View Article Online

Green
View Journal

Chemistry
Cutting-edge research for a greener sustainable future
Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: I. Borghol, C.
Queffelec, P. Bolle, J. Descamps, C. Lombard, O. lepine, D. kucma, C. Lorentz, D. Laurenti, V.
Montouillout, E. chailleux and B. bujoli, Green Chem., 2018, DOI: 10.1039/C8GC00094H.

Volume 18 Number 7 7 April 2016 Pages 1821–2242 This is an Accepted Manuscript, which has been through the

Green Royal Society of Chemistry peer review process and has been
accepted for publication.
Chemistry
Cutting-edge research for a greener sustainable future Accepted Manuscripts are published online shortly after
www.rsc.org/greenchem

acceptance, before technical editing, formatting and proof reading.

Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

author guidelines.

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s
ISSN 1463-9262 standard Terms & Conditions and the ethical guidelines, outlined
CRITICAL REVIEW
in our author and reviewer resource centre, still apply. In no
G. Chatel et al.
Heterogeneous catalytic oxidation for lignin valorization into valuable
chemicals: what results? What limitations? What trends?
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.

rsc.li/green-chem
 Page 1 of 8 PleaseGreen
do notChemistry
adjust margins
View Article Online
DOI: 10.1039/C8GC00094H

Journal Name

ARTICLE

Biosourced analogs of elastomer-containing bitumen through

hydrothermal liquefaction of Spirulina Sp. microalgae residues

Green Chemistry Accepted Manuscript

Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.

Received 00th January 20xx, a,f a a a b

Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Ilef Borghol, Clémence Queffélec,* Patricia Bolle, Julie Descamps, Christophe Lombard,
b c d d e
Olivier Lépine, Delphine Kucma, Chantal Lorentz, Dorothée Laurenti, Valérie Montouillout,
f a
Emmanuel Chailleux,* and Bruno Bujoli
The hydrothermal liquefaction of Spirulina sp. microalgae (i.e. cyanobacteria) byproducts was investigated for the
production of road binders from renewable sources. In the 220-300 °C temperature range, a water-insoluble viscous
material was obtained in a ca. 50% yield, which consisted of an oily fatty acid-based fraction mixed with organic and
inorganic solid residues (ca. 20%). More interestingly, this material exhibited viscoelastic properties similar to elastomer-
containing bitumen, when operating the hydrothermal liquefaction between 240 and 260 °C. This is the first example of a
bio-sourced product showing such properties. At higher temperature, fragmentation of species of high molecular weight
occurred, resulting in less viscous materials that were no more thermorheologically simple. The reactor filling ratio was
also allowed to vary (i.e. 30 % versus 60%), showing very little influence in terms of yield and viscoelastic properties of the
resulting bio-binders.

potential of the HTL method for the transformation of such

1 Introduction
Bitumen is a non-renewable petroleum-derived product
mostly used in road construction. The recent past has shown
supply issues for this heavy fraction of petroleum distillation,
since its conversion to gasoline is attractive in case of high
level of the crude oil market. Anticipation of the replacement
of this material is thus of high importance, in particular
prospection of efficient routes to produce bitumen substitutes
from renewable biomass sources. Among the different options
investigated up to now, most of them include vegetable oils in
their composition, either mixed with additives or chemically
modified to reach appropriate mechanical properties.1–4
Interestingly, many reports in the literature have recently
focused on the production of biofuels through the
hydrothermal liquefaction (HTL) of different biomass
resources, in particular microalgae.5–15 Nevertheless, the
renewable feedstocks in biobitumen has not been investigated
16
so far, if we except a few reports, with for example the cases
17 18 19
of swine manure , Japanese cedar, waste wood resources
20
and whole (i.e. not residues) microalgae, that have been
studied for the HTL-mediated production of additives to
bitumen. Upon blending of the resulting oily materials with a
petroleum binder, a drop in the viscosity of the binder was
most often observed.
21
In a recent proof of concept study, we have shown the high
potential of microalgae residues (i.e. material obtained after a
first extraction of high value compounds) for the production of
biobinders, using Scenedesmus sp. as a model system.
Microalgae can be grown on non-arable lands, with production
yields per hectare higher than in conventional agricultures.
Assuming that an industrial sector in this area is developed
(the technology for algal biorefinery has been accelerated
22–24
during the last 10 years), the generation of significant
amounts of microalgae waste is very likely and valorization of
these residues will be critical for the viability of the developed
processes. In this context, we have demonstrated that the
hydrothermal liquefaction is a particularly attractive approach
to transform microalgae residues into a viscoelastic
hydrophobic material with properties similar to that of
petroleum-based bitumen. By contrast with previous results
from the literature (vide supra), the whole hydrophobic
fraction could be used directly, with no need to blend it with a
conventional binder.
Interestingly, HTL can convert wet biomass and thus obviates
energy-consuming drying steps which are needed in other
25,26
thermochemical conversion processes. Our preliminary

