9 April 2015, UNESCO-IHE, Delft, The NetherlandsNicolas Bernet, Amandine Galès, Jérôme Hamelin,...

Integration of microalgae and anaerobic digestion

within a biorefinery approach for reuse and/or recovery of human residues

Nicolas Bernet, Amandine Galès, Jérôme Hamelin, Arnaud Hélias, Eric

Latrille, Bruno Sialve, Jean-Philippe Steyer, Michel Torrijos, Eric Trably,

Violette Turon, Nathalie Wery

Laboratoire de Biotechnologie de l’Environnement – INRA

Narbonne - France

1st International Seminar on Algal Technologies for Wastewater Treatment and Resource Recovery, 9 April 2015, UNESCO-IHE, Delft, The Netherlands

Laboratoire de Biotechnologie de l’Environnement

(INRA-LBE Narbonne)

http://www.montpellier.inra.fr/narbonne

Production

solid

liquid

gas

Use

Environnemental Biorefinery: From WWTP to WRRF

w

Water

Fertilizer

High-value Molecules

Energy

transformation


Objective: To give value to our waste

The next generation of WWTP – Towards WRRF

Source: VEOLIA, 2012

TODAY Energetic balance < 0

TOMORROW Energetic balance > 0

Anaerobic Digestion in France


60

80

100

120

140

2005 2006 2007 2008 2009 2010 2011 2012 2013

28 nationalities 35 permanent positions (16 researchers)

141 persons in 2014 (80+ FTE)

Molecular Microbiology

Biochemical Engineering

Process Engineering

Applied Mathematics and Multicriteria Analysis

Technological Transfer

Project Engineering

Microbial Ecology

w Mixed

ecosystems

Bioprocesses


The processes (mainly with bacteria)

500+ BMP and BHP tests / year 60+ digesters (1 liter to 1 m3) in operation Pretreatments (°C, US, O3, mechanical,…)


Few open and closed microalgae cultivation systems

are also running (up to 450 liters PBR and 28 m3 raceway)

Volta discovering the « marsh gas » in 1776…

Combined gas/liquid/solid treatment with bioenergy

In the bottom: Anaerobic Digestion

When looking at Nature

CO2 CH4

CO2

O2

O2

+ CO2

N, P

Minéralisation

Hydrolyse/AcidogeneseMéthanisation

Microalgues

Bacteries aerobies

Bacteries anaerobies

CO2

O2

Sédimentation

CO2

Corg

CO2 CH4

CO2

O2

O2

+ CO2

N, P

Minéralisation


Microalgues

Bacteries aerobies


CO2

O2

Sédimentation

CO2

Corg

In the top layer :

• Production of biomass

• Fixation of CO2

• Synergy between algae and bacteria

A large part of nitrogen and phosphorus is recycled through the « microbial loop »

Microalgae

culture

Anaerobic

Digestion

nutrients biomass biogas

CH4

CO2

Organic matter

Wastewater

CO2 CH4

CO2

O2

O2

+ CO2

N, P

Minéralisation


Microalgues

Bacteries aerobies


CO2

O2

Sédimentation

CO2

Corg

CO2 CH4

CO2

O2

O2

+ CO2

N, P

Minéralisation


Microalgues

Bacteries aerobies


CO2

O2

Sédimentation

CO2

Corg

Inspired from Nature, gas/liquid/solid treatment + bioenergy production !

When looking at Nature

From Goeluke & Oswald (1959)

Microalgae in Environmental biorefineries : an old story

Anaerobic Digestion of Microalgae

Microalgae features Impact on Anaerobic Digestion

Single/grouped cell or organic residue Specific surface for hydrolysis

High protein content Low C/N -> NH3 . toxicity on acetoclastic methanogens

Thick/hard cell wall for some species Low biodegradability; intracellular content access limited.

Presence of sodium for marine species Toxicity for acetoclastic methanogen

Effect of growth conditions on biochemical composition Increase of biogas yield

Concentration depends on harvesting process Organic loading rate

Silica structure for diatoms Potentially siloxans in biogas Biogas quality and post-processing

