Algal Research 27 (2017) 206–214

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Enhancement of biogas production from Ulva sp. by using solid-state MARK

fermentation as biological pretreatment
Nesrine Ben Yahmeda, Hélène Carrereb, M. Nejib Marzoukia, Issam Smaalia,⁎
a
Laboratoire LIP-MB INSAT, LR11ES26, Université de Carthage, INSAT-BP 676, Centre urbain nord, 1080 Carthage Cedex, Tunisie
b
LBE, INRA, Avenue des Etangs, 11100 Narbonne, France

A R T I C L E I N F O A B S T R A C T

Keywords: The green macroalgal biomass corresponds to an emerging and promising biofuel feedstock. Their biological
Pretreatment pretreatment and energetic conversion to biomethane were investigated and the enhancement of biogas pro-
Methane potential duction using the solid-state fermentation (SSF) as an eco-friendly innovative pretreatment of Ulva sp. was
Green-macroalgae precisely assessed. Compared to conventional acid and alkali pretreatments, the highest methane potential of
Solid-state fermentation
153 ± 3 mL CH4 g− 1VS with an anaerobic biodegradability of 57% was obtained using SSF pretreatment with a
Anaerobic digestion
locally isolated Aspergillus fumigatus SL1 strain. It was 132 ± 2 mL CH4 g− 1VS for raw Ulva sp. with biode-
Biogas
gradability of 49%. Acid pretreatment with 4% HCl at 150 °C had a negative effect on Ulva sp.'s methane po-
tential while alkali pretreatment with 4% NaOH at 20 °C showed a significant effect. The proposed SSF-based
pretreatment enhanced therefore biogas production of 21% and permitted an eco-friendly valorization of large
amounts of abundant macroalgae.

1. Introduction other algal or lignocellulosic biomasses at industrial large scale due to

Today, in fast-paced society, the rising number of world population
brings about a massive energy consumption characterized by the de-
crease of oil resources which are considered to be the main require-
ments for using renewable energies. The utilization of biomass as sus-
tainable source for energy production represents therefore a promising
alternative for the substitution, at least in part, of fossil fuels con-
sumption. Beside the energetic conversion of lignocellulosic biomass,
macroalgae biomass received a considerable attention as a third-gen-
eration biofuel feedstock due to its prolific growth in eutrophic coastal,
water fouling beaches and coastal waterways [1–3]. The use of mac-
roalgae biomass allows to benefit from several advantages including:
(a) high biomass production and high photosynthetic efficiency com-
pared to terrestrial crops [4]; (b) easy cultivation since it doesn't require
agricultural additives such as fertilizer and pesticides [5]; (c) low cost
of collection without environmental damage [6]; (d) no need of arable
land and fresh water resources [5,7]; (e) particular chemical composi-
tion characterized by absence/very low lignin content, high carbohy-
drates and low lipid levels that make it suitable for biogas production
[5,8]. Undoubtedly, the Ulva genus is one of the worldwide abundant
green macroalgae that is most known for generating a considerable
biomass issued either from eutrophication problems or cultures
[4,9,10]. However, this macro-algal biomass is still less valued than
the seasonality of the seaweed availability and the high H2S content of
the biogas, especially for high sulfur algae such as Ulva sp. [11]. Among
the few works describing the energetic conversion of macroalgal bio-
mass, few studies concerned the bioethanol production [12], whereas
the other deal with methanisation that provides a variability on biogas
yields [2,4,13,14]. In order to enhance the biofuels production, pre-
treatment of biomass was described as a crucial and important step.
These pretreatments were widely applied in the case of lignocellulosic
and microalgal biomasses with a large range of innovative ones
[15,16]. However, few works dealing with the pretreatment of green
macroalgal biomass aimed at the production of biofuels were cited
[14,17]. Indeed, to disrupt the thallus structure of algae and to increase
the availability of substrate for anaerobic digestion, different pretreat-
ment techniques have been assayed in recent years such as mechanical,
thermal, chemical, thermo-chemical and enzyme-based ones [8].
Nevertheless, these pretreatments have some disadvantages such as the
high energy demand especially for mechanical and thermal ones [18],
the formation of inhibitory compounds and the corrosion/environ-
mental problems for chemical pretreatment [19]. The formation of re-
calcitrant compounds that reduce the anaerobic degradability, and the
high cost of hydrolytic enzymes were also described as major problems
[8,14,20].
Among biological pretreatments of biomass, the direct use of

⁎
Corresponding author at: LR11ES24 - INSAT, Centre Urbain Nord, University of Carthage, BP 676, 1080 Tunis cedex, Tunisia.
E-mail address: Issam.Smaali@insat.rnu.tn (I. Smaali).

