Abomohra et al , 2020

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
A close-loop integrated approach for microalgae cultivation and efficient
utilization of agar-free seaweed residues for enhanced biofuel recovery
Abd El-Fatah Abomohraa,c,⁎, Adel W. Almutairib
a Department of Environmental Engineering, School of Architecture and Civil Engineering, Chengdu University, Chengdu 610106, China
b Biological Sciences Department, Faculty of Science & Arts, King Abdulaziz University, Rabigh 21911, Saudi Arabia
c Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt
G R A P H I C A L A B S T R A C T
A R T I C L E I N F O
Keywords:
Biodiesel
Biogas
Biorefinery
Circular economy
Seaweed wastes
A B S T R A C T
The aim of this work was to evaluate a novel integrated biorefinery route for enhanced energy recovery from
seaweeds and microalgae. Agar extraction prior to anaerobic digestion recorded the highest biogas productivity
of 32.57 L kg−1 VS d−1. Supplementation of the microalgal growth medium with anaerobic digestate from agar-
extracted biomass enhanced the microalgal growth, recording the highest dry weight of 4.57 g L−1 at 20%
digestate ratio. In addition, lipid content showed the highest value of 25.8 %dw. Due to enhancement of growth
and lipid content, 20% digestate ratio showed the highest lipid productivity and FAMEs recovery (65.2 mg L−1
d−1 and 123.3 mg g−1dw, respectively), with enhanced biodiesel characteristics. The present study estimated
annual revenue of 1252.7 US$ ton−1 from the whole Gracilaria multipartita biomass conversion into biogas,
while that through agar extraction deserved 36087.0 US$ ton−1, with enhanced annual biodiesel yield by 69.7%
over the control medium.
1. Introduction
Nowadays, biomass utilization as an energy source represents an
auspicious alternative approach to substitute fossil fuels. Algae, in-
cluding seaweeds and microalgae, are categorized as the 3rd generation
biofuel feedstocks, owing many advantages comparing to the terrestrial
plants as the 1st generation or lignocellulosic wastes as the 2nd gen-
eration biofuel feedstocks. For instance, algae don’t compete on fresh-
water, human food, or arable land, which minimizes the food-versus-fuel
debate associated with the 1st generation feedstocks. Among different
kinds of biofuels, microalgae have received increasing attention as a
feedstock for biodiesel due to their relatively high lipid productivity
and the increasing demand of liquid fuel (Almarashi et al., 2020).
However, microalgal cultivation is costly due to the excessive need of
nutrients and water for algal growth (Tan et al., 2020). Thus, utilization
of wastewater effluents for microalgal cultivation is an optimal
strategy, which brings other advantages such as nutrient recycling,
pollutant removal, and oxygenation of wastewater at low energy inputs.
In that context, anaerobic digestate might be a suitable growth medium
for microalgae due to its high nutrient content.
Beside the potential of microalgae to produce energy, marine bio-
mass has an estimated annual energy potential of about 100 EJ, which
https://doi.org/10.1016/j.biortech.2020.124027
Received 3 July 2020; Received in revised form 12 August 2020; Accepted 13 August 2020
⁎ Corresponding author at: Department of Environmental Engineering, School of Architecture and Civil Engineering, Chengdu University, Chengdu 610106, China.
E-mail addresses: abomohra@science.tanta.edu.eg, abomohra@cdu.edu.cn (A.E.-F. Abomohra).
Bioresource Technology 317 (2020) 124027
Available online 17 August 2020
0960-8524/ © 2020 Elsevier Ltd. All rights reserved.
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is much higher than that of terrestrial biomass and municipal solid
wastes, 22 and 7 EJ year−1, respectively (Chynoweth et al., 2001).
Marine biomass consists mainly of seaweeds (macroalgae), which grow
abundantly along the coastline of Red Sea (Omar et al., 2013). Mac-
roalgae consist mainly of carbohydrates (25–60 dw%), while protein
content ranges between 5–47 dw%, and lipids represent the lowest
proportion of macromolecules (2.7–12.5 dw%). The biochemical com-
position depends strongly on the species, the growth season, and the
geographical area (Abomohra et al., 2018b). In addition to biofuel
production, conversion of wild-seaweeds will reduce the environmental
nuisance of the extensive natural growth at the sea shores (Wang et al.,
2020). Due to the relatively low lipid content and high carbohydrate
content comparing to microalgae, seaweeds have been widely discussed
as a potential feedstock for biogas and bioethanol production. Cur-
rently, seaweeds processing industry for agar production generates
abundant amounts of solid waste residues, which are not properly
processed (Hessami et al., 2019). Typically, the biomass residues after
agar extraction contain a considerable amount of carbohydrates, which
can be converted further into biofuel. One possibility to enhance the
process feasibility is the energy recovery from the residual biomass by
anaerobic digestion. Another possibility is the efficient recycling of the
vast amount of the produced anaerobic digestate for different eco-
nomical purposes.
Anaerobic digestate has been widely used as a biofertilizer because
of its high nutrient content. However, transportation of liquid digestate
is not economically feasible nowadays due to the growing distance
between agriculture farms and digesters. In addition, digestate utiliza-
tion for that purpose depends on the seasonal variations in fertilizer
demand, which requires a large land area for digestate storage or dis-
posal (Ai et al., 2020). Consequently, there is an urgent need for better
and safe sustainable routes to dispose or reuse the digestate. Recently,
application of anaerobic digestate for microalgal cultivation was eval-
uated. Studied revealed that efficient biomass and lipids production by
cultivation of microalgae on anaerobic digestate suffers from several
barriers. Mainly, it requires mitigation of ammonia toxicity, which is a
detrimental factor for microbial growth at elevated concentrations
(Uggetti et al., 2014). Digestate dilution with wastewater, synthetic
growth medium, or seawater was discussed as a preferred strategy to
enhance the microalgal growth through mitigating ammonium inhibi-
tion, enhancing C/N ratio and lowering the medium turbidity (Xia and
Murphy, 2016). To the best of authors’ knowledge, no previous studies
investigated the possible application of digestate from anaerobic di-
gestion of seaweeds residues for microalgal cultivation and biodiesel
production. In that context, integration of microalgal-biodiesel pro-
duction with agar and biogas production from seaweeds may sig-
nificantly enhance the overall revenue and provide a more sustainable
energy generation system with efficient nutrient recovery and bio-based
valuable compounds production. In the present study, a novel suggested
integrated biorefinery route was designed. The aim was to evaluate the
impact of agar extraction on biomethane yield from the Rhodophyte
Gracilaria multipartita, as one of the important feedstocks for industrial
agar production. In addition, the impact of dilute-acid pretreatment of
the whole and residual biomass on biomethane production was studied.
