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