An overview of the activities of the Biomass and Bioenergy Lab:

Biological hydrogen production as a promising 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.it

Outline
Microalgae tecnology Waste to energy Dark fermentation Exploring microbial diversity: some examples Crude glycerol : from lab to industry

 Microalgae production have a potential of

100 t/ha/y dry biomass; 1/3 is bio-oil, 2/3 are bioproteins and sugars. From this last fraction it Microalghe around 90 m3/day of is possible to obtain methane.

An estimate based on 2008 statistical data shows that the yearly biomass coming from Agroindustrial chain and urban organic waste have an energy potential of around 100 TWh /year.

Animal manure: the energy potential at a national level is around 19 TWh

From waste to hydrogen energy

Hydrogen production by dark anaerobic fermentation of organic wastes is a promising strategy to obtain renewable and clean energy in a sustainable way

It can lead to the conversion of organic waste and feedstock into a host of valuable chemicals and energy The Approach: One way to improve the efficiency of H2 production is to explore the potentials offered by the microbial biodiversity, both in natural and artificial environment, identify and select bacterial strains with high H2 producing abilities from different substrates, and to characterize the microbial metabolism, in order to understand and optimize the whole process.

Wood

Vegetal waste

Animal manure

Glycerol

Dark Fermentation
VFA

Light fermentation

Acetate MEC

Methane Production

H2

H2 + CH4

H
2

Isolation of H2-producing mesophilic bacteria from the Averno lake

0 0 50 100 150 200 Vertical profile of phisical parameters in Averno lake

CSF4

250

300

0,00 0,00 5,00

50,00

100,00

10 P rofondit (m )

NO210,00 15,00 20,00

NO3-

Nitrification 150,00 200,00 + + 1/2O NO - + H+ + NH4 2 2 179 H 2O -185 NO2- + 1/2O2 NO3-

15

Denitrification 2NO3- + 10e- + 12H+ N2 + 6H2O

-298

Eh (mV)

20

25,00
25

30,00 35,00 -350,00

NH4+
-250,00 -150,00 -50,00
Eh (mV)

Sulfate Reduction 4H2 + SO42- + H+ HS- +4H2O H2 + S0 HS- + H+

50,00

30

Metanogenesis 150,00 CO2+ 3H2 250,00 4 +H2O CH

CH4
10000

35 0

H2S
2000

-305

Pot.Redox 4000

T C 6000

pH 8000 % D.O.

12000

Characterization of the microbial pool obtained from an eutrophic lake for mesophilic hydrogen fermentation
EXPERIMENTAL PROCEDURE

Sampling from Averno lake

DGGE

1 m

Samples from water column

3 m 5 m 6 m 9 m 15 m 21 m

Water samples
DGGE

PCR 16S rDNA

Sedimentwater interface
DGGE

DNA extraction

Niskin bottle

27 m 32 m 33 m Sequencing of single bands

Sediment sample at the interface sedimentwater

PCR products: -Eubacteria (495 bp) -Archaea (478 bp)

Sediment 34 m (AV1)

Genetic finger printing

ENEA Horizontal corer

Adapted from Paganin et al., 2007, IX Annual Congress of the Italian Federation of Life Sciences (FISV)

Isolation of H2-producing bacteria from vegetable waste:

Vegetable Waste
Isolation of bacterial strains

AutoAuto-Fermentation
Serial dilutions of extracts were plated on the basal fermentation medium (BFM) in phosphate buffer 0.1M, pH 6,7, 28C. 28 Single colonies were picked up from BFM plates and inoculated in 25 ml bottles containing 10 ml of BFM and tested for H2 production

63 distinct single colonies were obtained

11 strains out of 63 isolated were selected on the basis of H2 production(> 0,4 moli production(> Selezione degli H2/mole glucosio) idrogenoproduttori

Taxonomic identification of selected strains

DNA Extraction PCR amplification of 16S rDNA gene Sequencing of amplified DNA

(a) (b)

Sequence similarities between rDNA gene sequences of strain and those of the closest relatives in the NCBI database. Identification performed with RDP Classification Algorithm. Bootstrap confidence values are given between brackets (classification is well supported for confidence > 80%).

Improvement H2 production from Autofermentation 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

Inoculum:
Paenibacillus polymyxa ISSDS-851 1- Pantoea sp.57917 2- Endophyte bacterium SS10 3- Raoultella ornithinolytica Artificial MIX made by 1+2+3

Selected from: Avernos Lake

Vegetal Waste

Improvement H2 production from Autofermentation of Vegetal Waste

H2 production yields of anaerobic batch reactors treating waste

Feedstock Wheat straw Wheat straw Corn stover Cabbage Carrot Rice bran Food waste Food waste Sewage sludge Dairy manure Garbage slurry Seed Cow dung compost Cow dung compost Heated sludge Anaerobic digested sludge Anaerobic digested sludge Soy bean meal grass compost seed sludge Sewage sludge Dairy manure Pretreatment inoculm 105C 2h 100C 15 min 100C 15 min Pretreatment feedstock Yield temperature (C) (ml H2 /gVS) 68 1 49 62 71 61 77 59,2 16,26 31,5 31,1 29,25 21,95 18,46 85,65 66,69 Reference Fan et al., 2006 Fan et al., 2006 Datar et al., 2007 Okamoto et al. 2000 Okamoto et al. 2000 Noike & Mizuno, 2000 Lay et al., 2005 Hang et al., 2003 Xiao & Liu, 2009 Xing et al., 2010 Ohnishi et al., 2010 Yokoyama et al., 2007 This study This study This study This study HCL 2% + microwave heating 6 3 36 220C 3 min 35 37 37 35 35 35 37 36 37 60 28 28 28 28

100C 15 min heat 180*C 3h 90C 10 min Sterilized (121C) Sterilized (121C) 0,2% HCl 30 min infrared oven 2h boiled -

Garbage slurry enrichment Cow waste slurry Cow waste slurry Vegetal waste Vegetal waste Vegetal waste + Vegetal waste + potatoes peels potatoes peels Unique microflora Vegetal waste enrichment Vegetal waste+ Unique microflora potatoes peels enrichment

Continuous Flow Reactor using a mixed pool

l H2/ l day Continuum 1300 h 7-8

mol H2/mol gluc. 2-2,5

max rate (l H2/l h) 0.5- 0.6

H2 (%) 54-59

Effect of the artificial consortium on vegetal waste

Microalgae

Lipids Carbohydrates

Proteins

Glycerol Biodiesel 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 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 ) US biodiesel production and its impact on crude glycerol prices (www.thejacobsen.com). Taken from: Yazdani and Gonzalez; www.thejacobsen.com) from: Gonzalez; Current Opinion on Biotechnology (2007), 18 : 213-219. 2007), 213-219.

Contest

Crude Glycerol fermentation

Statistical 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 balance
Biomass 7% Gas 13% Ethanol

53% 27% Others

Energy balance
Hydrogen
7% 14%

Consumption
2%

Others

77%

Ethanol

THANK YOU FOR YOUR ATTENTION