D elhi India Dr Karen Trchounian PhD in Biophysics and Biotechnology Deputy Director of ScientificResearch Institute of Biology YEREVAN STATE UNIVERSITY 0025 YEREVAN ARMENIA ID: 907967
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Slide1
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 H
2
production by
Escherichia coli
Slide2Biohydrogen 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).H
2
is very effective energy carrier; it is ~3 time more effective than fuel and gas
Slide3Over the world
European Union Hydrogen HighwayThe 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 2Belgium 1Copenhagen 1Czech Republic 1Denmark 14Finland 2France 5Germany 41Greece 2Greenland 1Iceland 2
Italy 21Luxembourg 1Norway 10
Portugal 1
Spain 4
Sweden 5
Switzerland 2
The Netherlands 4Turkey 3United Kingdom 20http://www.hydrogencarsnow.com/eu-hydrogen-highway.htm
Slide4Over the worldNowadays in United States, Japan, United Kingdom, Netherlands, Germany, India etc. already H
2 filling stations are exploited and different cars, buses and other motor vehicles are working on hydrogen.
http://www.hydrogencarsnow.com/eu-hydrogen-highway.htm
Slide5Over 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 H
2
costs 2-4$.
~65 million
tonnes
/
yr
and yearly increase in 10-15%
Slide6Biohydrogen as an alternative energy source of future
H2 is produced chemically and biologically:Disadvantages of chemical productionHigh temperature (heating)Limited yield not renewableShort-term technologyHigh Energy DemandBiological method of H2 production is possible through special enzymes named hydrogenases catalyzing the simple redox reaction
2H+ +2
e-
H
2
(Trchounian et al. (2012) Crit. Rev. Biochem. Mol. Biol. 47, 236-249)Advantages of biological productionlow temperature (without heating)RenewableLong-term technologyPossibility of further improvement of technology for cheap H2 production
Slide7Glycerol 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).
SIGNIFICANCE
glycerol is very cheap carbon source (crude glycerol cost
s
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.
Slide8Mixed acid fermentation and H
2 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-
di
phosphate
ATP
ADP + P
i
Dihydroxyacetone
phosphate
Glyceraldehyde-3-phosphate
1,3-bisphoshpogycerate
NAD
+
+ P
i
NADH + H
ADP + P
i
ATP
Phosphoenolpyruvate
CO
2
ADP + P
i
ATP
Oxaloacetate
Malate
2H
Pyruvate
GLYCEROL
4H
Lactate
2H
Fumarate
Succinate
2H
Acetyl -CoA
Formate
2H + CO
2
Acetyl phosphate
H
2
ADP + P
i
ATP
Acetate
Acetaldehyde
Ethanol
2H
GLYCEROL
(!)
Insufficient knowledge on H
2
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)
Slide9Hydrogenases and formate hydrogen lyases
in E. coliSchematic 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)
H
2 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).H
+
translocation
?
Hyd-1
Hyd-2
FHL-1
FHL-2
Slide10Glycerol 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).
Slide11Glycerol fermentation,
hydrogenases and H2 production by E. coli at slightly alkaline pHThe 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 H
2
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).
Slide12Glucose and glycerol fermentation and hydrogenase activity by E. coli at different
pHsHyd-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. A
B
Slide13Glucose and glycerol fermentation and hydrogenase
activity by E. coli at different pHsIdentification 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)
Slide14Hyd-4
Hyd-3
Hyd-1
Hyd-2
2H
+ +2e- H2VH2 = {V(Hyd-3) – {V(Hyd-1) + V(Hyd-2) + V(Hyd-4)}
H
2
2H
+
+2e
-
GLYCEROL
pH 5.5
GLUCOSE
Hyd-4
Hyd-3
Hyd-1
Hyd-2
2H
+
+2e
-
H
2
V
H
2
= {V(Hyd-2) +V(Hyd-1)} – {V(Hyd-3) + V(Hyd-4)}
H
2
2H
+
+2e
-
GLYCEROL
pH 7.5
Different H
2
producing and H
2
uptaking
Hyd
-enzymes expressed by
E. coli
under glycerol fermentation at pH 7.5 or pH 5.5. V
H2
is H
2
producing rate by whole cells; V(
Hyd
) is H
2
producing or H
2
oxidizing rate by appropriate
Hyd
-enzyme. Arrows are for direction of enzyme operation to produce and/or to oxidize H
2
. 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)
Slide15Interaction between hydrogen and proton cycles at neutral and slightly alkaline pH
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 F0F
1
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, 1-7Questions:What is the F0F1-activity depending on pH? How is a model for acidic pH?
P
roton cycle
H
2
cycle
Slide16By 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 H
2 formation either at slightly acidic or slightly alkaline pH. Only at pH 5.5 H2 production decreased ~1.6 fold.H2 production during glucose or glycerol fermentation at different pHs
Slide17H2 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.
Slide18H2 production during glucose or glycerol fermentation at different pHs
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).
Slide19H2 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. Interestingly, at pH 5.5 no H
2 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).
Slide20H2 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% glucoseTrchounian, K. et al., (2014). Int, J. Hydrogen Energy 39, 6419-6423. 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 H
2
evolution
Slide21H2 production during glycerol and formate fermentation
at different pHsH2 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.
Slide22H2 production during glycerol and formate fermentation at different pHs
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.
Slide23H2 production during acetate fermentation at different pHs
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/lB - 2g/lC – 5g/l
Slide24H2 production during glycerol and acetate fermentation at different pHs
Delayed H2 production detection in the mixture of 5g/l acetate and 10g/l glycerolContinuos H2 production during 96 hAt 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.
Slide25Glucose and glycerol fermentation and hydrogenase
activity by E. coli at different pHTaken together, our findings Glucose concentration is distinctive for the activity of Hydrogenase 4New functions of Hyd enzymes were determined when glycerol was present in the growth medium during fermentationAll Hyd enzymes are reversible and function for maintaining H2 recycling: only absence of three hydrogenases disturbs the H
2 recyclingDifferent mixtures of carbon sources
enhances H
2
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.
Slide26Acknowledgements
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 lab
s
for collaboration
Slide27WELCOME TO ARMENIA
Slide28THANK YOU FOR YOUR ATTENTION