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
Laboratoire de Biotechnologie de l’Environnement
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
Laboratoire de Biotechnologie de l’Environnement
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
Laboratoire de Biotechnologie de l’Environnement
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,…)
Laboratoire de Biotechnologie de l’Environnement
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
Hydrolyse/AcidogeneseMéthanisation
Microalgues
Bacteries aerobies
Bacteries anaerobies
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
Hydrolyse/AcidogeneseMéthanisation
Microalgues
Bacteries aerobies
Bacteries anaerobies
CO2
O2
Sédimentation
CO2
Corg
CO2 CH4
CO2
O2
O2
+ CO2
N, P
Minéralisation
Hydrolyse/AcidogeneseMéthanisation
Microalgues
Bacteries aerobies
Bacteries anaerobies
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
Anaerobic Digestion of Microalgae
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 Batch 34 25 0.44 65
Chlorella Batch 34 45 0.60 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
Anaerobic Digestion of Microalgae
Anaerobic Digestion of Microalgae
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…
Life Cycle Assessment and Ecodesign
Life Cycle Assessment and Ecodesign
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
The Algotron process
Dr. Benoit Guieysse
The Algotron process
►Impact of process operation on biological activity ►Claim for hydro-biological modelling
The Algotron process
In collaboration with
The Algotron process
►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
The Algotron process
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 !
Ecological Engineering for biotic control
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
Ecological Engineering for biotic control
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
Link between microalgae and bacteria
µmax
Maximum growth rate per day
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
Link between microalgae and bacteria
µmax
When adding one by one 78 bacteria living with D. salina in natural environments :
Maximum growth rate per day
0
0.1
0.2
0.3
0.4
0.5
0.6
Link between microalgae and bacteria
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
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
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
Time (days) Time (days)
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
Growth on butyrate (0.1 gC/L)
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
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
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)
Growth on butyrate (0.1 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)
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)
Time (days) Time (days)
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
Growth on butyrate (0.25 gC/L)
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
0.3
0.4
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
0.4
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
Heterotrophic microalgae growth on non sterile fermentation medium
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
Heterotrophic microalgae growth on non sterile fermentation medium
0
0.1
0.2
0.3
0.4
0.5
0.6
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
Heterotrophic microalgae growth on non sterile fermentation medium
• 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
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