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Renewable Energy
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CELL FACTORIES, New perspectives for biotechnologies
Mireille BRUSCHILaboratoire 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,Systemsbiology …
• New biocatalysts, biofuel cells
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
BIOMASS
Cell factories
ENERGY
PRODUCTS OF INTEREST
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
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
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
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
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%
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?
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... »
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
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.
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
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
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
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
• 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
HYDROGENASE
H2 2 H+ 2 e-+
Fe-Hydrogenase Ni-Fe-Hydrogenase
NiFe active center
H+
e-
H2 H2
H2
Gas channels
[[NiFeNiFe] HYDROGENASE] HYDROGENASE
FeS clusters
Hydrogenase activity
H2 oxidation
H2
H2
H+H+
e-e-
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
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
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)
-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
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
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
-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
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)
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
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