Fuel 117 (2014) 537–543

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Fuel
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Scenedesmus obliquus as feedstock for biohydrogen production

by Enterobacter aerogenes and Clostridium butyricum
Ana Paula Batista, Patrícia Moura ⇑, Paula A.S.S. Marques ⇑, Joana Ortigueira, Luís Alves, Luísa Gouveia
Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugal

h i g h l i g h t s

 Biohydrogen is produced from Scenedesmus obliquus biomass.

 Enterobacter aerogenes and Clostridium butyricum are used to ferment dried and wet microalgal biomass.
 E. aerogenes produces 57.6 mL H2/g VS from 2.5 g/L microalgal biomass (wet).
 C. butyricum produces 113.1 mL H2/g VS from 50 g/L microalgal biomass (dried).

a r t i c l e i n f o a b s t r a c t

Article history: Hydrogen (H2) gas is seen as an ideal future energy carrier because it is easily converted into electricity in
Received 8 August 2013 fuel cells, liberates a large amount of energy per unit mass, and generates no air pollutants. In this work,
Received in revised form 20 September biological hydrogen (bioH2) was produced from the microalgal biomass of Scenedesmus obliquus which
2013
was used as a substrate for the fermentation by Enterobacter aerogenes ATCC 13048 and Clostridium butyr-
Accepted 24 September 2013
Available online 7 October 2013
icum DSM 10702. The bioH2 produced by each strain was assessed for different S. obliquus biomass con-
centrations, using both dried (5% moisture) and ‘‘wet’’ (69% moisture) biomass. The highest bioH2
production yields obtained were 57.6 mL H2/g VSalga from 2.5 galga/L by E. aerogenes and 113.1 mL H2/g
Keywords:
Biohydrogen
VSalga from 50.0 galga/L by C. butyricum. The bioH2 production rates, and biogas purity attained by using
Scenedesmus obliquus the wet biomass as a fermentation substrate were similar or higher than those obtained with the dried
Dark fermentation microalga. This means that the drying step is not needed and therefore saves considerable energy as this
Enterobacter aerogenes is one of the highest energy demanding stages when using this feedstock in fermentations for biofuels
Clostridium butyricum production.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Renewable, sustainable and carbon–neutral energy production
is needed to fulfil the growing energy demand and consequential
climate changes. Hydrogen (H2) is an ideal future energy carrier
which has technical, socio-economic and environmental benefits.
It has the highest energy content per unit weight of any known fuel
(142 kJ/g) [1]. It can also be transported for domestic/industrial
consumption through conventional means and is easily converted
into electricity in fuel cells, generating no air pollutants. H2 is
therefore being explored to be used in combustion engines and
fuel-cell electric vehicles and it is expected that its demand in-
creases significantly in the near future [2].
H2 can be produced through a number of conversion technolo-
gies such as thermochemical technologies (e.g. gasification, steam
reforming of methane, partial oxidation of oil), water electrolysis
⇑ Corresponding authors. Tel.: +351 210924600; fax: +351 217167195.
E-mail addresses: patricia.moura@lneg.pt (P. Moura), paula.marques@lneg.pt
(P.A.S.S. Marques).
and photolysis [3], and using a variety of, either fossil (e.g. coal)
or renewable (e.g. biomass) feedstock. H2 can also be produced
through biological conversion by photosynthesis, photo-heterotro-
phic, and dark fermentation [4]. There is an increased interest in
biological hydrogen (bioH2) production due to the fact that tradi-
tional ways of producing H2 are still costly and display a negative
environmental impact. The photosynthetic production of bioH2 is
based on the uptake of carbon dioxide and water by photosyn-
thetic organisms. Its major drawback is the need for a constant
light source to supply the reactor [5] and the fact that low yields
are obtained [6]. The photo-heterotrophic fermentation is based
on a similar process to anaerobic digestion however it is performed
by photo-fermentative bacteria which also need light. The process
of dark fermentation to produce bioH2 requires carbohydrate-rich
substrates and fermentative bacteria [6,7].