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1

Please do not adjust margins

 PleaseGreen
do notChemistry
adjust margins
ARTICLE
findings highlighted the critical influence of HTL experimental
conditions on the rheological behavior of the isolated
hydrophobic material, in particular the reaction temperature.
The purpose of the current paper was to investigate the
possible extension of these results to other microalgae species,
as well as the influence of the HTL experimental conditions
(temperature, reactor filling ratio [i.e. the amount of water
and biomass loaded versus total volume of the reactor]) on the
composition and viscoelastic properties of the hydrophobic
component of the HTL products. While our previous study was
focused on an eukaryotic microalgae (Scenedesmus sp.), a
prokaryotic species which is produced worldwide for its
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.
valorization as food supplements, pharmaceuticals and
nutraceuticals, was selected for this work (Spirulina sp.). In
particular, the biomass used in this study consisted of
industrial residues obtained once extraction of water soluble
proteins was performed. Interestingly, treatment of these
residues by HTL under suitable conditions led to the formation
of an hydrophobic fraction, showing viscoelastic properties
similar to that of elastomer-containing bitumen. The HTL
experimental conditions were found to influence the
molecular weight distribution in this oily material, which in
turn affected its rheological properties.
2 Materials and Methods
2.1 Materials
The microalgae residue used for this study was obtained from
AlgoSource (Alpha-Biotech, Asserac, France) which grows
Spirulina sp.. Although the composition of the crude dried
Spirulina can vary depending on the season, a representative
example is: proteins (ca. 65%), carbohydrates (ca. 20%) and
lipids (ca. 15%). AlgoSource markets Spirulina sp. extracts. In a
typical process, the crude Spirulina biomass is harvested,
freezed, brought back to room temperature and centrifuged.
The aqueous supernatant which contains about 50 wt.% of the
initial dry biomass is mainly composed of proteins,
carbohydrates and mineral salts, but no lipids. After a
purification process which is kept confidential, the resulting
drinkable solution is commercialized as food complement with
a guaranteed phycocyanin content (0.08 wt.%). After the
centrifugation step, a solid residue is obtained which is usually
eliminated as a waste. The latter was used for this study and
was freeze-dried prior to use.
The moisture and ash contents were measured by measuring
the weight loss of a 300 mg sample placed in an oven at 110 °C
for 2 hours or in a tubular furnace at 900 °C for 2 hours,
respectively.
Adapted procedures from the literature were used to
27,28 29 30
determine the lipid, protein and carbohydrate contents
in the biomass. The C, H, N, and S content of the sample was
measured using a ThermoScientific FLASH 2000 Series CHNS/O
Analyzer.
2.2 HTL apparatus and procedures
The HTL experiments were performed using a 300 mL Parr
reactor fitted with a heating mantle, allowing a maximum
operating temperature of 350 °C and a maximum pressure of
2 | J. Name., 2012, 00, 1-3
Please do not adjust margins
Page 2 of 8
View Article Online
DOI: 10.1039/C8GC00094H
Journal Name
200 bars. For each condition, the experiments were performed
in triplicate to ensure repeatability of the assays.
In a typical experiment, microalgae (21 g or 39 g) were
suspended in deionized water (4 mL g-1) and charged in the
autoclave that was then flushed with nitrogen (0.2 L min-1 for
5 minutes). The biomass / water ratio was kept constant
during all experiments i.e. 0.8 (80 wt.% of water). After
reaching the selected reaction temperature (220, 240, 260,
280 or 300 °C), the autoclave was heated for 1 hour, and then
allowed to cool down. The aqueous phase was poured out in a
beaker and the remaining water insoluble fraction was then
Green Chemistry Accepted Manuscript
removed from the reactor using a spatula. The autoclave was
rinsed with dichloromethane, the washings were combined
with the water insoluble fraction, and the resulting mixture
was finally concentrated under reduced pressure.
2.3 Analysis of HTL products
2.3.1 Analysis of the water-insoluble fraction
GC×GC-MS chromatograms were recorded using an Agilent
6890 apparatus with a liquid nitrogen cryogenic jet modulation
from Zoex Corporation coupled with a 5975B qMS (Scan
parameters: from 45 to 300 u at 22 scan/s) detector. The first
column was a moderately polar VF1701 column (30 m x 0.25
mm x 0.25 μm) and the second column was an apolar DB1
column (2 m x 0.1 mm x 0.1 μm). The temperature program of
the first oven started at 50 °C for 5 min and then was heated
up at 1.75 °C/min until 300 °C. The second oven started at 50
°C for 0 min and then heated up at 1.8 °C/min until 307 °C and
at 1.8 °C/min until 320 °C. The modulation time was 12 s and
the modulator hot jet temperature was set to 280 °C. This
technique only allowed to identify the lighter fraction of bio-
binders constituents, on the basis of their retention time and
mass in comparison with the preinstalled NIST (National
Institute of Standards and Technology) mass spectra database.
Gel-permeation chromatography (GPC) analyses were
performed by using an Agilent apparatus (1200 series)
equipped with two PL gel columns (50 and 500 Å) and a
differential refractive index (DRI) and UV detectors. Analyses
were carried out at 35 °C using THF as eluent at a flowrate of 1
mL/min. Bio-oil samples were diluted at around 1 wt % in THF
before injection. The GPC system was calibrated with
polystyrene standards with molecular weights from 162 to
55100 g.mol-1. Depicted chromatograms were normalized
according to the highest peak.
2.3.2. Analysis of the solid residues in the water-insoluble
fraction
FT-IR spectra were recorded on a Bruker IR-TF Vector 22, and
the samples were studied in KBr pellets (1 wt.% in KBr).
31 1 13
The P MAS and { H}- C CP-MAS NMR spectra were recorded
respectively on a Bruker Advance I spectrometer operating at
1 31
7.0 T ( H and P Larmor frequencies of 300 and 121.5 MHz)
1 13
and 9.4T ( H and C Larmor frequencies of 400 and 100.5
