Energy Conversion and Management 197 (2019) 111907

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Energy Conversion and Management

journal homepage: www.elsevier.com/locate/enconman

Evaluation of bioethanol and biodiesel production from Scenedesmus T

obliquus grown in biodiesel waste glycerol: A sequential integrated route for
enhanced energy recovery
⁎
Shannan Xua, Mahdy Elsayedb,e, Gehan A. Ismailc, Chunhou Lia, Shuang Wangd, ,
⁎
Abd El-Fatah Abomohrad,c,
a
Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs, South China Sea Fisheries Research Institute,
Chinese Academy of Fishery Sciences, 510300 Guangzhou, China
b
Department of Agricultural Engineering, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt
c
Botany Department, Faculty of Science, Tanta University, 31527 Tanta, Egypt
d
New Energy Department, School of Energy and Power Engineering, Jiangsu University, 212013 Jiangsu, China
e
College of Engineering, Huazhong Agricultural University, 430070 Wuhan, China

A R T I C LE I N FO A B S T R A C T

Keywords: The present study aimed to evaluate the energy recovery through biodiesel and bioethanol production from
Microalgae Scenedesmus obliquus in a sequential route of lipid extraction followed by fermentation, with recycling of waste
Biofuel glycerol (WG) as a nutrient supplement into the culture. Low WG concentrations significantly enhanced the
Biorefinery cellular dry weight over the control, recording the maximum significant value of 3.63 g L−1 using 2.5 g L−1 of
Fermentation
WG (WG2.5). In addition, the maximum carbohydrate and lipid contents were recorded in WG2.5, which re-
Transesterification
presented 16.4% and 21.7%, respectively, over the corresponding control, with simultaneous reduction in
protein content. Moreover, total fatty acid methyl esters (FAMEs) recovery from biomass increased after WG2.5
supplementation, recording an increase of 24.6% over the control. Fermentation of lipid-free biomass increased
the rate of bioethanol production, reaching its peak of 4.82 g L−1 at the 27th day. However, using of WG2.5 for
microalgal growth and the residual lipid-free biomass for fermentation showed the highest bioethanol pro-
duction peak of 5.58 g L−1 at day 27. Due to the accumulation of carbohydrates under WG, the biomass treated
with WG2.5 showed increase in maximum bioethanol productivity up to 0.185 g L−1 h−1. However, sequential
fermentation after lipid extraction enhanced the maximum bioethanol productivity by 32.3% and 15.1% over
the whole cells from synthetic wastewater (WW) and lipid-free biomass from WW, respectively. The highest
gross energy output of 21.4 GJ ton−1 dry microalgae was estimated from the integrated route where S. obliquus
was grown in WG-enriched medium and sequential fermentation was applied for the residual biomass after lipid
extraction, with the highest recorded energy conversion efficiency of 62.9%. These findings provide an in-
novative practical integrated approach for waste recycling and high conversion efficiency of microalgal biomass
for liquid biofuel production.

1. Introduction
The global population is dramatically increasing and projected to
exceed 9 billion by 2050 [1], which resulted in energy shortage and
negative environmental impacts due to the emission of greenhouse
gasses (GHGs) from excessive fossil fuel consumption [2]. Exploring
alternative energy resources is a critical concern in order to minimize
the dependence on fossil reserves, reduce the GHGs emissions, and to
maintain the environmental sustainability [3–6]. Biofuels can be
produced from different biomass feedstocks, and currently are dis-
cussed as a promising alternative energy source due to several ad-
vantages [7–11]. Based on the feedstock, biofuels can be classified into
four main generations [12]. The first-generation feedstocks include the
edible biomass such as corn, soybean and sunflower [13]. Using of
edible biomass requires significant amounts of fertilizers, water, and
large areas of cropland which competes with food crops and raised the
‘‘food versus fuel” debate [10,14,15]. The second generation of biofuels
depends on utilization of inedible lignocellulosic biomass such as rice

⁎
Corresponding authors at: New Energy Department, School of Energy and Power Engineering, Jiangsu University, 212013 Jiangsu, China.
E-mail addresses: alexjuven@ujs.edu.cn (S. Wang), abomohra@science.tanta.edu.eg (A.E.-F. Abomohra).