In recent years, bioH2 production through dark fermentation
has received increased attention due to its many advantages, such
as the high hydrogen production rates, the potential to convert
biomass or bio-wastes into hydrogen, and the feasibility of the
effective process design and control [8]. During dark fermentation,

0016-2361/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fuel.2013.09.077
538 A.P. Batista et al. / Fuel 117 (2014) 537–543
bioH2 can be produced by microbial consortia, where the prevail-
ing feedstock are unsterilized waste materials, or through more
controlled fermentation conditions by specific and highly H2-
producing strains. Good H2-producers include mesophiles, such
as species of clostridia and Enterobacter, and thermophiles such
as Thermotoga neapolitana [9]. Clostridia in particular are known
for their ability to use a wide range of feedstock, including complex
polysaccharides as cellulose and hemicelluloses. Some butyrogen-
ic, acetogenic, and cellulolytic strains are also recognised as excel-
lent H2 producers [10]. Several studies on the fermentation of
lignocellulosic residues by C. butyricum have reported high H2
yields, as for example the work of Liu et al. [11]. Enterobacter aer-
ogenes, an anaerobic facultative bacterium, has also been described
as a good H2 producer in fermentations with the most diverse sub-
strates, such as organic urban solid wastes [12], biodiesel residues
containing glycerol [13] and cyanobacterial and microalgal bio-
mass, such as Anabaena [6] and Nannochloropsis [14] biomass.
Microalgal biomass can be used as an interesting alternative
substrate for bioH2 production as it grows faster compared to high-
er plants, does not need arable land (it can be grown in marginal
areas, such as salines and deserts), nor potable water (brackish
and saline water can be used, as well as wastewater). Scenedesmus
obliquus in particular is a robust microalga that has good produc-
tivity rates [15] in moderate climates. It represents a good source
of sugars of around 10–34% on dry weight basis [16,17], and starch
is its storage carbohydrate. The microalgal cell wall polysaccha-
rides may also contribute as a carbohydrate substrate for bioH2 fer-
mentations. Due to the lack of hemicellulose and lignin, most
microalgae cell walls are more easily degraded than conventional
lignocellulosic biomass. This fact makes microalgal biomass prone
to milder pre-treatments prior to fermentation [18].
The main objective of this work was to evaluate and compare
the bioH2 production by E. aerogenes and C. butyricum in batch dark
fermentation using S. obliquus biomass as source of carbon feed-
stock. Dried and wet microalgal biomasses were tested as fermen-
tative substrates at different concentrations. Hydrogen production
yields, production rates, as well as biogas purity (H2/CO2) were
compared. The results obtained are an important contribution to
increase the know-how on bioH2 production from microalgal bio-
mass using two classical and robust hydrogen producing bacteria,
one being a facultative anaerobic bacteria (E. aerogenes) vs. the
other which is a strict anaerobic (C. butyricum) bacteria. To our
knowledge, hydrogen production by these strains has not been sys-
tematically compared under the same experimental conditions
using microalgal feedstock. The best combination of bacterial
strain/feedstock concentration for the production of bioH2 was
established for further optimisation studies.
2. Materials and methods
2.1. Microalga feedstock
2.1.1. Microalga production
The microalga used in this work was S. obliquus obtained from
Coimbra University Algotec (Portugal). The culture was grown in
Bristol culture medium (pH 7) [19]. Firstly, in 1 L glass air lift
reactors, with bubbling air, at a constant temperature of 25 ± 1 °C
under low light intensity (150 lE/m2 s, measured at the surface
of the photobioreactor using a Phywe luxmeter), and then
scaled-up into two outdoor open raceway ponds (300 L capacity,
2 m2 exposed area each). These were agitated by paddle–wheels
at approximately 5 m/min with natural light (with light/dark cycles).
These cultures were produced at the LNEG’s Lumiar Campus in the
city of Lisbon, located on the west coast of Portugal (38°420 N,
9°110 W).