Furthermore, application of anaerobic digestate for microalgae culti-
vation and biodiesel (fatty acid methyl esters, FAMEs) production was
evaluated, taking Scenedesmus obliquus as a model biodiesel-promising
microalga. Moreover, the impact of digestate supplementation on the
fatty acid profile, biodiesel characteristics, andprocess economy was
studied.
2. Materials and methods
2.1. Experimental design
In the present study, the whole seaweed and the agar-extracted
residual biomass were anaerobically digested for biogas and
biomethane production. The anaerobic digestate was applied further at
different ratios in the growth medium for cultivation of S. obliquus, and
biodiesel production was evaluated.
2.2. Biomass collection
Gracilaria multipartita was collected during March 2019 from the
Red Sea coast near Khumrah town, about 31 km south Jeddah city,
Saudi Arabia (21° 33′ 54″ N and 39° 10′ 10″ E). At the collection site,
the biomass was thoroughly washed with salt water to remove con-
taminants, then transported to the laboratory in sterilized plastic con-
tainers under iced conditions. The biomass was washed again with
distilled water to remove salt residues. In order to facilitate the milling
process, the biomass was placed on absorbent papers and air dried at
room temperature until constant weight, then milled into ≤3 mm fine
powder using electric coffee bean grinder and stored in vacuumed bags
under dry conditions until further use.
2.3. Agar extraction and pretreatment
In order to prepare the biomass residues in simulation to the in-
dustrial practice, agar was extracted from the dried samples. Agar ex-
traction was performed according to the adapted method of Craigie
(1978) as described by Givernaud et al. (1999). Briefly, 100 g of sea-
weed powder were impregnated overnight in 3 L distilled water. For
extraction, the mixture was boiled for 2 h with continuous stirring. The
extraction was performed twice and the residual biomass was milled
and re-extracted again for another two times, the first conducted at
100 °C for 2 h, while the second conducted for 1 h at 121 °C. The hot
extracted agar fluid was filtered through 0.45 µm pore-size filter,
freeze-thawed twice to remove impurities, washed with hot distilled
water, and freeze-dried to determine its weight. The residual biomass
was washed thoroughly using hot distilled water (80 °C), and air dried
at room temperature until constant weight. The whole and agar-free
(residual) biomasses were analyzed for volatile solids (VS), total solids
(TS), proteins, carbohydrates, lipids, ash, and the main elemental
composition.
For acid pretreatment, a certain volume of distilled water was added
to the dried whole or residual biomass (1:1, v/w), and 5 M of HCl was
added slowly with mixing to adjust the pH at 2 (Hessami et al., 2019).
The acidified samples were then boiled in a water bath at 100 °C for 1 h.
The mixtures were kept to cool down at room temperature, then pH was
adjusted to 7. For anaerobic digestion, untreated and pretreated whole
and residual seaweeds were studied.
2.4. Biomass characteristics
TS and VS of the whole and residual biomass were measured as
described by the American Public Health Association (APHA, 2005).
The elemental composition was performed using elemental analyzer
(Vario EL/micro cube Elementar, Germany). For carbohydrate and
protein estimation, 200 mg of the dried biomass were extracted in a
boiling water bath using 10 mL of 1 N NaOH for 2 h (Payne and
Stewart, 1988). Bradford method (Bradford, 1976) was used to estimate
the protein concentration using bovine serum albumin as a standard
reference. Total carbohydrates were determined quantitatively using
phenol-sulfuric acid method (Kochert, 1978) with glucose as a standard
reference. For total lipids, 500 mg of the dried biomass were extracted
with 15 mL of 2:1 (v/v) chloroform:methanol and 100 µL of 1 M HCL
(Folch et al., 1957). The mixtures were vortexed and incubated over-
night with shaking. After incubation, non-lipid components (mainly
pigments) were removed from the mixtures by washing with 3 mL of
0.9% NaCl. Lipid extracts were dried under a stream of argon followed
by incubation at 80 °C for 30 min, cooled down in a desiccator and
determined gravimetrically.
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
2
2.5. Anaerobic digestion
Batch anaerobic digestion of the untreated and pretreated whole
and residual biomass was performed in 1 L glass bottles. Activated
anaerobic sludge from a running anaerobic digestion experiment was
used as inoculum. The VS content of the inoculum was 1.4%, with total
ammonia nitrogen and C/N ratio of 1.8 g L−1 and 13.2, respectively.
Each reactor was filled with 1:1 inoculum to feedstock ratio (VS basis).
The initial pH of all reactors was adjusted to 7, and the final TS content
was adjusted to 5.0% (Costa et al., 2012) using deionized water (pH 7).
The reactors were flushed with nitrogen gas for 3 min, before capping
with rubber stoppers, to ensure the anaerobic conditions. To adjust
blank treatments, reactors with inoculum and distilled water only were
used (without biomass). The digestion experiments were performed
under mesophilic conditions at 37 °C and lasted for 29 days. Biogas
production was measured daily and samples were collected using 10 mL
gas-tight syringe (1161X40, Thomas Scientific, USA) to determine
methane ratio using gas chromatography as discussed in Section 2.8.
Cumulative and daily biogas/biomethane production were estimated.
At the end of digestion experiment (the 29th day), the maximum bio-
methane potential (Ps), peak rate of daily biomethane production (Rm),
and time of the peak rate of daily biomethane production (TRm) were
determined. In addition, technical digestion time (T80) was calculated
as the time at which 80% of the final biomethane yield produced.
Biogas productivity (GP) and biomethane productivity (MP) at T80 were
calculated as L kg−1 VS d−1 by dividing the cumulative biogas or
biomethane yield by the corresponding time (days).