http://dx.doi.org/10.1016/j.algal.2017.09.005
Received 8 April 2017; Received in revised form 5 September 2017; Accepted 5 September 2017
2211-9264/ © 2017 Elsevier B.V. All rights reserved.
N. Ben Yahmed et al.
Table 1
Pretreatment conditions.
Chemicala/fungib Concentration
Chemical HCl 4 g/100 g TS
NaOH 4 g/100 g TS
Biological Aspergillus fumigatus SL1 7 mL/100 g TS
a
In the case of chemical pretreatment the concentration is expressed as g chemical per 100 g TS of algae (washed and dried) and the time of the pretreatment is expressed by hours.
b
The concentration of fungi is expressed as mL of fresh conidia suspension (1 × 107 conidia mL− 1) per 100 g TS of algae and the time of pretreatment is evaluated by days.
filamentous fungi can overcome these limits cited above. In fact, fungi
pretreatments are inexpensive and eco-friendly techniques, especially
when they are deployed as aerobic solid-state fermentation (SSF). The
latter allows high biomass loading, requires low reactor volumes and
amounts of water. The fungi solid-state fermentation (SSF) was often
described as a good option for the pretreatment of anaerobic digestion
feedstock [21]. In this context, several lab-scale studies investigated the
impact of fungal pretreatment on various lignocellulosic biomasses
methane potential [22,23], however its effect on macroalgal biomass
has not been evaluated up to now.
Thus, the objective of this study is to evaluate the enhancement of
biogas production from Ulva sp. biomass after fungal SSF pretreatment
using locally isolated fungus from algae and to compare with conven-
tional acid/alkali chemical pretreatments. Associated BMP tests, bio-
degradability and chemical composition were also assessed.
2. Materials and methods
2.1. Biological materials
The green macroalgae Ulva sp. is growing in Tunis lagoon from
autumn to early spring. This macroalgal biomass is considered as a
waste that generates intense marine pollution. Towards the end of
spring, the lagoon is usually cleaned to prepare the tourist season [24].
Samples used in the present study were collected in March 2015 from
this lagoon (GPS: 36.790612, 10.259602, salinity: 37.4 psu) suffering
from eutrophication problem. These samples were washed in order to
dilute the concentration of salts and to eliminate sand and other im-
purities collected with the seaweed when removed from the lagoon.
Then, they were dried at ambient temperature, under the sun, for one-
two weeks to remove water which speeds up the decay of the algae and
ensure therefore long term storage of this biomass. Once dried these
algae were finely ground with a kitchen blender to facilitate its de-
gradation and stored until use.
Aspergillus fumigatus SL1 was isolated from an heterogenic matrix
that consists of stranded-decomposed Ulva sp. and soil. An aliquote of
this matrix was diluted and vortexed in sterile water and then plated in
PDA solid medium supplemented with carboxymethyl cellulose (CMC)
at 1% (w/v) and three antibiotics: Ampicillin (100 μg mL− 1),
Kanamycin (50 μg mL− 1) and Streptomycin (50 μg mL− 1).
Filamentous mycelia were separately isolated by successive plating
operations in the same medium described above under continuous
microscopic control. The purified fungal strains were afterwards
screened for their cellulolytic capability by plating on PDA-CMC
medium followed after a 3 days - growth by the Congo red coloration
test according to Kasana et al. [25]. This test allowed revealing strains
with halo zone corresponding to CMC hydrolysis. The strain giving the
largest halo was chosen. It was designed SL1 and was retained for the
subsequent studies of identification and pretreatment.
Genomic DNA of SL1 was extracted in accordance to the method
described by Moller et al. [26]. Its internal transcribed spacer (ITS)
region was amplified using PCR protocol. Universal primers ITS1 (5′-
TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATA-
TGC-3′) were used for the amplification. PCR reactions were performed
in 50 μL containing MgCl2 (2 mM), DNA (50 ng), dNTPs (200 μM),
Algal Research 27 (2017) 206–214
Temperature Time initial pH Moisture ratio (solid:liquid)
150 °C 0.5 h 2.3 1:28
20 °C 24 h 11.9 1:28
50 °C 2; 4; 6; 8 d 5 1:3
primers (0.6 μM each), and GoTaq DNA polymerase (1.25 U). The fol-
lowing PCR program was employed: 1 cycle of initial denaturation at
94 °C for 5 min followed by 32 cycles (denaturation at 94 °C for 45 s,
annealing at 50 °C for 45 s and extension at 72 °C for 1.30 min) and a
final elongation cycle at 72 °C for 7 min. The purified PCR products
were automatically sequenced using an Applied Biosystems 3130 DNA
sequencer (FST, Tunisia). DNA sequence analysis and editing were
performed using the BioEdit program v.7.2.5. Sequence similarities
were performed by the BLASTn algorithm at the NCBI server that
showed 100% identity (with 98% sequence cover) with Aspergillus fu-
migatus. Hence, after microscopic confirmation, the isolated strain was
identified as Aspergillus fumigatus SL1, its sequenced ITS region was
submitted to GenBank database under the accession number of
KU587041.
2.2. Pretreatment
The pretreatment conditions are presented in Table 1. HCl and
NaOH pretreatments were achieved as described and optimized for
algae by Jard et al. [27]. Biological pretreatment was performed by
solid-state fermentation (SSF) of the isolated fungus Aspergillus fumi-
gatus SL1. Conidia production was obtained from culturing A. fumigatus
SL1 on potato dextrose agar (PDA) for 4 days at 40 °C. Conidia were
harvested by washing the plate with 10 mL sterile water containing
0.9% (w/v) NaCl. The obtained conidia suspension was adjusted to the
concentration of 107 conidia mL− 1 per collected tube.
Solid-state fermentation was carried out in separate sets consisting
of 250 mL Erlenmeyer flasks. Each experiment used 35% of dried and
ground Ulva sp. macroalgae moistened at the ratio of 1:3 (solid:liquid)
with Mandels' salts solution (0.3 g L− 1 urea, 1.4 g L− 1 (NH4)2SO4,
2.0 g L− 1 KH2PO4, 0.3 g L− 1 CaCl2, 0.3 g L− 1 MgSO4, 0.25 g L− 1 yeast
extract, 0.75 g L− 1 peptone, 5 mg L− 1 FeSO47H2O, 20 mg L− 1 CoCl2,
1.6 mg L− 1 MnSO4 and 1.4 mg L− 1 ZnSO4) [28]. Flasks were auto-
claved at 120 °C for 20 min to decontaminate the algae feedstock by
eliminating endogenous microorganisms and therefore stimulating the
growth of the isolated fungal strain. Then, they were cooled and in-
oculated with conidia suspension at 7% (v/w) ratio relative to dry
ground algae. All the flasks were incubated at 50 °C for different times.
BMP (Biochemical Methane Potential) tests were carried out on the
pretreated samples and raw macroalgae. The remaining pretreated
samples were filtered to separate the solid fraction, which was used for
further chemical characterization.
2.3. Chemical composition
Pretreated and untreated Ulva sp. were analyzed for TS (Total
Solids) and VS (Volatile Solids) in accordance with APHA standard
methods [29].The carbohydrate (glucose, xylose, arabinose and rham-
nose) and uronic acids in solid phases were measured in duplicate using
the strong acid hydrolysis protocol adapted from Effland (1977) [30].
All pretreated and untreated samples were milled and samples (100 mg)
were hydrolyzed with 12 M H2SO4 for 2 h at room temperature, then
diluted to reach a final acid concentration of 1.5 M and kept at 100 °C
for 3 h. The mixture was filtered through paper fiberglass (GFF,
WHATMAN). The analysis of monosaccharide sugars was perform by
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N. Ben Yahmed et al. Algal Research 27 (2017) 206–214