MHz) using a 4 mm double-resonance MAS probe head and a
31
spinning frequency of respectively 3 and 10 kHz. The P
quantitative MAS spectra were recorded using single pulse
(π/10) acquisition; 1024 scans were co-added with a recycle
delay of 60 s to ensure complete recovering of the longitudinal
This journal is © The Royal Society of Chemistry 20xx
 Page 3 of 8 PleaseGreen
do notChemistry
adjust margins
Journal Name
31
magnetization. The P chemical shift anisotropy (CSA) was
determined from the spinning sideband intensities in the MAS
31 1 13
spectrum using DMfit software. The { H}- C CP-MAS
spectrum was acquired using a contact time of 1 ms with a
32 1
linear amplitude ramp on the H channel. ~82000 transients
were co-added with a recycle delay of 1s (experiment duration
of about 23 h).
1 33
For all experiments, H SPINAL-64 decoupling with a nutation
31
frequency of 70 kHz was applied during signal acquisition. P
13
and C chemical shifts were referenced relative to H3PO4 85%
and TMS, respectively
Qualitative elemental analyses were performed by energy
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.
dispersive X-ray spectroscopy using a scanning electron
microscope JEOL JSM 5800 LV, equipped with a SDD energy
dispersive spectrometer SAMx instrument.
2.4 Rheology
The rheological properties of bio-binders were measured with
a Kinexus pro+ rheometer in shear mode, according to the
European Standard EN 14770. The water insoluble fractions
were loaded, in the melted state at 80 °C, on a plate-plate
geometry (with a diameter of 8 mm and/or 25 mm) with 1 mm
gap. Dynamic shear rheometry (DSR) takes measures of phase
angles d (°) and of norm of complex modulus (|G*|) at
different temperature values: at high temperatures (from 20
°C to 80 °C) and low temperatures (from 20 °C down to -20 °C),
in various frequencies (from 0.01 Hz to 10 Hz). Measurements
were performed in the small strain domain in order to remain
in the linear viscoelasticity theory framework.
3 Results and discussion
In our pioneering work, we have demonstrated that a binder
showing similar rheological properties than a conventional
petroleum-based asphalt could be obtained by hydrothermal
21
liquefaction of Scenedesmus sp. residues. Interestingly, this
proof of concept study proposed an option that could
potentially enhance the economic viability of the microalgae
industry, by converting microalgae waste, generated after a
first valorization, process into bio-asphalt. Although
encouraging, these preliminary results were obtained on a
single biomass source. In order to investigate whether this
biorefinery concept might be a general route for the
production of biobitumen, the suitability of residues issued
from diverse microalgae species as feedstock for that
application has to be explored. Therefore, a microalgal strain
very different from that used in our previous work (eukaryotic
Scenedesmus sp.) was selected for the present study:
prokaryotic Spirulina sp., which is produced worldwide for its
valorization as food supplements, pharmaceuticals and
nutraceuticals.
3.1 Characterization of the initial biomass
The initial biomass was obtained after a water extraction of
water-soluble proteins performed on freshly harvested
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins
View Article Online
DOI: 10.1039/C8GC00094H
ARTICLE
Spirulina sp.. After extraction, a solid/liquid separation step
was performed, leading to a wet paste which was dried before
for HTL trials. The chemical composition of the microalgae
residue is described in Table 1. The low protein content in the
residue, when compared to data from the literature for whole
7–9,34,35
Spirulina sp. (ca. 50-60 wt%), denotes that ca. 60% of
the protein has been removed during the first valorization
step. When heating the residue at 900 °C to combust the
organic fraction, the main detected elements in the ashes
were calcium, phosphorus and magnesium, with a content of
ca. 23, 12 and 2 atomic %, respectively. These elements are
Green Chemistry Accepted Manuscript
indeed present in the microalgae culture medium and are thus
trapped during the centrifugation process.
Table 1. Chemical composition of the Spirulina sp. residues
a
used in this study. Values are based on the freeze dried
biomass and corresponds to experiments run in triplicate.
a
Moisture content (wt. %) 7.7 ± 0.3
a
Ash content (wt. %) 7.3 ± 0.4
a
Protein content (wt. %) 20.5 ± 0.4
a
Lipid content (wt. %) 35.2 ± 0.6
Carbohydrate content (wt. %)
a
16 ± 5
Elemental analysis (wt. %)
C 48.6
H 7.0
N 9.7
S < 0.1
3.2 Hydrothermal liquefaction of Spirulina sp. residues
3.2.1 Hydrothermal liquefaction
In addition, we have investigated whether the amount of
biomass loaded in the reactor can affect its thermochemical
transformation, as a result of a pressure increase in the
autoclave due to decarboxylation reactions, which might in
turn influence the rheological properties of the water-
insoluble HTL product. In this context, the loading factor (LF)
was defined as following:
LF = (Vwater + mmicroalgae) / Vreactor, where Vwater is the
volume of water (mL), Vreactor is the total volume of the
reactor (mL) and mmicroalgae is the dry mass of microalgae
residues (g), making the approximation of a direct
correspondence between the mass and the volume occupied
by the biomass, for simplification. The water/microalgae ratio
(4 mL/g) was fixed, and two different loading factors (i.e. 0.30
and 0.60) and six different temperatures (i.e. 220, 240, 260,
280 and 300 °C) were studied. After each experiment and
release of the gas pressure, two phases were separated: a
yellow to orange colored aqueous phase and a dark brown to
black viscous phase insoluble in water.
3.2.2 Analysis of the liquefaction products
For each experiment, the data related to the 3 phases
(gaseous, aqueous and non-water soluble fractions) formed
during the liquefaction process are summarized in Table 2.
The internal pressure in the reactor was slightly higher in the
260-300 °C temperature range when the loading factor was
J. Name., 2013, 00, 1-3 | 3
 PleaseGreen
do notChemistry
adjust margins Page 4 of 8
View Article Online
DOI: 10.1039/C8GC00094H
ARTICLE Journal Name