2.6. Microalgal growth
To study the effect of digestate on the growth and lipid production,
S. obliquus was cultivated at initial OD680 of 0.113 ± 0.002 in 750 mL
tubular glass cylinders containing 500 mL of synthetic waste water
(SWW). The SWW was prepared as describe by Ma et al. (2016) and
contained (mg L−1); MgSO4·7H2O 400, NH4Cl 230, K2HPO4 200,
Na2HPO4 100, NaNO3 100, NaCOOCH3 100, CaCl2·2H2O 100, and
KH2PO4 50 at pH 7. SWW with different substitutions of anaerobic
digestate (0 as a SWW control, 5%, 10%, 20% and 40%, v/v) was tested.
A sterile-filtered air containing 1.5% (v/v) CO2 was bubbled to the
cultures at a rate of 0.2 vvm. Cultures were incubated at 25 ± 1.3 °C
and average light intensity of 110 μmol photons m−2 s−1 at a photo-
period of 18:6h light:dark cycle. The light intensity applied in the
present study was selected based on the study of Dewez et al. (2005).
2.7. Biomass, lipids and FAMEs analysis
2.7.1. Biomass production
Cell count was performed every other day to monitor the algal
growth using a haemocytometer (Bright-Line, Reichert, Buffalo, USA).
The dry weight (dw) was determined at the start and the end of ex-
ponential growth phase, then the biomass productivity (g L−1 d−1) was
calculated from Eq. (1);
= − −Biomass productivity dw dw t t( )/( )e s e s (1)
where dws and dwe represent the dry weight (g L−1) at the start and the
end of exponential growth phase at times ts and te, respectively.
2.7.2. Lipid estimation
At the start and the end of exponential phase, 20 mL of culture were
centrifuged at 3000× g for 10 min. Total lipids were extracted from the
collected cells using chloroform:methanol (2:1, v/v) (Bligh and Dyer,
1959). The solvent was evaporated from lipid extracts under a stream of
argon, followed by drying at 80 °C for 30 min. Lipids were determined
gravimetrically per cellular dry weight, and lipid productivity (mg L−1
d−1) was calculated from Eq. (2);
= − −Lipid productivity L L t t( )/( )e s e s (2)
where Ls and Le represent volumetric lipid production (mg L−1) at the
start and the end of exponential phase at time ts and te, respectively.
2.7.3. FAMEs analysis
The effect of anaerobic digestate on FAMEs recovery and fatty acid
profile was estimated at the end of exponential growth phase. Briefly,lipids were extracted from 15 mL of the culture according to Abomohra
et al. (2016). As an internal standard, 16 µg of 1,2,3-trinonadeca-
noylglycerol were added to the cell pellet prior to extraction. After lipid
extraction, fatty acids were transesterified to FAMEs followed by gas
chromatographic analysis using GC-FID (Varian 3900, USA) manu-
factured with a Select Fame (50 m × 0.25 mm) capillary column as
described previously (Abomohra et al., 2020a). FAMEs characteristics
were estimated based on fatty acid profile according to Hoekman et al.
(2012).
2.8. Other analytical methods
Water salinity was determined by a portable conductivity meter
(Oakton, Eutech Instruments, Singapore), while pH and temperature
were measured using a temperature/pH meter (Milwaukee MW102,
USA). Biological oxygen demand (BOD), chemical oxygen demand
(COD), dissolved oxygen (DO), ammonia nitrogen (NH4-N), nitrate ni-
trogen (NO3-N), nitrite nitrogen (NO2-N), total nitrogen (TN), ortho-
phosphate (PO4-P), and total phosphorus (TP) were analysed following
the standard protocols of the American Public Health Association
(APHA, 2005). Digestate optical density was measured spectro-
photometrically at 680 nm. Volatile fatty acids (VFAs) in the anaerobic
digestate were measured by gas chromatography (GC 2010, Tokyo,
Japan) manufactured with a capillary column (FFAP, HP-INNOWAX,
30 m × 0. 25 mm). Both detector and injector temperatures were ad-
justed at 250 °C, while oven temperature was increased at a rate of
20 °C min−1 up to 200 °C. Methane ratio in the biogas was measured by
gas chromatography GC9790II (Lanzhou Atech Technologies Co, China)
manufactured with TCD detector and 1.5 m HayeSep Q packed column.
The injector, oven, and detector temperatures were set at 100 °C, 50 °C,
and 55 °C, respectively.
2.9. Statistical analysis
Experiments were carried out in triplicates and the results were
calculated as the mean ± standard deviation (SD). The statistical
software SPSS (IBM, v. 20) was used to analyze the results of different
treatments by performing one-way analysis of variance (ANOVA) fol-
lowed by least significant difference (LSD) test at a probability level
(P) ≤ 0.05.
3. Results and discussion
3.1. Characteristics of G. multipartita
Seaweeds are currently used as enormous source for industrial
production of many phytochemicals such as agar, alginate, and carra-
geenan. Out of the global seaweed biomass, the genus Gracilaria re-
presents 60%, and the agar yield can reach as high as 40.7% (Noura
et al., 2014). Therefore, the aim of the present section was to study the
changes in G. multipartita composition after agar extraction and the
consequent impact on biogas production. Table 1 shows the main
characteristics of G. multipartita before and after agar extraction. The
dry weight reduced by 41.3% (w/w) after agar extraction, from 100 g
dw to 58.7 g dw residues. The whole biomass before agar extraction
contained 58.12, 12.94, and 4.82 dw% of carbohydrates, proteins, and
lipids, respectively. However, the extraction process reduced the con-
tents of carbohydrates and lipids by 4.6% and 58.9%, respectively,
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
3
while protein content increased by 13.8% with respect to the whole
biomass. In agreement with the present results, Hessami et al. (2019)
recorded 88% reduction in carbohydrate and protein contents of G.
persica after agar extraction, with the maximum recorded loss in lipid
content. The reduction in carbohydrate was attributed to the biomass
treatment for agar extraction, which results in swelling of biomass and
increases the cellular pore size, facilitating the release of agar, lipids,
and sugars upon extraction. In addition, the extraction process was
reported as an effective step to enhance lipid hydrolysis (Kendel et al.,
2015), which resulted in more pronounced reduction in lipid content.