For the recovery of the microalgal biomass from the raceway
ponds, the agitation was stopped and the biomass settled down.
Most of the liquid phase was poured out and the collected biomass
was submitted to subsequent centrifugation (10,000 rpm; 10 min)
(Avanti J25, Beckman). Half of the biomass was kept with 69%
moisture and was named the ‘‘wet biomass’’, whereas the other
half was dried at 80 °C overnight (16 h) and was named ‘‘dried bio-
mass’’. Both wet and dried biomass were used as carbon and en-
ergy sources in the fermentations for H2 production.
2.1.2. Microalga chemical analysis
The microalgal biomass was characterised in terms of its chem-
ical composition, according to the A.O.A.C. [20], and all analyses
were performed in duplicate. Moisture was determined by drying
the sample in an oven at 105 °C until it reached a constant weight.
Total ash was determined by incineration at 550 °C in a muffle fur-
nace. The average moisture and ash content was further used for
calculating the volatile solids (VS) content. Protein content was
also estimated by the Kjeldahl method using 6.25 as the conversion
factor of total nitrogen to crude protein. The Soxhlet method was
used for total lipid determination through the extraction with
petroleum ether over 6 h and after the removal of the solvent.
The total sugar content was evaluated by the phenol–sulphuric
method [21], on filtered samples previously submitted to quantita-
tive acid hydrolysis. This method consists in a two stage hydroly-
sis: the first acid hydrolysis (H2SO4 72% w/w, 30 °C, 1 h) attacks
the polysaccharide fibres making them soluble and the second
one (H2SO4 4% w/w, 120 °C, 1 h) hydrolyses the sugar polymers,
yielding sugar monosaccharides with minimum degradation.
2.2. Fermentative bacteria
The fermentations were performed by two bacterial strains: E.
aerogenes ATCC 13048 Sputum (American Type Culture Collection,
Manassas, USA) and Clostridium butyricum DSM 10702 (German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany). E. aerogenes, harvested from exponentially grown cul-
tures, was used for the fermentation experiments. The original cul-
ture was kept at 4 °C in solid CASO Agar. The bacterial synthetic
growth media used was a 20 g/L peptone solution. The fermenta-
tion medium for the bioH2 production assays contained K2HPO4
(7.0 g/L), KH2PO4 (5.5 g/L), tryptone (5 g/L), yeast extract (5 g/L),
(NH4)2SO4 (1.0 g/L), MgSO47H2O (0.25 g/L), CaCl22H2O (0.021 g/
L), Na2MoO42H2O (0.12 g/L), nicotinic acid (0.02 g/L), Na2SeO3
(0.172 mg/L), NiCl2 (0.02 mg/L), with a pH of 6.8. C. butyricum
was pre-cultured in a Reinforced Clostridial Medium (RCM, Oxoid).
The basal modified medium (BM1) used in the fermentation exper-
iments was adapted from Moura et al. [22], and prepared in 50 mM
phosphate buffer (pH 6.8) under N2 atmosphere.
2.3. Biohydrogen production experiments
Batch fermentation assays were performed in 120 mL serum
bottles which were closed with butyl rubber stoppers and crimped
with aluminium seals. Each contained 20 mL basal fermentation
medium and microalgal biomass that were sterilised in an auto-
clave at 121 °C/15 min (2 atm) before starting the fermentative
process. The sterilised bioreactors (and fermentation medium)
were aseptically purged with bubbling N2 to eliminate O2, before
inoculation with exponentially grown E. aerogenes at 10% (v/v).
The fermentation was carried out under orbital shaking
(220 rpm) for 6 h at 30 °C. In the case of C. butyricum, the microal-
gal biomass was added to the individual serum bottles before the
anoxic distribution of the fermentation medium and sterilisation.
A preculture grown overnight was inoculated at 1% (v/v) and the
fermentation was conducted at 37 °C for 48 h, 150 rpm.