HPLC using a combined Water/Dionex system equipped with BioRad
HPX-87H column kept at 50 °C. The mobile phase consisted of 0.005 M
H2SO4 was run at a flow-rate of 0.3 mL min− 1. The refractive index
detector (Water R410) was used to quantify carbohydrates. The HPLC
system was calibrated with glucuronic acid, galacturonic acid, glucose,
xylose, arabinose and rhamnose (Sigma–Aldrich).
Kjeldahl nitrogen (TKN) was titrated using a Buchi 370-K after
mineralisation of the samples. The protein content was calculated by
using a nitrogen conversion factor of 6.25 [31].
The lipid content was determinate using the protocol described in
the standard NF V 03-713 [32].
Carbon/nitrogen ratio and sulfur content were analyzed by ele-
mentary analysis using Flash Smart analyser (Thermo Fisher Scientific
analyser).
2.4. FTIR-ATR analysis
Cellulose(%TS) = Glucose(%TS) 1.11 (1)
Hemicelluloses(%TS) = [Xylose(%TS) + Arabinose(%TS)
+ Rhamnose(%TS)] 1.13; (2)
with 1.11 the conversion factor for glucose-based polymers (glucose) to
monomers and 1.13 the conversion factor for xylose based polymers
(arabinose and xylose) to monomers [34].
Theoretical methane potential (TMP) of Ulva sp. was computed
using the constant values [crude protein = 0.49 LCH4 g− 1VS, crude
lipid = 0.85 LCH4 g− 1VS, crude carbohydrates = 0.39 LCH4 g− 1VS]
considered to estimate the methane potential of feedstock as suggested
by Powell B. Marquez et al. [35]. Actual protein (P), lipid (L) and
carbohydrates (C) composition obtained from Ulva biomass were also
used as shown in the formula:
TMP = [(0.49 LCH 4 g−1VS) × (P)] + [(0.85 LCH 4 g−1VS) × (L)]