Nevertheless, the extracted residue still contained a substantial fraction
of carbohydrates (55.5 %dw) which is a considerable amount for
anaerobic digestion. Due to agar extraction and the significant reduc-
tion in macromolecules, the ash content of the extracted residue in-
creased by 12.7% over that of the whole biomass (Table 1). In addition,
the extracted sample showed 18.7% and 41.4% reduction in the total
carbon and hydrogen, respectively. However, lower reduction of 11.3%
in nitrogen was recorded, which might be correlated to the more loss of
lipids and carbohydrates than proteins. Digestion of nitrogen-con-
taining feedstocks (with C/N ratio below 15) was reported as a pro-
blematic issue due to digestion inhibition at the elevated levels of
ammonia (Meng et al., 2020). Interestingly, the current results showed
C/N ratio of 17.6 and 16.1 for the whole and extracted biomass, re-
spectively.
3.2. Anaerobic digestion
In this section, anaerobic digestion performance of the whole and
extracted biomass was compared, with or without acid pretreatment.
Fig. 1 shows the cumulative biomethane yield and the daily biomethane
production during 29 days of anaerobic digestion. For the whole bio-
mass, the cumulative biomethane yield at the end of the anaerobic
digestion was 192.8 and 225.5 L kg−1 VS for the untreated and pre-
treated samples, respectively (Fig. 1A). However, agar extraction en-
hanced the biomethane yield up to 253.4 and 278.8 L kg−1 VS, re-
spectively. The cumulative biogas yield showed different trend, where
the pretreated residual biomass had insignificant difference with the
untreated residues (Fig. 2A). Thus, the increase in biomethane yield
after pretreatment can be attributed mainly to enhancement of bio-
methanation, leading to higher biomethane content. Enhancement of
biogas yield in the agar-extracted biomass might be attributed to the
reduction of lipids, as the long chain fatty acids were reported to have
inhibitory effect due to instability of the digestion system (Salama et al.,
2019). Similarly, Hessami et al. (2019) reported the highest cumulative
biomethane yield for the pretreated residues of G. persica which in-
creased from 0.148 Nm3 Kg−1 VS in the untreated sample to 0.237 Nm3
Kg−1 VS after pretreatment. They attributed the enhancement of bio-
methane yield after acid pretreatment to the loosening of biomass
structure. Concerning daily biomethane production, anaerobic diges-
tion of the untreated whole biomass showed a short peak of 26.3 L kg−1
VS d−1 at the 9th day of digestion (Fig. 1B). However, pretreatment
showed insignificant changes in the biomethane peak (26.4 L kg−1 VS
d−1), with a shift of the peak time to earlier time (7th day). The pre-
treated residual biomass showed the highest recorded biomethane peak
of 32.2 L kg−1 VS d−1 at the 7th day, which was 10.3% higher than that
of the untreated residues (Fig. 1B). Similarly, the pretreated residual
biomass showed the highest recorded biogas peak of 52.4 L kg−1 VS
d−1 at the 7th day (Fig. 2B). Thus, the present results indicated that
dilute acid pretreatment and agar extraction are efficient methods to
hydrolyze the complex carbohydrate molecules into simple fermentable
sugars (Hessami et al., 2019), which could be easily assimilated by
microbes resulting in better digestion rate (Elsayed et al., 2019; Wang
et al., 2019).
Higher biomethane content in the produced biogas and the higher
biomethane production peak, as well as the shorter time to reach the
peak, are advantageous approaches from an industrialization aspect.
T80 is an efficient parameter to specify the rate of biomethanation and
the substrate digestibility for further economic biogas production
(Elsayed et al., 2018). Anaerobic digestion of the untreated and the
pretreated whole biomass showed the heights T80 of 12 days (Table 2).
However, agar extraction prior to anaerobic digestion reduced the T80
to 11 days, which decreased further to 10 days after pretreatment of the
extracted biomass. It is noteworthy to mention that the recorded T80 in
the present study is much shorter thanthat recorded for anaerobic di-
gestion of lignocelluloses. For instance, a shortest T80 of 15 days was
recorded during anaerobic digestion of rice straw (Meng et al., 2020).
However, Elsayed et al. (2020) stated 21 days as the T80 for anaerobic
digestion of raw rice straw, which reduced to 14 days after pretreat-
ment with anaerobic digestate. The shorter T80 of seaweeds comparing
to lignocelluloses might be attributed to the simpler biochemical
composition and absence of lignin.
At T80, methane content of the untreated agar-extracted biomass
was 63.4%, which represented 5.9% lower than that of the whole
biomass (Table 2). However, pretreatment of the extracted biomass
significantly enhanced biomethane content to 68.5%. Due to the in-
crease in biomethane yield, shortening the peak time, and enhancement
of biomethane content, the highest GP of 32.57 L kg−1 VS d−1 was
recorded from the pretreated extracted biomass, which represented
49.7% higher than that of the whole pretreated biomass (Table 2). In
addition, it showed 48.4% higher MP, with the highest significant Ps
and Rm of 278.8 L kg−1 VS and 32.2 L kg−1 VS d−1, respectively. En-
hancement of biomethane yield from the extracted biomass might be
attributed to the processing steps of agar extraction, which was re-
ported to reduce the complex polysaccharide macromolecules, making
carbohydrates more accessible to biological degradation (Hessami
et al., 2019). In addition, many seaweeds are rich in phenolic com-
pounds which are difficult to be degraded and can inhibit the metha-
nogenic bacteria (Murphy et al., 2015). Furthermore, most of seaweeds,
especially those belong to the genus Gracilaria, contain high metal ions
and sulphur contents which were reported as inhibitors to the anaerobic
digestion (Freile-Pelegrín and Robledo, 1997), which may be released
from the biomass during agar extraction. In that context, agar extrac-
tion from G. cornea using NaOH concentrations between 0.5% and 1.0%
resulted in significant reduction in sulphate content up to 4% (Freile-
Pelegrín and Robledo, 1997). In addition, up to 90% reduction in the
metal ions was recorded after pretreatment of seaweeds by weak acids
(Cao et al., 2019). Moreover, increasing of viscosity due to the presence
of agar in the whole biomass reduced the substrate accessibility to
enzymes and decreased the degradation efficiency of the biomass, re-
sulting in lower biogas yield in both untreated and pretreated whole
Table 1
Characteristics of Gracilaria multipartita before and after agar extraction.
