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CELL FACTORIES, New perspectives for biotechnologies Mireille BRUSCHI Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, CNRS, Marseille, France

Mireille BRUSCHI

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Page 1: Mireille BRUSCHI

CELL FACTORIES, New perspectives for biotechnologies

Mireille BRUSCHILaboratoire de Bioénergétique et Ingénierie des Protéines,

Institut de Microbiologie de la Méditerranée, CNRS, Marseille, France

Page 2: Mireille BRUSCHI

Biology and renewable energies• Increasing interest in biotechnology to develop green energy• Make use of living cells (microorganisms,bacteria,algae..) or

of their components (enzymes) to produce new sources of energy. Concept of Cell Factories

• Development of new biotechnological processes:• Screening the biodiversity to find new microorganisms

showing useful potentials for the production of methane, hydrogen and lipids…

• Use of the potential of genomic studies to characterize the metabolic pathways involved. Genetic engineering,Systemsbiology …

• New biocatalysts, biofuel cells

Page 3: Mireille BRUSCHI

Biomass Energy or bioenergy

Biomass is the first source of renewable energy of ourplanet.It represents all the non fossil material coming fromliving cells (animal or vegetal).172 Bt/year of dry material

• Bioenergy is the production of energy from biomass.Itconsists of recovering the energy released from the degradation of the biomass in CO2 et H2O (elements fromwhich biomass has been constituted )

BiomassResources

Agricol productsand wastes

Liquid wastes

Animals residusUrban solid wastes

Forest productsand wastes

Industrial residusand wastes

Page 4: Mireille BRUSCHI

BIOMASS

Cell factories

ENERGY

PRODUCTS OF INTEREST

Page 5: Mireille BRUSCHI

Optimizing the first generation

Improving ,energetic yield and cost of the processValorisation of valuable co-products

FIRST GENERATION BIOFUELS

Rape seed oilSunflower oil

Sugar beetSugar cane

CornMaizePotatoes

Biodiesel

Mixedto gazoil

Mixedto petrol

StarchSugar Ethanol

Page 6: Mireille BRUSCHI

CONCEPT OF BIOREFINERIES

Carbon cycle

Biomass

Fossil energiesProducts

Energy, human andanimal food

wastesAgriculturalcoproducts

An integrated biomass plant, at the same location, could produceliquid fuel, edible oil, sugars, animal feed, power and polymers or

chemical intermediates.

Fuel,heat

and newbioproducts

Page 7: Mireille BRUSCHI

Biomass consists of cellulose, hemicelluloses and ligninAcidic hydrolysis and enzymatic treatment are necessarybefore the fermentation of sugars into ethanol

European project « New Improvement for LignocellulosicEthanol » involves 28 industrial companies and researchlaboratories (CNRS, l’INRA …)

Technological bottlenecks

Pretreatment stepsTo find a method that does not degrade hemicelluloseinto an inhibitor of fermentation

Enzymatic hydrolysis steps:Decrease the costImprove catalytic enzyme efficiency

Fermentation stepTo succeed in pentoses fermentationTo improve alcool concentration before distillationTo obtain efficient yeast strains even in the presence offermentation inhibitors

2 t of biomass give 400l of ethanol per day

Distillation

Pretreatment with hot gas

Lignocellulose

Lignin

enzymatichydrolysis

enzymatichydrolysis +fermentation

fermentation

Page 8: Mireille BRUSCHI

TOWARDS A THIRD GENERATION• Microorganism cultures do not compete with

arable lands.• Production of biogas and lipids from anaerobic

fermentative bacteria .• Hydrogen production from water and solar

energy by the functioning of photosyntheticmicroorganisms.

• Lipid production from autotrophic microalgae• Optimizing bioprocessing conversion• Exploiting microbial genomes for energy

production• Fuel cells and electricity

Page 9: Mireille BRUSCHI

Use of biomass for Biogaz production by anaerobic fermentation

CHCH44

HH22

Anaerobic fermentative Bacteria(Clostridium, Bacillus.)

Coupling of substrates oxidationto H2, CO2 and acetate formationClostridium, Sulfate reducingbacteria

Production of CH4 and CO2(methanogens using acetateand H2)

In produced biogaz, CH4 55-85%

Page 10: Mireille BRUSCHI

Inoculum not necessary

Existence of hydrogen potential (7ml / g DCO biodegradable waste )

Hydrogen potential of biomassANR Promethee

Wastes

4mm

Physico chemical conditions (pH,°C…) increase H2 production (Patent INRA-CNRS-VEOLIA)

Instability of H2 Production : role of the interactions intra/inter species?