 A.P. Batista et al. / Fuel 117 (2014) 537–543
The effect of the substrate concentration was studied by using
different concentrations of the microalgal biomass, ranging from
2.5 g/L to 50 g/L (dry weight). BioH2 production assays comparing
the use of dried and wet S. obliquus biomass were also carried
out, for the concentrations which previously led to the highest
H2 yields (2.5 g/L for E. aerogenes and 50 g/L for C. butyricum).
All the experiments were performed in triplicate and the results
are expressed as average ± standard deviation. Control fermenta-
tion assays, without microalgal biomass, were also prepared.
2.4. Analytical methods
2.4.1. Analysis of the gaseous phase
The gaseous phase samples were collected directly from the
headspace of the serum bottles by using a gas-tight syringe. The
H2 and CO2 content was analysed by gas chromatography, at atmo-
spheric pressure, in a Varian 430-GC equipped with TCD and a
fused silica column (Select Permanent Gases/CO2-Molsieve 5A/Bor-
abound Q Tandem #CP 7430). The injector and column were oper-
ated at 80 °C and the detector at 120 °C. Argon was the carrier gas
at a rate of 32.4 mL/min.
The hydrogen production yields (mL H2/g VSalga) were calcu-
lated by dividing the total volume of hydrogen produced by the
amount of algal biomass used as fermentation substrate, in terms
of its volatile solids content. In order to compare with the results
published by other authors (Table 2) the yields were also calcu-
lated in terms of dry weight of algal biomass (mL H2/g (dw)) as
well as the productivities in terms of mL H2/L/h.
2.4.2. Analysis of the liquid phase
The supernatants of the fermentation trials, obtained by centri-
fugation (15000 rpm; 2 min) and filtration of the liquid phases
(0.2 lm), were analysed by HPLC in terms of sugars, ethanol, and
organic acids concentration. The analysis was performed in a
Merck Hitachi HPLC system (Darmstadt, Germany) equipped with
an Aminex HPX-87H column (BioRad) and a refraction index detec-
tor. The temperature of the column was set to 50 °C and the eluent
consisted of H2SO4 5 mM at a flow rate of 0.5 mL/min.
3. Results and discussion
3.1. Microalga biomass characterisation
The chemical composition of the dried alga is presented in Ta-
ble 1. The total sugar content is quite high (30.7% (w/w)) which
is a positive feature regarding the potential use of this biomass
as a fermentative substrate for the production of hydrogen. This re-
sult is also in accordance with a previous study by Miranda et al.
[17], which reported a total sugar content of 31.8% (w/w) (mainly
glucose) for S. obliquus. These values are far higher than the re-
ported by Becker [16], 10–17% (w/w), although recently values of
39% (w/w) have been reported [23]. This alga has also proved to
be a good source of single cell oil for biodiesel production, with a
total lipid content of 17.1% (Table 1). This is in agreement with pre-
Table 1
Chemical composition of Scenedesmus obliquus dried biomass (80 °C/16 h).
Item Unit Value
Moisture content % 5.4 ± 0.1
Total minerals % dry weight 20.2 ± 0.5
Crude protein % dry weight 20.4 ± 0.02
Crude fat % dry weight 17.1 ± 0.2
Total sugars % dry weight 30.7 ± 0.8
Volatile solids (VS) % 75.5
539
vious studies in which an oil content of 12–18% was reported
[15,16]. In some cases, lipid contents around 40% are reported
under specific stress conditions [24,25] namely high nitrogen
depletion and CO2 concentrations (up to 50%). This alga can there-
fore be regarded as a versatile feedstock for biofuels production. It
is possible to produce either oil-derived (e.g. biodiesel, jet-fuel) or
sugar-derived (e.g. bioH2, bioethanol) biofuels either in separate
processes or in sequential steps in an integrated sustainable pro-
cess (biorefinery concept). In addition to the oil and sugar content,
S. obliquus also contained 20.4% (w/w) of crude protein and 20.2%
(w/w) of total minerals (Table 1). The total VS was 75.5% (w/w)
and this value was used to calculate the bioH2 yield. This can then
be compared to values reported in the literature which were
obtained using other complex substrates. Moreover, microalgae
mineral content (ash) varies depending on the composition of the
culture medium and the harvesting process.