Infrared reflectance vibrational spectra were carried out on pow- + [(0.395 LCH 4 g−1VS) × (C)] VS(g) of Ulva sp. (3)
dered samples using a Bruker's Alpha-P FTIR spectrometer, which in- All methane potentials were expressed in mL CH4 g −1
initial VS, so
cludes a single bounce diamond ATR. Spectra were collected at 4 cm− 1 the eventual losses of organic matter during pretreatments were con-
resolution intervals in the wave region of 4000–400 cm− 1 at room sidered in the results.
temperature. The biodegradability of Ulva sp. before and after pretreatment can
be determined as follows:
2.5. Biochemical methane potentials
BD(%) = [Experimental methane potential (mL CH 4 g−1VS)
Methane production was assessed in batch BMP tests in mesophilic TMP (mL CH 4 g−1VS)] ∗100 (4)
conditions (35 °C), as described by Jard et al. [2]. The inoculum ori-
ginated from the outlet of an up flow anaerobic sludge blanket reactor Data was analyzed by a one-way analysis of variance (ANOVA)
(UASB) treating wastewater from a sugar industry; it had a volatile using the software Kaleidagraph v.4.0 at a 95% confidence limit
suspended solid concentration of 23.3 g L− 1. The Substrate/Biomass (α = 0.05). The ANOVA was based on F-testing and was followed by
ratio applied was 0.5 gVS of algae per gVS of inoculum. Thus, each Tukey's pairwise comparisons between pretreatments' averages. For p-
bottle contained 4 gVS of inoculum and 2 gVS of ground dry algae (raw) values < 0.05, the difference between pretreatments averages was
or pretreated algae. Bottles were filled to 400 mL with a bicarbonate estimated statistically significant while for p-values > 0.05 the dif-
buffer complemented with nutrients (Table S1). Control trials con- ference between pretreatments' averages was not statistically sig-
taining a fully biodegradable substrate (ethanol) and a blank (no sub- nificant.
strate) were used. The ethanol control was used to check the inoculum
activity and the blank control to measure endogenous methane pro- 3. Results and discussion
duction which was subtracted from the methane production of each
sample. Bottles were rapidly sealed using butyl-rubber stoppers and 3.1. Chemical composition of Ulva sp.
held with clamped aluminum collars. Nitrogen gas was flushed into
airspace in order to evacuate oxygen and maintain anaerobic. Once The approximate chemical composition of Ulva sp. is summarized in
prepared, bottles were shaken thoroughly and incubated at 35 °C with Table 2. The monosaccharides composition after acid hydrolysis was
continuous agitation. All BMP tests were carried out in duplicate. also analyzed and the total carbohydrate content was quantified as the
During incubation, biogas production was regularly monitored by sum of each individual sugar. This carbohydrate content was found at
pressure measurement of the headspace using a manometer (Keller, 33.2 ± 0.8% containing 12.4 ± 0.2% of glucose based on total solids
LEO 2). The concentration of CH4 in biogas was determined by gas which is favorable to anaerobic digestion. In fact, soluble glucose is
chromatography (PerkinElmer, Clarus 480). BMP test was carried out
until biogas production ceased. Methane yields were calculated by di- Table 2
viding the corrected methane volume (standard pressure and tem- Chemical composition of Ulva sp. after drying and grinding.
perature 105 Pa and 273.15 K, respectively) by the weight of sample VS
Characteristics Mean ± S.D
added to each bottle.
TS (%wet weight) 84.1 ± 0.1
2.6. Microscopic observations VS (%TS) 67.8 ± 0.1
Total carbohydrates (%TS)a 33.2 ± 0.8
Glucose (%TS) 12.4 ± 0.2
Scanning electron microscopy (SEM) analysis of untreated and Xylose (%TS) 3.9 ± 0.1
chemical treated Ulva sp. was performed in a JEOL JSM-6100 Scanning Rhamnose (%TS) 9.8 ± 0.8
Microscope (University of Reims Champagne Ardennes, France). Arabinose (%TS) 7.1 ± 0.4
Microscopic observations of biological pretreated Ulva sp. was achieved Uronic acids (%TS) 5.7 ± 0.1
Proteins (%TS)b 11.4 ± 0.5
using a Leica M205FA stereomicroscope (Leica Microsystems GmbH, Lipids (%TS) 1.8 ± 0.05
Wetzlar, Germany). C/N 7.3 ± 0.03
S (%TS) 4.2 ± 0.04
2.7. Calculations and statistical analysis
a
Total carbohydrate content was quantified as the sum of each in-
dividual sugar (glucose, xylose, rhamnose, arabinose) measured in dupli-
The cellulose and hemicelluloses content in raw and pretreated cate using the strong acid hydrolysis protocol.
algae was determined on the basis of the monomeric sugar content as b
The protein content was calculated by using a nitrogen conversion
described by Monlau et al. [33] following theses equations: factor of 6.25.