https://doi.org/10.1016/j.enconman.2019.111907
Received 4 June 2019; Received in revised form 31 July 2019; Accepted 1 August 2019
0196-8904/ © 2019 Elsevier Ltd. All rights reserved.
S. Xu, et al.
straw, wheat straw, sawdust, and municipal wastes [7,16]. Although
lignocellulosic wastes are discussed as the first candidate for biofuel
production due to their abundance [17], the challenge in their utili-
zation is to remove lignin and overcome the recalcitrant structure in
order to improve the accessibility of cellulose and hemicellulose.
Therefore, pretreatment is an essential step [18,19], which is energy
intensive, time consuming and/or costly. Recently, wild-type algae
have attracted enormous attention in third-generation biofuel produc-
tion [20], while genetically modified microalgae are currently dis-
cussed as a fourth-generation biofuel feedstock [12].
Algae are more viable as a biomass feedstock due to their growth at
harsh conditions in arid lands using wastewater or marine water
without competing with human food [21–24]. In addition, some mi-
croalgal species have a short doubling time of few hours with high CO2
fixation efficiency of more than 10-fold comparing to crop plants [25].
Amongst, Scenedesmus obliquus showed the superior biomass and lipid
production and was recommended for biodiesel production in many
previous screening studies. For instance, Abomohra et al. [22] screened
different freshwater microalgae and suggested S. obliquus as a pro-
mising candidate for biodiesel production due to its high biomass
productivity which resulted in high biodiesel yield. Recently, El-Sheekh
et al. [26] compared eight species of Scenedesmus for their efficiency in
biodiesel production and nominated S. obliquus as a promising candi-
date. In addition to the high lipid production, some microalgae have
high carbohydrate content, which can be used as bioethanol feedstock
[27,28]. Moreover, the absence of lignin in microalgae and the nature
of carbohydrates (composed mainly of starch and cellulose) make it
easy to be converted into usable monosaccharides [29].
Despite the aforementioned merits, the feasibility of biofuel pro-
duction from microalgae is not yet well established [30,31]. Due to the
high production cost, microalgal biofuels are not yet employed on in-
dustrial scale [32,33]. Cell disruption and components extraction are
the significant bottleneck in biodiesel and bioethanol production from
microalgal biomass. Many studies have been conducted to enhance the
production of the target compounds by selecting the microalgal species
[34], adjusting the extraction method [35] or optimization of micro-
algal growth conditions [36–40]. However, many of these methods are
inefficient and/or require a long-duration, unsustainable chemical/
physical processing leading to many environmental burdens and ele-
vated cost [41]. Recycling of byproducts from biodiesel production was
discussed as a cost-effective approach for enhancement of biomass and
lipid production in microalgae [42,43]. In addition, biorefinery of mi-
croalgae by conversion of the whole cellular components into valuable
compounds was discussed as one solution to maximally utilize the
biomass constituents [44–47]. In that context, the lipid-free biomass
with high carbohydrate content can be used as a bioethanol feedstock
by further fermentation. In addition, lipid extraction is considered as a
pretreatment step which concentrates and eases the availability of
carbohydrates for further conversion.
After biodiesel production by transesterification of lipids, each
100 kg of the produced biodiesel generates about 10 kg of waste gly-
cerol (WG) as a by-product [48]. Garlapati et al. [49] reported that the
estimated global WG production from biodiesel industry is 4 billion
gallons. Due to the high cost of WG purification, it is not desired to be
used by the industry [50] and, therefore, unrefined WG has become a
potential environmental pollutant. Therefore, new innovative technol-
ogies need to be developed in order to provide a full use of the WG to
reduce its environmental impacts from one side and the biofuel pro-
duction cost from the other side. Alternatively, the WG from algae-
based biodiesel production can be recycled as a supplemental organic
carbon source for the microalgal culture. This kind of waste recycling
and sequential biofuel production might improve the efficiency of mi-
croalgae-based biofuel industry. There is a gap in literature concerning