Parameters Whole biomass Extracted residue
Initial weight (g, dw) 100 ± 0.01 100 ± 0.02ns
Final weight (g, dw) na 58.7 ± 1.05*
TS (%wt)w 23.24 ± 1.17 15.24 ± 1.69*
VS (%wt)w 18.70 ± 1.23 11.44 ± 1.49*
Carbohydrates (dw%) 58.12 ± 1.41 55.45 ± 1.46*
Proteins (dw%) 12.94 ± 0.82 14.73 ± 1.19*
Lipids (dw%) 4.82 ± 0.41 1.98 ± 0.40*
Ash (dw%) 24.35 ± 1.47 27.44 ± 1.48*
Elemental composition (%TS) C 46.35 ± 0.45 37.66 ± 1.49*
H 5.90 ± 1.58 3.46 ± 0.65ns
N 2.65 ± 0.28 2.35 ± 0.27ns
S 1.76 ± 0.16 1.42 ± 0.14ns
Od 18.99 ± 0.12 25.67 ± 0.25*
na Not applied, TS Total solids, VS Volatile solids.
ns and * represent insignificant and significant differences, respectively, be-
tween the extracted value and the corresponding parameter of the whole bio-
mass (at P ≤ 0.05).
w Wet basis, d Calculated by difference O = 100 - (C + H + N + S + Ash) on
TS basis.
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
4
biomass as shown in Fig. 2A. Therefore, digestate from the pretreated
residual biomass was used further for microalgal cultivation. Table 3
shows a comparison of the main characteristics of anaerobic digestate
from the pretreated whole and residual biomass.
3.3. Microalgal growth and lipid production
The growth pattern of S. obliquus grown for 24 days on SWW sup-
plemented with different ratios of anaerobic digestate is shown in
Fig. 3A. Supplementation of the medium with anaerobic digestate up to
40% enhanced the growth over SWW. However, the highest cell
number was recorded with 20% digestate ratio, which represented
12.2% higher than that of 40% at the 24th day. In addition, the control
culture reached the stationary phase at the 18th day, while digestate
supplementation increased the exponential growth up to 20 days. It
might be attributed to the additional nutrients in the digestate-enriched
medium which supports the growth for longer time. Light is a limiting
factor for the dense cultures grown under photoautotrophic conditions,
while enrichment of growth medium with organic carbon could com-
pensate the light limitation and achieve higher biomass production
(Tan et al., 2020). Similarly, the highest significant dry weight of 4.57 g
L−1 was recorded in 20% digestate-supplemented medium, which was
31.3% higher than the control (Fig. 3B). However, increasing of di-
gestate ratio to 40% resulted in decline in the dry weight to 4.07 g L−1,
which was still 17.0% higher than the control.
Due to growth stimulation, the highest recorded biomass pro-
ductivity of 0.252 g L−1 d−1 was obtained using 20% digestate, which
was 31.3% and 15.1% higher than the control and 40% digestate-sup-
plemented medium, respectively (Fig. 3B). In accordance with the
present results, Tan et al. (2020) confirmed that optimization of
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m
et
ha
ne
 p
ro
du
c
on
 (L
 k
g-
1
VS
 d
-1
)
Diges on me (days)
B
Fig. 1. Cumulative biomethane yield (A) and daily biomethane production (B) from anaerobic digestion of untreated (Un-) and pretreated (Pre-) whole and agar-
extracted Gracilaria multipartita.
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
5
Fig. 2. Cumulative biogas yield (A) and daily biogas production (B) from anaerobic digestion of untreated (Un-) and pretreated (Pre-) whole and agar-extracted
Gracilaria multipartita.
Table 2
Biogas and biomethane production through anaerobic digestion of the whole biomass and extracted residues of Gracilaria multipartita with and without acid pre-
treatment.
Treatments T80 (days) Methane content (%) GP* MP* Ps** Rm** TRm (days)
Untreated Whole 12 67.4 ± 0.7a 19.08 ± 0.47a 12.86 ± 0.23a 192.8 ± 2.95a 26.3 ± 1.62a 9
Extracted 11 63.4 ± 0.7b 29.08 ± 1.39b 18.43 ± 0.68b 253.4 ± 4.67b 29.2 ± 1.32b 7
Pretreated Whole 12 69.1 ± 0.8c 21.75 ± 0.83a 15.03 ± 0.53c 225.5 ± 6.24c 26.4 ± 1.26a 7
Extracted 10 68.5 ± 0.7ac 32.57 ± 1.74c 22.31 ± 0.97d 278.8 ± 3.58d 32.2 ± 1.39c 7
Values with the same letter in the same column showed insignificant differences (at P ≤ 0.05).
T80 was calculated based on biomethane production, while methane content represents the content at T80.
*GP and MP are biogas and biomethane productivities, respectively, as L kg−1 VS d−1 calculated at T80 as the cumulative value (L kg−1 VS) divided by time (days).
**Ps is the maximum biomethane potential (L kg−1 VS) at the end of the digestion time (the 29th day), Rm is the peak rate of daily biomethane production (L kg−1 VS
d−1), and TRm is the time of the peak rate of daily biomethane production (days).
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
6
acidified starch wastewater supplementation to the growth medium is
necessary in order to enhance lipid production and pollutants removal
by microalgae. Growth retardation at high digestate ratios might be
attributed to the high ammonia concentration and the dark background
color in the digestate which limits the light penetration into the culture.
In that context, Sayedin et al. (2020) attributed the growth inhibition of
three microalgal species, namely S. obliquus, Chlorella sacchrophila, and
C. sorokiniana in three of the tested growth media using anaerobic di-
gestate (raw digestate, two-timesdigestate dilution, and struvite re-
moved digestate) to the high ammonia concentration, however, higher
dilutions of digestate showed enhanced growth. In addition, Bohutskyi
et al. (2016) studied the growth of four microalgae, namely S. dimor-
phus, S. acutus, C. vulgaris, and C. sorokiniana in the digestate from
anaerobic digestion of municipal wastewater. Results showed en-
hancement of the growth rate (up to 0.9 d−1) at 10% digestate ratio
using secondary wastewater. However, increasing of digestate ratio to
20% resulted in reduction of the growth rate to 0.6 d−1. The negative
impact of digestate was attributed to the high ammonia content (nearly
800 mg L−1) and/or the high propionate concentration (with a spike of
1000 mg L−1). In the present study, enhancement of growth at rela-
tively higher digestate ratio of 20% might be attributed to the lower
ammonia content, especially in the digestate of the residual biomass
(167.5 mg L−1), and the lower propionate concentration of 194.96 mg
L−1 (Table 3). In agreement with the present elucidation, Uggetti et al.
