VH2) by E. coli BW25113 wild type and mutants with defects in Hyd-1 and Hyd-2 (A), Hyd-3 and Hyd-4 (B) during mixed

carbon fermentation in assays supplemented with glycerol or formate at pH 7.5.

A

During glycerol fermentation when external formate was supplemented all Hyd enzymes function in H2 producing mode. Deletion of each of the

Hyd enzymes is compensated by the other one towards H2 production Trchounian K. & Trchounian A (2015). Renewable Energy, 83, 345-351.

H2 production during glycerol and formate fermentation at different pHs

wt hyaB hybC selC

hyaB hybC hycE

selC hyaB hybC hycE0

10

20

30

40

50

60pH 7.5 glycerol pH 7.5 formate pH 6.5 glycerol pH 6.5 formate

H2

pro

du

ctio

n, m

V E

h/m

in/m

g d

ry w

eigh

t

H2 production rate (VH2) by E. coli BW25113 wild type and mutants with defects in Hyd-1, Hyd-2 , Hyd-3, Hyd-4 and formate dehydrogenases

during mixed carbon fermentation in assays supplemented with glycerol or formate at pH 7.5 and pH 6.5 Trchounian K. & Trchounian A

(2015). Renewable Energy, 83, 345-351.

Only deletion of three Hyd enzymes disturbs H2 production in the assays supplemented with glycerol at both pHs.

At pH 6.5 in the formate supplemented assays deletion of three Hyd enzymes only by 50% affects H2 production the rest is produced by Hyd-4.

H2 production during acetate fermentation at different pHs

pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.5 pH 7.5 pH 6.5 pH 5.5 A B C

0

1

2

3

4

5

6

H2

pro

du

ctio

n y

ield

, mm

ol L

-1

At pH 7.5 and pH 6.5 H2 yield was highest when cells were grown in the

presence of 5g/l acetate. Trchounian K et al. (2015) Int. J. Hydrogen Energy 40,

12187-12192.

A – 1g/l

B - 2g/l

C – 5g/l

H2 production during glycerol and acetate fermentation at different pHs

Delayed H2 production detection in the mixture of 5g/l acetate and 10g/l glycerol

Continuos H2 production during 96 h

At pH 5.5 H2 production yield was ~2.7 fold compared to the cells grown on acetate only. Trchounian K et al. (2015) Int. J.

Hydrogen Energy 40, 12187-12192.

Glucose and glycerol fermentation and hydrogenase activity by E. coli

at different pH

Taken together, our findings Glucose concentration is distinctive for the activity of

Hydrogenase 4

New functions of Hyd enzymes were determined when glycerol was present in the growth medium during fermentation

All Hyd enzymes are reversible and function for maintaining H2 recycling: only absence of three hydrogenases disturbs the H2 recycling

Different mixtures of carbon sources enhances H2 production

Proposal

Hydrogenase enzymes have a key role in proton sensing to regulate the cytoplasmatic pH by producing H2 and to maintain proton motive force by having cross talk with proton-ATPase.

Acknowledgements

Prof. Dr. A. Trchounian, Drs. A. Poladyan, A.

Vassilian and other people in the lab,

for discussion and some comments

The study was done as a part of Basic support and Research Grants from the Ministry of Education and Science of the

Republic of Armenia (#11-1F202, 13-F002) and supported by ANSEF (USA) Research Award (biotech-3460), FEBS

Research Fellowship, DAAD Research Scholarship

Prof. Thomas Wood

(Penn State University, University Park, USA)

Prof. R. Gary Sawers

(Martin Luther University of Halle-Wittenberg,

Germany)

Prof. Dr. Ramon Gonzalez (Rice University,

Houston, USA) and members of their labs for

collaboration

WELCOME TO ARMENIA

THANK YOU FOR YOUR ATTENTION