the sequential biodiesel and bioethanol production from microalgae
using the recycled WG to further enhance the biomass, lipids and car-
bohydrate production. Therefore, the present study aimed to evaluate
2
Energy Conversion and Management 197 (2019) 111907
the growth, lipid and carbohydrate production by S. obliquus grown in
wastewater supplemented with WG. In addition, the performance of
biomass conversion into fatty acid methyl esters (FAMEs) and/or
bioethanol using sequential lipid transesterification and fermentation of
lipid-free biomass was studied. In total, the present study evaluated
seven conversion routes. The gross bioenergy output (GBO) and energy
conversion efficiency (ECE) using the studied conversion routes were
evaluated.
2. Materials and methods
2.1. Microalga and growth conditions
Scenedesmus obliquus Kützing SAG276-10 was grown in 2 L tubular
glass cylinders containing 1.5 L/each of synthetic wastewater (WW) at
initial OD680 of 0.25 ± 0.07. The WW was composed of (mg L−1);
MgSO4·7H2O 400, NH4Cl 230, K2HPO4 200, Na2HPO4 100, NaNO3 100,
CaCl2·2H2O 100, NaCOOCH3 100, and KH2PO4 50, with adjusting the
pH value at 7 [51]. A sterile filtered air was continuously applied to the
cultures through an aeration tube (Φ = 3 mm) connected to the bottom
of each glass cylinder. The WG was obtained from lipid extraction and
transesterification of microalgal biomass harvested by flocculation from
lab-scale tubular photobioreactor [52]. The WW was enriched by dif-
ferent concentrations of WG (0, 2.5, 5, 10 and 20 g L−1), namely con-
trol, WG2.5, WG5, WG10, and WG20, respectively. Cultures were il-
luminated by tubular fluorescent lamps (PHILIPS Master TL-D 85 W/
840) at light intensity of 100 μmol m−2 s−1 and a photoperiod of 18:6h
light: dark cycle at 25 ± 1 °C. At 2 days interval, cell number was
monitored using Neubauer hemocytometer (Bright-Line, Reichert,
Buffalo, USA). In addition, the dry weight (DW) was measured at the
early exponential phase, late exponential phase (LEP), and stationary
phase (STP). The values of DW were used to calculate the biomass
productivity using Eq. (1);
Biomass productivity (g DW L−1d−1)
= (DWS − DWi )/(tS − ti )Biomass productivity(gDWL−1 per day)
−1
= (DWS − DW)/(t
i S − ti )Biomass productivity(g DW L per day)
= (DWS − DW)/(t
i S − ti) (1)
−1
where DWS and DWi are the DW (g L ) at the time of late exponential
phase or stationary phase (tS) and the early exponential phase (ti), re-
spectively.
2.2. Routes design
Bioethanol and biodiesel production, as well as GBO and ECE of S.
obliquus, were evaluated through seven different routes as summarized
in Fig. 1. The first two routes (P1 and P2) represent biodiesel or bioe-
thanol production, respectively, from the whole biomass grown in WW.
P3 represents bioethanol production from the residual biomass grown
in WW. P4 and P5 represent biodiesel and bioethanol production, re-
spectively, from microalgae grown in WG-enriched medium. P6 and P7
represent the integrated pathways for biodiesel plus bioethanol pro-
duction from biomass grown in WW and WG-enriched medium, re-
spectively.
2.3. Determination of biochemical composition of S. obliquus
After cell harvest from 5 mL of microalgal culture by centrifugation
at 3000×g for 10 min, cells were firstly disrupted by acid hydrolysis
using 5 mL of 1 N NaOH solution in a boiling water bath for 2 h. Total
carbohydrates were determined by phenol–sulfuric acid method [53].
Briefly, 1 mL of 5% phenol was added to 1 mL of the hydrolyzed sample,
and then 5 mL of conc. sulphuric acid was added slowly to the mixture.
The samples were incubated at room temperature for 30 min, then the
S. Xu, et al.
Fig. 1. Process diagram of dual biodiesel and bioethanol production from Scenedesmus obliquus grown in wastewater and WG-enriched medium. Biodiesel or
bioethanol production from the whole biomass grown in WW (P1 and P2, respectively). Bioethanol production from the lipid-free biomass from WW (P3). Biodiesel
and bioethanol production from microalgae grown in WG-enriched medium (P4 and P5, respectively) and the integrated pathways for biodiesel plus bioethanol
production from biomass grown in WW and WG-enriched medium (P6 and P7, respectively).
absorbance was measured at 490 nm. Glucose (0.1–1 mg mL−1 at an
interval of 0.1 mg mL−1) was used to generate the standard curve for
the calibration.
Protein concentration was determined according to Bradford