Sulfate in seawater Competition between SRB and methanogens H2S toxicity


Organism Mode T (°C) HRT (d) L CH4 g VS-1 % CH4

Chlorella and Scenedesmus Batch 35-50 0.17-0.32 62-64

Tretraselmis CSTRa 35 14 0.31 72-74

Spirulina Semicontinuous 30 33 0.26 68-72

Dunaliella Batch 35 28 0.44 ns

Chlorella vulgaris Batch 28-31 64 0.31-0.35 68-75

Chlorella Batch 34 14 0.35 65



Chlorella and Scenedesmus CSTRa 35 10 0.09-0.136 69

Arthrospira Platensis Batch 38 32 0.29 61

Chlamydomonas reinhardtii Batch 38 32 0.39 66

Chlorella Kessleri Batch 38 32 0.22 65

Dunaliella salina Batch 38 32 0.32 64

Euglena Gracilis Batch 38 32 0.32 67

Scenedesmus obliquus Batch 38 32 0.18 62

Chlorella and Scenedesmus Batch 35 40 0.16 70

0

50

100

150

200

250

300

350

400

450

500

0 10 20 30 40 50 60

mLC

H4/g

MV

Time (days)

Chorella ajout 1

Spiruline ajout 1

Dunaliella ajout 1



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

L C

H4. g

VS-1

►Species (Resistant cell-wall, biochemical composition) ►Growth conditions

Pretreatment of Microalgae

T0 T30 min T60 min T180 min

Solubilisation Exopolymers and

intracellular content

Coagulation Raw biomass

22 % 48 % Anaerobic biodegradability

► Phytoplanktonic ecosystem dominated by Scenedesmus littoralis

► Sytox Green staining (Entire cells / Disrupted cells )

Recycling Digestate to Microalgae

Element Microalgae composition

mg/g

N-NH4 10-140

P-PO4 0.5-33

K 1-75

Na 0.4-47

Mg 0.5-75

Ca 0-80

Fe 0.2-0.34

Cu 0.006-0.3

Zn 0.005-1

Co 0.0001-0.2

Mn 0.02-0.24

Grobbelaar (2004) & Wu (1981)

► Digestate liquid phase can cover the nutrients requirements of

microalgal biomasses (Goeluke & Oswald, 1959; Wu, 1981)

► Some features can influence growth :

► Turbidity : light

► Nitrogen : toxicity

► Heavy metals, LCFA, organic compounds : toxicity and

primary nutrition

► Defiency of micro-elements

Consortia microbiens de Thau

Temps (j)

0 2 4 6 8

D.O

.

0,0

0,2

0,4

0,6

0,8

1,0

100%

78%

56%

22%

Conway

Consortia microbiens de Mèze

Temps (j)

0 2 4 6 8

D.O

.

0,0

0,2

0,4

0,6

0,8

1,0

100%

78%

56%

22%

Z8

Microalgae from sea water Microalgae from fresh water

Recycling Digestate to Microalgae

Time (d) Time (d)

Modeling Anaerobic Digestion of Microalgae

Modeling Anaerobic Digestion of Microalgae

Met

han

e yi

eld

(m

3 C

H4/

kg C

OD

)

__ Model output

● Experimental data

Passos et al., Algal Research (in press)

HRT 15 days 0.10 L CH4/g

COD

HRT 20 days 0.17 L CH4/g COD (+70%)

Monoraphidium sp.

Oocyistis sp.

Stigeoclonium sp.

Nitzschia sp.

Only the microalgae biochemical

characteristics had to be

modified to use the model !

A most interesting aspect of the ecology of airborne algae is their interference with human health: There is

abundant information on allergenic properties of airborne algae which probably act on the respiratory

system in a similar way to pollen. Most strikingly, data indicate that algae could be a cause of more medical problems than pollen, since in contrast to pollen, algae are able to actively accumulate pollutants, such as heavy metals or radioactive dust material (Bergman et al. 1983, Bernstein and Safferman 1965, 1972, Lunceford 1968, McElhenney et al. 1965, McGovern et al. 1965, McGovern et al. 1966, Mittal et al. 1979, Schlichting 1974b, 1986, Tiberg 1989).

7.800 m

2.400 m

PetroSun, Rio Hondo, Texas, US.

Bioaerosols: in… and out !

Mixing

Gas injection

Wind Rain Sun (convection)

Scum & Foam

Wave

Meteorological, physical and biological factors of aerosolization, dispersal and deposition

Diversity

Dispersion

Aerosolization

Rain

Wet deposition

Dry deposition

Diversity

Microbial communities from open water spread to the environment through bioaerosols Bioaerosols are sources of contaminations at different spatial scales Processes for intensification of algal ponds culture increase bioaerosol emissions (Bubble bursting is considered to be the major generator of aerosols ) Aerosolization should be taken into account to design and improve pond management

Bioaerosols: in… and out !

