Fuel 329 (2022) 125362

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Fuel
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Full Length Article

Experimental investigation on simultaneous production of bioethanol and

biodiesel from macro-algae
Nagarajan Jeyakumar a, *, Anh Tuan Hoang b, *, Sandro Nižetić c,
Dhinesh Balasubramanian a, d, e, *, Sriram Kamaraj f, Prakash Lakshmana Pandian a,
Ranjna Sirohi g, h, Phuoc Quy Phong Nguyen i, Xuan Phuong Nguyen i, *
a
Department of Mechanical Engineering, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India
b
Institute of Engineering, HUTECH University, Ho Chi Minh City, Viet Nam
c
Univeristy of Split, FESB, Rudjera Boskovica 32, 21000 Split, Croatia
d
Mechanical Engineering, Faculty of Engineering, Khon Kaen University, Khon Kaen, Thailand
e
Center for Alternative Energy Research and Development, Khon Kaen University, Khon Kaen, Thailand
f
Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India
g
Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea
h
Centre for Energy and Environmental Sustainability, Lucknow-226 029, Uttar Pradesh, India
i
PATET Research Group, Ho Chi Minh City University of Transport, Ho Chi Minh City, Viet Nam

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

Keywords: The present work compares the biofuel production efficiency of Padina tetrastromatica and Sargassum swartzii
Padina tetrastromatica macro-algae biomass by fermentation with Saccharomyces cerevisiae and Zymomonas Mobilis. The optimal pre­
Sargassum swartzii treatment factors were identified by studying the effect of temperature, pH, and enzyme concentration on the
Bioethanol
conversion of macroalgae biomass to fermentable reducing sugars. The Low concentration of enzyme did not
Biodiesel
Macro-algae
help conversion of biomass to reducing sugars even after 15 h of treatment with enzyme, but when the enzyme
Lipid concentration was increased, the desired conversion was completed within 4 h of enzyme treatment. Both Padina
tetrastromatica and Sargassum swartzii had an ethanol yield of above 80% irrespective of the difference in the
species used for the fermentation. The highest possible ethanol yield (95.12%) was achieved for Padina tetra­
stromatica biomass when it was fermented with Zymomonas Mobilis. From the above results, it is clear that both
Padina tetrastromatica and Sargassum swartzii have the potential to be used as reliable feedstock for biofuel
production. The spent biomass after the fermentation process was used for the lipid extraction process and the
extracted lipid was used for biodiesel production. The percentage of biodiesel yield obtained by means of the
Soxhlet extraction process from Padina tetrastromatica and Sargassum swartzii macro-algae with the conventional
NaOH catalyst was found to be 84% and 89% respectively. MgO.La2O3 was synthesized by means of the co-
precipitation method. With the introduction of MgO.La2O3 nano-catalyst biodiesel yield was improved up to
88% and 92% for Padina tetrastromatica and Sargassum swartzii macro-algae, respectively.

1. Introduction [3,4]. Initially, the first-generation biofuels were obtained from edible
biomass like potato, corn, wheat, barley, sugarcane, sugar beet, and
Increased energy consumption and environmental issues such as air vegetable oils which can replace the usage of fossil fuel combustion and
pollution and global warming arising out of fossil fuels combustion have also reduce atmosphere CO2 emission into the environment [5]. How­
necessitated the search for bio-based resources from renewable fuels ever, the usage of first-generation fuel sources was believed to create
[1,2]. As per the current growth rate, the energy demand of the world is food or fuel crisis. To overcome this problem second-generation biofuels
projected to increase from 12,300 mtoe (2020) to 16,700 mtoe by 2035 were produced from non-edible feedstock like agricultural wastes, wood

* Corresponding authors at: Mepco Schlenk Engineering College (N. Jeyakuma and D. Balasubramanian); HUTECH University (A.T. Hoang); Ho Chi Minh City
University of Transport (X.P. Nguyen).
E-mail addresses: nagarajmepcoeng@gmail.com, nagarajan@mepcoeng.ac.in (N. Jeyakumar), hatuan@hutech.edu.vn (A.T. Hoang), dhineshbala91@gmail.com,
dhineshbala91@mepcoeng.ac.in (D. Balasubramanian), phuong@ut.edu.vn (X.P. Nguyen).

https://doi.org/10.1016/j.fuel.2022.125362
Received 4 January 2022; Received in revised form 26 May 2022; Accepted 18 July 2022
Available online 27 July 2022
0016-2361/© 2022 Elsevier Ltd. All rights reserved.
N. Jeyakumar et al. Fuel 329 (2022) 125362

Fig. 1. Critical steps for bioethanol and biodiesel production from algae [17].

residual waste, and energy crops [6]. The quantity of carbon emitted or attention in terms of its economic prospectus. The pathway of algal
consumed for the second-generation biofuels is negative or neutral [7]. liquid biofuel production includes mass cultivation, biomass harvesting
However, the seasonal dependence on raw materials is the major and drying, and extraction of lipid and finally the lipid extracted was
drawback of the second-generation fuels. Third-generation biofuels chemically converted to biofuel [16]. The bioethanol and biodiesel
include algae which is an effective alternative renewable source for synthesis obtained from algae biomass consists of the following steps as
biofuel production which overcomes the disadvantage of the first and given in Fig. 1.
the second-generation biofuels [8,9]. The algal biomass can be culti­ Bioethanol produced from algal sources has gained importance
vated by any of three mass cultivation techniques namely mixotrophic, because of increased heat of vaporization and octane rating in com­
heterotrophic, and autotrophic methods [10,11]. Algal species unlike parison with the conventional fuels [18]. Bioethanol that is considered
aquatic organisms lack true stems and roots and they contain chloro­ to be clean and renewable energy, obtained from various feedstocks is a
phyll pigments. Based on morphology they can be classified as micro­ top alternative to gasoline fuel [19,20]. Bioethanol obtained from the
algae and macro-algae also they vary from unicellular blue-green algae first-generation feedstock when produced in large quantity may lead to
to multicellular form [12,13]. The growth rate of algae is about 20 to 30 food shortage globally whereas the production of bioethanol from the
times higher than the fodder crops and also about 30 times more oil second-generation lignocelluloses materials is not found to be viable on
content than conventional feed stock was observed. The lipid content of the industrial scale [21,22]. Macro-algae have an increased potential in
the macro-algae ranges from 1 to 5% and the oil from macro-algae has bioethanol production as they possess a superior growth rate and have
high quality [14]. Various methods of offshore and onshore cultivation the capability to make huge biomass in comparison with any other
were adopted for macro-algal species. Kelp growth, floating and raft terrestrial plant in the world [23,24]. Among the bioethanol production
cultivation comes under offshore cultivation techniques. On shore methods, biomass fermentation employs ethanologenic microbes in
cultivation using sea water has the benefit of increased yield [15]. anaerobic or semi anaerobic conditions which come under a biological
Indeed, the mass growth of algae nowadays is drawing considerable approach. The fermentation includes the bioconversion of

