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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