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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


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,Systems biology … • New biocatalysts, biofuel cells


Biomass Energy or bioenergy Biomass is the first source of renewable energy of our planet.It represents all the non fossil material coming from living cells (animal or vegetal). 172 Bt/year of dry material •

Bioenergy is the production of energy from biomass.It consists of recovering the energy released from the degradation of the biomass in CO2 et H2O (elements from which biomass has been constituted )

Liquid wastes

Forest products and wastes Agricol products and wastes

Biomass Resources Industrial residus and wastes

Animals residus

Urban solid wastes


ENERGY BIOMASS

PRODUCTS OF INTEREST

Cell factories


FIRST GENERATION BIOFUELS Mixed to gazoil

Rape seed oil Sunflower oil Biodiesel Sugar beet Sugar cane

Mixed to petrol Corn Maize Potatoes

Starch

Optimizing the first generation Improving ,energetic yield and cost of the process Valorisation of valuable co-products

Sugar

Ethanol


CONCEPT OF BIOREFINERIES

Carbon cycle Biomass

wastes Fossil energies Products

Fuel, heat and new bioproducts Agricultural coproducts

Energy, human and animal food An integrated biomass plant, at the same location, could produce liquid fuel, edible oil, sugars, animal feed, power and polymers or chemical intermediates.


Biomass consists of cellulose, hemicelluloses and lignin Acidic hydrolysis and enzymatic treatment are necessary before the fermentation of sugars into ethanol European project « New Improvement for Lignocellulosic Ethanol » involves 28 industrial companies and research laboratories (CNRS, l’INRA …) Lignocellulose Technological bottlenecks

Pretreatment with hot gas

enzymatic hydrolysis + fermentation

enzymatic hydrolysis

Lignin

fermentation

Distillation

Pretreatment steps To find a method that does not degrade hemicellulose into an inhibitor of fermentation Enzymatic hydrolysis steps: Decrease the cost Improve catalytic enzyme efficiency Fermentation step To succeed in pentoses fermentation To improve alcool concentration before distillation To obtain efficient yeast strains even in the presence of fermentation inhibitors 2 t of biomass give 400l of ethanol per day


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 photosynthetic microorganisms. • Lipid production from autotrophic microalgae • Optimizing bioprocessing conversion • Exploiting microbial genomes for energy production • Fuel cells and electricity


Use of biomass for Biogaz production by anaerobic fermentation Anaerobic fermentative Bacteria (Clostridium, Bacillus.) Coupling of substrates oxidation to H2, CO2 and acetate formation Clostridium, Sulfate reducing bacteria

H2 Production of CH4 and CO2 (methanogens using acetate and H2) In produced biogaz, CH4 55-85%

CH4


Hydrogen potential of biomass ANR Promethee

4mm

Wastes

Inoculum not necessary Existence of hydrogen potential (7ml / g DCO biodegradable waste ) Physico chemical conditions (pH,°C‌) increase H2 production (Patent INRA-CNRS-VEOLIA) Instability of H2 Production : role of the interactions intra/inter species?


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... »


Synthetic microbial ecosystem Sulfate reducing  bacteria

Clostridium Clostridium acetobutylicum ATTC824 (Cab)

Desulfovibrio vulgaris Hildenborough(DvH) Anaerobic Mesophile Genome sequenced

Gram+  Fermentation  ABE

H2 , Ethanol , production

Gram‐ Sulfate respiration (BSR)

Production/consumption of H2


H2 produced by the synthetic consortium 2500

SRB + C.a.

H2 Production (Âľmol)

2000

1500

1000

C.a.

500

SRB

0 0

20

40

Times (h.)

60

80

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


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


PHOTOSYNTHESIS Hydrogen, lipid production, CO2 capture Triglycerides optimized production for biodiesel

Microalgae Photosynthetic bacteria

Improve

H2 photoproduction from H2O and solar energy by hydrogenases CO2 capture

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


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 need to be improved


(1) Geologists claim that much crude oil comes from diatoms. (2) Diatoms do indeed make oil. (3) Agriculturists claim that diatoms make 10 times as much oil per hectare as oil seeds, with theoretical estimates reaching 200 times

Classification of lipids in diatoms.