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N. Jeyakumar et al. Fuel 329 (2022) 125362

carbohydrates into organic acids or alcohols through glycolytic inter­ specific method in the pre-treatment of macro-algae biomass but it is a
mediate. There are two main steps involved in the bioprocessing of costlier method in comparison with acid treatment. The combination of
carbohydrate-carrying feedstock for bioethanol production. The pri­ acid treatment followed by enzyme treatment provides the synergic ef­
mary step includes polysaccharides hydrolysis into fermentable sugars fect in the synthesis of reduced sugar from the macro-algae biomass. The
which are accompanied by the conversion of fermentable sugars into proper optimization of the pre-treatment method is still lacking in the
bioethanol by deploying suitable microorganisms for the fermentation macro-algae bioethanol production research. Hence, the current work
process [25]. This is followed by the purification and concentration of focuses on the optimization of the pre-treatment method to enhance
bioethanol by means of the distillation process [26]. Several methods bioethanol production from the macro-algae. Also, limited biofuel syn­
are available for the pretreatment of macro-algae species to increase the thesis works have been performed with macro-algae.
reducing sugar extraction [27]. Among them, mechanical pre- In the present work, the bioethanol yield produced from two macro-
processing accompanied with acid and enzymatic hydrolysis was algae species Padina tetrastromatica and Sargassum swartzii is studied
found to enhance the saccharification efficiency in comparison with the after the treatment of these macro-algae with different acid treatment
other pretreatment processes [28]. Mechanical pre-processing of macro- and enzyme treatment methods. The difference in ethanol production is
algal species includes processes such as washing, drying & milling. In the also compared with the Saccharomyces cerevisiae yeast species and
preliminary preprocessing step, the newly collected algal biomass is Zymomonas Mobilis fungal species. The spent biomass after the fermen­
cleaned by water to remove the dust, sand, epiphytes, salt, and adhering tation process is used for the lipid extraction process and the extracted
harmful materials. This is followed by a drying process to remove the lipid is used for biodiesel production.
water content to increase the shelf life of biomass done by a hot air oven
or in the presence of sunlight. The last step is milling to reduce the size of 2. Materials and methods
the macro-algal biomass followed by seizing. This can be attributed to
increased reaction efficiency due to smaller-sized particles during Biodiesel is produced from algal biomass by means of trans­
anaerobic fermentation for bioethanol production [29]. During the acid esterification process. By adopting saccharification and fermentation
hydrolysis of macro-algal polysaccharides under strongly acidic condi­ methods bioethanol can be obtained from macro-algae.
tions, the inhibitory products like 5-hydroxy methyl furfural, formic
acid, aldehydes and levulinic acid are produced which will negatively 2.1. Macro-algae
impact the fermentation process [30,31]. To hydrolyze the wide variety
of complex macro-algal polysaccharides such as starch, cellulose, and Padina tetrastromatica which is fan shaped yellowish-brown marine
agar into monomeric reducing sugar like galactose and glucose, com­ algae is found largely in the shallow waters of mid and lower coastal
mercial enzymes like cellulose, amylase, and agarase are used for parts of India. This biomass is found to contain a substantial quantity of
enzymatic pretreatment [32–34]. The potential factors that affect the carbohydrate and lipid thus it is considered to be an excellent feedstock
pretreatment process are temperature, pH, time for treatment, substrate for biofuel production. In our current work, Padina tetrastromatica was
concentration, and chemical or biological agents used for the conversion collected from Rameshwaram, Mandapam region (09o17lN Lat, 79o08lE
[35]. Macro-algae cultivated through large ocean ecosystem do not Long) palk pH bay, Tamil Nadu, India. Another potential macro-algae
require land, sunlight and fertilizers [36]. species selected for our studies for biofuel production is Sargassum
Biodiesel is one of the most widely used fuel sources for the auto­ swartzii. It was collected from Mandapam (9o16l54.03llN;
mobile industry [37]. Biodiesel production costs can be reduced for 79o11l20.50llE) located at the Gulf of Mannar coast, Tamilnadu, India.
commercialization by exploration of non-edible cost-effective feed­
stocks. A wide variety of naturally grown macro-algal species with high 2.2. Biomass selection for bioethanol production
lipid content is available for biodiesel production which can reduce the
cost of production of biodiesel [38]. Macro-algae species are free from Sargassum swartzii and Padina tetrastromatica are two macro-algae
Sulphur and highly biodegradable. Many works cited in the literature species selected for ethanol production which belong to the brown
have concluded that algae biodiesel is environmentally safe and can be algae family. The reason for the selection of these macro-algae for our
directly used in diesel engines [39,40]. study is they contain high carbohydrate content such as glucose, starch,
As the carbohydrate content in the macro-algae is high, it attracts as cellulose, mannitol, glucan, alginate, laminarin, galactose, etc. which
a potential source for bioethanol production. However, the major could be effectively used for ethanol production [42]. The collected
obstacle in the utilization of these marine macro-algae comes from the macro-algae were washed with distilled water to remove the impurities.
presence of stronger hydrocolloid cell walls. Hence, the pre-treatment is After the washing, the macro-algae were dried in the normal sunlight for
the necessary step for the breakdown of these hydrocolloid-protected 24 h to remove the residual moisture. The dried algae biomass was
cellular structures and the conversion of carbohydrates into sugar. powdered then it was sieved with the help of 300-µm mesh to collect
Bioethanol from macro-algae involves pretreatment methods which uniform size biomass particles. Finally, the prepared biomass powder
include washing, drying, milling, and homogenization accompanied by was stored in the air-tight container for further processing. Experimental
physicochemical & enzymatic pretreatment to obtain the monomeric results have shown that sulfated polysaccharides obtained from
sugars for fermentation. Then the fermentation of biomass algae could Sargassum swartzii can be used as a better source of antioxidants [43].
be carried out using baker’s yeast called Saccharomyces cerevisiae [41].
The required macro-algal samples have been collected from the destined 2.3. Extraction of crude lipid
area. Preprocessing is followed by pretreatment methods like mechan­
ical, chemical, and enzymatic hydrolysis. Fermentable sugars are pro­ Dried and grounded biomass of about 30 g was taken in a 250 ml
duced from complex carbohydrates by the above process. In the final conical flask and added with 150 ml of distilled water, which was son­
steps, the monomeric sugars are transformed to bioethanol utilizing icated for about 5 min. The crude lipid extraction was carried out by
specific microbes [29]. The effective pre-treatment method reduces the means of the Soxhlet apparatus. The solid material consisting of
cost of ethanol production significantly. A wide range of pre-treatment powdered algae was kept inside the thimble made of thick filter paper
methods such as ultra-sonication, ozone treatment, microwave treat­ which was loaded to the main chamber of the Soxhlet extractor equip­
ment, and acid hydrolysis are available. Among these methods, acid ped with a condenser. The extractor was kept inside the flask having the
hydrolysis is cost-efficient in comparison with the other methods. extraction solvent. The solvent employed in the extraction process was
However, the release of the by-products reduces the efficiency of car­ chloroform and methanol (2:1). Once the solvent was heated it evapo­
bohydrates into fermentable sugars. The enzyme hydrolysis is the more rated and moved up of the distillation arm and flooded into the chamber

