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
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