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


Technological key locks for production of hydrogen •

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 process engineering, physiology and genetics, is needed

H2 Collect and Storage


HYDROGENASE H2 Fe-Hydrogenase

2 H+ + 2 e Ni-Fe-Hydrogenase


[NiFe] HYDROGENASE

H2

NiFe active center H+ e-

FeS clusters H2

H2 Gas channels


Hydrogenase activity H2 oxidation

e- e H++ H

H2 H2


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

Fe Ni

Valine 74

Leucine 122

Liebgott et al. Nat. Chem. Biol, 2009

Volbeda et al. IJHE, 2002


Photobio Hydrogene H2

2 H+

Hydrogenase

NADP+

NADPH2

H2

FNR Qa

LHC 2 H2 O

PSII P680

O2 + 4 H+

PQ(H)2

Fd

cytb6

PSI

cytf

P700 Pc

LHC

2e-

Photosynthetic organism coupling water photolysis to hydrogen production


Aquifex aeolicus

-Optimal growth temperature 85째C (most hyperthermophilic bacterium) -Exceptional phylogenetic position -Completely sequenced genome - Growth on H2/O2/CO2 , inorganic compounds with a sulfur compound (S째, thiosulfate, or H2S)


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


Hydrogenases as biocatalysts for biofuel cells ?

Chemical catalyst

H2

2H+ + 2eBiochemical catalyst V Hydrogenase H2

e-

e-

2 H+

H2 O

Anode

Platinum Cost Inhibited by CO Availability Weak specificity Degradability Membrane

1/2 O2

Cathode

Hydrogenases Turn over

Resistant to Specificity T°, pH, Biodegradable CO, O2‌ Bioavailability


Efficient immobilization of hydrogenases at the electrode Control of hydrogenases orientation: environment of the distal FeS cluster Mesophilic, Anaerobic, Desulfovibrio fructosovorans

Thermophilic, Microaerophile, Aquifex aeolicus

R

R S

S

Au

R

S

+ hydrogenase

R

S

S

S

Au

Au H+

H2

Direct or Mediated electron transfer ? X.Luo et al. JBIC 14(2009)1275; P.Infossi et al.Int.J.Hydrogen Energy 35(2010)10778


Increase of connected hydrogenases at the electrode CO2H

Use of carbon nanotube networks 65

SWCN deposited Onto PG surface

+ 110 µM MV2+ 45

HO2C

Current µA/cm2

50 nm

100 nm

N2

25

5

25 °C H2 atm.

-15 -0.8

-0.6

-0.4

-0.2

Potential V vs Ag/AgCl Df NiFe hydrogenase

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

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


Institut de Microbiologie de la Mediterranee Laboratory of Bioenergetic and Protein Engineering Energetic metabolism of extremophiles bacteria –Anaerobic fermentation of biomass Scientific leader Marie-Therese Giudici-Orticoni ANR : PROMETHEE, INGECOH PIE : multiresistant hydrogenase production Molecular ecology and hydrogen metabolism Scientific leader Marc Rousset ANR:DIVHYDO,HYLIOX, Engineering H2cyano,AlgoH2 CO2 fixation and lipid production (Chlamydomonas reinhartii, diatom Asterionella formosa) Scientific leader Brigitte Meunier-Gontero ANR:Galactolipase PIE: DIALOG Fuel cells Scientific leader Elisabeth Lojou ANR:BIO-CAT H2, BIOPAC PIE:InHaBioH2 Biophysics of metalloproteins Scientific leader Bruno Guigliarelli ANR:CAFE, SPINFOLD

BRGM Orléans CREED, VEOLIA, Paris Société des eaux de Marseille

LBE Narbonne CEA Cadarache G. Peltier CEA IBS Grenoble J. Fontecilla EIPL F. Carriere LCP Denoyel CEA Saclay Chauvat INSA Toulouse

W.Lubitz (MaxPlanck Institut Mülleum) V.Fernandez (Madrid) S.Maberly (CEH England)


The French Competitiveness Clusters Programme Programme launched in 2004 by the French Government To foster cooperation between industry, R&D labs and universities To develop highly competitive industrial sectors 71 french competitiveness clusters so far Capenergies , cluster for non-greenhouse gas energy sources, has been accredited in 2005


Strategy Facilitating partnerships between: 3 Cluster components (research, training, industry) to foster innovation from research to development 9 thematic fields Since the beginning: 220 projects submitted 174 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


Mireille BRUSCHI