3
N. Jeyakumar et al. Fuel 329 (2022) 125362

Fig. 2. Process layout in bioethanol and biodiesel production from algae; (a) - Flow chart of Sargassum swartzii and Padina tetrastromatica macro-algae species powder
conversion; (b) - Distillation of fermented enzymatic hydrolysate to collect alcohol.

housing the solid thimble. The condenser condensed the vapor which A fraction of the non-volatile compound dissolves in the solvent
dripped down back into the main chamber containing the solid material. during each cycle. After the extraction was over, the obtained crude lipid
Thus, the chamber contained the solid material filled with warm solvent sample was filtered by means of Whatman filter paper to separate the
at a slower rate. When the Soxhlet chamber became almost full, the solid particles entrained during extraction. This was followed by a
chamber was emptied automatically by means of a siphon sidearm, with simple distillation process to separate the crude algal lipids from the
the solvent running back again to the distillation flask. This cycle was solvents. The non-soluble part of the extracted solid remained in the
repeated many times over hours. thimble which was then discarded. After the pre-extraction of lipid,

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N. Jeyakumar et al. Fuel 329 (2022) 125362

2.5. Fermentation of algal hydrolysate

The pH level of the hydrolysate obtained after the acid and the
enzyme treatment was adjusted by using sodium hydroxide solution.
The resulting biomass was sterilized in the autoclave for 15 min to
ensure the removal of the microbial contamination and inhibitors pro­
duced during the hydrolysis step. The sterilized biomass was taken with
the supplements that were required for the fermentation process. The
ethanol fermentation was performed by the Saccharomyces cerevisiae
(yeast strain) and Zymomonas mobilis (bacterial strain). The preparation
of media was performed as follows [51]. Briefly, 2.5 g of Luria-Bertani
medium was prepared and then autoclaved at a temperature of 121℃.
After autoclaving, 5 g of glucose solution was added as the initial carbon
source. Then the prepared medium was inoculated with 0.5 g/Lt of
Zymomonas Mobilis culture [52]. For the fermentation of biomass with
Saccharomyces cerevisiae, 2 wt% yeast peptone dextrose medium was
used. The prepared medium was autoclaved at 121℃ for 20 min. After
autoclaving, 5 g of glucose solution was added as the initial carbon
source. Then the prepared medium was inoculated with 0.5 g/Lt of
Fig. 3. FTIR of catalyst MgO.La2O3. Saccharomyces cerevisiae culture [53]. Fig. 2a represents the flow chart
of Sargassum swartzii and Padina tetrastromatica macro-algae species
quality biomass rich in carbohydrates by-products was thrown away as powder synthesis, and Fig. 2b shows the distillation of fermented
waste. This can be alternatively used as a biofertilizer for agricultural enzymatic hydrolysate to collect alcohol.
purposes. Also, the residual biomass rich in carbohydrates can be The ethanol content after the fermentation process was estimated by
recycled for bioethanol production as a potential substrate [44–46]. the chemical method [54]. Briefly, 100 μL of the fermented medium was
added to 1 ml of the dichromate solution (9 wt%) and 2.9 ml of the
2.4. Optimization of acid and enzyme hydrolysis concentrated perchloric acid in a 10 ml Erlenmeyer flask. The resulting
solution was mixed properly and kept at 25 ◦ C for 20 min and then
The optimization of the acid hydrolysis was performed for the macro- diluted to the mark with water. Then the absorbance was measured at
algae biomass with three different acids, namely hydrochloric acid, 267 nm to find the concentration of ethanol existing in the medium of
phosphoric acid, and sulfuric acid. The optimization was performed to fermentation. Ethanol yield was calculated by the following formula.
find the optimal concentration of acid solution required for hydrolysis
Concentration of Ethanol (g/L)
[47]. The preliminary study of acid hydrolysis with three acids sug­ Ethanol yield (%) = × 100%
Initial concentration of sugar(g/L)
gested that hydrochloric acid is effective against Padina tetrastromatica
and Sargassum swartzii. Hence, the hydrolysis process was proceeded
with hydrochloric acid for further analysis to find the optimal acid 2.6. Synthesis of nano catalyst
concentration for the hydrolysis of two different macro-algae strains.
Briefly, 0.5 mg of powdered algae was added to 5 ml of acid solutions MgO.La2O3 was synthesized using the co-precipitation method.
with various concentrations (2–8 wt%) and the resulting solution was Required quantities of aqueous Mg (NO3)2⋅6H2O (1 M) and La (NO3)
subjected to a temperature of 121℃ in the autoclave for 20 min. The 3⋅.6H2O (1 M) were premixed and the obtained solution was heated to
acid pretreatment reaction was stopped by raising the solution pH with about 343 K in a round bottom flask equipped with a condenser. To the
the help of 2 N sodium hydroxide solution [48]. The acid-treated mixed nitrate solution, aqueous Na2CO3 (1 M) was added drop by drop
biomass was taken for the centrifuge at 8000 rpm for 10 min to sepa­ with constant stirring and temperature maintained at 30 ◦ C till a pH