208
N. Ben Yahmed et al.
rapidly transformed by microorganisms during the anaerobic digestion
[13]. It is also important to report that Ulva sp. used in this work was
characterized by a high protein content (11.4 ± 0.5%TS) compared to
a low content of lipids (1.8 ± 0.05%TS) and an absence of lignin,
making it a suitable substrate for biogas production. These results are in
agreement with Jard et al.'s study that described Ulva lactuca as a
substrate rich on volatile solids, carbohydrates and proteins and cor-
responds to a good candidate for biogas production [2]. However, the
C/N ratio of Ulva sp. was low (7.3) and the sulfur content of Ulva sp.
was high (4.2% TS) which means that high levels of H2S must be ex-
pected in the biogas produced by anaerobic fermentation such as also
reported by Briand and Morand [13]. To improve the C/N ratio, Ulva
sp. could be co-digested with another substrate poor in nitrogen as
described by Allen et al. [10].
209
Algal Research 27 (2017) 206–214
Fig. 1. FTIR spectra (400–4000 cm− 1) of untreated (A) and
fungal pretreated Ulva sp. by SSF with Aspergillus fumigatus
SL1 (B). Arrows indicated bands with the major changes.
Fig. 2. Effect of SSF pretreatment time on BMP of Ulva sp. bio-
mass. Values correspond to means of two replicates of in-
dependent values ± standard deviations (error bars). All values
are significantly different (p < 0.0001).
It is also important to note that the biochemical composition of
macroalgae depends wildly on the growth conditions and thereby
season and the pollution of water bodies [36]. Approximately, ac-
cording to Ortiz et al., the total solids (TS) of U. lactuca collected in
November, from the coastal area of Northern Chile, consist of 27%
protein, 0.3% lipids and 62% carbohydrates [31].
Furthermore, according to Briand and Morand [13], the chemical
composition of Ulva sp. collected from North Brittany green tides,
during the drifting season (from June to September), varies throughout
the harvest season. In fact, the VS and carbohydrates content decreased
from 83% to 67% and from 61% to 41%, respectively, during this
harvest period. However, the proteins and lipids content increased from
12% to 17% and from 2.8% to 3.5% respectively. Besides, Briand and
Morand demonstrated that the macroalgae Ulva sp.is characterized by a
N. Ben Yahmed et al. Algal Research 27 (2017) 206–214

