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An overview of the activities of the An overview of the activities of the “Biomass and Bioenergy Lab”:“Biomass and Bioenergy Lab”:
Biological hydrogen production as a promising Biological hydrogen production as a promising Biological hydrogen production as a promising Biological hydrogen production as a promising source of renewable energy source of renewable energy
Giulio Izzo, Floriana Fiocchetti, Giulia Massini, Antonella Signorini, Fabrizio De Poli, Antonella Marone, Chiara Patriarca, Silvia Rosa, Cristiano Varrone.
Laboratory of Biomass and Bioenergy
Head of Laboratory: Dott. Giulio Izzo / giulio.izzo@enea.itHead of Laboratory: Dott. Giulio Izzo / giulio.izzo@enea.it
Outline Outline
•• Microalgae tecnologyMicroalgae tecnology
•• Waste to energyWaste to energy
•• Dark fermentationDark fermentation
•• Exploring microbial diversity: some examplesExploring microbial diversity: some examples
•• Crude glycerol : from lab to industryCrude glycerol : from lab to industry
MicroalgheMicroalghe
•• Microalgae Microalgae production have a potential of production have a potential of 100 t/ha/y dry biomass; 1/3 is bio100 t/ha/y dry biomass; 1/3 is bio--oil, 2/3 are oil, 2/3 are proteins and sugars. From this last fraction it proteins and sugars. From this last fraction it is possible to obtain around 90 mis possible to obtain around 90 m33/day of /day of methane.methane.
An estimate based on 2008 An estimate based on 2008 statistical data shows that statistical data shows that the yearly biomass coming the yearly biomass coming from Agroindustrial chain from Agroindustrial chain and urban organic waste and urban organic waste have an energy potential of have an energy potential of around 100 TWh /year.around 100 TWh /year.
Animal manure: the energy potential at a national level is around 19 TWh
Hydrogen production by dark anaerobic fermentation of organic wastes is a Hydrogen production by dark anaerobic fermentation of organic wastes is a promising strategy to obtain renewable and clean energy in a sustainable waypromising strategy to obtain renewable and clean energy in a sustainable way
FromFrom wastewaste toto hydrogenhydrogen energyenergy
The Approach:The Approach:
One way to improve the efficiency of HOne way to improve the efficiency of H22 production is to explore the potentials production is to explore the potentials offered by the microbial biodiversity, both in natural and artificial environment, offered by the microbial biodiversity, both in natural and artificial environment, identify and select bacterial strains with high Hidentify and select bacterial strains with high H22 producing abilities from different producing abilities from different substrates, and to characterize the microbial metabolism, in order to understand substrates, and to characterize the microbial metabolism, in order to understand and optimize the whole process.and optimize the whole process.
It can lead to the conversion of organic waste and feedstock It can lead to the conversion of organic waste and feedstock into a host of valuable chemicals and energyinto a host of valuable chemicals and energy
Wood Vegetal waste Animal manure Glycerol
Dark FermentationDark
Fermentation
VFA
H2
Light fermentationMEC
Methane Production