The percentage of water was residual in the dried biomass
(5.4%, Table 1), whereas it reached 68.6% in the microalga that
was harvested, concentrated, and used directly as a substrate feed-
stock in the fermentations. The critical steps of using microalgae as
fermentation substrates are the low cell densities attained and the
consequentially high operational costs which are needed to in-
crease biomass concentrations through harvesting and de-water-
ing or drying. If the water removal stage could be omitted, this
would be an important step forward to reduce costs in the produc-
tion of third generation biofuels using wet algal biomass.
3.2. Biohydrogen production
3.2.1. Effect of S. obliquus concentration
The effect of the initial substrate (microalga) concentration on
hydrogen production was studied in the fermentations by E. aerog-
enes and C. butyricum (Fig. 1).
For both bacteria, the bioH2 yields were influenced by the
increase in substrate concentrations. In the fermentations by E.
aerogenes, a decrease from 56.5 to 10.8 mL H2/g VSalga was ob-
served when the initial S. obliquus biomass concentration increased
from 2.5 to 50 g alga/L, respectively (Fig. 1a). However, higher
cumulative hydrogen volumes (2.8–10.8 mL) and production rates
(0.47–1.80 mL/h) were attained when the microalgal biomass
concentration increased (Fig. 1b). Gas purity (H2/CO2) however in-
creased only slightly from 1.13 to 1.40, showing that it was less
influenced by the substrate concentration. This behaviour was also
observed by Yang et al. [26] for lipid extracted S. obliquus biomass
fermented by an anaerobic sludge, where a decrease in the hydro-
gen yield from 40.3 to 25.6 mL/VS was reported when the initial
substrate concentration increased from 4.5 to 45 gVS/L. In addition,
recent studies with Nannochloropsis sp. [14] also reported a reduc-
tion of around 45% in the hydrogen yields obtained by using 10 g/L
alga instead of 2.5 g/L of alga as a substrate for E. aerogenes dark
fermentation. García-Peña and co-authors [27] also observed the
decreasing tendency in the hydrogen yields due to high glucose
concentrations of 30 g/L, as a substrate in a semi-continuous
biological system with seed sludge. This concentration effect can
be attributed to the fact that the experiments were conducted in
batch mode and in fixed headspace bottles, suggesting an inhibi-
tory effect by increased H2 partial pressure [28]. In fact, in previous
studies with E. aerogenes using glycerol as substrate, a significant
increase in the value of the H2/CO2 volumetric ratio (from 2 to 8)
was observed, due to the continuous removal of the produced
gases which was done through a peristaltic pump [13].
The highest hydrogen production attained by C. butyricum was
113.1 mL/g VSalga by using 50 g/L of dried S. obliquus biomass,
and this value decreased to 94.3 mL/g VSalga in the fermentations
with 2.5 g/L of S. obliquus biomass (Fig. 1a). This difference is not
as expressive as the decrease in the cumulative hydrogen volume
540 A.P. Batista et al. / Fuel 117 (2014) 537–543

Table 2
Survey of hydrogen yields and productivities using microalgal biomass as substrate.

Microalga Pre-treatment Innocula Yields and Ref.