rate the fine acid-treated biomass particles from the acidic solution. The value of 12 was reached. The obtained precipitate was kept within the
excess acid and fermentation inhibitory materials developed during the medium for about 20 min. and finally, the gel suspension was filtered
acid pretreatment were removed by the consequent centrifugation fol­ and washed with warm distilled water many times. Following that the
lowed by the washing of collected biomass with distilled water until the precipitate was dried in an oven for about 16 h at 353 K and the product
wash liquid reaches the neutral pH. The neutralized acid-treated obtained was then calcined in a furnace to derive the final catalyst i.e.,
biomass was subjected to the enzyme hydrolysis by treating it with MgO.La2O3 [55]. The wavelength peak at 531 cm− 1 corresponded to
α-amylase enzyme [49]. The effect of enzyme concentration on the MgO stretching bond [56]. The presence of broad absorption bands in
conversion of biomass into sugar was studied with the different con­ the wavelength spectra of 648–663 cm− 1 corresponded to the presence
centration ranges of enzymes. of LaO nanoparticles. The existence of absorption bands in the spectral
A 10 ml of the acid hydrolyses sample was taken for the enzyme wavelength of 1363 cm− 1 confirms the presence of COO– stretching
hydrolysis optimization study. The concentration of the enzyme was [57]. FTIR and SEM images of MgO.La2O3 were shown in Fig. 3 and
taken in the range of 5 mg/ml to 75 mg/ml. The time for enzyme hy­ Fig. 4. From the SEM image, the particle size was found to be around
drolysis was also taken as a variable and it was increased from 4 h to 42 300 nm.
h. After the enzyme hydrolysis, the Total Reducing Sugar (TRS) content
produced by the biomass was identified with the help of the DNS (dinitro 2.7. Lipid extraction and biodiesel production
salicylic acid) method using glucose as standard [50]. This array formed
a colored complex with ethanol at 550 nm. The ethanol concentration The spent solids after ethanol production were subjected to the
after the fermentation was measured by noting the absorption at 550 nm Soxhlet extraction process for the separation of lipid for biodiesel syn­
in the spectrometry analysis. The effect of enzyme concentration and thesis. The free fatty acid content of lipid extracted from algal biomass
time of reaction were studied to find the optimal enzyme concentration was found to be less than 2%. To synthesize biodiesel from the collected
and the time required for the enzyme hydrolysis. After optimum process lipid, about 0.5 g of NaOH pellets was added to 50 ml of methanol to
parameters were identified, the bulk macro-algae biomass was hydro­ produce sodium methoxide solution. Then the sodium methoxide solu­
lyzed in this condition and it was taken for the fermentation process. tion was mixed slowly with algal oil. The reaction conditions were

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N. Jeyakumar et al. Fuel 329 (2022) 125362

Fig. 4. SEM image of MgO.La2O3.

Fig. 5. Soxhlet extraction process for lipid synthesis.

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N. Jeyakumar et al. Fuel 329 (2022) 125362

Fig. 6. FTIR of lipid samples from spent biomass.

and glycerol at the bottom. The biodiesel was separated and washed
Table 1
with distilled water. The percentages of biodiesel yield obtained from
FTIR spectra of lipid synthesized from Sargassum swartzii and Padina
Padina tetrastromatica and Sargassum swartzii macro-algae with con­
tetrastromatica.
ventional NaOH catalyst were found to be 84% and 89% respectively by
Sargassum swartzii Padina tetrastromatica Reference Stretching means of the Soxhlet extraction process [58].
3345 3357 3010 =C–H Fig. 5 shows the spent biomass collected after bioethanol production
2944 2957 2925 –CH2 by fermentation followed by the Soxhlet Extraction process for lipid
2850 2846 2855
synthesis for biodiesel production. With the introduction of MgO.La2O3
–CH3
1416 1423 1460 CH2
1028 1026 1070–1250 C–O–C nano-catalyst biodiesel yield was improved up to 88% and 92% for
– 756 720 CH2 rocking Padina tetrastromatica and Sargassum swartzii macro-algae respectively.
The FTIR of lipid samples extracted from the spent biomass of Padina
tetrastromatica and Sargassum swartzii macro-algae were shown in Fig. 6.
maintained such as methanol to oil ratio of 8:1, the reaction speed of The lipid content can be identified by the presence of spectral peaks
450 rpm, and reaction temperature of 60 ◦ C with the reaction time of 90 corresponding to wavelength using FTIR. Table 1 shows the comparison
min. The entire mixture collected after the transesterification process of wavelength with the reference spectral peaks (FTIR spectroscopy for
was then transported to a separating funnel and kept for about 72 h. The evaluation and monitoring of lipid – Kristin) [59].
mixture was separated into two distinct layers with alkyl esters at the top

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N. Jeyakumar et al. Fuel 329 (2022) 125362

Fig. 7. The concentration of total reducing sugar after acid hydrolysis; (a) - For Padina tetrastromatica; (b) – For Sargassum swartzii.