increased from 0.30 to 0.60. Moreover, in the case of the increasing the temperature above 240 °C resulted in a gradual
highest loading factor, the pressure steadily increased during decrease of the amount of water-soluble compounds which
the HTL once the temperature plateau was reached and was approximately divided by two at 300 °C (see Table 2, entry
stabilized after 45 minutes (i.e. + 7 bars at 220 °C, + 17 bars at 5). This might likely result from a decomposition of these
300 °C, see Table 2, entries 2-3). After cooling the reactor to compounds with a concomitant enrichment of the gas phase.
room temperature at the end of the HTL (see Table 2, entry 4), Finally, as regards the yield of the water-insoluble fraction (see
a residual pressure was present. Given that the main Table 2, entry 6), a stable value of ca. 46% was observed over
component in the gas fraction was carbon dioxide, it is very the 220-300 °C temperature range, for a loading factor of 0.30.
likely that the formation of gaseous products results from Similar yields of ca. 48% were obtained for the highest loading
decarboxylation reactions. ratio (0.60).
By contrast, no effect of the loading factor was observed on

Green Chemistry Accepted Manuscript

the yield of products present in the aqueous phase. However,
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.

Table 2. Experimental data related to the HTL of Spirulina sp. residues.

Entry Loading factor (LF) 0.60 0.30

1 Reaction temperature (°C) 220 240 260 280 300 220 240 260 280 300
2 Initial pressure (bar) once temperature 30±0 41±1 60±0 86±1 120± 29±1 40±1 56±1 80±3 108±
plateau reached 1 4
3 Final pressure (bar) at the end of the 36±3 48±2 69±5 106± 133± 28±1 39±1 56±3 78±3 108±
temperature plateau 0 1 1
4 Final pressure (bar) at room temperature ca. 8-9 ca. 13-16 ca. 2-3 ca. 4-5
5 Yield for the water-soluble fraction (%)a 40±0 40±0 32±0 27±0 20±1 38±0 36±1 32±0 26±0 22±0
6 Yield for the water-insoluble fraction (%)a 44±1 48±2 48±1 51±3 54±1 49±1 45±0 48±2 47±0 46±1
7 Amount of solid residues in the water- 43 19 18 20 19 38 nm 16 nm 18
insoluble fraction (%)b
8 Fraction of solid residues insoluble in 6M 54 28 14 11 10 50 nm 17 nm 10
HCl (%)c
a
calculated on the basis of the dry biomass; b percentage of the water-insoluble fraction, which is insoluble in dichloromethane
and isolated by filtration (pore size: 0.22 µm); c percentage of the water-insoluble fraction, which is insoluble in both
dichloromethane and 6M HCl, and isolated by filtration (pore size: 0.22 µm). nm: not measured.