(2014) cultivated a microalgal consortium dominant with Scenedesmus
sp. in different ratios of anaerobic digestate with different NH4-N
contents of 50–260 mg L−1. Results showed significant reduction in the
growth rate of microalgae by increasing of NH4-N content above 50 mg
L−1. Likewise, C. sorokiniana growth enhanced by 200% due to re-
duction of the digestate from palm oil mill effluent dosage from 100%
to 20% (Khalid et al., 2018).
3.4. Lipid production
Results showed that lipid content significantly increased by in-
creasing the applied ratio of digestate up to 40%, with the highest
significant value of 25.8 %dw at 20% (Fig. 3C). Lipid productivity is the
most precise parameter that indicates lipid production per unit volume
in a specific time, which is an essential factor for large-scale microalgal
biodiesel production. Generally, stress conditions, such as nitrogen and
phosphorus deficiency, have been reported to enhance the lipid content
of microalgae, with simultaneous reduction in the growth, which results
in reduction or insignificant changes in the lipid productivity. However,
heterotrophic and mixotrophic microalgal growth conditions were re-
ported to simultaneously enhance the growth and lipid content, leading
to significant increase in lipid productivity (Almarashi et al., 2020).
Due to enhancement of growth and lipid content at 20% digestate, it
Table 3
Characteristics of digestate slurry after anaerobic digestion of the whole and
agar-extracted pretreated Gracilaria multipartita.
Parameters Whole biomass Extracted residue
TS (g L−1) 23.41 ± 0.61 27.47 ± 0.87*
VS (g L−1) 14.32 ± 0.76 12.32 ± 0.83*
ODa 1.43 ± 0.14 1.21 ± 0.13ns
COD (g L−1) 9.29 ± 0.83 9.03 ± 0.52ns
pH 6.93 ± 0.09 7.17 ± 0.05ns
NH4-N (mg L−1) 478.61 ± 18.26 167.48 ± 21.77*
NO3-N (mg L−1) 23.57 ± 0.58 12.77 ± 0.68*
NO2-N (mg L−1) 3.42 ± 0.13 nd*
TP (mg L−1) 107.51 ± 12.44 41.32 ± 7.58*
PO4-P (mg L−1) 86.32 ± 3.83 19.43 ± 1.72*
Mg (mg L−1) 31.55 ± 6.60 174.36 ± 11.09*
Na (mg L−1) 1151.39 ± 46.81 1156.61 ± 44.64ns
S (mg L−1) 35.97 ± 0.98 36.46 ± 0.69ns
Ca (mg L−1) 6.43 ± 0.62 7.67 ± 0.39ns
K (mg L−1) 1771.97 ± 40.63 1723.52 ± 57.73ns
Mn (mg L−1) 0.51 ± 0.05 0.29 ± 0.03*
Fe (mg L−1) 2.94 ± 0.21 2.12 ± 0.29ns
VFAs (mg L−1) 2464.45 ± 103.80 1562.06 ± 93.09*
Acetate (mg L−1) 2114.20 ± 71.45 1343.67 ± 63.28*
Propionate (mg L−1) 301.43 ± 27.52 194.96 ± 27.29*
Butyrate (mg L−1) 48.82 ± 4.83 23.43 ± 2.52*
nd Not detectable, aOptical density at 680 nm, TS Total solids, VS Volatile so-
lids, COD Chemical oxygen demand, VFAs Total volatile fatty acids.
ns and * represent insignificant and significant differences, respectively, be-
tween the extracted value and the corresponding parameter of the whole bio-
mass (at P ≤ 0.05).
Fig. 3. Growth curves (A), biomass production (B), and lipid content/pro-
ductivity (C) of Scenedesmus obliquus grown on synthetic wastewater (SWW)
with different ratios of anaerobic digestate from agar-extracted Gracilaria
multipartita. Columns of the same series with the same letter showed insignif-
icant difference (at P ≤ 0.05).
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
7
showed the highest recorded lipid productivity of 65.2 mg L−1 d−1,
which was 82.6% higher than that of the control (Fig. 3C). In agreement
with these results, Tan et al. (2020) recorded the lowest lipid pro-
ductivity of 20.8 mg L−1 d−1 in C. pyrenoidosa grown in a synthetic
medium, which increased by 2.3 and 4.2 times due to cultivation in a
mixture of acidified starch wastewater to sludge anaerobic digestate
ratios of 0.5:1 and 1:1, respectively. In addition, significant increase in
lipid productivity of microalgae was previously reported using organic
wastes, such as sugar beet pulp (Wang et al., 2019), lipid-free micro-
algal biomass residues (Abomohra et al., 2018a), and kitchen lipidic
wastes (Abomohra et al., 2020b). Therefore, anaerobic digestate can be
used efficiently as a promising supplement to the microalgal growth
medium for enhanced lipid production.
3.5. FAMEs profile and characteristics
Biodiesel characteristics and quality are affected mainly by FAMEs
composition, where degree of unsaturation and carbon chain length of
the produced FAMEs are the key parameters to govern the other char-
acteristics such cetane number, iodine value, oxidative stability, and
higher heating value (HHV). Table 4 shows the FAMEs profile of S.
obliquus grown in SWW supplemented with different ratios of anaerobic
digestate. A total of thirteen fatty acids were detected in all treatments,
with a carbon chain length of C14-C18. Palmitic acid (C16:0) was the
dominant saturated fatty acid (SFA) in all treatments, while oleic acid
(C18:1n-9) was the dominant monounsaturated fatty acid (MUFA).
However, linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) formed
the majority of polyunsaturated fatty acids (PUFAs). Although all
treatments showed the same fatty acids, the total FAMEs recovery and
the percentage of different fatty acids were significantly affected by
digestate ratio. The highest significant FAMEs recovery of
123.3 mg g−1 dw was recorded at 20% digestate, which was 29.4%
significantly higher than that of the control (Table 4). Overall, in-
creasing of digestate ratio enhanced the degree of fatty acid saturation
with the maximum SFAs proportion of 23.3% of total fatty acids using
20% digestate. In addition, MUFAs showed the highest proportion of
31.2% of total fatty acids at 20% digestate. SFAs and MUFAs propor-
tions increased in favor of PUFAs, which decreased from 51.7% in the
control to the minimum value of 45.5% of total fatty acids in 20% di-
gestate. Previous studies reported a significant increase in SFAs of mi-
croalgae due to growth supplementation with organic carbon (Rai et al.,
2013; Tan et al., 2020). In that context, microalgal cell physiology tends
to accumulate SFAs under mixotrophic or heterotrophic conditions,
while accumulate PUFAs (mainly C16:3 and C18:3) under autotrophic
conditions (Perez-Garcia et al., 2011).
Reduction of PUFAs proportion in the produced microalgal FAMEs
is advantageous to improve the oxidation stability. Specifically, low
content of fatty acids with double bonds equal or greater than 4 is
desirable to provide oxidation stability for the produced biodiesel (Liu
et al., 2019). The present results showed relatively low proportion of
C18:4n-3 in all treatments, within the range of 3.1–3.8% of the total
fatty acids and the minimum value after supplementation with 20%
digestate. However, higher ratio of MUFAs to the summation of PUFAs
and SFAs M/(P + S) is important to obtain proper compromise between
oxidative stability and cold flow properties (Esakkimuthu et al., 2020).
In the present study, the M/(P + S) ratio significantly increased from
0.372 in the control to 0.453 and 0.417 at 20% and 40% digestate,
respectively (Table 4). Table 5 shows the main biodiesel characteristics
of S.obliquus grown on synthetic wastewater with different ratios of
anaerobic digestate from agar-extracted G. multipartita in comparison to
Table 4
Fatty acid methyl esters (FAMEs, mg g−1 dw) of Scenedesmus obliquus grown on synthetic wastewater (SWW) with different ratios of anaerobic digestate from the
pretreated agar-extracted Gracilaria multipartita, and harvested at the late exponential phase.
FAMEs SWW 5% 10% 20% 40%
C14:0 0.15 ± 0.00 0.18 ± 0.00 0.24 ± 0.01 0.25 ± 0.01 0.36 ± 0.03
C16:0 16.89 ± 0.37 16.35 ± 0.83 19.97 ± 0.64 22.78 ± 0.80 20.96 ± 0.71
C16:1n-10 0.97 ± 0.03 1.56 ± 0.32 1.71 ± 0.08 1.44 ± 0.06 1.88 ± 0.08
C16:1n-7 1.21 ± 0.06 1.05 ± 0.30 1.09 ± 0.09 1.20 ± 0.10 1.19 ± 0.10
C16:2 2.12 ± 0.07 2.47 ± 0.17 3.03 ± 0.21 3.35 ± 0.24 3.17 ± 0.22
C16:3 4.33 ± 0.18 4.24 ± 0.05 5.21 ± 0.06 3.95 ± 0.08 5.44 ± 0.06
C16:4n-3 6.58 ± 0.56 6.19 ± 0.48 6.98 ± 0.55 6.36 ± 0.52 6.41 ± 0.51
C18:0 3.17 ± 0.16 4.24 ± 0.18 4.61 ± 0.09 5.75 ± 0.16 4.52 ± 0.11
C18:1n-9 23.68 ± 0.73 26.43 ± 0.59 30.80 ± 0.69 35.78 ± 0.80 31.27 ± 0.70
C18:2n-6 11.84 ± 0.32 12.83 ± 0.61 14.17 ± 0.41 15.57 ± 0.44 16.35 ± 0.47
C18:3n-3 19.83 ± 1.10 16.73 ± 0.82 19.50 ± 0.96 21.76 ± 1.05 19.80 ± 0.97
C18:3n-6 0.90 ± 0.04 1.65 ± 0.09 1.34 ± 0.07 1.34 ± 0.11 1.36 ± 0.07
C18:4n-3 3.62 ± 0.17 3.45 ± 0.18 4.02 ± 0.21 3.77 ± 0.21 4.08 ± 0.21
SFAs 20.21 ± 0.53a 20.77 ± 0.67a 24.82 ± 0.72b 28.77 ± 0.93c 25.84 ± 0.85b
MUFAs 25.85 ± 0.81a 29.04 ± 1.21b 33.60 ± 0.78c 38.42 ± 0.89d 34.34 ± 0.80c
PUFAs 49.22 ± 1.83a 47.57 ± 1.90a 54.25 ± 2.09b 56.09 ± 2.00b 56.60 ± 2.12b
FAMEs Recovery 95.27 ± 3.14a 97.38 ± 3.29a 112.67 ± 3.28b 123.28 ± 3.50c 116.78 ± 3.47b
M/(P + S) 0.372 ± 0.001a 0.425 ± 0.017b 0.425 ± 0.012b 0.453 ± 0.013c 0.417 ± 0.012b
SFAs Saturated fatty acids, MUFAs Monounsaturated fatty acids, PUFAs Polyunsaturated fatty acids, M/(P + S) Ratio of MUFAs to the summation of PUFAs and SFAs.
Values with the same letter in the same row showed insignificant differences (at P ≤ 0.05).
Table 5
The estimated biodiesel characteristics of Scenedesmus obliquus grown on synthetic wastewater (SWW) with different ratios of anaerobic digestate from the pretreated
agar-extracted Gracilaria multipartita, and harvested at the late exponential phase.
Characteristics SWW 5% 10% 20% 40% Europe (EN 14214) US (ASTM D6751-08)
Degree of unsaturation 1.78 1.71 1.69 1.61 1.67 – –
Kinematic viscosity (mm2 s−1) 4.08 4.13 4.14 4.19 4.15 3.5–5.0 1.9–6.0
Specific gravity 0.88 0.88 0.88 0.88 0.88 – 0.85–0.9
Cloud point (°C) −3.80 −2.78 −2.55 −1.45 −2.32 – –
Cetane number 51.00 51.50 51.62 52.17 51.74 51–120 Min. 47
Iodine value (g I2/100 g oil) 145.21 139.55 138.22 132.10 136.96 Max. 120 –
Higher heating value (MJ kg−1) 41.67 41.54 41.50 41.36 41.47 – –
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
8
the values recommended by European (EN 14214, 2008) and American
(ASTM D6751-08, 2008) international standards. In general, values of
kinematic viscosity, specific gravity, and cetane number for all treat-
ments showed agreement with those of the recommended international
standards. Application of anaerobic digestate up to 20% showed the
highest cetane number and the lowest iodine value, which provides an
advantage for better combustion. Thus, digestate from anaerobic di-
gestion of agar-extracted G. multipartita provides a promising medium-
supplement to achieve, not only higher biodiesel yield, but also a de-
sirable FAMEs profile.