HRAP Shallow Lagoons and Algal Ponds

0%

25%

50%

75%

100%

Combustion

Heat

Electricity

Emissions

Input

Fertilizer

Infrastructure

0%

25%

50%

75%

100%

Combustion

Transformation

Dewatering

Culture

Fertilizer

Need to improve the electricity consumption and/or origin

Life Cycle Assessment and Ecodesign

Increase algae concentration in the pond

To decrease the volume of water to be stirred

and to be removed

Increase the productivity

By using system control, algae strains, etc…

Use of renewable energy

E.g. solar panels and wind turbines

Energy efficient harvesting process

Decrease the use of fertilizer

Reuse of N, P, etc…


Hydrolysis/

Acidogenesis Methanogenesis Culture of

microalgae

CH4

Biogas

Organic

residues

CO2

(Gaseous waste) CO2

Nutrients

Organic acids + nutrients

Algal biomass

anr-symbiose.org

Concept of "biologically mediated solar battery« (patent INRA/INRIA/Ifremer)

The Symbiose project (2009-2012)

The Algotron process

200

220

240

260

280

300

320

340

0 10 20 30 40 50 60 70

Depth (mm)

Time (days)

(1) Height evolution (2) Meteorological data

qin qin qin qinqrain qrain

►Water losses modelling :

Knowledge of water footprint and

process design and operation

(2) Pond features


Dr. Benoit Guieysse

►Impact of process operation on biological activity ►Claim for hydro-biological modelling


In collaboration with


►Ecosystem Dynamic : Molecular Ecology approach

► Diversity : bacterial >> eucaryotic

► Highly dynamic system

Sampling

16S rDNA

18S rDNA

►Scenedesmus ecosystem

► Synthetic medium for growth

► 2.5 months experiments (July-August)

CE-SSCP : 1 color = 1 specie (1 sample every 3 days)

0%

10%

20%

30%

40%

50%

60%

70%

80%

0 20 40 60 80

Eucaryotic abundancy (%)

0%

5%

10%

15%

20%

25%

30%

35%

0 20 40 60 80

Bacterial abundancy (%)

CE-SSCP

CE-SSCP


0%

10%

20%

30%

40%

50%

60%

70%

80%

0 10 20 30 40 50 60 70 80

Eucaryotic abundancy (%)

Resilience of Scenedesmus littoralis

Predatory events

Time (days)

Chytrids Dileptus mucronatus

Sialve B., Hamelin J., Steyer J.P. (2015) in prep.

Let‘s go back to bacteria

Macro-molecules

Monomers

Organic acids,

alcohols

Acetate CO2 + H2

Hydrolysis B.

Acidogenesis B.

Acetogenesis B.

Monoacetogenesis B.

CH4 + CO2 CH4

Acetoclastic

methanogenesis B.

Hydrogenotrophic

methanogenesis B.

Hydrolysis and

acidogenesis

Acetogenesis

Methanogenesis

Dark fermentation : low pH, short HRT favoring H2 – and metabolites – production !

Ecological Engineering for biotic control

raw

heated

raw

heated

raw

heated

Mix of all

In each reactor, same operating conditions (feed=glucose, HRT=10h, T=37°C, pH=5.5)

In steady state in each reactor (after several HRTs), only one major specie (difference only in minor species)

BDA

Cæ

Cæpth

Man

Manpth

Mix

BDApth

Rela

tive

Au

on

da

nce

of

ba

cte

ria

Link between structure and function of the ecosystem !


Identical major bacteria, so identical performance, isn’t it ?…. NO !!!

BDA

Cæ

Cæpth

Man

Manpth

Mix

BDApth

Re

lative

Au

on

da

nce o

f b

acte

ria

Clostridium sp.

Desulfovibrio sp.

Influence on the metabolism

[H2] x 2.5 ! Change in Metabolic flux

Study of the interactions : a model


New microbial interactions !

Link between microalgae and bacteria

µmax = 0.25 d-1

µmax

With Dunaliella salina in axenic conditions

Maximum growth rate per day

0

0.1

0.2

0.3

0.4

0.5

0.6


µmax


0

0.1

0.2

0.3

0.4

0.5

0.6

When adding one by one 78 bacteria living with D. salina in natural environments :

Cytophaga-

Flavobacterium-

BacteroidesGram + α Protéobactéries γ Protéobactéries

Halomonas sp.

Marinobacter sp.

Rhodococcus fascians

Thalassococcus sp.

Alteromonas sp.Maribacter sp.

Muricauda sp.

