Biohydrogen and Biodiesel

from microalgae December 18, 2014

G. Torzillo
Istituto per lo Studio degli Ecosistemi
Sede di Firenze
Main factors accounting for the recent global boom in
bio-energy production.

___________________________________________

• Strategic aspect: Developed countries want to

reduce their dependence on fossil energy imports,
particularly from politically instable countries.

• Ecological aspect: Renewable resources can

help to reduce the threat of global warming.
 Changes in Atmospheric Concentration
CO2, CH4, and N20 – A Thousand Year History

Atmospheric concentration CO2 (ppm)

Atmospheric concentration CH4 (ppb)

360
1750
340 CH4
1500
CO2 320
1250
300

280 1000

260 750

1000 1200 1400 1600 1800 2000 1000 1200 1400 1600 1800 2000

Atmospheric concentration N2O (pbb)

310

N2O 290

270

250
1000 1200 1400 1600 1800 2000
Source: IPCC Third Assessment Report (2001)
Loser—Ice Sheets
 Who Is Using What ?
Size of the Country Shows Relative Proportion of Indicated Parameter

Fuel
Population Use

Fuel Fuel Use

Imports Increase
http://www.sasi.group.shef.ac.uk/worldmapper/index.html
 The 20-20-20 EU policy
By 2020

100%

-20% -20%

+20%
+20%
Energy
Greenhouse efficiency
Renewables in
gas levels energy mix
improvement
 Definitions; what counts as “renewable”

 Renewable energy is energy from non-fossil

sources: wind, solar, geothermal, aerothermal,
hydrothermal, ocean energy, hydropower,
biomass, landfill and sewage treatment plant gas
and biogases

 Nuclear power is not a renewable energy source !

 Fossil fluel Renewable
reserves energy
potential

Biomass

Energy potential/Global annual consumption Energy potential/Global annual consumption

Energy Utilizable with Percentage of Potentially available

potential present state of utilizable energy with respect to annual
Energy source (amount per the art technology consumption
year) EJ EJ
Solar radiation ~ 1.111.500 ~ 1.482 ~ 0.13% ~ 2150
Wind Energy ~ 78.000 ~ 195 ~ 0.25 % ~ 150
Biomass ~ 7.800 ~ 156 ~ 2.0% ~ 15
Geothermal ~ 1.950 ~ 390 ~ 20 % ~ 4
Hydro/tide power ~ 1.170 ~ 78 ~ 6.7% ~ 2
Energy Annual
consumption (2008) 517.7 ● Exa = 1018
~1
Source:
(Hessen-Nanotech, 2008)
Scenario of the development of global energy demand
(source:www.solarwirtschaft.de)
Why hydrogen?

1) Excellent energy carrier: Lower heating value of H2 = 120 MJ/kg; Oil ≈ 43

MJ/kg; Biogas = 27.2 MJ//kg)

2) Some vehicles manufacturers have demonstrated that H2 can be used

directly in an internal combustion engine

3) The combustion of H2 produces water as the only by-product, while

burning of fossil fuels generates CO2 and a variety of pollutants
In principle, if H2 could promptly replace fossil fuels,
both the energy and environmental problems of our
planet could be simultaneously solved.
But:
1) In the Earth atmosphere H2 represents 1 ppm (by
volume)
2) Its production it is a strongly energy demanding
process.
Biological routes for hydrogen production.

Rupprecht et al. Appl. Microbiol. Biotechnol.

2006
Metabolic pathways in Chlamydomonas reinhardtii

DCMU

- Gaffron and Rubin, 1942

 PSII PSII Contribution up to 80 %

carbohydrate

Scoma et al. 2012 (Int. J. Hydrogen Energy)

The process must be carried out under strict anaerobiosis
conditions, caused by the sulfur-stavation.

I II III
IV

Faraloni C. and Torzillo G. ( 2010). J. Phycol.

 Main topics

1) Selection of Chlamydomonas reinhardtii strains

with a high hydrogen output rate

2) Optimization of the cultures conditions in laboratory

photobioreactors

3) Testing hydrogen production in photobioreactors

outdoors.
 PsbA gene – D1 protein

153
294

(Courtesy of U. Johanningmeier)
D1
Chlamydomonas reinhardtii mutants screened for the H2 production at ISE-FI
 L159I-N230Y

WT leucine 159 isoleucine

asparagine 230 tyrosine
0,014

0,012
Mutant
0,01

0,008

a* (λ) (m2 mg chla -1)

0,006

0,004

WT
0,002

0
400 450 500 550 600 650 700 750

Wavelength (nm)