Table 3 peak around 1417 cm− 1 is due to the CeC stretching vibration of
Biochemical methane potentials and assessed biodegradability of untreated and pre- aromatics [43] and the peak at 1075 cm− 1 is characteristic of the CeN
treated washed and dried Ulva sp. Values mentioned in the table are significantly different
stretching vibration of aliphatic amines, which confirms the presence of
(p < 0.0001).
proteins [44]. Untreated macroalgae have strong stretching vibration
Pretreatment conditions Methane BMP (mL CH4 Increase BD (%)a peaks corresponding to the OeH and NeH groups, but those trans-
content (%) g− 1VS) BMP (%) mittances decrease in the pretreated macroalgae indicating the de-
composition of carbohydrates and proteins. Similarly, the decrease of
Untreated 63 132 ± 2 – 49
Acid pretreatment (4% 61.5 77 ± 5 − 55 29 the peaks of 1638 cm− 1 and 1075 cm− 1 and the appearance of the
HCl at 150 °C) peak of 2350 cm− 1 (in the treated macroalgae) assigned to the C^N
Alkali pretreatment (4% 57 148 ± 11 16 55 group of nitriles [44] orient to the degradation of proteins by the fungi
NaOH at 20 °C) during the biological pretreatment. The decomposition of algae organic
Fungal pretreatment (SSF 58 153 ± 3 21 57
matter is due to the capability of this fungal strain to metabolise the
with Aspergillus
fumigatus SL1) Ulva-based substrate. Indeed, the filamentous fungi Aspergillus fumigatus
SL1 was able to hydrolyze the complex structure of the Ulva sp. thallus
The fungal pretreatment gave the best results compared to the "untreated", acid pre- as shown by microscopic observations (Fig. 5), and therefore metabo-
treatment or alkali pretreatment. lizing the organic nutrients to ensure a correct growth. In fact, ac-
a
BD (%): biodegradability calculated from the Eq. (4).
cording to Singhania et al. fungal growth as well as morphology can be
strongly correlated to cellulase production [38]. Obviously, filamentous
very low content of lignin associated to a high fraction of hemi- fungi considered as strong cellulases and xylanase secreting strains
celluloses and ulvanes which give a specific structure, favorising ac- perform better efficient using SSF since the solid medium simulates a
cessibility to hydrolytic enzymes with a good hydrolysis output [13]. fungal natural habitat [45–47]. Consequently, the used A. fumigatus SL1
Moreover, Ulva sp. provides cells with a thinner and simpler morpho- strain was grown in the absence of free water and has secreted specific
logical structure, and a large surface area, making it easier to break hydrolytic enzymes (cellulolytic, hemicellulolytic and proteolytic ac-
down and digest compared to other macroalgae [37]. tivities…) degrading the cell walls of algae [5,45]. Indeed, the fungal
metabolism can transform structural carbohydrates into to non-struc-
tural soluble carbohydrates using cellulolytic enzymes [22]. It can also
3.2. Effect of Aspergillus fumigatus SL1 based SSF pretreatment on Ulva sp.
hydrolyze proteins and modify the amino acid composition of the
structure and methane potential
substrate [48,49]. The penetration of fungi mycelium as shown by
microscopic observations of fungal pretreated algae by SSF with As-
Filamentous fungi belonging to Aspergillus genus in particular, are
pergillus fumigatus SL1 (Fig. S1) can increase pore sizes and surface
well known as efficient producers of plant cell wall-degrading enzymes,
areas. This open up a greater available surface area in target substrates
allowing the decomposition of organic matter in general and of cellu-
facilitating the accessibility to enzymatic attacks. Thus, the growth of
losic substrates particularly [21,38]. The newly isolated fungus from a
mycelium by transforming the algae and making it more digestible
complex matrix consisting of decomposed Ulva sp. and soil was iden-
improved the biochemical methane potential (BMP). As shown in Fig. 2,
tified as Aspergillus fumigatus SL1 by both macroscopic and molecular
a significant increase (p < 0.05) in BMP after 8 days of fermentation
tools (GenBank Accession No.·KU587041). This filamentous fungus was
which reached 153 ± 3 mL CH4 g− 1VS was observed. Moreover, it is
cultivated under solid-state fermentation (SSF) using the dried and
important to note that the poor digestibility of sulfated polysaccharides
ground Ulva sp. as substrate. The SSF culture, usually employed for
(ulvan) may cause the poor methane production for untreated Ulva sp.
enzymes production [39,40], was used here as a biological pretreat-
compared to pretreated ones. The ulvan is not well digested by me-
ment step of Ulva sp. biomass aimed at biogas production. FTIR spectra
thanogenic bacteria but it could be metabolized by the fungi during the
(Fig. 1) and microscopic observations (Fig. 5 and Fig. S1) were achieved
biological pretreatment [50,51]. For biological pretreatment by SSF
to describe the structural changes of algal biomass by the SSF fungal
with A. fumigatus SL1 the found BMP is higher than the one described in
pretreatment. The fingerprint regions of the obtained FTIR spectra for
Japan for Ulva spp. biomass pretreated only by biological hydrolysis
untreated and fungal-pretreated (SSF of eight days) Ulva sp. are pre-
under freshwater conditions (77.6 mL CH4 g− 1VS) [35]. Besides, com-
sented in Fig. 1. The broad band around 3240 cm− 1 is assigned to the
pared to enzymatic pretreatment of macroalgae using crude hydrolytic
stretching vibrations of hydrogen bonded OeH groups and NeH groups
broths of Aspergillus niger [14], the proposed SSF based-pretreatment
indicating the presence of polysaccharides carbohydrates and proteins
with high substrate loading (35% of algae) allowed not only valoriza-
in Ulva thallus [41]. The peak at 1638 cm− 1 is assigned to the C]O
tion of a larger amounts of green macroalgae but also overcomes
group of amides, which arises due to the presence of proteins [42]. The

Fig. 3. Carbohydrates composition of raw macroalgae and of the

solid residue after acid and alkali pretreatments. Values corre-
spond to means of two replicates of independent values ±
standard deviations (error bars).