2.8. Characterization of the synthesized biodiesel using GC–MS chromatogram obtained with Padina tetrastromatica and Sargassum
swartzii biodiesel was recorded for fatty acid content analysis [60].
The synthesized biodiesel from macro-algae species was collected
and stored. Gas chromatography (7890A model, Agilent technologies 3. Results and discussion
make) provided with a flame ionization detector was employed to
measure the fatty acid content. The silica capillary column (HP-88, USA 3.1. Effect of acid concentration on total reducing sugar yield
model, Agilent make) with film thickness of 300 m × 0.25 mm × 0.20
µm was employed in this study. In the present analysis, helium was used The selection of pre-treatment method and amount of total reducing
as a carrier gas. Hexane and methyl heptadecanoate were employed as a sugars (TRS) released from the macro-algae species are based on their
solvent and internal standard with the biodiesel analysis. The bio-compositions. The bio composition of marine algae is based on

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N. Jeyakumar et al. Fuel 329 (2022) 125362

Table 2
Concentration of TRS after enzyme hydrolysis for Sargassum swartzii and Padina tetrastromatica.
Reducing sugar obtained from Sargassum swartzii (g/L) Reducing sugar obtained from Padina tetrastromatica (g/L)

Time/concentration of enzyme 5 Units/ml 10 Units/ml 15 Units/ml 5 Units/ml 10 Units/ml 15 Units/ml

15 h 4.78 ± 0.12 5.01 ± 0.1 4.71 ± 0.15 4.53 ± 0.2 5.13 ± 0.15 4.74 ± 0.16
21 h 4.53 ± 0.07 4.95 ± 0.14 4.99 ± 0.3 6.13 ± 0.06 6.15 ± 0.15 6.12 ± 0.1
42 h 5.98 ± 0.13 5.20 ± 0.3 5.53 ± 0.14 5.74 ± 0.12 5.57 ± 0.15 5.95 ± 0.12

Time/concentration of enzyme 25 Units/ml 50 Units/ml 75 Units/ml 25 Units/ml 50 Units/ml 75 Units/ml

4h 22.9 ± 0.12 26.4 ± 0.1 28.6 ± 0.15 26.4 ± 0.08 21.2 ± 0.15 24.4 ± 0.05
5h 26 ± 0.17 21.2 ± 0.12 21.8 ± 0.15 29.4 ± 0.13 26.2 ± 0.2 27.4 ± 0.18

Fig. 8. Reducing sugar and ethanol yield from algae after using various fermentation methods; (a) - Zymomonas mobilis using Sargassum swartzii; (b) - Zymomonas
mobilis using Padina tetrastromatica; (c) - Saccharomyces cerevisiae using Sargassum swartzii; (d) - Saccharomyces cerevisiae using Padina tetrastromatica.

salinity, temperature, light availability of the water medium through algae species is less than one percent which makes the gentle pre-
which macro-algae is grown. The pre-treatment process is essential for treatment methods enough for the release of sugars in comparison
the macro-algae as they contain complex polysaccharides such as with terrestrial plants [63]. The acid hydrolysis results for Padina tet­
hemicelluloses, cellulose, agar, alginate, mannitol, laminarin, ulvan, rastromatica and Sargassum swartzii treated with hydrochloric acid for
and carrageenan which cannot be used directly as an energy source by 20 min duration are shown in Fig. 7.
microorganisms for the fermentation process. Hence, these poly­ The pretreatment reaction time and the acid concentration range
saccharides need to be converted into fermentable sugars such as were fixed based on literature reading from previous related articles
glucose, xylose, arabinose, galactose, mannose, and fucose for the con­ [64]. From Fig. 7a, the acid hydrolysis results in the range of concen­
version of algae biomass into bioethanol [61]. Commonly, brown algae tration from 2 to 8 wt% Show that the maximum achievable TRS content
species contain hemicelluloses as a major component which made the (6.12 mg/ml) has been obtained at 6 wt% Hydrochloric acid pre-
acid treatment essential for the release of reducing sugar for the treatments. When the acid concentration is increased beyond 6%, the
fermentation process [62]. However, the lignin content in the brown TRS content after hydrolysis from Padina tetrastromatica is reduced to

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N. Jeyakumar et al. Fuel 329 (2022) 125362

Table 3
Comparison of ethanol yield with other previous works.
Biomass algae Species Initial pH Temperature Time Reference Ethanol yield%
(oC) (hr)

Sargassum swartzii Saccharomyces cerevisiae 4 40 60 Present work 86.27 ± 1.5

Sargassum swartzii Zymomonas mobilis 4 40 60 85.85 ± 1.5
Padina tetrastromatica Zymomonas mobilis 4 40 60 95.12
Padina tetrastromatica Saccharomyces cerevisiae 4 40 60 62.13 ± 1.5
Sargassumspp Saccharomyces cerevisiae 4.5 40 72 [42] 89.1
Padina tetrastromatica Saccharomyces cerevisiae 4 30 72 [71] 83.4
Kappaphycusalvarezii Saccharomyces cerevisiae 5 30 60 [76] 92.2
Porphyridiumcruentum Saccharomyces cerevisiae 4.8 30 6 [77] 70.3
Gelidiumamansii Saccharomyces cerevisiae 4.8 37 12 [78] 84.9

Fig. 9. FTIR spectra of bioethanol from algal biomass.