method [54] in 0.1 mL of the hydrolyzed sample using Quick StartTM
Bradford 1x Dye Reagent (BIO-RAD). The absorbance was determined
by spectrophotometer at 595 nm. The standard curve was generated by
using bovine serum albumin in the concentration range of
0.1–1 mg mL−1 at an interval of 0.1 mg mL−1.
Total lipids were determined gravimetrically according to Bligh and
Dyer method [55] using chloroform:methanol (2:1, v/v). Lipid extracts
were dried under a stream of argon gas in pre-weighed glass vials. The
glass vials containing lipid extracts were dried at 80 °C for 30 min,
cooled in a desiccator and weighed again. Lipids and carbohydrates
productivities (P) were calculated using Eq. (2).
P (mgL−1d−1) = (CS − Ci )/(tS − ti ) (2)
where CS and Ci are the lipid or carbohydrate production (as mg L−1) at
the time of late exponential phase or stationary phase (tS) and the early
exponential phase (ti), respectively.
2.4. Characteristics of WG
The concentrations of nitrate (NO3–N), nitrite (NO2–N), and am-
monia (NH4-N) nitrogen were determined using salicylic acid [56],
naphthylethylenediamine-sulfanilamide (NEDASulfa) [57], and phe-
nate [58] methods, respectively. Total phosphorus (TP) and total ni-
trogen (TN) were determined using phosphorus TNT845 and nitrogen
HR-TNT test kits (Hach, USA), respectively, following the manu-
facturer’s manual. Glycerol concentration was analyzed spectro-
photometrically according to Bondioli and Della Bella [59]. Each
sample was properly diluted with distilled water, then 5 mL of ethanol
(47.5%, v/v) were added into 5 mL of the diluted sample. The resulting
solution was mixed with 1.2 mL of 10 mM sodium periodate solution
and 1.2 mL of 0.2 M acetylacetone solution. Mixtures were incubated at
70 °C in a water bath for 1 min, and immediately cooled down to 20 °C
using a cold-water bath. The absorbance of the solution was determined
at 410 nm, and was compared to that of the standard calibration curve
using pure glycerol. The ash content was measured in 1 mL of the WG
by ignition at 550 °C in a muffle furnace until constant weight.
3
Energy Conversion and Management 197 (2019) 111907
2.5. FAMEs preparation
In order to convert lipids into FAMEs, 10 mL of culture were col-
lected at LEP for lipid extraction as described by Abomohra et al. [35].
As an internal standard, 8 µg of the triglyceride trinonadecanoylgly-
cerol was added to each sample prior to extraction. Transesterification
of fatty acids were performed using 333 µL methanol:toluene (1:1, v/v)
and 167 µL of 0.5 M sodium methoxide. The resultant FAMEs were
extracted by hexane and re-concentrated in 50 µL of acetonitrile. A
volume of 1 µL was injected and analysed by gas chromatography (GC-
FID, Varian 3900, USA) equipped with 50 m length and 0.25 mm in-
ternal diameter Agilent J&W Select Fame capillary column (Agilent
Technologies, Wilmington, USA). The oven temperature program
started at 140 °C for 3 min, increased at 1.5 °C min−1 until 187 °C, then
increased at 5 °C min−1 until 220 °C and was kept constant at this
temperature for 5 min. The velocity of helium as a carrier gas was kept
constant at 1.0 mL min−1. Injector and detector temperatures were
adjusted at 275 °C and 300 °C, respectively.
2.6. Hydrolysis and fermentation
Sugars were extracted from S. obliquus biomass using 2 N H2SO4
(1%, w/v) followed by heating in autoclave at 120 °C for 30 min as
modified from Miranda et al. [60]. The pH of the hydrolysate was ad-
justed to 5.5 using Mg(OH)2 crystals [61] and used directly for Sac-
charomyces cerevisiae fermentation at an initial OD600 of 0.25. Biomass
fermentation was performed in 500 mL glass bottles containing 300 mL
working volume. To provide anaerobic environment, nitrogen gas was
used to flush the headspaces of the bottles for 3 min, then sealed with
rubber stoppers. The bottles were incubated for 48 h on a shaking water
bath at 30 °C and 120 rpm.
2.7. Sugars and bioethanol estimation
At regular intervals, the residual sugars were determined using 80%
hot ethanol extraction as described by Chow and Landhäusser [62]. A
0.1 mL of the extract was mixed with 200 μL of 4% (w/v) phenol, fol-
lowed by rapid addition of 0.8 mL concentrated sulfuric acid. After
30 min of color development in the dark at room temperature, absor-
bance was measured at 490 nm as described in Section 2.3. In addition,
bioethanol was analyzed by injection of 2 µL of the sample into GC-FID
S. Xu, et al. Energy Conversion and Management 197 (2019) 111907