210
N. Ben Yahmed et al.
A B
substrate inhibition and limitation during enzymatic saccharification. It
presents therefore several advantages over enzymatic pretreatment,
especially the reduction of the steps' number of an enzymatic-based
process. It permitted to avoid the cost of enzyme production/recovery
step and the additional sterilization stage of the enzymatic crude ex-
tract. Indeed, when enzymatic hydrolysis is applied upstream anaerobic
digestion, there are strong risks that released sugars are consumed by
endogenous microorganisms. A sterilization step which might be too
costly within the biogas plant context may thus be required to eliminate
endogenous microorganisms [21]. Furthermore, SSF is more advanta-
geous since it has greater volumetric productivities, higher product
stability, low contamination risk, lower instrumental cost and reduces
the need of expensive nutrient medium [45]. Besides, inhibitor con-
centrations are reduced owing to the fungal metabolism [22]. Never-
theless, during fungi pretreatment, a careful control of fungi growth is
necessary and their solid-state fermentation on biomass should be op-
timized to ensure efficient pretreatment with maximum algae de-
gradation while avoiding matter's loss due to the carbohydrate con-
sumption [21].
3.3. Comparison of fungal and conventional chemical pretreatments
To more investigate the effect of fungal pretreatment on the biogas
production it was compared to conventional acid and alkali pretreat-
ments. Methane potentials obtained after different pretreatments are
summarized in the Table 3. The p-value found by One-way Anova
analysis was < 0.05 (p < 0.0001) meaning that the pretreatment had
a significant effect on the methane production. Acid pretreatment,
using 4% HCl at 150 °C had a significant negative impact on BMP
suggesting an important hemicelluloses and cellulose removal (Fig. 3).
A loss of matter such as volatile fatty acids as well as Maillard or other
caramelisation reactions were occurred due to the high temperature
applied during this pretreatment. Similarly, this decrease of BMP was
also observed by Jard et al. and Karray et al. using acid pretreatment at
160 °C and at 100 °C respectively [14,27].
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Algal Research 27 (2017) 206–214
Fig. 4. Scanning Electron Microscopy picture of Ulva sp.
without pretreatment (A) and with alkali pretreatment (B).
Fig. 5. Microscopic observations of Ulva sp. without
SL1 mycellium
pretreatment (A) and after eight days of fungal pre-
treatment by SSF with Aspergillus fumigatus SL1 (B).
On the other hand, as shown by the Table 3, alkali pretreatment (4%
NaOH) allowed an increase of BMP from 132 ± 4 mL CH4 g− 1VS
(untreated Ulva sp.) to 148 ± 11 mL CH4 g− 1VS (alkali treated Ulva
sp.). This increase can be explained by the biochemical composition
changes occurred during this pretreatment especially the solubilization
of the cell wall sugars such as cellulose (25%) and hemicelluloses (35%)
as illustrated in Fig. 3. These results are in accordance with Jard's et al.
study [27] which demonstrated that the addition of 0.04 g NaOH g− 1
TS at 20 °C led to a release of carbohydrates and proteins and, therefore,
induced an increase in the BMP of Palmaria palmata. The increase of
BMP after alkali pretreatment can also be explained by the strong de-
gradation of the algae thallus structure with the NaOH as shown by
Scanning Electron Microscopy (Fig. 4). Indeed, alkali pretreatment
swells the fibers and increases the pore size, facilitating the diffusion of
the hydrolytic enzymes and the degradation of algae by methanogenic
bacteria [5].
The Tukey's pairwise comparisons of the methane production after
different pretreatments showed that there is significant difference be-
tween BPM issued from untreated algae and those issued from alkali
(p < 0.0001) and fungi (p < 0.0001) pretreated ones. Moreover,
there is a significant difference between BMP of alkali and fungi pre-
treated algae (p = 0.007). In fact, as shown in the Table 3, fungal
pretreatment by SSF improved the overall biodegradability of Ulva sp.
(57%) leading to 153 ± 3 mL CH4 g− 1VS. It seems that these two
latter pretreatments have a different mechanisms when acting to Ulva
biomass. The alkali pretreatment consists of a simple destructuration of
macroalgae thallus by NaOH leading to the solubilization of cell wall
sugars. However, the proposed fungal SSF-based pretreatment allowed
the decomposition of algae thallus coupled to an increase of biomass
degradability due to the bioconversion of nutrients molecules related to
the growth of mycelium.
Consulting the bibliography, similar or higher values of BMP from
Ulva species were reported (Table 4). Nevertheless, they didn't use SSF
as biological pretreatment, which is to our knowledge the first study
describing it. The variability of recorded methane potential could be
N. Ben Yahmed et al. Algal Research 27 (2017) 206–214

Table 4
Comparison of methane yields in batch experiments at mesophilic conditions reported for Ulva sp. in different publications with present study.

Macroalgae Place of harvest Season of harvest Composition (%, w/ Pretreatment conditions BMP (mL CH4 References
w)a g− 1VS)