5.16 mg/ml. This result has shown that an increase in the acid content difference in the optimal pre-treatment acid concentration for the Padina
from 2% to 6% increases the conversion of macro-algae biomass to TRS. tetrastromatica and Sargassum swartzii may be due to the biomass
Further increase in acid concentration reduces the TRS concentration. composition difference of these species. The fiber content in the
This may be due to the occurrence of side reactions that facilitate the Sargassum swartzii is less than that of Padina tetrastromatica which causes
production of 5-hydroxy-methyl-furfural and organic acids in extreme the reduction in the concentration of acid required for the pretreatment
pH condition [65,66]. When the results were compared with the alkali of Sargassum swartzii biomass [67].
hydrolysis of Padina by Ashok et al. [71], it could be observed that the
acid hydrolysis requires more concentration (6% of HCl) of acid in 3.2. Effect of α-amylase enzyme concentration on hydrolysis
comparison with the alkali treatment (2.5% of NaOH) for the production
of reducing sugar. The enzyme pretreatment is essential for the hydrolysis of algae
The acid hydrolysis results for Sargassum swartzii are given in Fig. 7b. biomass at a lower temperature. Several enzymes such as amylases,
The results show that the TRS concentration obtained after the hydro­ xylanases, and cellulases have been successfully used for the pretreat­
lysis is high at a low acid concentration (2 wt%). However, an increase ment of algae biomass for the extraction of reducing sugar. Kim et al.
in the acid content reduces the total reducing sugar yield from the acid [68] used the cock tail enzyme with the combination of α-amylase,
hydrolysis. The gradual reduction in the concentration of TRS is cellulase, and β-glucosidase which is extracted from aspergillus fungus
observed for the increase in the acid concentration from 2 to 8 wt%. This for the pretreatment of Enteromorpha intestinalis. Table 2 shows the
is because of the activation of more by-product formation mechanisms concentration of total reducing sugar after enzyme hydrolysis of
which leads to the formation of other organic compounds with the in­ Sargassum swartzii and Padina tetrastromatica with different concentra­
crease in the concentration of acid solution for hydrolysis. The optimal tions of α-amylase enzyme and pretreatment time. The sugar release
concentration for the pre-treatment of Padina tetrastromatica is 6 wt% from the biomass is very low for both Sargassum swartzii and Padina
But for the Sargassum swartzii the optimal concentration it is 2 wt%. The tetrastromatica treated with a low concentration of enzyme in the range

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N. Jeyakumar et al. Fuel 329 (2022) 125362

enzyme associated with ultra-sonification for the pre-treatment of Ulva

rigida macro-algae biomass. This result was similar for both Sargassum
swartzii and Padina tetrastromatica macro-algae. Hence, the enzyme
concentration of 25 Units/ml and 5 hrs of enzyme hydrolysis time were
fixed as an optimal pretreatment condition for the fermentation of
macro-algae biomass. The enzyme hydrolysis enhances the release of the
sugars from the macro-algae biomass. The combination of acid hydro­
lysis and enzyme treatment enhances the sugar release from the macro-
algae [70].

3.3. Ethanol fermentation by batch cultivation

In earlier studies such as Borines et al. [42] dilute (3.4–4.6%) H2SO4

and cellulase enzyme for the pretreatment of Padina tetrastromatica
macro-algae and the hydrolysate was taken for the fermentation at the
temperature and pH condition of 40 ◦ C and 4.5 respectively for 72 hr by
Saccharomyces cerevisiae, which led to the ethanol yield of 89%. Several
works have been reported based on the simultaneous extraction of both
biodiesel and bioethanol from the algae biomass. Ashok et al. [71] re­
ported an ethanol yield of 83% for the spent biomass that was used for
the oil extraction process for the biodiesel synthesis. Harchi et al. [70]
have prepared bioethanol with the yield of 73% by yeast strain Pachy­
solen tannophilus using Ulva rigida macro-algae as a sole carbon source.
These previous studies provide the anticipation that the macro-algae
biomass could be used as a great source for the production of biofuels
with a high yield. In our current study, Sargassum swartzii and Padina
biomasses were acid hydrolyzed for 20 min with 2 wt% and 6 wt% HCl
respectively, then enzyme-treated for 4hr to produce optimum reducing
sugar concentration. The dilute acid and enzyme pretreated biomasses
were taken for the fermentation process. The sugar content change and
the conversion of the ethanol during the fermentation process were
monitored for 60 hrs of operation. The final ethanol yield obtained after
60 hrs of fermentation of Sargassum swartzii by Zymomonas mobilis and
Saccharomyces cerevisiae were 85.85 ± 1.5% and 86.27 ± 1.5% respec­
tively. Similar kinds of bioethanol yield results were obtained by Borines
et al. [42] and Ardalan et al. [72] for the fermentation of Sargassum
swartzii and Sargassum angustifolium biomass using Saccharomyces cer­
evisiae. The percentage yield of ethanol produced from Padina by
Zymomonas mobilis and Saccharomyces cerevisiae were 95.12% and 62.13
± 1.5% respectively. The reducing sugar consumption and yield of
ethanol during the different time intervals of the fermentation process
are given in Fig. 8(a-d).
Fig. 8a represents reducing sugar and ethanol yield for the fermen­
tation of Sargassum swartzii using Zymomonas mobilis bacterial culture.
The reducing sugar content decreases with the increase in the fermen­
tation time. The consumption rate of reducing sugar is almost constant
up to 38 hrs and reached its stationary phase. The sugar content of the
Sargassum swartzii biomass medium gradually reduced from the initial
concentration of 26 ± 0.17 g/L to 4.9 ± 0.15 g/L during the fermenta­
tion process for 60 hrs using Zymomonas mobilis. The rate of increase of
ethanol yield is less up to 24 h. However, the ethanol yield increases
rapidly from 24 hrs to 48 hrs and it reaches saturation point. Fig. 8b
represents reducing sugar and ethanol yield for the fermentation of
Padina tetrastromatica using Zymomonas mobilis bacterial culture. The
Fig. 10. GC/MS analysis after fermentation of; (a) - Sargassum swartzii oil; (b) - reducing sugar content decreases linearly up to 48 hr. and reached its
Padina tetrastromatica oil. stationary phase. For Padina tetrastromatica, the sugar concentration of
medium reduces to 2.4 g/L from the initial concentration of and 29.4 ±
from 5 Units/ml to 15 Units/ml even for the long enzyme treatment 0.13 g/L for the initial 38 hrs of the fermentation process and the rate of
period. However, once the enzyme concentration is increased to 25 consumption of the reducing sugar is linear during this phase. In a later
Units/ml, the sugar concentration rapidly increased to 26 ± 0.17 g/L period, the rate of sugar consumption reaches its stationary phase and
and 29.4 ± 0.13 g/L for Sargassum swartzii and Padina tetrastromatica the sugar content is reduced to 1.71 g/L.
respectively, within 5 hrs of pre-treatment time. The increase of enzyme The rate of increase of ethanol yield is rapid up to 24hrs of fermen­
concentration above 25 Units /ml does not show reasonable improve­ tation to reach 76.2 ± 2.7% and it reached the max yield of 95.12 ±
ment in the conversion of biomass into the reducing sugar. Korzen et al. 1.5% in 60 hrs. Fig. 8c represents reducing sugar and ethanol yield for
[69] also obtained similar kinds of results when they used α-amylase the fermentation of Sargassum swartzii using Saccharomyces cerevisiae
yeast culture. The rate of decrease of reducing sugar is almost constant