Page 11: Mireille BRUSCHI

Objectives

« … comprehension, building and study of microbial consortia to establish

parameters governing networks of metabolic correlations with the objective

of optimizing the production of hydrogen... »

Page 12: Mireille BRUSCHI

Production/consumption of H2

Anaerobic

Mesophile

Genome sequenced

Sulfate reducing bacteria

Desulfovibrio vulgarisHildenborough(DvH)

Gram‐Sulfate respiration (BSR)

Synthetic microbial ecosystem

Gram+ Fermentation  ABE

Clostridium

Clostridium acetobutylicumATTC824 (Cab)

H2 , Ethanol , production

Page 13: Mireille BRUSCHI

In the consortium:-the hydrogen production is 3 fold higher thanclostridium alone-modification of the metabolic pathways(modification in butyrate and in lactate pathway)

H2 produced by the synthetic consortium

Times (h.)0 20 40 60 80

H2 P

rodu

ctio

n (µ

mol

)

0

500

1000

1500

2000

2500

SRB

C.a.

SRB + C.a.

Page 14: Mireille BRUSCHI

Conclusions

Metabolic model of the consortium

This implies a different experimental design from the one that could be appropriate for studying an enzyme mechanism.

-Complete reaction mixture (all substrates, all products, all effectors).

- Reversible conditions, as close to physiological as possible.

- Always take into account product inhibition, inhibition by other metabolites that are present in the system, interactions between enzymes

- Rate equation must be thermodynamically correct

Modeling of the bioreactor is built at present on the basis of the metabolic model

Page 15: Mireille BRUSCHI

PHOTOSYNTHESISHydrogen, lipid production, CO2 capture

MicroalgaePhotosynthetic bacteria

Improve metabolism (triglycerides synthesis as regard to nutriments depletion)oxygen inhibition (Hydrogenase)

Triglycerides optimizedproduction for biodiesel

H2 photoproduction fromH2O and solar energy byhydrogenases

CO2 capture

Page 16: Mireille BRUSCHI

Microalgal Biofuels

• One of the most promising feedstocks for biofuel• Resurgence of algal biofuels research and industrial and oil companies investment

• Microalgae are unicellular photosynthetic microorganisms abundant in fresh water and marine environments everywhere on earth.They are capable of utilising carbon dioxide and sunlight to generate the complex biomolecules necessary to their survival.Under certain conditions (deprivation and stress), they can accumulate significant amounts of lipids (more than 50% of their cell dry weight.

• High per ha productivity compared to typical terrestrial oil-seed crops

• Use of otherwise non-productive, non arable land

• Production of both biofuels and valuable co-products

• For cost and energy reduction and maximization of lipid productivity, cell properties, open or closed cultivation systems, bioreactor design, efficiency in supply and use of nutriments needto be improved

Page 17: Mireille BRUSCHI

A pennate diatom, Navicula sp.,showing an oil droplet

Classification of lipids in diatoms.

(1) Geologists claim that much crude oil comes fromdiatoms.

(2) Diatoms do indeed make oil.

(3) Agriculturists claim that diatoms make 10 times as muchoil per hectare as oil seeds, with theoretical estimates reaching 200 times

Page 18: Mireille BRUSCHI

• Most of H2 producing bacteria also use it.

• Knowledge of metabolism is required

• Hydrogenases are sensitive to temperature, pH, oxygen

• Optimized Biocatalysts

• For scale-up processes, integrated approach from processengineering, physiology and genetics, is needed

• H2 Collect and Storage

Technological key locks for production of hydrogen

Page 19: Mireille BRUSCHI

HYDROGENASE

H2 2 H+ 2 e-+

Fe-Hydrogenase Ni-Fe-Hydrogenase

Page 20: Mireille BRUSCHI

NiFe active center

H+

e-

H2 H2

H2

Gas channels

[[NiFeNiFe] HYDROGENASE] HYDROGENASE

FeS clusters

Page 21: Mireille BRUSCHI

Hydrogenase activity

H2 oxidation

H2

H2

H+H+

e-e-

Page 22: Mireille BRUSCHI

Towards engineering O2 tolerance in Ni-Fe hydrogenases in reducing diffusion rate and accelerating reactivation rate

Volbeda et al. IJHE, 2002

NiNiFeFe

Leucine 122Leucine 122

Valine 74Valine 74

Liebgott et al. Nat. Chem. Biol, 2009

Page 23: Mireille BRUSCHI

P680

Qa

PSII

O2 + 4 H+

2 H2O

LHC

Pc

P700

cytb6

cytfPSI LHCPQ(H)2

Hydrogenase

2 H+H2

FNRFd

NADP+ NADPH2

2e-2e-2e-

H2H2H2

PhotobioPhotobio HydrogeneHydrogene

Photosynthetic organism coupling water photolysis to hydrogenproduction

Page 24: Mireille BRUSCHI

Aquifex aeolicus

-Optimal growth temperature 85°C (most hyperthermophilic bacterium)

-Exceptional phylogenetic position

-Completely sequenced genome

- Growth on H2/O2/CO2 , inorganic compoundswith a sulfur compound (S°, thiosulfate, or H2S)

Page 25: Mireille BRUSCHI

-The Oxygen-Tolerant Hydrogenase I from Aquifex aeolicus Weakly Interacts with Carbon Monoxide: An Electrochemical and Time-Resolved FTIR Study.Pandelia ME, Infossi P, Giudici-Orticoni MT, Lubitz W.Biochemistry. 2010, 49(41):8873-8881.