0.09 d-1 µmax 0.48 d-1

-63% +100%

Effect + : 12

Effect neutral : 27

Effect - : 39


µmax

When adding one by one 78 bacteria living with D. salina in natural environments :


0

0.1

0.2

0.3

0.4

0.5

0.6

Dark fermentation

Microalgal heterotrophy

(mixotrophy)

Industry effluent

BioH2

Fermentation by-products Acetate, butyrate, propionate, lactate,

ethanol

N and P

Treated water

Biolipids (biofuel)

Abiotic parameters (pH, T°, etc.)

Biotic parameters (bacteria, algae

community)

Coupling dark fermentation with heterotrophic microalgae

PhD of Violette Turon

co-supervised with Eric Fouilland

vv

Agitation: 150 rpm

Inoculation : 10%

Daily measures of acids (HPLC, GC) Cellular dry weight (OD, MS)

Agitation: 150 rpm Black Erlenmeyer flasks (125 mL)

Working volume: 40 mL

100 µmol photons.m-2.s-1

Solid medium

Coupling dark fermentation with heterotrophic microalgae

Specie GenBank 18 S % Lipids T° Specificity

Auxenochlorella protothecoide

CCAP 211 7A X56101 14.6 – 57.8

25-28

Very close to strict

heterotrophs

Chlorella sorokiniana

CCAP 211 8K

X62441

19.0 – 61.5 %

35-37

(20 - 42) Heat tolerance

0

0.1

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0.6

0

0.05

0.1

0.15

0.2

0.25

0.3

0 2 4 6 8 10 12 14

0

0.05

0.1

0.15

0

0.05

0.1

0.15

0 2 4 6 8 10 12 14

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Growth on butyrate (0.25 gC/L)

Time (days) Time (days)

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Bu

tyra

te c

on

cen

trat

ion

(g

C.L

-1)

Bu

tyra

te c

on

cen

trat

ion

(g

C.L

-1)

Growth on acetate & butyrate


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 2 4 6 8 10 12 14


INHIBITION (Haldane)

Diauxic behaviour

Growth on acetate (1.2 gC/L)

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.5 1 1.5 2 2.5 3

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Ace

tate

co

nce

ntr

atio

n (

gC

.L-1

)

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

NO INHIBITION (Monod)

Growth of heterotrophic microalgae on VFAs

Mathematical Modeling

0

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1

1.2

0

0.2

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1

1.2

1.4

0 0.5 1 1.5 2 2.5 3

0

0.05

0.1

0.15

0

0.05

0.1

0.15

0 2 4 6 8 10 12 14

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Growth on acetate (1.2 gC/L)



Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Bu

tyra

te c

on

cen

trat

ion

(g

C.L

-1)

Bu

tyra

te c

on

cen

trat

ion

(g

C.L

-1)

0

0.1

0.2

0.3

0.4

0.5

0.6

0

0.05

0.1

0.15

0.2

0.25

0.3

0 2 4 6 8 10 12 14

Growth on acetate & butyrate

Cel

lula

r D

ry W

eig

ht

(g.L

-1)


Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Ace

tate

co

nce

ntr

atio

n (

gC

.L-1

)

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 2 4 6 8 10 12 14


Turon et al., Bioresource Technology (2015)

Characterization of heterotrophic microalgae growth on unsterile fermentation effluent : interactions with facultative anaerobic bacteria?

With medium from dark fermentation?

Turon et al., Algal Research (submitted)

Inoculation Hydrogen production

glucose consumption

Anaerobic sludge

(mixed bacterial culture) Anaerobic batch (5 g/L glucose and ammonium, 37 °C)

pH neutralization

(6.5)

Sterilization

(filtration 0.2 µm)

Microalgae Inoculum (100 µmol photons.m-2.s-1 ; 150 rpm; 25 °C)

Acetate: 0.3 gC/L Butyrate: 0.7 gC/L

Microalgae inoculation

Microbial monitoring: OD, CDW, qPCR

(Chlorophyta and bacteria) and CE-SSCP

Inoculation:

10 % V/V

Heterotrophy: 25 °C, aerobic conditions

Sterile/unsterile medium from dark fermentation

0

0.1

0.2

0.3

0.4

0.5

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10

Cel

lula

r D

ry W

eig

ht

(g.L

-1)

Time (days)

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

Butyrate (gC.L-1)

Acetate (gC.L-1)

Microalgae (g.L-1)

0.44 0.42

0

0.1

0.2

0.3

0.4

0.5

Microalgae growth and organic acids uptake Yield on acetate

no effect of raw medium on growth on acetate Too high concentration of butyrate to support growth

(gC

bio

mas

s/g

C a

ceta

te)

Sterilized

fermentation medium

Synthetic medium

Heterotrophic microalgae growth on sterile fermentation medium

0

0.1

0.2

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0.5

0.6

0.7

0.8

0 2 4 6 8 10

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

Time (days)

Butyrate (gC.L-1)

Acetate (gC.L-1)

Consumption of both substrates by which microorganisms?