11
N. Jeyakumar et al. Fuel 329 (2022) 125362

Table 4
Fatty acid composition of Padina tetrastromatica biodiesel.
Peak Peak time/ Area, Fatty acid methyl ester Fatty acid Carbon PubChem CID/ IUPAC name Class compound
no. min % number CAS

1 7.108 1.01 Phenol, 2,4-bis (1,1- Myristic Acid C14:0 000096–76-4 (3,5-ditert-butylphenoxy)- –
dimethylethyl trimethyl silane
2 11.694 8.47 n-Hexadecenoic acid Oleic acid C18:1 000112–79-8 octadec-9-enoic acid Saturated fatty acid
3 11.779 3.81 Pentadecanoic acid Pentadecanoic C15 001002–84-2 Pentadecanoic acid –
acid
4 11.883 10.27 Tridecanoic acid Tridecanoic acid C13 000638–53-9 Tridecanoic acid –
5 13.273 0.57 6-Octadecenoic acid, Oleic acid C18:0 000593–39-5 (Z)-octadec-6-enoic acid Saturated fatty acid
(Z)-
6 13.528 51.09 cis-Vaccenic acid Oleic acid C18:2 000506–17-2 (Z)-octadec-11-enoic acid Poly-unsaturated
fatty acid
7 13.67 9.29 cis-Vaccenic acid Oleic acid C18:2 000506–17-2 (Z)-octadec-11-enoic acid Poly-unsaturated
fatty acid
8 16.327 3.71 trans-13-Octadecenoic Oleic acid C18:1 000693–71-0 (E)-octadec-13-enoic acid Mono-unsaturated
acid fatty acid
9 16.516 0.93 cis-9-Hexadecenoic acid Palmitic acid C16:1 1000333–19-5 (E)-hexadec-9-enoic acid Saturated fatty acid
10 16.639 1.69 Petroselinic acid, methyl Octadecenoic C19 052380–33-3 methyl (Z)-octadec-11-enoate –
ester acid
11 17.773 3.61 Cinnamyl cinnamate – C18 000122–69-0 [(E)-3-phenylprop-2-enyl] (E)-3- –
phenylprop-2-enoate
12 17.981 1.03 1H-Indole, 1-methyl- Nethylindole C9 000603–76-9 1-methyl-2-phenylindole –
13 18.17 4.54 13-Tetradecen-1-ol Palmitic acid C16:1 056221–91-1 tetradec-13-enyl acetate Saturated fatty acid
acetate

Table 5
Fatty acid composition of Sargassum swartzii biodiesel.
Peak Peak time/ Area Fatty acid methyl ester Fatty acid Carbon PubChem CID/ IUPAC name Class compound
no. min % number CAS

1 5.462 0.34 Cyclododecane – C12:0 000294–62-2 2,3-dihydroxypropyl (E)-

octadec-9-enoate
2 7.098 0.85 Phenol, 2,4-bis(1,1- Myristic acid C14:0 000096–76-4 (3,5-ditert-butylphenoxy)- –
dimethylethyl trimethylsilane
3 7.911 0.48 1-Octadecene Oleic acid C18:1 000112–88-9 octadec-1-ene Mono-unsaturated
fatty acid
4 10.086 0.47 1-Pentadecanol acetate Heptadecanoic C17:0 000629–58-3 pentadecyl acetate –
acid
5 11.873 1.23 n-Hexadecanoic acid Palmitic acid C16:1 000057–10-3 hexadecanoic acid Saturated fatty acid
6 12.1 0.8 1-Octadecene Oleic acid C18:1 000112–88-9 octadec-1-ene Saturated fatty acid
7 12.554 0.37 7,11-Hexadecadienal Palmitic acid C16:1 1000130–85-7 (7E,11E)-hexadeca-7,11- Saturated fatty acid
dienal
8 13.131 0.8 Octadec-9-enoic acid Linolenic acid C18:2 000593–39-5 octadec-9-enoic acid Poly-unsaturated
fatty acid
9 13.613 1.63 6-Octadecenoic acid Stearic acid C18:0 000593–39-5 (Z)-octadec-6-enoic acid Saturated fatty acid
10 13.944 1.75 Bromoacetic acid, – C12:0 018992–03-5 octadecyl 2-bromoacetate –
octadecyl ester
11 15.182 3.96 6-Octadecenoic acid Stearic acid C18:0 1000336–66-8 (Z)-octadec-6-enoic acid Saturated fatty acid
12 16.355 4.6 Benzo[h]quinoline, 2,4- – C15 000605–67-4 2,4-dimethylbenzo[h] –
dimethyl quinoline
13 17.575 18.83 Pyridine – C5 005048–08-8 Pyridine –
14 17.924 10.28 2-Ethylacridine – C15 055751–83-2 2-Ethylacridine –
15 18.917 25.89 2-Methyl-7-phenylindole – C15 001140–08-5 2-methyl-7-phenyl-1H-indole –
16 20.194 11.34 2,3-Dihydroxypropyl – C21 002716–53-2 2,3-dihydroxypropyl (E)- –
elaidate octadec-9-enoate