Membrane-bound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance.Pandelia ME, Fourmond V, Tron-Infossi P, Lojou E, Bertrand P, Léger C, Giudici-Orticoni MT, Lubitz W.J Am Chem Soc. 2010, 132(20):6991-7004

Guiral et al, J. Proteome Res, 2009

S0

-3 Ni-Fe Hydrogénases-Actives at 90°C-High stability for thermal and chemical denaturation-Oxygen, CO, NO resistant

Aquifex aeolicus

Page 26: Mireille BRUSCHI

Chemical catalystH2 2H+ + 2e-

Biochemical catalyst

1/2 O2

Anode Cathode

H2O

HH22

2 H2 H++

e-e-

HydrogenaseHydrogenase

V

Hydrogenases

Turn over SpecificityBiodegradableBioavailability

Resistant to T°, pH,

CO, O2…

HydrogenasesHydrogenases as biocatalysts for as biocatalysts for biofuelbiofuel cells ?cells ?

Platinum

Inhibited by COWeak specificity

Membrane

CostAvailability

Degradability

Page 27: Mireille BRUSCHI

Efficient immobilization of Efficient immobilization of hydrogenaseshydrogenases at the electrode at the electrode

Control of hydrogenases orientation: environment of the distal FeS cluster

Mesophilic,Anaerobic,

Desulfovibriofructosovorans

Thermophilic,Microaerophile,

Aquifexaeolicus

SAu

S

R

H2H+

Direct or Mediated electron transfer ?Direct or Mediated electron transfer ?

SAu

S

RR

SAu

S

+

hydrogenase

R

X.Luo et al. JBIC 14(2009)1275; P.Infossi et al.Int.J.Hydrogen Energy 35(2010)10778

Page 28: Mireille BRUSCHI

-15

5

25

45

65

-0.8 -0.6 -0.4 -0.2

+ 110 µM MV2+

Potential V vs Ag/AgClC

urre

nt µ

A/c

m2

25 °CH2 atm.

GraphiteGraphite

Increase of connected Increase of connected hydrogenaseshydrogenases at the electrode at the electrode

Use of carbon nanotube networks

E. Lojou et al., JBIC 13 (2008) 1157-1167

SWCN depositedOnto PG surface

N2

Df NiFe hydrogenase

50 nm

CO

2HH

O2C

100 nm

A. Ciaccafava et al., Langmuir (2010) under press

Page 29: Mireille BRUSCHI

Institut de Microbiologie de la MediterraneeLaboratory of Bioenergetic and Protein

Engineering

Energetic metabolism of extremophiles bacteria –Anaerobicfermentation of biomass

Scientific leader Marie-Therese Giudici-OrticoniANR : PROMETHEE, INGECOHPIE : multiresistant hydrogenase production

Molecular ecology and hydrogen metabolismScientific leader Marc RoussetANR:DIVHYDO,HYLIOX, Engineering H2cyano,AlgoH2

CO2 fixation and lipid production (Chlamydomonas reinhartii, diatom Asterionella formosa)

Scientific leader Brigitte Meunier-GonteroANR:Galactolipase PIE: DIALOG

Fuel cellsScientific leader Elisabeth LojouANR:BIO-CAT H2, BIOPAC PIE:InHaBioH2

Biophysics of metalloproteinsScientific leader Bruno GuigliarelliANR:CAFE, SPINFOLD

BRGM OrléansCREED, VEOLIA, ParisSociété des eaux de Marseille

LBE Narbonne CEA Cadarache G. PeltierCEA IBS Grenoble J. FontecillaEIPL F. CarriereLCP DenoyelCEA Saclay ChauvatINSA Toulouse

W.Lubitz (MaxPlanck Institut Mülleum)

V.Fernandez (Madrid)S.Maberly (CEH England)

Page 30: Mireille BRUSCHI

Programme launched in 2004 by the French GovernmentTo foster cooperation between industry, R&D labs and universitiesTo develop highly competitive industrial sectors71 french competitiveness clusters so far

Capenergies , cluster for non-greenhouse gas energy sources, has been accredited in 2005

The French Competitiveness Clusters Programme

Page 31: Mireille BRUSCHI

Since the beginning: 220 projects submitted174 certified projects distributed over 9 thematic fields, concerning 130 differentmembers,

A high rate of SMEs involved for a total investment of 1.066 M€.101financed (112 M€ subsidies obtained / 260 M€ total budget)

International R&D partnerships Euro - Mediterranean countries (mission in Israël Nov.2007)

International commercial and industrial partnerships

StrategyFacilitating partnerships between:

3 Cluster components (research, training, industry) to fosterinnovation from research to development

9 thematic fields