exhibits a broad resonance with its associated spinning

3.2.3 Characterization of the water-insoluble HTL fraction
3.2.3.1 Characterization of the dichloromethane-insoluble
component
Attempts to solubilize the water-insoluble HTL fraction in
dichloromethane showed the presence of an insoluble solid
that was isolated by filtration on a membrane with a pore size
of 0.22 µm. Indeed, filtration using standard filter paper was
found to be poorly efficient, due to the small size of these
particles. It is worth noting that while the amount of this
dichloromethane-insoluble phase was the same over the 240-
300 °C temperature range (ca. 20%, see Table 2, entry 7), a
significantly higher content of solid was present at 220 °C for
the two loading factors (ca. 40%).
Analysis of the isolated solid by different techniques showed
the presence of organic and inorganic components. Indeed,
EDX analysis of this solid showed the presence of phosphorus
and calcium as the major heavy elements, with a small amount
of magnesium. It is very likely that these inorganic species
were initially present in the culture medium of the microalgae,
and have been trapped in the spirulina wet residue obtained
by centrifugation (final water content: 80 wt.%). Interestingly,
the 31P MAS NMR spectra of this solid were comparable
whatever the HTL temperature. At low MAS spinning rate the
31
P MAS spectrum (Figure S1 in supplementary information)
sidebands characteristic of the anisotropy of the chemical shift
interaction (CSA). The signal is centered at 1.1 ppm and the
CSA has been estimated at ~30 ppm in agreement with the
presence of poorly crystalline calcium / magnesium
orthophosphates.36 Moreover, the {1H}-13C CP-MAS NMR
spectrum (Figure S2 in supplementary information) was
consistent with the concomitant presence of a high molecular
weight biopolymer, very similar to that we found in the bio-oil
formed by HTL of residues of Scenedesmus sp..21 The spectrum
consisted of intense resonances centered around 30 ppm
assigned to (CH2)n chains, as well as weaker broad peaks
corresponding to carbon-carbon insaturations (130-140 ppm)
and carbonyl functions (ca. 170 ppm), respectively. This
suggests the presence of a fatty acid-based polyester
structure. Interestingly, the inorganic component was shown
to be fully soluble in a 6M hydrochloric acid solution, and the
FT-IR spectrum of the remaining organic component exhibited
intense absorption bands at 1622, 1694, 2849 and 2917 cm-1,
fully consistent again with an aliphatic carbonyl-containing
polymeric compound (Figure S3 in supplementary
information). As a result, it was thus possible to determine the
relative ratio between the organic and inorganic components
by difference (see Table 2, entry 8). Upon rising the
temperature, the organic content in the solid dramatically