up to the first 48 hrs of fermentation and a further increase in time

Table 6 reduced the sugar consumption marginally. The sugar content of the
Comparison of biodiesel yield obtained from different feedstock. Sargassum swartzii biomass medium gradually reduced from the initial
Algae feedstock Catalyst Biodiesel yield Reference concentration of 26 ± 0.17 g/L to 5.1 ± 0.15 g/L during the fermenta­
(wt. %) tion process for 60hrs using Zymomonas mobilis. The rate of increase of
Cyanobacteria MgAl–LDO/ZSM 41.1 [79] ethanol yield is linearly increased up to 48 h of fermentation to reach
− 5 81.27 ± 0.7% and it increases up to 86.27 ± 1.5% in 60 hr. The results
Chlamydomonas β Zeolite 43.8 [80]
are similar to those of the previous work done on Kappaphycus alvarezii
debaryana
Ulva prolifera HY 41.3 [81] macro-algae for the production of bioethanol by Saccharomyces cer­
Saccharina japonica HMSZ – 5 39.05 [82] evisiae [73]. Fig. 8d represents reducing sugar and ethanol yield for the
Spirulina (Arthrospira) Fe/HMSZSM-5 37.77 [83] fermentation of Padina tetrastromatica biomass using Saccharomyces
plantensis cerevisiae yeast culture. The initial reducing sugar content is 29.4 g/L
Padina tetrastromatica MgO.La2O3 88 Present
Sargassum swartzii 92 study
and decreases rapidly to the concentration of 13.2 g/L after the initial
lag period for the first 24 hrs. of fermentation. After 24hrs the reducing

12
N. Jeyakumar et al. Fuel 329 (2022) 125362

sugar concentration is almost constant and reached the final concen­ biodiesel synthesis which will reduce the amount of waste released to
tration of 8.3 g/L. The rate of conversion of reducing sugar into bio­ the environment. MgO.La2O3 has been synthesized employing the co-
ethanol is high at the initial period up to 24 hrs. of fermentation process precipitation method. With the introduction of MgO.La2O3 nano-
but later the conversion rate declines and the final ethanol yield after 60 catalyst biodiesel yield has been improved up to 88% and 92% for
hrs. of the fermentation process is 62.13 ± 1.5%. In another previous Padina tetrastromatica and Sargassum swartzii macro-algae respectively.
work, Harun et al. [74] produced bioethanol with a yield of 52% from The future scope of the present study can be accomplished by improving
the sulfuric acid pretreated Chlorococcum humicola microalgae species the biofuel yield by adopting new techniques. Macro-algae biofuel
by the fermentation process using Saccharomyces cerevisiae yeast culture. production can be enhanced by immobilization of cellulose using
Prasad et al. [75] produced bioethanol from the dilute sulfuric acid nanoparticles that reduce the hydrolyze enzyme consumption. Various
pretreated wheat straw biomass and the maximum achievable bio­ integrated techniques like hydrothermal liquefaction, fermentation and
ethanol yield was only about 5.29%. These results indicate that the pyrolysis methods are used to increase the yield. Microwave pyrolysis
bioethanol yield from the current study by using Padina tetrastromatica create optimum environment conditions which can be adopted to pro­
and Sargassum swartzii macro-algae biomass is higher than the micro­ duce biofuels for aviation sector [14]. Biohydrogen can be synthesized
algae and other terrestrial lignocellulosic biomass. Table 3 shows the employing combined microwave and pretreatment method. Chemo
comparison of ethanol yield with the other previous works. disperser treatment can be employed to extract bio methane from algal
The bioethanol produced from the macro-algae biomass was biomass. Various methods such as pyrolysis, micro wave treatment and
analyzed by the FTIR. Fig. 9 shows the FTIR for the ethanol sample. In hydrothermal liquefaction can be used for bio oil extraction from macro-
the figure, the broad peaks at 3337 cm− 1 represent –OH functional algae [84].
group, and peaks at 2973 cm− 1 represent -C–H functional groups. The
peaks at 1086 and 1044 cm− 1 correspond to the -C–O- functional CRediT authorship contribution statement
groups present in bioethanol. The characteristic peaks indicate the
presence of ethanol in the distillated fermentation broth. The amounts of Nagarajan Jeyakumar: Conceptualization, Methodology, Valida­
ethanol produced from Sargassum by Zymomonas mobilis and by tion, Writing-Original Draft. Anh Tuan Hoang: Conceptualization,
Saccharomyces cerevisiae are 12 ml/50gm and 15 ml/50gm respectively. Methodology, Writing-Reviewing and Editing. Sandro Nižetić:
The amounts of ethanol produced from Padina by Zymomonas mobilis Reviewing and Editing. Dhinesh Balasubramanian: Methodology,
and by Saccharomyces cerevisiae are 14 ml/50gm and 7 ml/50gm Writing-Reviewing and Editing. Sriram Kamaraj: Reviewing and Edit­
respectively. ing. Prakash Lakshmana Pandian: Reviewing and Editing. Ranjna
Sirohi: Reviewing and Editing. Phuoc Quy Phong Nguyen: Reviewing
3.4. GC–MS results for biodiesel prepared from the lipid of spent algae and Editing. Xuan Phuong Nguyen: Data curation, Writing-Reviewing
and Editing.
Biodiesel samples obtained from the spent feedstock of Padina tet­
rastromatica and Sargassum swartzii after fermentation were studied by
GC–MS analysis. The chromatogram obtained with Padina tetrastroma­ Declaration of Competing Interest
tica and Sargassum swartzii is shown in Fig. 10. The percentages of fatty
acid components present in the biodiesel extracted from Padina tetra­ The authors declare that they have no known competing financial
stromatica and Sargassum swartzii lipids are given in Table 4 and Table 5 interests or personal relationships that could have appeared to influence
respectively. the work reported in this paper.
The major fatty acid content present in Padina tetrastromatica spent
biomass biodiesel are oleic acid (73.13%), Trideconic acid (10.27%), Acknowledgment
and palmitic acid (5.47%). The major fatty acid content present in
Sargassum swartzii oil spent biomass biodiesel is 0.48% oleic acid, 0.85% The authors would like to thank the Management of Mepco Schlenk
Myristic acid, 0.47% Hepto deconic acid, 1.23% palmitic acid, 0.8% Engineering College, Sivakasi for providing laboratory facilities to
Linolenic acid, and 5.59% Stearic acid. The fatty acid present after the perform this study.
fermentation process is higher for Padina tetrastromatica spent biomass
in comparison with Sargassum swartzii spent biomass. This result leads to References
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