productivitiesa
Scenedesmus obliquus Autoclave (15 min) Clostridium butyricum 113.1 mL H2/g VS Present
90.3 mL H2/g (dw) work
84.6 mL H2/LFM/h
Scenedesmus obliquus (wet) Autoclave (15 min) Enterobacter 57.6 mL H2/g VS Present
aerogenes 45.1 mL H2/g (dw) work
22.6 mL H2/LFM/h
Scenedesmus obliquus Autoclave (30 min) Clostridium butyricum 2.9 mol/moltotal sugars [7]
Chlorella vulgaris Acid; alkaline; autoclave; enzymatic Clostridium butyricum 81 mL H2/g (dw) [11]
Chlamydomonas reinhardtii Clostridium butyricum 17.29 mL H2/LFM/h [34]
Nannochloropsis Thermal + acid + pressure Clostridium 3.39 mL H2/LFM/h [35]
acetobutylicum
Anabaena sp. Autoclave (15 min) Enterobacter 15.2 mL H2/g (dw) [6]
aerogenes
Nannochloropsis sp. Autoclave (15 min) Enterobacter 60.6 mL H2/g (dw) [14]
aerogenes
Thalassiosira weissflogii Mechanical pressing; sonication; French press; freeze–thaw; Thermotoga 36.2 mL H2/LEXT/h [9]
stirring + sonication neapolitana
Chlamydomonas reinhardtii Sonication; methanol; autoclave + acid; enzymatic Termotoga neapolitana 35.83–53.3 mL H2/ [36]
LFM/h
Arthrospira maxima Enzymatic Anaerobic activated 49.7–78.7 mL H2/g [37]
sludge (dw)
Arthrospira maxima (wet) Boiling; bead milling; ultrasonication; enzymatic Anaerobic activated 38.5–92 mL H2/g [33]
sludge (dw)
Chlorella vulgaris and Dunaliella Anaerobic digested 10.8 and 12.6 mL H2/ [38]
tertiolecta sludge g VS
Scenedesmus Alkaline; thermal; alkaline + thermal Anaerobic digested 16.89–45.54 mL/g VS [39]
sludge
Scenedesmus Thermal Anaerobic digested 25.64–40.27 mL/g VS [26]
sludge
Scenedesmus obliquus Ultrasonication Anaerobic consortia 7.06–8.40 mL H2/ [40]
LFM/h
a
FM = fermentation medium; EXT = extract.

and production rate. It can be seen, from Fig. 1b, that the cumula-
tive hydrogen production decreased from 85.9 to 3.8 ml when 2.5
instead of 50 g/L of S. obliquus biomass was used. These values
point out that C. butyricum can extensively use the microalgal sug-
ars, up to approximately 15.4 gtotal sugars/L, for bioH2 production.
In terms of soluble metabolite production, in the case of E. aer-
ogenes, it was observed that negligible concentrations of ethanol,
succinic, formic and acetic acid (0.10–0.16 g/L) were produced.
This is in accordance with the constant pH value of the medium
(6.6–6.8). C. butyricum mainly produced butyrate (2.5 g/L) and ace-
tate (1.4 g/L), and small amounts of formate (0.4 g/L). In the case of
C. butyricum, the accumulation of these acids led to a decrease in
pH to 5.1. In spite of a pH value of 5.5 being cited in the literature
[29] as optimal for hydrogen production by this strain, the exten-
sive acidification that rapidly occurs in closed systems may inhibit
further bacterial growth [30]. This fact favours short fermentations,
with optimised substrate load for each biomass type and bacterial
strain, similar to the ones carried out in this study.
3.2.2. Effect of wet vs. dried biomass
In order to reduce the energy used for bioH2 production with mic-
roalgal biomass as feedstock, the fermentation assays were performed
with wet S. obliquus biomass. The experiments were conducted by
using the optimal biomass concentrations previously determined

Fig. 1. Effect of Scenedesmus obliquus biomass concentration on (a) the fermentative hydrogen yield and (b) cumulative H2 production (open symbols) and rate (filled
symbols) by Enterobacter aerogenes (squares) and Clostridium butyricum (triangles).
 A.P. Batista et al. / Fuel 117 (2014) 537–543
for each bacterial strain (section 3.2.1). One of the critical steps of
using microalgae for biofuels production is the need to remove water
from the harvested biomass in order to concentrate the algal slurry for
further transport, storage and bioconversion [31]. However, if the
microalgae production ponds are located in close proximity to the fer-
mentation facility, there is the possibility of eliminating the biomass
drying step. This would avoid additional heating requirements and
would be important to increase the efficiency of the process.