3.6. Biomass balance and gross energy yield
From the economic aspect, extraction of valuable compounds, such
as agar, is a promising approach to enhance the economic feasibility of
seaweeds conversion. In addition, using the whole seaweeds for anae-
robic digestion has many technical challenges due to high levels of
sulphated polysaccharides, sand deposition in the digesters, presence of
halogens, and high salinity (Hessami et al., 2019). Using the residual
biomass for anaerobic digestion offers additional advantage due to re-
moval of these hurdles. As discussed in the previous sections, agar ex-
traction resulted in reduction of the dry weight and VS by 41.3% and
38.8%, respectively, while simultaneously enhanced the biomethane
productivity by 48.4% over the whole biomass digestion. Therefore,
biomass balance, gross energy yield and revenue estimation are im-
portant parameters in order to evaluate the overall process. Fig. 4
shows the biomass, agar, biomethane and biodiesel production by the
suggested integrated route of anaerobic digestion of 1 ton of dry G.
multipartita followed by cultivation of S. obliquus on SWW supple-
mented with different ratios of anaerobic digestate. Anaerobic digestion
of 1 ton of the whole seaweed was estimated to produce 4107 m3 y−1 of
biomethane, which decreased by 17.4% due to agar extraction,
reaching 3392 m3 y−1 (Fig. 4). Considering the HHV of biomethane as
39.82 MJ m−3 (Wu et al., 2020), the estimated energy output from the
dry whole seaweed and agar-extracted biomass could be 6722 and
4624 MJ ton−1, respectively. From an economic perspective, agar is
more valuable product than biogas, with an estimated average market
price of 109.4 US$ kg−1 (Amazon, 2020) and 0.305 US$ m−3
(International Renewable Energy Agency, 2017), respectively. Thus,
the estimated annual revenue from the whole seaweed conversion is
1252.7 US$ ton−1 seaweeds, while that from conversion of G. multi-
partita through agar extraction deserves 36087.0 US$ ton−1 (1034.6 US
$ ton−1 seaweeds for biogas plus 35052.4 US$ ton−1 seaweeds for
agar). In addition, application of 20% anaerobic digestate to the was-
tewater as a growth medium enhanced the estimated annual biodiesel
yield to 11.3 kg m−3 of the cultivated medium, which was 69.7%
higher than that of the control medium (Fig. 4). Considering the HHVs
of biodiesel produced from cultivation of S. obliquus on SWW and 20%
digestate ratio as 41.67 and 41.36 MJ kg−1, respectively (Table 5), the
estimated energy output could be 3969.9 and 5098.9 MJ ton−1 of dry S.
obliquus. It is noteworthy to state that the energy recovery from lipid
portion of S. obliquus biomass in the present study using the two routes
represented 24.7% and 31.7%, respectively, of the total HHV of S. ob-
liquus biomass, 16.1 MJ kg−1 (Choi et al., 2019). Thus, agar extraction
prior to anaerobic digestion followed by digestate recycling for micro-
algal cultivation is a novel integrated approach with the potential to
enhance the biomass utilization and economic feasibility of biofuel
production from algae.
4. Conclusion
In the present study, a maximum biomethane potential of 278.8 L
kg−1 VS was recorded with the pretreated residual biomass, re-
presenting 23.6% higher than that of the whole biomass. Enrichment of
SWW with 20% of anaerobic digestate for S. obliquus cultivation
showed the highest biomass and lipid productivities of 0.252 g L−1 d−1
and 65.2 mg L−1 d−1, respectively. In addition, 20% digestate en-
hanced FAMEs recovery, showing the highest value of 123.3 mg g−1
dw, with enhanced FAMEs profile. Further research is required to study
the life cycle assessment of the suggested approach in a pilot- and large-
scale system.
Fig. 4. Biomass, agar, biomethane and biodiesel produced from 1 ton of dry Gracilaria multipartita and Scenedesmus obliquus grown on synthetic wastewater (SWW)
with different ratios of anaerobic digestate.
A.E.-F. Abomohra and A.W. Almutairi Bioresource Technology 317 (2020) 124027
9
CRediT authorship contribution statement
Abd El-Fatah Abomohra: Conceptualization, Funding acquisition,
Investigation, Methodology, Project administration, Validation, Writing
- review & editing. Adel W. Almutairi: Data curation, Formal analysis,
Funding acquisition, Methodology, Validation, Visualization, Writing -
original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
AcknowledgmentsAuthors are grateful to Chengdu University (start-up funds for high-
end talents 2081920048) and King Abdulaziz University for providing
the required financial support and facilities to complete this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2020.124027.
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	A close-loop integrated approach for microalgae cultivation and efficient utilization of agar-free seaweed residues for enhanced biofuel recovery
	1 Introduction
	2 Materials and methods
	2.1 Experimental design
	2.2 Biomass collection
	2.3 Agar extraction and pretreatment
	2.4 Biomass characteristics
	2.5 Anaerobic digestion
	2.6 Microalgal growth
	2.7 Biomass, lipids and FAMEs analysis
	2.7.1 Biomass production
	2.7.2 Lipid estimation
	2.7.3 FAMEs analysis
	2.8 Other analytical methods
	2.9 Statistical analysis
	3 Results and discussion
	3.1 Characteristics of G. multipartita
	3.2 Anaerobic digestion
	3.3 Microalgal growth and lipid production
	3.4 Lipid production
	3.5 FAMEs profile and characteristics
	3.6 Biomass balance and gross energy yield
	4 Conclusion
	CRediT authorship contribution statement
	Declaration of Competing Interest
	Acknowledgments
	Appendix A Supplementary data
	References