Acetate and butyrate uptake

Heterotrophic microalgae growth on non sterile fermentation medium

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0

0.1

0.2

0.3

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0.5

0.6

0.7

0.8

0 2 4 6 8 10

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

Microalgae : qPCR analysis (specific Chlorophyta 18S rDNA)

Time (days)

Butyrate (gC.L-1)

Acetate (gC.L-1)

Log

(co

pie

s o

f 1

8S

rDN

A/µ

L )

Microalgae (Log(18S rDNA/µL))

Growth of microalgae on acetate


Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

Microalgae : qPCR analysis (specific Chlorophyta 18S rDNA)

Time (days)

Butyrate (gC.L-1)

Acetate (gC.L-1)

Log

(co

pie

s o

f 1

8S

rDN

A/µ

L )

Microalgae (Log(18S rDNA/µL))

Growth of microalgae on acetate Same behavior on sterile and unsterile fermentation medium Acetate uptake seemed to be due to microalgae assimilation

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10

Unsterile medium

Sterile medium


0

0.1

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0.4

0.5

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0.7

0.8

0 2 4 6 8 10

5.5

6

6.5

7

7.5

8

8.5

Log

(co

pie

s o

f 1

6S

rDN

A/µ

L )

Time (days)

Bacteria: qPCR analysis (generic bacteria 16S rDNA)

Met

abo

lite

con

cen

trat

ion

(g

C.L

-1)

Bacteria grew during acetate assimilation by algae amensalism with algae ?

Bacterial growth can explain the butyrate uptake

CE-SSCP analysis 3-4 bacterial species

1 peak = 1 species


• Microalgae diversity implies diversity of energetic potential

• Biodegradability is low for adapted species to waste treatment

• Pretreatment improve energy and nutrient recovery

• Digestate covers nutrients needs • Turbidity might not be an issue • Complex ecosystems improve

treatment efficiency

• Experiments confirm interactions between hydrodynamic (pond design) and biological activity

• Adapted ecosystems offer resilience • Pilot scale step is necessary to understand and improve

Algal biomass

AnaerobicDigestion

Microalgaeculture

NutrientsCO2 fromBiogas

Multidisciplinarity is the key condition for the successfull development of biorefinery

To conclude

To conclude Anaerobic digestion in algal biorefineries

N2 fixing Cyanobacteria

AD Stripping Razon (2012) (NH4)2SO4

Microalgae

Microalgae Fuel Cell

Microalgae AD Methanotrophs PHA

Digestate Biomass

AD

AD

CH4+CO2 CH4+O2

Van der Ha et al. (2012)

Digestate

Biogas

Electricity

De Schamphelaire & Verstraete (2009)

Biomass

► Integration of AD with microalgae culture for several applications and others to discover…

► Evolution of AD from organic waste treatment to bioenergy production towards biorefinery

Biomass Organic fertilizer

Energy

Biogas

To conclude

Basic Research Applied Research

Anaerobic Digestion

• Improve anaerobic biodegradability • Process optimization (anaerobic biofilter) • Digestate management for optimal

nutrients recovery

Microalgae • Search for local and adapted species

and ecosystems • Microalgae/bacteria interactions

• Microbial engineering for specific functional response (product, treatment…)

Culture • Open systems design optimization • Process engineering and automation

(optimization of the energetic needs)

LCA

• N2O emissions (biological and physical mechanisms)

• Aersolization of phytoplanktonic population (mechanisms involved)

• Control of GHG emissions and aerosolization of microorganisms

Challenges should be tackled on all involved aspects !

Looking for best species Limiting

contaminations Optimization of operating conditions

Best use of light and energy (eg., for mixing)

Optimization of recovery/extraction

Recycling of nutrients Valorization of biomass

A vision of the whole chain

Optimization of energy production

Together with modeling, control, LCA, economic analysis, legislation, social acceptance, landscape impact

To conclude

http://www.montpellier.inra.fr/narbonne [email protected]

Thank you very much for your attention

9 April 2015, UNESCO-IHE, Delft, The NetherlandsNicolas Bernet, Amandine Galès, Jérôme Hamelin,...

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Transcript of 9 April 2015, UNESCO-IHE, Delft, The NetherlandsNicolas Bernet, Amandine Galès, Jérôme Hamelin,...