WT L159I-N230Y
Cell diameter (μm) 7.32 9.43
Chl per cell (µg x 10-6) 4.27 3.32
a* (m2 mg chl a-1) 0.0041 0.0059
Productivity (ml H2/Litre) 29 ± 9 504 ± 22
Energy Conversion Efficiency - 2.5 %
 Photosynthetic activity Carbohydrate accumulation

140

Carbohydrates (mg/L)
1500
90

(µmol O2 /mg Chl/h)

L159I-N230Y

Oxygen evolution
1250
40 CC124
L159I-N230Y 1000
-10
-60 CC124 750

-110 500
-160
250
-210
0 100 200 300 400 500 600 700 0
0 20 40 60 80 100 120 140 160 180
PFD (µmol/m2/s)
Time (h)
H2 production rate (mL L -1 h-1)

15 15 0.18
L159I-N230Y CC124
12 12 0.15
ΔF/F´m ΔF/F´m

(  F/F'm)
0.12
9 9
0.09
6 6
0.06
H2 rates
3 3
H2 rates 0.03

0 0 0.00
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Time (h) Time (h) J. Biotechnol. (2012),157:613-619
Main features of an ideal C. reinhardtii strain
for H2 production
1) Lower Chl content per cell

2) Higher respiration to phosynthesis ratio,

which reduces the length of the aerobic phase

3) High carbohydrate accumulation

4) Longer hydrogen production phase

Higher Hydrogen Output

 After Tsygankov et al. (2002).

1) Photobioreactor;2) Intermediate bottle for gas-liquid conversion;3) Liquid-

accumulating bottle; 4) Electronic balance; 5) PC with interface for data
recording and storage; 6) - 7) - 8) Probes: Temp, pH, Eh; pO2; PAM-2100; 9)
Magnetic mixing system.
 Optimizing the light
Magnetic bar utilization efficiency
by means of different
mixing systems

Impeller

Giannelli L., Scoma A. Torzillo G. (2009).

Biotechnol. Bioeng. 204: 76-90
 Center-to-Wall Top-Down
Turbine
positions
Time Speed (m Time Speed (m
(s) s-1) (s) s-1)
Upper three-
bladed 0.053 0.47 ~0.00 < 0.10
radial turbine
Pitched Blades
0.100 0.25 0.20 0.13
Turbine n°1
Pitched Blades
> 0.500 <0.10 0.33 0.16
Turbine n°2
Pitched Blades
> 0.500 <0.10 0.12 0.21
Turbine n°3

Bottom six-
bladed 0.058 0.43 n.d. ~0.00
radial turbine
Conditions: Chl= 24 mg/L;
Giannelli et al. Biotechnol. Bioeng, 2009
PFD= 140+140 μmol m-2 s-1
a) b)

c) d)
 Tubular reactor (50 L volume)

Solar
light

Lab Experiments
18-20% of
what
expected

η = 0.05% Scoma et al., J. Biotechnol. (2012).

η = 0.21% (solar light) Vs 0.05% in the 50 L PBR

Int. J. Hydrogen Energy (2012), 371651-61

18 s

Crit. Rev. Biotechnol. (2014).

 CONCLUSIONS
1) A number of promising C. renardtii strains for H2
production have been selected.

2) At present the hydrogen output outdoors using sulfur starved C.

reinhadtii cultures is about 20% of that achieved under laboratory
conditions.

3) The necessity of using sulfur starvation strongly complicates the

technology (e.g. necessity of culture centrifugation) and limits the
scale-up.

4) With present state of art technology based on S-starvation

the LCE is still < 1%, that is less than the maximum attainable.
Title: Design, construction and demonstration of solar biofuel production
novel(photo)synthetic cell factories.
Objective:
CyanoFactory brings together ten selected leading, highly complementary European
partners with the aim to carry out integrated, fundamental research aiming at applying
synthetic biology principles towards a cell factory notion in microbial biotechnology. The
vision is to build on recent progress in synthetic biology and develop novel photosynthetic
cyanobacteria as chassis to be used as self-sustained cell factories in generating a solar
fuel.
Cyanofactory
FP7-ENERGY-2012-1-2STAGE, Grant Agreement nº
308518
Strategies for improving light-driven hydrogen production

Challenges for implementation Possible workarounds

O2 inhibition of hydronenase Create O2 tolerant H2-ase.

Competition for reductant by CO2 Engineering photosynthetic

fixation pathways
Low light conversion efficiency to H2
pathways that override competition
with CO2 fixation
Reduce antenna size, and replace
nitrogenase with H2-ase;
Improve performance of the PBR
 Algal
Biomass

Biodiesel
Why Biodiesel?