Ulva sp. Tunis lagoon March TS 84.1 Washed and dried (untreated) 132 ± 2 Present study
VS 67.8 Washed and dried treated with:
C/N 7.3 - Acid pretreatment 77 ± 5
CHD 33.2 - Alkali pretreatment 148 ± 11
Proteins 11.4 - SSF Fungal pretreatment 153 ± 3
Lipids 1.8
Ulva spp. Near the coast of Kirachō, June TS* 17.3 Washed with freshwater 77.66 [35]
Japan VS 69.3 + biological hydrolysis
C/N* 16
CHD 53.9 Washed with seawater + biological
Proteins 13.6 hydrolysis 180.9
Lipids 0.6
Ulva lactuca Argideen estuary, Ireland June TS 72 Fresh algae 183 [10]
VS 40
C/N 9.6 Washed and dried 250
CHD n.d.
Proteins 23.7
Lipids n.d.
Ulva lactuca Lézardrieux (Côtes d'Armor. July TS 83.3 Washed, dried at 40 °C for 24 h and 241 ± 10 [2]
Brittany), France VS 82.1 roughly chopped
C/N 15.1
CHD 31.4
Proteins 13.1
Lipids 0.1
Ulva lactuca Seden Beach (Odense Fjord), Late April TS * 9.8 Unwashed and roughly chopped 162 [4]
Denmark VS* 7.1
C/N 7.9
CHD 24
Proteins n.d.
Lipids n.d.
Ulva sp. Osaka Bay, Japan Summer season TS* 12 Fresh 126 [54]
(before the natural death of VS 82.7
algae) C/N 7.7 Washed and ground 180
CHD n.d.
Proteins n.d
Lipids n.d.
Ulva sp. North Brittany, France Drifting season (from June TS n.d. Unwashed 110 [13]
to September) VS 65–83 Washed 94
C/N 10.7–19.5 Not-ground 145
CHD 41–61 Washed and ground 177
Proteins 10.3–17.2
Lipids 1.6–3.5

a
All components measured in dry weight except with * (using fresh biomass).TS: total solids, VS: volatile solids, CHD: carbohydrates, n.d. not determined.

explained by the difference of chemical composition between Ulva 4. Conclusion

species collected from different coasts and at different seasons. As
shown by Table 4, most of these algae were harvested at summer season
that could improve their chemical composition and therefore their
BMP. Besides, yields of biomethane greatly varied according to pre-
treatment method and experimental conditions. The anaerobic in-
oculum-sludge represents also a crucial parameter that commands the
final methane yield. The role of a microbial ecology investigation ap-
plied to anaerobic digestion of Ulva biomass should be assessed. It could
orient the choice of inoculum sludge type [9]. Nevertheless, it is im-
portant to note that fungal SSF pretreatment newly employed in the
present work avoids the use of chemicals such as NaOH and/or ex-
pensive enzymes that cost 0.41 €·Kg− 1 TS [52] and 1.22 €·Kg− 1 TS
[53], respectively, for pretreatment. Aspergillus fumigatus SL1 based SSF
pretreatment represents an innovative biological eco-friendly pretreat-
ment using a specific fungal strain locally isolated from algal biomass
that considerably reduces the pretreatment cost, requiring heat energy
that can be produced on-site. It improves the biodegradability of Ulva
sp. biomass and lead to the valorization of large amount of seaweed,
source of eutrophication problems in several urban/industrial sites.
Therefore, this principle of biological pretreatment could represent a
bioremediation solution for these unbalanced ecosystems.
This study demonstrates the proof of using fungal SSF as effective
biological pretreatment method for enhancing biogas production from
green macroalgae such Ulva sp.. The utilization of this latter still quite
recent and needs further investigations to assess an optimal biomethane
yield, related to the both origin and composition of the algal biomass.
The enhancement of biogas production that reached
153 ± 3 mL CH4 g− 1VS, was statistically higher than values obtained
with chemical conventional pretreatments. Performing SSF with a
specific fungal strain, isolated from the same algal biomass, and
growing on it as the sole carbon source allowed to benefit from all the
advantages of SSF, notably the large biomass loading, the low chemical
risk related to the strong alkali and its high cost, facilitating therefore
the scale-up and the design of eco-friendly processes.
Authors' contributions
Ben Yahmed N. carried out the most of the experiments, partici-
pated to the interpretation of data and the redaction-correction of the
manuscript. Carrere H. planned and supervised the BMP experiments at
the LBE, participated to the interpretation of results and the correction
of the manuscript. Marzouki M.N. participated to the interpretation of

212
N. Ben Yahmed et al.
results. Smaali I. conceptualized the work, supervised the SSF experi-
ments at the LIP-MB Laboratory and participated to the redaction-cor-
rection of the manuscript.
Acknowledgements
The authors gratefully acknowledge the Tunisian Ministry of Higher
Education and Scientific Research (LR11ES24) - University of Carthage
(Tunisia) for concession of a research grant which has permitted to
Nesrine Ben Yahmed to carry out this research at INRA Narbonne
(France). This work integrates the bilateral research Project TUNGER-
72. We express our gratitude to Marie-Lou Hillion and Hélène Thomas
(INRA Narbonne, France) who helped to the determination of the
macroalgae chemical composition and to Pr. Faouzi Bouachir for the
FTIR analysis. The authors would also like to thank Mr. Dominique
Chevalier, from Thermo Fisher Scientific, Courtaboeuf - France for the
elementary analysis.
Competing interests
The authors declare that there are no competing interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.algal.2017.09.005.
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