equipped with J&W DB-624 column (30 m, 0.53 mm, 3 µm, Agilent
Technologies, USA). Nitrogen was used as a carrier gas at a flow rate of
5 mL min−1. The oven temperature was adjusted at 70 °C, while both
injector and detector temperatures were adjusted at 270 °C. The con-
centration of ethanol (g L−1) was determined using pure ethanol as a
standard. Bioethanol yield (Y, g g−1 biomass) was calculated at dif-
ferent growth points using Eq. (3);
g
Ethanol concentration ( L )
Y= g
Biomass ( L ) (3)

In addition, the accumulative bioethanol productivity (Pe,

g L−1 h−1) was calculated according to Eq. (4);
Pe = (Et − Ei )/ t (4)
−1
where Ei and Et represent the initial ethanol concentration (g L ) and
that after time t (h), respectively.

2.8. Statistical analysis

All experiments were performed in repeatability using three ex-
perimental sets. Results were presented as the mean ± SD. One-way
ANOVA followed by LSD test were applied to compare the statistical
differences among different pretreatments using SPSS (v. 20, IBM).
3. Results and discussion
3.1. Characteristics of WG
Table 1 shows the main parameters of WG used for microalgae
cultivation. It was slightly acidic and composed mainly of glycerol
(688 g L−1). In addition, it contained some impurities, where the total
nitrogen content represented 53.0 mg L−1, and low phosphorous con-
tent of 9.33 mg L−1. However, carbohydrates were not detected, which
is the reason for the low ash percentage. Sadhukhan and Sarkar [63]
estimated 12.9 wt% of non-glycerol organic matter in the crude glycerol
byproduct from lipid transesterification. In the present study, the re-
corded ash content (1.8 wt%) was 68.8% lower than that reported by
Sadhukhan and Sarkar [63]. Thus, the effect of WG supplementation on
growth and biochemical composition of S. obliquus can be attributed
mainly to the glycerol content, as an organic carbon source.
3.2. Growth and biomass composition
Results showed that S. obliquus cell number at different WG con-
centrations was slightly affected (Fig. 2). The highest cell number was
recorded using WG2.5 which represented 4.4% increase over that of
WW at STP (day 24). In addition, low WG concentrations significantly
enhanced the cellular dry weight over the control, recording the max-
imum significant value of 3.63 g L−1 at STP using WG2.5 (Table 2).
Thus, WG enhanced the cell multiplication and accumulation of storage
compounds. This finding can be confirmed by the recorded
Table 1
Main parameters of the used glycerol byproduct from transesterification of
Scenedesmus obliquus lipids.
Parameters Concentration
pH 6.4 ± 0.06
Glycerol (g L−1) 688.0 ± 3.0
Total nitrogen (TN, mg L−1) 53.0 ± 6.0
Ammonia–nitrogen (NH4-N, mg L−1) ND
Nitrate–nitrogen (NO3-N, mg L−1) 10.8 ± 2.3
Nitrite–nitrogen (NO2-N, mg L−1) ND
Total phosphorus (TP, mg L−1) 9.3 ± 1.5
Total carbohydrate (TC, mg L−1) ND
Ash (wt%) 1.8 ± 0.17
Fig. 2. Effect of different concentrations of waste glycerol (WG; 2.5, 5, 10 and
20 mg L−1) on the growth of Scenedesmus obliquus grown in synthetic waste-
water (WW).
enhancement of carbohydrates and lipids at low concentrations of WG
over the control, with simultaneous reduction in proteins (Table 2).
Similar results were reported by Abomohra et al. [64] who confirmed
the accumulation of lipids and carbohydrates in microalgal cells grown
in WG-enriched medium on the expense of proteins. The maximum
carbohydrate and lipid contents were recorded in WG2.5 at STP (32.99
dw% and 25.11 dw%), representing 16.4% and 21.7%, respectively,
over the corresponding control. However, the increase in the dry
weight, carbohydrate and lipid contents in the present study were
higher than that found by Abomohra et al. [64] who reported maximum
values of 3.56, 30.41 and 23.84 g L−1, respectively, using 5 mg L−1 of
WG. These results confirm that cells prefer lower concentrations of WG.
However, the effect of WG concentrations lower than 2.5 g L−1 needs
further investigation. The retardation in growth at high WG con-
centrations might be attributed to the free fatty acid residues in the
soap [63] which were confirmed to restrict the microalgal growth [51].
Due to lipid and carbohydrate accumulation with enhancement of
biomass production under WG supplementation, their productivities
increased significantly as well (Fig. 3). At all treatments, carbohydrate,
protein and lipid contents at LEP showed insignificant differences with
those at STP (Table 2). However, lipid and carbohydrate productivities
were significantly higher in the LEP than STP, due to the constant
content over time (Fig. 3). The highest lipid and carbohydrate pro-
ductivities were recorded at WG2.5 representing 26.5% and 22.3%,
respectively, over the control. These results confirmed that WG assim-
ilation enhanced the accumulation of lipids and carbohydrates in the
cells. Fig. 4 represents a simplified metabolic pathway for a model
unicellular photoautotrophic microalgal cell showing the suggested
metabolic pathways of WG. Glyceraldehyde 3-phoshate (GAP) is the
end-product of photosynthesis, which enters the glycolysis pathway
generating glucose 6-phosphate (the precursor of carbohydrates). GAP
might be transformed also to pyruvic acid, which is then invested either
for free fatty acids (FFAs) synthesis through fatty acid synthesis (FAS)
cycle or in the tricarboxylic acid (TCA) cycle to produce the energy
compounds and other necessary intermediates. The WG might bind
directly to FFAs producing monoacylglycerides (MAG), or binds with
acylglycerides to form diacylglycerides (DAG) or triglycerides (TAG,
the storage lipids). The present results suggested that WG stimulates
FAS cycle in favor of TCA cycle to form lipids rather than proteins.
Another scenario might be stimulation of proteins degradation to α-
ketoglutarate to form phosphoenolpyruvate which results in accumu-
lation of FFAs (Fig. 4). The present results are in conformity with those

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S. Xu, et al. Energy Conversion and Management 197 (2019) 111907