• To reduce their dependence on fossil energy

imports.

• Biodiesel fuel can be added directly to gasoline with

no need to adapt the distribution system or vehicle.

• Biodiesel emits fewer greenhouse gas (GHG) that

petrodiesel
Crop Oil yield (L ha-1 y-1)
Corn 172
Soybeans 446
Canola 1190
Jatropha 1892
Coconut 2689
Oil palm 5950
Microalgae 22,000-33,000 *

Produzione potenziale di olio 15 tons/ha/anno

Rodolfi L, Chini-Zittelli, Tredici M., et al, 2010
 1) Production per area of microalgae cultures could
greatly exceed the yield of the best oilseed crops
Algal
Biomass 2) Microalgae cultivation requires less amount of
water than terrestrial plants

3) Microalgae can be cultivated in seawater or

brackish water on areas not used for conventional
agriculture ( NO FOOD VS FLUES CONFLICTS)

4) Microalgal biomass production may be associated

to direct use of flue gas, waste CO2 ( 1 Kg of biomass
requires about 2 Kg of CO2)

5) Cultivation of algae does not require pesticides;

Biodiesel 6) Nutrients for microalgae (N and P) can be obtained

from waste-water
Biodiesel dalle microalghe Dr. Chini Zittelli (CNR-ISE)

 Selezione di ceppi microalgali oleaginosi

 Valutazione del contenuto in lipidi e resa in

biomassa

 Induzione della sintesi di lipidi mediante carenze Nannochloropsis sp.

nutrizionali: valutazione della produzione
potenziale in olio dei ceppi selezionati

 Coltura massiva all’aperto di Nannochloropsis sp.

in fotobioreattori chiusi a basso costo ed in
carenza di azoto

S ill = 10 m2 Volume coltura = 0,6 m3

+N -N
Green Wall Panel: Brevetto WO2004/074423
 Limitations

1) Difficulty to growth the selected species

under pure culture outdoors;

2) Limited information on their

performance in commercial plant
operation;

3) The high energy input for culture

mixing, CO2 supply, and harvesting-
dewatering.

4) High cost of Photobioreactor

Transesterification
 Biodiesel by Direct Transesterification

Overall scheme
Courtesy of Molina Grima
Biomass slurry,
150 g/l

Hexane

· Methanol
Homogenizer
· Sulfuric acid Hexanic phase

Centrifuge Biodiesel

Methanol

Waste biomass
Extraction
Hydroalcoholic phase

Water
Sulfuric acid

Advantages: Disavantages:
No dry biomass required High volume of solvents required
Similar to processes actually used Distillation required for solvent recycling
Suitable to be adapted to biodiesel plants Spent biomass highly degraded
 1 Barrel of Oil (150 litres)

300 Kg Biomass with 50% TAG

Oil cost = 60 US& $/Barrel

Acceptable biomass cost =

60/300 = 0.2 US $
 Actual production cost US$ 10 Kg-1

> 50 times
higher

Cost required for biodiesel production

US$ 0.2 Kg-1
 Constraints on oil production
Availability of CO2

• Need concentrated CO2: 120-fold atmospheric concentration

• If emissions of CO are used, they must be co-located with: water, cheap land, suitable climate conditions
2

Imposed limits

48 million tons of algal crude oil:

≈ 14-days’ worth of annual US consumption of petroleum (2011)
 Other major limits

Supply of N and P

To produce 48 million tons of algal crude, need:

~ 3.7 million tons of P (i.e., 2.5-fold the current US production for agriculture)

~ 12.2 million tons of N (i.e., 85 % of the US consumption in agriculture)

Only option

Recycle nutrients ► anaerobic digestion

Need methods for concentrating CO2 at low cost
 Improving the energy prospects of microalgae

Genetic and metabolic engineering issues

1. Increase the biomass yield
2. Increase the oil content in biomass
3. Engineer N-fixation in algae

Technological issues

1. Reduce the cost of the photobioreactors

2. Reduce cost for CO2 concentration
 Gracias por su atención

2H2 + O2

2H2O
 Acknowledgments
Dr. Faraloni Cecilia
Dr Scoma Alberto
Dr Ing.Giannelli Luca
Mr Pinzani Edoardo
Mr Cicchi Bernardo
Guests
Dr.Oncel Suphi (Ege University, Izmir, Turkey)
Prof Silva Margarita (Costa Rica University)
Support Prof Hugo Perales (UNAM Mexico)

EU (2015-2018: Cost Action)

EU (2012-2015): CYANOFACTORY

Regione Toscana (EBH2)

MIUR (Italian Ministry of Education) FISR (HYDROBIO)