Acetate
H2 H2 + CH4
0
5
10
0 50 100 150 200 250 300
NitrificationNitrificationNHNH44
++ + 1/2O+ 1/2O22→→ NONO22-- + H+ H++ + +
HH22OONONO22
-- + 1/2O+ 1/2O22→→ NONO33--
NO3-NO2
-
179
-185
CSF4
Isolation of HIsolation of H22--producing producing mesophilic bacteria from mesophilic bacteria from
the Averno lakethe Averno lake
Vertical profile of phisical parameters in Averno lake
0,00
5,00
0,00 50,00 100,00 150,00 200,00
10
15
20
25
30
35
0 2000 4000 6000 8000 10000 12000
Pro
fondità (m
)
CH4
DenitrificationDenitrification2NO2NO33
-- + 10e+ 10e--+ 12H+ 12H++ →→ NN22 + +
6H6H22OO
Sulfate ReductionSulfate Reduction44HH22 + SO+ SO44
22-- + H+ H++ �������� HSHS--+4H+4H22O O
HH22 + S+ S00 �������� HSHS-- + H+ H++
MetanogenesisMetanogenesisCOCO22+ 3H+ 3H22�������� CHCH4 4 +H+H22OO
H2S
NO3
NH4+
-298
-305
Eh
(mV)
10,00
15,00
20,00
25,00
30,00
35,00
-350,00 -250,00 -150,00 -50,00 50,00 150,00 250,00
Eh (mV)
Pot.Redox T�C pH D.O. %
CharacterizationCharacterization ofof the the microbialmicrobial pool pool obtainedobtained fromfrom ananeutrophiceutrophic lakelake forfor mesophilicmesophilic hydrogenhydrogen fermentationfermentation
DGGE
EXPERIMENTAL PROCEDURE
Sampling from Averno lake
Samplesfromwater column
Water samples
Sediment-water DNA
PCR 16S rDNA
DGGE
1 m
3 m
5 m
6 m
9 m
ENEA Horizontal corer
Niskin bottle
column
Sedimentsample at the interface sediment-water
DGGE
waterinterface
DNA extraction
Genetic finger printing
Sequencing of single bands
PCR products:-Eubacteria (495 bp)-Archaea (478 bp)
9 m
15 m
21 m
27 m
32 m
33 m
Sediment 34 m (AV1)
Adapted from Paganin et al., 2007, IX Annual Congress of the Italian Federation of Life Sciences (FISV)
VegetableVegetable WasteWaste
Isolation of HIsolation of H22--producing bacteria from producing bacteria from
vegetable waste:vegetable waste:
IsolationIsolation ofof bacterialbacterial strainsstrains
63 63 distinctdistinct single single coloniescolonies werewereobtainedobtainedAutoAuto--FermentationFermentation
Selezione degli Selezione degli
idrogenoproduttoriidrogenoproduttori
11 strains out of 63 isolated were selected 11 strains out of 63 isolated were selected on the basis of H2 productionon the basis of H2 production(> 0,4 moli (> 0,4 moli HH22/mole glucosio)/mole glucosio)
Serial dilutions of extracts were Serial dilutions of extracts were
plated on the basal fermentation plated on the basal fermentation
medium medium (BFM) in phosphate (BFM) in phosphate
buffer 0.1M, pH 6,7, 28buffer 0.1M, pH 6,7, 28°°C.C.
obtainedobtained
Single colonies were picked up Single colonies were picked up
from BFM plates and inoculated in from BFM plates and inoculated in
25 ml bottles containing 10 ml of 25 ml bottles containing 10 ml of
BFM and tested for HBFM and tested for H22 productionproduction
DNA DNA ExtractionExtraction
SequencingSequencing ofofamplifiedamplified DNADNA
PCR PCR amplificationamplificationofof 16S 16S rDNArDNA genegene
Taxonomic identification of selected strainsTaxonomic identification of selected strains
(a) Sequence similarities between rDNA gene sequences of strain and those of the closest relatives in the NCBI database.
(b) Identification performed with RDP Classification Algorithm. Bootstrap confidence values are given between brackets
(classification is well supported for confidence > 80%).