Table 2
Cellular dry weight and main components of Scenedesmus obliquus grown in synthetic wastewater (WW) and wasteglycerol-supplemented (WG) media. Cells were
harvested at late exponential phase (LEP, 18th day) and stationary phase (STP, 24th day).
Parameter Growth phase Control (WW) WG (g L−1)

2.5 5 10 20

Dry weight (g L−1) LEP 3.48 ± 0.05Aa 3.59 ± 0.04Ab 3.54 ± 0.06Abc 3.51 ± 0.05Aabc 3.49 ± 0.04Aac
STP 3.52 ± 0.04Aa 3.63 ± 0.04Ab 3.57 ± 0.04Aa 3.54 ± 0.05Aa 3.52 ± 0.06Aa
Carbohydrate content (dw%) LEP 26.42 ± 1.46Aa 31.03 ± 1.14Ab 30.66 ± 0.79Ab 30.13 ± 0.54Ab 29.62 ± 2.43Ab
STP 28.35 ± 1.09Aa 32.99 ± 0.63Ab 31.08 ± 1.11Abc 30.74 ± 1.44Ac 29.65 ± 1.08Aac
Protein content (dw%) LEP 39.20 ± 0.96Aa 35.11 ± 1.44Ab 34.04 ± 1.69Abc 32.79 ± 0.71Acd 31.90 ± 1.13Ad
STP 38.36 ± 1.37Aa 34.16 ± 0.47Ab 33.20 ± 1.00Ab 32.34 ± 1.27Abc 31.31 ± 0.14Ac
Lipid content (dw%) LEP 19.80 ± 0.37Aa 24.03 ± 0.52Ab 22.84 ± 0.41Ab 23.31 ± 0.69Ab 23.16 ± 0.45Ab
STP 20.64 ± 0.51Aa 25.11 ± 0.71Ab 23.30 ± 0.27Ac 23.18 ± 0.26Ac 23.70 ± 0.68Ac

Values of the same measured parameter with the same capital letter at different growth phases in the same column showed insignificant difference (at P ≤ 0.05).
Values with the same small letter in the same row showed insignificant difference (at P ≤ 0.05).

Fig. 4. Simplified metabolic pathways of model unicellular microalgae showing

Fig. 3. Effect of different concentrations of waste glycerol (WG; 2.5, 5, 10 and
20 mg L−1) on lipids productivity (A) and carbohydrate productivity (B) of
Scenedesmus obliquus at late exponential phase (LEP, 18th day) and stationary
phase (STP, 24th day). Columns of the same treatment with different capital
letters showed significant difference (P ≤ 0.05), while columns of the same
series using different treatments with the same small letter showed insignificant
difference (at P ≤ 0.05).
obtained by Dourou et al. [65] who found that Yarrowia lipolytica and
Umbelopsis isabellina under nitrogen depletion tend to use glucose as
organic carbon source to accumulate lipids and polysaccharides rather
than proteins. In addition, Bellou and Aggelis [66] found that TCA was
disrupted under nitrogen limitation, and acetyl-CoA was directed to-
wards lipids biosynthesis through inhibition of mitochondrial isocitrate
dehydrogenase (ICDH) that catalyzes the conversion of isocitrate to α-
ketoglutarate, resulting in migration of citrate to the cytosol. In the
the probable role of waste glycerol. FAS: fatty acid synthesis, TCA: tricarboxylic
acid, GPAT: glycerol-3-phosphate acyltransferase, Lyso-PA: lysophosphatidic
acid, LPAAT: lysophosphatidic acid acyltransferase, PA: phosphatidic acid,
DAG: diacylglyceride, TAG: triacylglyceride, PAP: phosphatidic acid phoapha-
tase, DAGAT: diacylglyceryl hydroxymethyl trimethyl-β-alanine.
cytoplasm, citrate was turned to oxaloacetate and acetyl-CoA by ATP-
citrate lyase, using NADPH as energy source in FAS cycle leading to
lipids accumulation. Based on the results of maximum carbohydrates
and lipids productivities, 2.5 g L−1 of WG was chosen to supplement the
growth medium of microalgae and cells were harvested at the LEP for
biodiesel and bioethanol production.
3.3. Biodiesel production
Table 3 shows FAMEs profile of S. obliquus grown in both control
(WW) or WG2.5 and harvested at LEP. Obviously, WG2.5 significantly
enhanced the glycerol yield from 6.52 dw% to 11.02 dw%.