4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Page 5 of 8 PleaseGreen
do notChemistry
Journal Name
decreased, from ca. 50% at 220 °C to ca. 10% at 300 °C. This
suggests that the HTL-mediated deconstruction of the
microalgae residue is incomplete at 220 °C leading to a non-
homogeneous oily fraction, and that, above this temperature,
the biopolymer is then gradually fragmented into species of
smaller size that the filtration membrane cut-off.
3.2.3.2 Characterization of the dichloromethane-soluble
component
Gel permeation chromatography (GPC) was also used to
investigate the influence of the temperature and loading
factor on the distribution of molecular masses in the part of
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.
the water-insoluble fraction which is soluble in
dichloromethane.
100
90
80
70
60
Norm.
50
40
30
20
10
0
150 1 500
Molar Mass (PS eq.)
Figure 1. Molecular weight distribution curve measured by
GPC, for the water-insoluble fraction: black (T = 260 °C, LF =
0.30), orange (T = 260 °C, LF = 0.60), blue (T = 300 °C, LF = 0.60)
Figure 1 presents the molecular weight distribution curve
recorded for samples prepared at 260 °C (with LF = 0.60 and
0.30, respectively) and 300 °C (LF = 0.60). For all samples, a
multimodal distribution was observed, with four broad main
contributions centered at ca. 350, 500-550, 800 and 2400
g.mol-1 in polystyrene equivalent (PS eq.), respectively.
Compared to the most abundant fraction (500-550 g.mol-1 PS
eq.), increasing the temperature from 260 to 300 °C at a given
loading factor (0.60) results in a decrease of the high molecular
weight components in favor of smaller species. Similarly, an
even more pronounced trend was observed when increasing
the loading factor from 0.30 to 0.60 at a given temperature
(260 °C). As a result, the average molecular weight values
(calculated as Mn, Mw, or Mw - see Table 3) of the water-
insoluble fraction are significantly lower when operating the
hydrothermal liquefaction at high temperature.
Table 3. Average molecular weight values (calculated as Mn,
Mw, or Mw) of the water-insoluble fraction obtained at
different temperature and loading factor.
Loading factor (LF) 0.30 0.60
Reaction temperature (°C) 260 260
Number average molecular 600 500
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins
adjust margins
View Article Online
DOI: 10.1039/C8GC00094H
ARTICLE
weight (Mn)
Mass average molecular weight 1670 1020 640
(Mw)
Size average molecular weight 10000 3900 1300
(Mz)
Although the amount of vaporizable constituents in the water-
insoluble fraction is rather low, as evidenced by GPC, 2D-GC
analyses showed the presence of three main families of
products with a similar distribution whatever the HTL
experimental conditions: (i) linear and branched long chain
Green Chemistry Accepted Manuscript
alkanes and alkenes; (ii) free fatty acids and their amide
derivatives; (iii) nitrogen-containing heterocyclic compounds,
including piperazine and morpholine-based derivatives (Figure
S4 in supplementary information). As reported in the
literature, the proteins still present in the biomass undergo
degradation during the HTL process, which results in nitrogen-
containing fragments forming heterocycles or condensation
products with the lipidic fraction.
3.3 Rheological properties of the water-insoluble HTL fractions
The rheological properties of the water-insoluble HTL fractions
were measured using the procedure described in the
experimental section. However, it was not possible to test the
material obtained at 220 °C, since deconstruction of the initial
15 000 biomass was not complete resulting in an inhomogeneous and
non-viscous product. The stiffness isochrones at 1Hz as a
function of temperature for both loading factors are presented
in Figure 2, and the rheological data for a conventional
bitumen are given for comparison.
Whatever the HTL processing conditions, the water-insoluble
HTL fractions appeared to be less sensitive to temperature
than conventional bitumen, especially in the high temperature
domain [40 °C – 80 °C] for which the bio-binders stiffness
stabilized whereas the bitumen tendency was to become even
softer. Consequently, even though the conventional bitumen
was always stiffer in the low temperature domain, the water-
insoluble fractions showed a higher modulus for high
temperature measurements.
For what regards the influence of the HTL conditions, the
higher the HTL temperature, the softer the resulting water-
insoluble fraction, as a result of a decomposition of high
molecular weight components in smaller species. This was
particularly true for a loading factor of 0.30. However, for a
loading factor of 0.60, even though a similar general trend was
observed, the stiffness decrease when rising the HTL
temperature was not monotonic.
300
380
J. Name., 2013, 00, 1-3 | 5
 PleaseGreen
do notChemistry
adjust margins Page 6 of 8
View Article Online
DOI: 10.1039/C8GC00094H
ARTICLE Journal Name

a) T= 240°C a)
T= 260°C 90 T= 240°C
1E+09 T= 280°C 80 T= 260°C
1E+08 70
T= 300°C T= 280°C
1E+07
|G*| (Pa)

60 T= 300°C
1E+06
1E+05 50

d (°)
1E+04 40
1E+03 30
1E+02
20
-20 0 20 40 60 80 100

Green Chemistry Accepted Manuscript

T (°C) 10
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.

b) T=240°C 0
T=260°C 1E+02 1E+04 1E+06 1E+08
|G*| (Pa)
1E+09 T=280°C
1E+08 b)
T=300°C 90
1E+07
|G*| (Pa)

1E+06 80 T=240°C
1E+05 T=260°C
1E+04 70 T=280°C
1E+03 T=300°C
60
1E+02
50
-20 0 20 40 60 80 100

T (°C)
Figure 2. Norm of the complex modulus as a function of
temperature (at 1 Hz) for the water-insoluble HTL fractions
prepared at 240, 260, 280 and 300 °C, with a loading factor of
(a) 0.60 and (b) 0.30. Data for a conventional bitumen are
given for comparison (black squares)
In Figure 3, the complex modulus of each water-insoluble HTL
fraction was plotted in Black spaces. This graphical
representation highlights the relation between the stiffness
|G*| and the phase angle d (°) for all temperature/frequency
couples chosen for the measurements. These “Black curves”
represent the linear viscoelastic signature of a material. When
an overlap of the isotherms is present, the Black curve is
continuous, thus meaning that one rheological state measured
at one temperature can be found at another temperature by
adjusting the frequency. This property refers as the “time to
temperature equivalence principle” and is linked to a stable
structural molecular organization over the temperature range.
Figure 3 showed that continuous Black curves were only
observed for the intermediate HTL temperatures, i.e. 240 °C
and 260 °C for LF = 0.60, and only 240 °C for LF = 0.30. On the
other hand, it should be noted that these water-insoluble HTL
fractions never reached a complete liquid state (i.e. d = 90°
whatever the frequencies). Indeed, both stiffness and phase
angle decreased when rising temperature, giving evidence of a
molecular network or a dispersed particles system which
provides elasticity to the material when the oily fraction is
melted.
d (°)
40
30
20
10
0
1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08
|G*| (Pa)
Figure 3. Complex modulus for the water-insoluble HTL
fractions prepared at 240, 260, 280 and 300 °C, plotted in the
Black space, with a loading factor of (a) 0.60 and (b) 0.30.
Interestingly, although different in terms of chemical
composition and molecular structure, the Spirulina-derived
biobitumen shows an overall viscoelastic signature very similar
to that typically observed for elastomer-containing petroleum-
based bitumen composed of ca. 8 % Poly(styrene-butadiene-
37
styrene) [SBS] dispersed in the bitumen (Figure 4).
Comparison of isochrones depicted in figure 4b shows that,
even though the stiffness are comparable in the low
temperature domain, Spirulina-derived biobitumen and
elastomeric-containing bitumen do not evolve like
conventional bitumen in the high temperature domain. In fact,
they remain stiffer as a result of a non-fusible fraction present
in the oily matrix which confers elasticity to the material. It is
worth noting that this type of SBS-modified bitumen has been
developed to improve the plasticity of road pavements
undergoing high heavy vehicle traffic.

