October 5, 2015, 6 th World Congress on Biotechnology, New - PowerPoint Presentation

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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

Slide2

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).H

2

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

Slide3

Over 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

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Over 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

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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 H

2

costs 2-4$.

~65 million

tonnes

/

yr

and yearly increase in 10-15%

Slide6

Biohydrogen 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

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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).

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.

Slide8

Mixed 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)

Slide9

Hydrogenases 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

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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).

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Glycerol 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).

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Glucose 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

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Glucose 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)

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Hyd-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)

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Interaction 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

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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 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

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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.

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H2 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).

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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. 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).

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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% 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

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H2 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.

Slide22

H2 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.

Slide23

H2 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

Slide24

H2 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.

Slide25

Glucose 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.

Slide26

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 lab

s

for collaboration

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WELCOME TO ARMENIA

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THANK YOU FOR YOUR ATTENTION