There were no significant differences in the H2 yield from the
dried or the wet biomass in the fermentation by E. aerogenes;
56.5 mL/g or 57.6 mL/g VSalga, respectively (Fig. 2). The volumetric
H2 production also increased from 2.8 to 3.6 mL and the production
rate from 0.47 to 0.60 mL/h. With respect to the volumetric H2 to
CO2 ratio, the variation was not significant. However, the hydrogen
yield of 113.1 mL/g VSalga (Fig. 2) obtained during the fermentation
of dried microalga biomass by C. butyricum was higher than the
one obtained with wet biomass (80.4 mL/g VSalga). This difference
may be due to a higher fragility of the dried microalgal cell walls.
Because of the formation of biomass aggregates at the end of the
drying process, a manual step of mechanical comminution by
grinding was necessary to homogenise the S. obliquus biomass. This
increases the specific surface area of the microalgal biomass and
the accessibility of the feedstock compounds. It may also promote
some degree of cellular disintegration, rendering the substrate
more amenable to a subsequent microbial attack [32].
To our knowledge, there is only one study reporting the use of
wet biomass from microalgae/cyanobacteria for bioH2 production
by fermentation [33]. In that study, Arthrospira (Spirulina) platensis
was submitted to several physical and enzymatic methods and fer-
mented by an adapted anaerobic sludge, resulting in a maximum
yield of 92 mL H2/g (dw). Wet A. platensis biomass without pre-
treatment was hardly fermented and, after subsequent boiling
and bead milling, a yield of 38.5 mL H2/g (dw) was attained.
Fig. 2. Hydrogen yield (a), maximum volumetric production (b), production rate (c) and gas purity (d) obtained from the fermentation of Scenedesmus obliquus dried (5%
moisture) and wet (69% moisture) biomass by Enterobacter aerogenes and Clostridium butyricum.
541
3.2.3. E. aerogenes vs. C. butyricum bioH2 production
The optimal bioH2 yields in the fermentation of S. obliquus bio-
mass by C. butyricum and E. aerogenes obtained in this work are de-
picted in Table 2 and compared with other authors.
C. butyricum yielded higher H2 production values than E. aerog-
enes. At the optimal substrate (microalga) concentration and con-
ditions (dry or wet) production yields of 57.6 and 113.1 mL H2/g
VSalga were obtained for E. aerogenes and C. butyricum, respectively
(Table 2). This can be related to the different degradation capabil-
ities of each microbial strain. In fact, the versatility and efficacy of
clostridia to utilise a broad range of carbohydrate sources is well
known, especially for the biological hydrogen production from
the most diverse biomass sources [10]. In this work, S. obliquus
was not submitted to any pre-treatment except the required auto-
clave sterilisation of the fermentation medium (supplemented
with the microalgal biomass). This exposure to a higher tempera-
ture and pressure may have released storage polysaccharides from
the microalgae cells. Furthermore, starch is the main storage poly-
saccharide in green algae. C. butyricum produces amylases and is
therefore able to assimilate starch, which is not possible by E. aer-
ogenes [41]. This could also explain the higher hydrogen yields ob-
tained by C. butyricum with higher substrate concentrations, as can
be observed in Fig. 1.
In general, the present results are significantly higher than the
ones obtained by other authors in the fermentation of microalgal
biomass, especially for C. butyricum, showing that the strains used
in this work were very efficient in metabolizing S. obliquus biomass
constituents (Table 2). The cell walls of this microalga are com-
posed of a trilaminar layer: a thick inner cellulose layer, a very thin
middle layer (unknown chemical composition) and an outer sporo-
pollenin-containing layer (resistant polymer formed from carote-
noids) [42]. Despite a gradual change in the cell walls’
composition with age, Scenedesmus has one of the most resistant
542 A.P. Batista et al. / Fuel 117 (2014) 537–543
cell walls amongst all microalgae [31,43]. Nonetheless, bioH2
production after alkaline, thermal or ultrasonication pre-treatment
of S. obliquus biomass was lower [39,26,40] than in the present
work (Table 2). For instance, S. obliquus lipid-extracted biomass
residues through pre-treatment (alkaline and thermal) yielded
only a maximum of 45.5 mL H2/g VS when inoculated with an
anaerobic digested sludge [39].