6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Page 7 of 8 PleaseGreen
do notChemistry
adjust margins
View Article Online
DOI: 10.1039/C8GC00094H
Journal Name ARTICLE

to be assessed by addressing different aspects: scaling-up of

the process, studies on bio-bitumen aging, life-cycle analysis,
a) T = 260 °C ; LF = and global cost analysis of the process compared to the
90 0.60 distillation of petroleum.
Standard bitumen
80
70 Elastomer-
60 containing bitumen Conflicts of interest
50 There are no conflicts to declare.
d (°)

40
30
20 Acknowledgements

Green Chemistry Accepted Manuscript

10
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.

0
The authors acknowledge support from the “Centre National
1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 de la Recherche Scientifique”. This work also received the
|G*| (Pa) financial support of Région Pays de la Loire and IFSTTAR
(doctoral grant of I.B.), and ANR (Algoroute project, grant ANR-
b) 16-CE08-0017). Frank Fayon at CEMHTI and Christophe
T = 260 °C; LF = 0,60 Geantet at IRCELYON are also gratefully acknowledged for
fruitful discussion.
Standard bitumen
1E+09
Complex modulus IG*I

1E+08 Elastomer-containing Notes and references

1E+07 bitumen
1E+06
1E+05
1E+04
1E+03
1E+02
-15 35
Temperature (°C)
Figure 4. Comparison of (a) the linear viscoelastic data plotted
in a black space, and (b) the norm of the complex modulus as a
function of temperature (at 1 Hz), for a water-insoluble HTL
fraction (T = 260 °C; LF = 0.60), a conventional bitumen and an
elastomer-containing bitumen.
4. Conclusion
The hydrothermal liquefaction of Spirulina sp. residues was
successfully used to yield an analogue of elastomer-containing
bitumen. This work confirms the potential of microalgae
residues as a renewable feedstock for the production of bio-
binders for road construction. Interestingly, while 300-350 °C
HTL temperatures are usually recommended for the
production of microalgal biofuel, the experimental results of
this study suggest that the 240-260 °C temperature range is
the most appropriate for the preparation of bio-bitumen. For
this particular application, it is indeed critical to preserve high
molecular species, which seem to play a role similar to
asphaltenes and resins in petroleum-based binders.
Efforts are now needed to investigate the composition of the
HTL water-insoluble fraction as a function of the microalgae
strain and rationalize how the main constituents and their
molecular weight distribution influence the final viscoelastic
properties. Then, once the best candidates of microalgae
residues has been determined, in terms of performance for
road paving, the economic viability of the technology will have
1 Colas, FR patent, FR2853647(A1), 2004.
2 Colas, FR patent, FR2932806(A1), 2008.
3 Colas, FR patent, FR2932797 (A1), 2009.
4 D. Sun, G. Sun, Y. Du, X. Zhu, T. Lu, Q. Pang, S. Shi and Z. Dai, Fuel,
2017, 202, 529–540.
85 5 M. P. Caporgno, E. Clavero, C. Torras, J. Salvado, O. Lepine, J.
Pruvost, J. Legrand, J. Giralt and C. Bengoa, Acs Sustain. Chem.
Eng., 2016, 4, 3133–3139.
6 W.-T. Chen, Y. Zhang, J. Zhang, G. Yu, L. C. Schideman, P. Zhang
and M. Minarick, Bioresour. Technol., 2014, 152, 130–139.
7 W.-T. Chen, L. Tang, W. Qian, K. Scheppe, K. Nair, Z. Wu, C. Gai, P.
Zhang and Y. Zhang, Acs Sustain. Chem. Eng., 2016, 4, 2182–2190.
8 B. E.-O. Eboibi, D. M. Lewis, P. J. Ashman and S. Chinnasamy,
Bioresour. Technol., 2014, 174, 212–221.
9 C. Gai, Y. Zhang, W.-T. Chen, P. Zhang and Y. Dong, Energy
Convers. Manag., 2015, 96, 330–339.
10 C. Jazrawi, P. Biller, Y. He, A. Montoya, A. B. Ross, T.
Maschmeyer and B. S. Haynes, Algal Res.-Biomass Biofuels
Bioprod., 2015, 8, 15–22.
11 H. Li, Z. Liu, Y. Zhang, B. Li, H. Lu, N. Duan, M. Liu, Z. Zhu and B.
Si, Bioresour. Technol., 2014, 154, 322–329.
12 X. Tang, C. Zhang, Z. Li and X. Yang, Bioresour. Technol., 2016,
202, 8–14.
13 C. Tian, B. Li, Z. Liu, Y. Zhang and H. Lu, Renew. Sustain. Energy
Rev., 2014, 38, 933–950.
14 C. Zhang, X. Tang, L. Sheng and X. Yang, Green Chem., 2016, 18,
2542–2553.
15 M. Jin, Y.-K. Oh, Y. K. Chang and M. Choi, ACS Sustain. Chem.
Eng., 2017, 5, 7240–7248.
16 Owerhall, E.; AU patent, AU2016100073 (A4), 2016.
17 E. H. Fini, E. W. Kalberer, A. Shahbazi, M. Basti, Z. You, H. Ozer
and Q. Aurangzeb, J. Mater. Civ. Eng., 2011, 23, 1506–1513.
18 S.-H. Yang and T. Suciptan, Constr. Build. Mater., 2016, 114,
127–133.
19 X. Yang, Z. You, Q. Dai and J. Mills-Beale, Constr. Build. Mater.,
2014, 51, 424–431.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Please do not adjust margins