5
S. Xu, et al. Energy Conversion and Management 197 (2019) 111907

Table 3
Fatty acid methyl esters (FAMEs) and glycerol yields (as mg g−1 dw) of
Scenedesmus obliquus grown in wastewater (WW) and medium-supplemented
with 2.5 g L−1 of waste glycerol (WG2.5). Cells were harvested at the 18th day
of growth (late exponential phase).
Fatty acids Control (WW) WG2.5

14:0 0.16 ± 0.00 0.21 ± 0.01

16:0 18.23 ± 0.59 21.65 ± 0.89
16:1n-10 1.02 ± 0.03 1.48 ± 0.06
16:1n-7 1.30 ± 0.06 1.23 ± 0.11
16:2 2.24 ± 0.07 3.04 ± 0.21
16:3 4.41 ± 0.16 5.26 ± 0.38
16:4n-3 7.91 ± 0.33 8.28 ± 0.65
18:0 3.27 ± 0.14 4.66 ± 0.06
18:1n-9 24.85 ± 0.81 37.06 ± 1.25
18:2n-6 12.61 ± 0.50 16.57 ± 0.98
18:3n-3 20.78 ± 1.03 21.98 ± 1.72
18:3n-6 1.02 ± 0.04 1.23 ± 0.08
18:4n-3 4.21 ± 0.19 4.46 ± 0.24
SFAs 21.66 ± 0.72Aa (21.23) 26.51 ± 0.94Ba (20.86)
MUFAs 27.18 ± 0.87Ab (26.64) 39.77 ± 1.36Bb (31.29)
PUFAs 53.17 ± 2.29Ac (52.13) 60.82 ± 4.22Bc (47.85)
Total FAMEs 102.01 ± 3.72Ad 127.10 ± 6.28Bd
Glycerol 65.23 ± 2.31 (6.52 dw%)A 110.21 ± 2.68 (11.02 dw%)B

Values in brackets represent the fatty acid proportion (as % of total fatty acids).
SFAs, MUFAs, and PUFAs represent saturated-, monounsaturated- and poly-
unsaturated fatty acids, respectively.
Values with different capital letters in the same row showed significant dif-
ferences (P ≤ 0.05).
Values with different small letters in the same column showed significant dif-
ferences (P ≤ 0.05).

Enhancement of glycerol yield, as a byproduct of transesterification

process, confirms the higher content of storage lipids in the biomass, as
discussed in Section 3.2. In addition, total FAMEs recovery from the
biomass increased by cultivation of S. obliquus in WG2.5, recording
24.6% increase over WW. The proportions of monounsaturated fatty
acids (MUFAs) using WG2.5 increased by 17.5% over that of WW,
which resulted in reduction of polyunsaturated fatty acids (PUFAs)
proportion by 8.2%. The increased percentage of MUFAs was re-
presented mainly by oleic acid (C18:1n9, 29.16%). Moon et al. [67]
confirmed that increasing of oleic acid proportion is desirable to en-
hance the fuel properties of FAMEs. Wilkes et al. [68] attributed the
better quality of biodiesel from plants grown in warmer and drier
seasons to the relatively higher proportions of oleic acid which results
in better cold flow and acceptable fuel properties. In addition, cetane
number is an important parameter that affects the biodiesel quality,
which depends on the fatty acids chain length and degree of un- Fig. 5. Time courses of sugars consumption and ethanol production by fer-
saturation. Thus, FAMEs with high PUFAs content are not desirable mentation of Scenedesmus obliquus intact cells grown in WW (A), lipid-free
biomass of cells grown in WW (B) and lipid-free biomass of cells grown in WW
because they are more susceptible to oxidation during storage [69].
enriched with 2.5 g L−1 waste glycerol (C).
These results implied that addition of low concentrations of WG to the
algal growth medium not only increases the total FAMEs recovery, but
also improves the quality of the produced biodiesel. cost pretreatment and rapid processes of saccharification and fermen-
tation [25,70]. In the present study, efficient bioethanol production was
recorded from lipid-free biomass without additional pretreatment faster
3.4. Bioethanol production
than its production from the whole biomass. Thus, performing lipid
extraction prior to fermentation accelerated the rate of sugar con-
Fig. 5A shows the ethanol production and the reduction in sugar
sumption leading to faster and higher bioethanol production. Due to the
concentration of cells grown in WW. The minimum significant con-
shift in the time of stationary ethanol production from the 33th day to
centration of residual sugars and the maximum significant bioethanol
27th day, it was important to represent the ethanol productivity (Fig. 6).
yield were achieved on the 33th day of fermentation. However, using of
The peak of ethanol productivity was recorded at 24 h in all treatments.
lipid-free biomass increased the rate of carbohydrate conversion,
A maximum ethanol productivity of 0.161 g L−1 h−1 was recorded from
reaching its peak of 4.82 g L−1 at the 27th day (Fig. 5B). Enrichment of
the biomass grown in WW (Fig. 6A). Due to the better assimilation of
growth medium with 2.5 g L−1 of WG and using the residual lipid-free
carbohydrates in lipid-free biomass, it showed increase in maximum
biomass for fermentation showed the highest bioethanol production
bioethanol productivity to 0.185 g L−1 h−1 (Fig. 6B). However, using
peak of 5.58 g L−1 at day 27 (Fig. 4C). As previously discussed, sup-
WG2.5 and sequential fermentation after lipid extraction enhanced the
plementation of culture with organic carbon source such as WG sti-
maximum bioethanol productivity by 32.3% and 15.1% over that of
mulated the metabolism of S. obliquus cells to accumulate more car-
intact cells and lipid-free biomass from WW, respectively (Fig. 6C),
bohydrates. Cost-effective ethanol production requires efficient low-