Table 2 also depicts several studies with other microalgae,
showing different morphologies and consequently different cell
wall compositions. These examples include some cyanobacteria
(e.g., Arthrospira and Anabaena) which do not have a cellulose cell
wall, and consequently should be easier to digest. However, our
results are still higher, or comparable, with the ones reported for
cyanobacteria [6,33,37].
The best results attained by Cheng and co-workers [33,37] were
reported with enzymatically treated A. maxima biomass, by an
adapted anaerobic sludge. In this case, the H2 yield obtained was
only 38.5 mL H2/g dw after boiling and bead milling of the micro-
algal biomass (Table 2). The severity of either of the referred pre-
treatments would suggest higher bioH2 yields. However, in the
present work, in which only autoclave sterilisation (15 min) was
used, comparable and better results were obtained. When using
pure cultures for bioH2 production, thermal treatment is manda-
tory and generally represents a huge operational cost in the whole
production process. The possibility of merging microalgae biomass
pre-treatment with culture medium sterilisation represents a clear
benefit in terms of the process cost and simplicity.
The fermentation by pure cultures can also be an advantage
over the use of microbial consortia, since even after inocula pre-
treatment [44], hydrogenotrophic microorganisms may persist
and cause a decrease in the H2 concentration [45]. Moreover, other
carbon source competitors, non-hydrogen producers, may co-exist
in the consortium, thus contributing to a lower overall hydrogen
yield.
In previous studies carried out by the authors [6,14], using E.
aerogenes and the biomass of Anabaena sp. and Nannochloropsis
sp., similar H2 production rates were obtained (0.43 and
0.53 mL H2/h, respectively) (Fig. 2), although some differences in
the yields were observed (Table 2). Regarding the fermentation
of microalgal biomass by Clostridium species the highest yield
found in the literature (Table 2) was reported by Liu et al. [11] with
81 mL H2/g alga (dw), which is slightly lower than the hydrogen
yield obtained in this work by C. butyricum (90.3 mL/g alga dw).
4. Conclusions
The results obtained in this work demonstrate the potential of
the S. obliquus biomass as feedstock for hydrogen production by
dark fermentation, using E. aerogenes and C. butyricum. The highest
hydrogen yield (113.0 mL H2/g VSalga) was obtained with C. butyr-
icum and a concentration of dried microalgal biomass of 50.0 g/L.
E. aerogenes was able to produce 57.6 mL H2/g VSalga with an initial
substrate concentration of 2.5 g/L after 6 h of fermentation. C.
butyricum requires anoxic conditions for both growth and H2 pro-
duction, while E. aerogenes is a facultative anaerobic bacteria (aer-
obic growth), resulting in both lower maintenance and operational
costs. The use of wet microalgal biomass for bioH2 production with
comparably high yields is an advantage in terms of energy and eco-
nomic efficiency, because it suppresses an intermediate drying
step, which is always time and energy consuming.
Acknowledgements
This research was part of ‘‘Microalgae as a sustainable raw mate-
rial for biofuel production (Biodiesel, Bioethanol, Bio-H2 and Biogas)’’
(PTDC/AAC-AMB/100354/2008) and ESIBITS ‘‘Evaluation of the Sus-
tainability of Industrial Biohydrogen production by microalgae, and
Integration on taxi/bus Transport Systems’’ (EXPL/EMS-ENE/1078/
2012) projects sponsored by the Portuguese Foundation for Science
and Technology (Fundação para a Ciência e Tecnologia – FCT). Ana
Paula Batista acknowledges the Post-Doc Grant SFRH/BPD/84812/
2012 from FCT. The authors would also like to thank Stephanie
Seddon-Brown (MSc) for the English proofreading.
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