ImprovementImprovement HH22 production from production from
Autofermentation of Vegetal WasteAutofermentation of Vegetal Waste
Vegetable refuses
V = green vegetables
VP = green vegetables +
potatoes
Auto-fermentation test of V and VP
biomasses
Auto-fermentation batch with
selected inocula� Paenibacillus polymyxa ISSDS-851
� 1- Pantoea sp.57917� 2- Endophyte bacterium SS10� 3- Raoultella ornithinolytica
� Artificial MIX made by 1+2+3
Averno’s Lake
Vegetal Waste
Inoculum: Selected from:
ImprovementImprovement HH22 production from production from
Autofermentation of Vegetal WasteAutofermentation of Vegetal Waste
H2 production H2 production yieldsyields ofof anaerobicanaerobic batchbatch reactorsreactors
treatingtreating wastewaste
Feedstock SeedPretreatment
inoculm
Pretreatment
feedstock
temperature
(°C)
Yield
(ml H2/gVS)Reference
Wheat straw Cow dung compost - HCL 2% + microwave heating36 68 Fan et al., 2006
Wheat straw Cow dung compost - - 36 1 Fan et al., 2006
Corn stover Heated sludge 105°C 2h 220°C 3 min 35 49 Datar et al., 2007
CabbageAnaerobic digested
sludge100°C 15 min - 37 62 Okamoto et al. 2000
CarrotAnaerobic digested
sludge100°C 15 min - 37 71 Okamoto et al. 2000
Rice bran Soy bean meal 100°C 15 min - 35 61 Noike & Mizuno, 2000
Food waste grass compost heat 180*C 3h - 35 77 Lay et al., 2005Food waste grass compost heat 180*C 3h - 35 77 Lay et al., 2005
Food waste seed sludge 90°C 10 min - 35 59,2 Hang et al., 2003
Sewage sludge Sewage sludge Sterilized (121°C) Sterilized (121°C) 37 16,26 Xiao & Liu, 2009
Dairy manure Dairy manure infrared oven 2h0,2% HCl 30 min
boiled36 31,5 Xing et al., 2010
Garbage slurryGarbage slurry
enrichment- - 37 31,1 Ohnishi et al., 2010
Cow waste slurry Cow waste slurry - - 60 29,25 Yokoyama et al., 2007
Vegetal waste Vegetal waste - - 28 21,95 This study
Vegetal waste +
potatoes peels
Vegetal waste +
potatoes peels- - 28 18,46 This study
Vegetal wasteUnique microflora
enrichment- - 28 85,65 This study
Vegetal waste+
potatoes peels
Unique microflora
enrichment- - 28 66,69 This study
Continuous Flow Reactor using a mixed poolContinuous Flow Reactor using a mixed pool
l H2/ l day mol H2/mol gluc. max rate (l H2/l h) H2 (%)
Continuum
1300 h
7-8 2-2,5 0.5- 0.6 54-59
EffectEffect ofof the the artificialartificial consortiumconsortiumon on vegetalvegetal wastewaste
Microalgae
Lipids
Carbohydrates
Proteins
Biodiesel
Glycerol
Fermentation H2
Will Biodiesel production increase ?
- EU biodiesel production was 6 million tons in 2006, and
is expected to increase to 10 million tons within this year
(2010)
In China:
- As world petroleum prices rise, and as China becomes
increasingly reliant on imported fuels, the government has
since decided to boost the biodiesel industry
- In 2004, the Ministry of Science launched its “Biofuel
Technology Development Project”
- In 2005 the government initiated a special agricultural and
forestry biomass development program, setting a
USUS biodieselbiodiesel productionproduction andand itsits impactimpact onon crudecrude glycerolglycerol pricesprices((wwwwww..thejacobsenthejacobsen..comcom)).. TakenTaken fromfrom:: YazdaniYazdani andand GonzalezGonzalez;;CurrentCurrent OpinionOpinion onon BiotechnologyBiotechnology ((20072007),), 1818 :: 213213--219219..
forestry biomass development program, setting a
nationwide target for annual biodiesel production of 2
million tons by 2010 and 12 million tons by 2020
-China has also intensified its research and development in
biodiesel technology with a series of government-led
research programs and a special fund dedicated to this
endeavor. Researchers have achieved several
technological breakthroughs, enabling producers to
diversify their feedstock and cut costs
Worldwatch Institute – Visiona for a Sustainable World
(http://www.worldwatch.org )
ContestContest
Crude Glycerol fermentation Crude Glycerol fermentation
Statistical optimization of the processStatistical optimization of the process
•At the end of optimization we obtained 2.150 L H2/L/day (yield > 0.94 mol H2 / mol crude
glicerol), 50% of glycerol is converted to Ethanol (yield = 1 mol EtOH/ mol glycerol), without
preatretment and substrate added.
• H2 concentration in biogas is 50%.
ENEA UTRINN-BIO
Mass balanceMass balance
53%
13%
7%
Biomass
Gas
Ethanol
53%
27%
Others
Energy balanceEnergy balance
14%
7%
2%
ConsumptionHydrogen
Others
77%Ethanol
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