6
S. Xu, et al.
Fig. 6. Time courses of bioethanol yield and accumulative ethanol productivity
by fermentation of Scenedesmus obliquus intact cells grown in WW (A), lipid-free
biomass of cells grown in WW (B) and lipid-free biomass grown in WW enriched
with 2.5 g L−1 waste glycerol (C).
which resulted in enhancement of bioethanol yield (0.558 g g−1 dw) by
15.8% and 7.9%, respectively. Nguyen et al. [71] recorded bioethanol
yield of 0.292 g g−1 dw from microalgae pretreated with thermal and
chemical methods, which is 47.7% lower than that recorded in the
present study. In addition, the maximum bioethanol yield in the present
study was 45.7% higher than that reported by Harun et al. [72] who
recorded bioethanol yield of 0.383 g g−1 dw from microalgal biomass
pretreated with supercritical CO2. Taking into account the energy
needed to apply the supercritical or thermal pretreatment conditions,
the present study suggested more energy-efficient approach for higher
bioethanol recovery from microalgal biomass.
3.5. Gross bioenergy output
GBO (as GJ ton−1 dry biomass) from each conversion route was
calculated by multiplying the generated quantity of each biofuel with
the corresponding calorific value. According to Fulke et al. [73],
7
Energy Conversion and Management 197 (2019) 111907
Fig. 7. The gross bioenergy output (GBO) and energy conversion efficiency
(ECE) of Scenedesmus obliquus biomass using the studied routes. P1 and P2 re-
present biodiesel or bioethanol production, respectively, from the whole bio-
mass grown in WW. P3 represents bioethanol production from the lipid-free
biomass from WW. P4 and P5 represent biodiesel and bioethanol production,
respectively, from microalgae grown in WG-enriched medium. P6 and P7 re-
present the integrated pathways for biodiesel plus bioethanol production from
biomass grown in WW and WG-enriched medium, respectively.
Sadhukhan et al. [74] and Chisti [75], the calorific values for S. obliquus
biomass, bioethanol and biodiesel are 34.0, 26.7 and 37.8 MJ kg−1,
respectively. The estimated GBO and ECE of the studied pathways are
summarized in Fig. 7. It is noteworthy that the present results are a
simplified analysis where energy input involved in algae cultivation,
harvest, fermentation, and transesterification were not considered. In
general, the GBO and ECE using P1 and P4 showed the lowest values,
confirming that conversion of S. obliquus biomass to bioethanol is more
favorable than biodiesel. Recycling of WG enhanced the GBO for all
conversion pathways. For example, using WG enhanced the biodiesel
production (P4) by 69.2% with respect to that of WW (P1). Similarly,
application of WG for bioethanol production from lipid-free biomass
(P5) enhanced the bioethanol yield by 15.7% over that of WW (P3).
However, the highest GBO of 21.4 GJ ton−1 was obtained from the
integrated seventh route (P7) where S. obliquus was grown in WG-en-
riched medium and sequential fermentation was applied for the re-
sidual biomass, with ECE of 62.9%. The recorded GBO using this con-
version pathway is about 2 times higher than that reported by Elsayed
et al. [76] who obtained GBO of 10.58 GJ ton−1 using sequential fer-
mentation and anaerobic digestion of rice straw. Hence, the current
study confirms the potential of microalgae as a promising feedstock for
biofuel production using sequential fermentation after biodiesel pro-
duction to enhance the energy recovery. Moreover, enrichment of
growth medium with recycled WG will enhance the energy yield and
eliminate the need for nutrients derived from fossil resources as well as
concurrent remediation of biodiesel byproducts.
4. Conclusion
Culture conditions, lipid extraction and biomass pretreatment are
very important factors for liquid biofuel production from microalgae. In
the present study, the green microalga S. obliquus was grown in WW
enriched with different concentrations of biodiesel-derived glycerol,
which significantly enhanced lipid and carbohydrate accumulation.
Biodiesel and/or bioethanol were produced from the biomass, and the
energy outputs of seven conversion pathways were studied. Results
confirmed that recycling of WG as a supplement of organic carbon in
the growth medium has a significant impact on energy recovery, with
highest biodiesel and bioethanol yields of 127.1 mg g−1 dw and
S. Xu, et al. Energy Conversion and Management 197 (2019) 111907

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