 PleaseGreen
do notChemistry
adjust margins Page 8 of 8
View Article Online
DOI: 10.1039/C8GC00094H
ARTICLE Journal Name

20 H. Dhasmana, H. Ozer, M. L. Al-Qadi, Y. Zhang, L. Schideman, B.

K. Sharma, W.-T. Chen, M. J. Minarick and P. Zhang, Transp. Res.
Rec., 2015, 121–129.
21 M. Audo, M. Paraschiv, C. Queffelec, I. Louvet, J. Hemez, F.
Fayon, O. Lepine, J. Legrand, M. Tazerout, E. Chailleux and B.
Bujoli, Acs Sustain. Chem. Eng., 2015, 3, 583–590.
22 Y. C. Sharma, B. Singh and J. Korstad, Green Chem., 2011, 13,
2993–3006.
23 B. Gilbert-Lopez, J. A. Mendiola, J. Fontecha, L. A. M. van den
Broek, L. Sijtsma, A. Cifuentes, M. Herrero and E. Ibanez, Green
Chem., 2015, 17, 4599–4609.
24 C. Yang, R. Li, C. Cui, S. Liu, Q. Qiu, Y. Ding, Y. Wu and B. Zhang,

Green Chemistry Accepted Manuscript

Green Chem., 2016, 18, 3684–3699.
Published on 18 April 2018. Downloaded by Fudan University on 23/04/2018 14:00:47.

25 S. S. Toor, L. Rosendahl and A. Rudolf, Energy, 2011, 36, 2328–

2342.
26 V. Budarin, A. B. Ross, P. Biller, R. Riley, J. H. Clark, J. M. Jones,
D. J. Gilmour and W. Zimmerman, Green Chem., 2012, 14, 3251–
3254.
27 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol., 1959, 37,
911–917.
28 J. Folch, M. Lees and G. H. S. Stanley, J. Biol. Chem., 1957, 226,
497–509.
29 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol.
Chem., 1951, 193, 265–275.
30 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith,
Anal. Chem., 1956, 28, 350–356.
31 D. Massiot, F. Fayon, M. Capron, I. King, S. Le Calve, B. Alonso, J.
O. Durand, B. Bujoli, Z. H. Gan and G. Hoatson, Magn. Reson.
Chem., 2002, 40, 70–76.
32 G. Metz, X. L. Wu and S. O. Smith, J. Magn. Reson. A, 1994, 110,
219–227.
33 B. M. Fung, A. K. Khitrin and K. Ermolaev, J. Magn. Reson., 2000,
142, 97–101.
34 P. Biller and A. B. Ross, Bioresour. Technol., 2011, 102, 215–225.
35 U. Jena, K. C. Das and J. R. Kastner, Appl. Energy, 2012, 98, 368–
375.
36 F. Pourpoint, C. Gervais, L. Bonhomme-Coury, F. Mauri, B.
Alonso and C. Bonhomme, Comptes Rendus Chim., 2008, 11, 398–
406.
37 G. D. Airey, Fuel, 2003, 82, 1709–1719.

Graphical abstract

Analogs of elastomer-
containing bitumen
80 Elastomer modified bitumen analog (T=260 °C ;
LF=0.60)
Elastomeric bitumen
70

From 60

Microalgae
220 to 50

residues 300 °C
d (°)

40

(20 %) 30

+ Loading 20

factor 0.3
Water (80 %) and 0.6
10

0
1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09
|G*| (Pa)

8 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins