Algal Research 42 (2019) 101606

Contents lists available at ScienceDirect

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

Review article

A critical review on production of bioethanol from macroalgal biomass T

a a b,⁎ a,⁎
Niyam Dave , Raja Selvaraj , Thivaharan Varadavenkatesan , Ramesh Vinayagam
a
Department of Chemical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India
b
Department of Biotechnology, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India

ARTICLE INFO ABSTRACT

Keywords: In order to combat emerging concerns due to global climate change and increasing conventional fuel prices,
Bioethanol bioethanol appears to be a sustainable green resource as co-fuel to meet the future energy demand for trans-
Macroalgae portation. As per the present scenario, bioenergy research emphasizes on bioethanol production from alternate
Pretreatment non-terrestrial substrates, like microalgae and macroalgae. Among them, macroalgae represent a rich source of
Microbial fermentation
carbohydrates for bioethanol production. The production of bioethanol from macroalgal biomass mainly in-
Response surface methodology
volves three steps namely mechanical pre-processing, pretreatment and microbial fermentation. Of these, the
pretreatment step is the most crucial that tends to improve the saccharification efficiency for efficient bioethanol
production. Currently, there are three main types of pretreatment methods used for macroalgae viz., physical/
physicochemical, chemical and biological. In addition, the two-step saccharification or combinational pre-
treatment approach is also utilized to enhance the yield of reducing sugars, which can be fermented to bioe-
thanol using suitable microbial strain(s) under restrained conditions. The current review provides a critical
assessment and detailed overview about the sequential process for bioethanol production from macroalgal
biomass. Additionally, this paper gives an insight on various statistical optimization approaches using response
surface methodology for the biomass pretreatment step and provides a viewpoint about the technical advances in
third-generation bioethanol production.

1. Introduction
First generation feedstocks have been the preferred choice as an
automotive co-fuel in many countries, as of now [1]. In spite of the
rising concern over the “Food and Fuel debate”, the second generation
resources are utilized as substrates for biofuel production [2]. These
involve the usage of lignocellulosic materials such as bagasse, cotton
stalk, corn stover, wood-chips, rice straw, organic waste and decaying
plant residues. But, the major limitation with second generation feed-
stocks is that it contains high amounts of lignin and requires large
agricultural land for its cultivation [2]. Hence, to overcome the above
limitations of these feedstock, algal biomass has been identified as the
third generation energy resource [3]. It includes the use of various
carbohydrate-rich strains of algal biomass for the production of bio-
fuels, such as biohydrogen, biodiesel and bioethanol [4]. Algal-derived
bioethanol has gained huge attention due to its higher octane rating and
higher heat of vaporization as compared to conventional fuels [5].
Furthermore, it reduces the release of greenhouse gases and serves as an
eco-friendly energy source. Hence, in the near future, algal bioethanol
seems to be an ideal sustainable energy resource for mankind [6].
Algae usually possess chlorophyll pigments but, unlike aquatic
plants, they are devoid of true stems and roots. In nature, they are the
most diverse forms of organisms that vary from unicellular blue green
algae to various multi-cellular forms, like kelp. Algae can be categor-
ized into two groups based on morphology: microalgae and macroalgae
[7]. Among them, macroalgae are aquatic eukaryotic organisms which
are largely found in marine or fresh water ecosystems. Macroalgae are
mainly photoautotrophic and widely distributed across the planet. They
play an important role in aquatic ecosystems and serve as a source of
nutrition at all trophic levels [8]. Macroalgae are classified into three
types based on pigment synthesis: Chlorophyceae, Phaeophyceae, and
Rhodophyceae [9].
Recently, bioethanol production from macroalgae has been given
due importance owing to the high carbon and low lignin content,
properties ideal for its conversion into bioethanol. Bioethanol produc-
tion from macroalgae involves various pretreatment steps followed by
microbial fermentation [10]. The conventional separate hydrolysis and
fermentation (SHF) method for macroalgal biomass involves mechan-
ical pre-processing (such as drying, milling and homogenization) fol-
lowed by physico-chemical and enzymatic pretreatment to get the
monomeric sugars for fermentation. Apart from this, other methods like
simultaneous saccharification and fermentation (SSF) and simultaneous

⁎
Corresponding authors.
E-mail address: ramesh.v@manipal.edu (R. Vinayagam).

https://doi.org/10.1016/j.algal.2019.101606
Received 29 November 2018; Received in revised form 26 June 2019; Accepted 30 June 2019
2211-9264/ © 2019 Elsevier B.V. All rights reserved.
N. Dave, et al. Algal Research 42 (2019) 101606

saccharification and co-fermentation (SSCF) have been employed to Table 1

increase the reducing sugar yield, the final ethanol titer and minimize Bioethanol production from various feedstock.
the time required for the bioethanol production [11]. Fermentation of Biomass Carbohydrate Bioethanol Reference
algal biomass is generally performed using baker's yeast (Saccharomyces content yield
cerevisiae) [12]. In addition, the seasonal variation in carbohydrate (% dw) (g/g
content among various macroalgal species has encouraged the use of substrate)

engineered yeast strains for enhanced bioethanol production [13]. First generation Cornmeal 70.8 0.34 [69]
Sorghum 52.5 0.25 [70]
2. Brief overview of bioethanol production Sweet potato 59.3 0.21 [71]
Second generation Sugarcane 71.8 0.29 [72]
bagasse
Bioethanol is widely produced using various chemical and biolo-
Wheat straw 67.2 0.30 [19]
gical approaches [14]. The biological approach involves the fermenta- Corn stover 69.7 0.30
tion of biomass using ethanologenic microbes under anaerobic or semi- Third generation Laminaria 51.9 0.40 [73]
anaerobic conditions [15,16]. Fermentation is an ancient technology japonica
(macroalgae)
known to mankind, which implies bioconversion of carbohydrate
Chlorella 51.0 0.23 [74]
through glycolytic intermediates into acid or alcohol. The bioprocessing vulgaris
of carbohydrate-containing feedstock is mainly carried out in two steps. (microalgae)
The first step involves hydrolysis of polysaccharides into fermentable Gracilaria 60.0 0.43 [75]
sugars followed by their conversion to bioethanol using suitable mi- verrucosa
(macroalgae)
croorganisms. Further, downstream processing includes purification
and concentration of bioethanol by distillation process. A major lim-
itation of the production process is the lower concentration of bioe-
thanol in the fermentation broth. Hence, a large amount of energy is
required for the separation and purification of bioethanol from the
fermentation broth [17].

2.1. Macroalgae as a feedstock – emergence of third generation bioethanol

production

The world faces an energy crisis due to the depletion of limited non-
renewable fuel resources and its environmental effects [18]. Hence, the
field of bioenergy has become a center of attention for researchers.
Current biomass resources for bioethanol production majorly include
edible vegetation, agricultural residues and algae. Recently, extensive
research has been carried out in the field of phycology to study the
process of microbial conversion of macroalgal biomass into bioethanol
for energy generation [19]. Macroalgae play an important role in
marine ecosystem by providing food and habitat for aquatic animals.
They are generally found in the coastal environments with suitable
substrate for attachment and as floating structures on sea surface [20].
Macroalgae contain substantial amounts of structural polysaccharides.
Fig. 1. Flow-chart of sequential steps in macroalgal bioethanol production.
However, the type and concentration of carbohydrate generally varies
among the various classes of macroalgae. It necessitates development of
new technologies for efficient bioconversion of macroalgal biomass into sugars from the complex carbohydrates by mechano/thermo-chemical
bioethanol [21]. Literature reveals that macroalgae can produce sig- and biological methods. In the final stage, the monomeric sugars are
nificantly high amounts of bioethanol (0.43 g/g substrate) as compared converted to bioethanol using specific microbes. An overview of all the
to terrestrial feedstock and require lesser production time (Table 1). three stages of macroalgal bioethanol production is summarized in
Since, the macroalgal biomass has negligible lignin content, it does not Fig. 1.
require an additional delignification step for its bioprocessing [22].
Apart from this, macroalgal biomass comprises a relatively higher 2.2. Steps in the production of bioethanol using macroalgal biomass
amount of holocellulose fraction than microalgae. Macroalgae can be
cultivated in larger quantities in marine environment as compared to 2.2.1. Collection and compositional analysis
microalgae. Hence, macroalgae is projected as a prospective candidate Macroalgae is considered as an ideal substrate for bioethanol
for bioethanol production. synthesis due to its high biomass productivity [25]. However, in terms
At present, there is a vital interest among the scientific community of growth pattern, various macroalgal strains vary significantly with
to study the conversion of macroalgal biomass to bioethanol. The respect to global diurnal climatic variables and marine habitats [26].
overall process involves three stages – collection and compositional Mainly, these climatic variables determine the rate of photosynthesis
analysis followed by pretreatment and microbial fermentation. The first for carbon accumulation and physiological response to stress conditions
stage begins with the artificial or on-shore cultivation of macroalgae [27]. Hence, it is necessary to identify suitable macroalgal strains with
followed by collection of biomass. In case of laboratory cultivation, the optimum growth conditions for bioethanol production. However, in
biomass is collected from the culture medium by means of sedimenta- practice, the collection of a specific indigenous macroalgal strain is a
tion and/or filtration. The on-shore cultivated macroalgal biomass is tedious process, therefore different ex-situ and in-situ techniques have
either manually or mechanically collected from the coastal or estuarine been adopted for macroalgal cultivation [28]. For example, the mac-
environment with respect to season. Subsequently, the washed, dried roalgal strain Ulva lactuca and Cystoseira amentacea were ex-situ culti-
and powdered biomass is subjected to compositional analysis [23,24]. vated using photobioreactor or dark sterile chambers under controlled la-
In the second stage, the biomass is pretreated to obtain the fermentable boratory environment by mimicking the natural growth conditions [29,30].

2
N. Dave, et al. Algal Research 42 (2019) 101606

Likewise, the in-situ on-shore cultivation approach has been developed
for macroalgal strain, e.g., Kappaphycus alvarezii and Gracilaria sp. using
raft or rope cultivation technique [31]. The on-shore cultivation tech-
nique is widely used for the commercial production of macroalgal
biomass that involves three-dimensional propagation of specific mac-
roalgae on nets attached to a large horizontal rope or raft in seawater
[31]. Overall, the macroalgal cultivation methods provide consistent
amounts of total biomass per batch on dry weight basis. However, the
major limitation of this method lies in terms of high production cost and
contamination with other marine epiphytes. Thus, in recent times, the
collection of macroalgal biomass directly from the natural resources is
more favored for bioethanol production.
In nature, several carbohydrate-rich strains of macroalgae are found
abundantly in aquatic ecosystems, which are primarily collected using
random sampling [32]. The common habitats for macroalgae include
estuaries, tidal pools, rocky shores and mangrove roots. As per the
phylogenetic classification, there are three groups of macroalgae –
green algae (Chlorophyta), brown algae (Phaeophyta) and red algae
(Rhodophyta) [33]. Among them, the green algae are majorly euryha-
line and usually grow in upper littoral zone of seawater. The red and
brown algae mainly grow in middle to lower littoral zone in deep-sea.
Thus, the sampling strategy is usually predetermined for the collection
of macroalgal biomass based on diversity assessment. Post collection of
the macroalgae, the biochemical characterization is performed using
proximate analysis [34,35]. There are mainly four components, which
are widely studied among the macroalgal strains, viz., carbohydrate,
protein, lipid and ash content (Table 2). As shown in the table, the
variation in the reported biochemical composition of macroalgal strains
may be due to influence of several environmental factors, such as,
temperature, wind, photoperiod, salinity and nutrient concentration.
Commonly, these ecological parameters fluctuate with reference to the
locality and the seasonal effect. Additionally, the tidal periods can also
indirectly affect the macroalgal biomass availability and biochemical
composition.
Macroalgae generally vary in relative carbohydrate concentration in
context to the site of collection (Table 3). Broadly, the total sugar
content of macroalgae can be categorized into two parts: easily hy-
drolyzable glucan part and the remaining non-glucan part. The mac-
roalgal strains belonging to the group Chlorophyta are known to contain
cellulose or starch as glucan and sulphated polysaccharides as non-
glucan content [36]. The macroalgal strains of the group Phaeophyta
and Rhodophyta usually comprise cellulose as glucan and other non-
glucan polysaccharides like laminarin, mannitol, alginate, carrageenan
and agar [37,38]. However, the major glucan content among all the
macroalgal strains is cellulose, which can be easily broken down to its
monomers using pretreatment processes and fermented to bioethanol.
According to [39], seasonal variations tend to play a major role in
macroalgal biomass carbohydrate composition. A study on Laminaria
digitata (Oarweed) found that the ratio of laminarin and mannitol
varied greatly between spring and summer seasons that consequently
affects final bioethanol yield [40]. Hence, the carbohydrate composi-
tion among the macroalgal strains is an important parameter in de-
signing the process for bioethanol production, as described in Table 3.
2.2.2. Pretreatment process
Macroalgal cell wall is composed of two layers viz., external (pri-
mary) and internal (secondary) [41]. In the bioprocessing of macro-
algae, the most essential step is the pretreatment that tends to break-
down the outer cell wall to release biomolecules like, cellulose and
other complex sugar polymers (e.g., glucan and galactan), which are
easily accessible for bioethanol production. The internal cell wall is
mainly composed of hemicellulose, pectin and sulfated sugar polymers.
Based on the biochemical composition of macroalgae, the external cell
wall can result in higher or lesser rigid structures [41]. As per the basic
principle of structural chemistry, the crystalline structure of macroalgal
cell wall tends to have greater stability. Consequently, these structures
require specific approach to hydrolyze the major macroalgal poly-
saccharides for bioethanol production. Hence, it is necessary to change
their structure through physical, chemical and biological means or
combinations of these pretreatment methods. There are mainly three
factors that determine the efficiency of pretreatment processes namely,
cellulose crystallinity, hemicellulose fraction and accessible surface
area for enzymatic hydrolysis [42]. Apart from it, an effective pre-
treatment method should also retain some portion of non-hydrolysable
hemicellulose and lessen the formation of fermentation inhibitors [42].
Till now, various pretreatment processes for the three major types of
macroalgae have been developed to maximize the reducing sugar
(majorly glucose and galactose) extraction [43]. There are certain
technical limitations in the existing pretreatment approaches that in-
clude the low-efficiency and high processing cost for macroalgal bio-
mass conversion. Consequently, there is a methodological void in terms
of selection of an appropriate pretreatment strategy for macroalgal
bioethanol production. Literature suggests that mechanical pre-pro-
cessing followed by acid and enzymatic hydrolysis tend to increase the

Table 2
Compositional analysis of various macroalgal strains.
Macroalgae strain Carbohydrate Protein Lipid Ash Reference
(%dw) (%dw) (%dw) (%dw)

I. Chlorophyta Ulva pertusa 59.07 ± 0.20 6.30 ± 0.25 2.39 ± 0.10 22.8 ± 0.39 [59]
Ulva fasciata Delile 43 ± 4.5 14.4 ± 2.2 1.83 ± 0 16 ± 2.7 [76]
Enteromorpha intestinalis 42.8 31.6 1.3 24.3 [77]
Ulva lactuca 23.8 ± 0.80 16.4 ± 0.14 1.0 ± 0.23 21.5 ± 0.29 [58]
Ulva rigida 53 ± 1 23.4 ± 0.51 1.2 ± 0.2 21.7 ± 1.12 [78]
II. Phaeophyta Laminaria japonica 54.5 ± 0.09 7.40 ± 0.06 1.37 ± 0.01 28.3 ± 0.01 [59]
Sargassum sp. 41.81 10.25 0.75 ± 0.02 26.19 ± 0.07 [34]
Ascophyllum nodosum 44.66 5.24 2.99 18.61 [79]
Fucus serratus 26.4 ± 0.75 9.6 ± 0.72 2.8 ± 0.38 18.8 ± 0.58 [58]
Laminaria digitata 46.6 12.9 1.0 26.0 [35]
Sargassum latifolium 20.1 5.7 4.2 25 [80]
III. Rhodophyta Gelidium amansii 77.2 13.1 1.1 8.6 [73]
Gelidium amansii 71.43 ± 0.08 10.47 ± 0.05 0.74 ± 0.04 2.8 ± 0.03 [59]
Kappaphycus alvarezii 59.58 ± 0.88 5.74 ± 0.89 0.75 ± 0.22 19.70 ± 0.09 [81]
Gracilaria sp. 76.67 16.0 1.2 6.13 [82]
Kappaphycus 51.6 ± 0.3 2.3 ± 0.3 0.2 ± 0.1 17.2 ± 0.1 [45]
alvarezii G11
Chondrus crispus 21.8 ± 1.57 19.9 ± 0.27 0.48 ± 0.25 19 ± 1.02 [58]
Palmaria palmata 39.4 ± 1.00 22.9 ± 0.16 3.3 ± 0.60 25.7 ± 0.31 [58]
Gracilaria sp. 56 9.5 0.7 3.0 [83]

3
N. Dave, et al.
Table 3
Comparison of carbohydrate composition of various macroalgae.
Macroalgae strain Place of collection/origin
I. Chlorophyta Ulva pertusa Oshima-south area of Hokkaido prefecture, Japan
Kjellman
Ulva rigida National Institute of Oceanography, IOLR, Haifa, Israel
Ulva fasciata Delile Adri coast of Gujarat, India
Ulva prolifera Coasts of Qingdao, China
Ulva rigida Tétouan coastal region, Azla, Morocco
II. Phaeophyta Alaria crassifolia Oshima-south area of Hokkaido prefecture, Japan
Kjellman
Sargassum sp. Coastal waters of Bolinao, Pangasinan, Philippines
Ascophyllum nodosum Broughty Ferry beach, Dundee, Scotland
Laminaria digitata Broughty Ferry beach, Dundee, Scotland
III. Rhodophyta Gelidium elegans SEA-LABO Corporation in Kochi prefecture, Japan
Kuetzing
Gracilaria verrucosa Chilika Lake, Odisha, India
Gelidiopsis variabilis Coastal region of Nochiyoorani, Veraval, Old Mandapam Jetty and
Valinokkam, India
Gelidium micropterum Coastal region of Veraval and Old Mandapam Jetty, India
Gelidium pusillum Coastal region of Veraval and Valinokkam, India
Kappaphycus alvarezii Buton Island, Southeast Sulawesi, Indonesia
Gracilaria fisheri Earthen pond cultures in Surat Thani Province, Thailand
saccharification efficiency as compared to other pretreatment processes
[44]. In general, mechanical pre-processing of macroalgal biomass
prior to saccharification involves three steps, viz., washing, drying and
milling. During the initial pre-processing step, freshly collected mac-
roalgal biomass is washed with water to remove sand, dust, salt, epi-
phytes and adhering detritus material. Further, drying step is performed
to remove water content, which also increases the shelf life of the
biomass for the subsequent steps. This may be done in a hot air oven or
under sunlight. In addition, the ratio of water content and dry cell
biomass is a critical parameter in bioprocessing. Finally, milling step is
performed for the size reduction of macroalgal biomass, which is then
subjected to sieving. Based on the principle of surface chemistry,
smaller particles tend to have higher reaction efficiency during anae-
robic fermentation for bioethanol production.
Mechanical, chemical and enzymatic pretreatment methods have
been proposed and investigated for bioethanol production. Different
examples of pretreatment processes and reducing sugar yield for bioe-
thanol production from macroalgae have been described in Table 4.
Majorly, dilute-acid or alkali treatment followed by enzymatic hydro-
lysis seems to be an economically viable pretreatment method for
bioethanol production. The holocellulose content of macroalgal bio-
mass generally varies between 46 and 68%dw (Table 3) and it does not
contain other complex structural components. Therefore, through di-
lute-acid treatment, the holocellulose content can be broken down to
simpler sugars (e.g., glucose and xylose, etc.) and further saccharifica-
tion efficiency can be increased using enzymatic hydrolysis.
The chemical method is widely accepted and it involves the various
steps like cellulose de-polymerization, hemicellulose-solvation and
structural modification using mild alkali or dilute-acid treatment. The
mild alkali treatment using hydroxyl derivatives of sodium and po-
tassium salts is most commonly used for the red algae [21,45]. A dis-
advantage of alkali treatment is the requirement of huge quantity of
water for desalting, which increases the production cost. In the acid
pretreatment, dilute sulphuric acid is mainly used for all the classes of
macroalgae [46]. The saccharified algal hydrolysate is neutralized with
sodium hydroxide or solid lime to pH 7 in order to eliminate fermen-
tation inhibitors. The insoluble salt precipitate is then separated by
centrifugation and filtration. Finally, the neutralized pretreated algal
hydrolysate is utilized for the bioethanol production. Under strong
acidic conditions, acid hydrolysis of macroalgal polysaccharides lead to
the production of inhibitory products such as, 5-hydroxymethylfurfural
4
Algal Research 42 (2019) 101606
Carbohydrate content for ethanol production (% Reference
dw)
starch, beta-1,3-glucan, and cellulose: 22.0 [84]
Cellulose: 23.8 ± 1.2 [85]
Starch: 7.6 ± 1.1
Cellulose: 11 [48]
Cellulose: 19.4 ± 0.5 [86]
Cellulose and hemicellulose: 30.69 [78]
Starch, beta-1,3-glucan, and cellulose: 24.5 [84]
Alpha-cellulose and hemicelluloses: 46.08, [34]
mannitol: 5.04
Alginate, laminarin, mannitol and cellulose: 57.84 [38]
Alginate, laminarin, mannitol and cellulose: 64.47
Starch, beta-1,3-glucan, and cellulose: 21.8 [84]
Alpha-cellulose and hemicellulose: 62–68 [75]
Cellulose: 11.38 ± 0.62 [37]
Cellulose: 10.63 ± 0.41
Cellulose: 12.20 ± 0.45
Cellulose: 7.11 [87]
Cellulose and hemicellulose: 64.51 [88]
(5-HMF), levulinic acid, formic acid and aldehydes [47]. Enzymatic
pretreatment involves commercial enzymes like cellulase (endo-gluca-
nase, cellobiohydrolase and cellobiase), amylase and agarase to hy-
drolyze the complex macroalgal polysaccharides (e.g., cellulose, starch
and agar) into monomeric reducing sugar (e.g., glucose and galactose)
[48–50]. The pretreatment of macroalgal biomass is represented in
Fig. 2.
As discussed above, a host of factors affect the pretreatment pro-
cesses include pH, temperature, treatment time, concentration of sub-
strate and the agents used for the pretreatment. A significant number of
experiments will have to be performed to arrive at the optimized pre-
treatment parameters for higher bioethanol yield. In order to reduce the
number of experiments, in recent times, Design of Experiments (DOE), a
statistical optimization method, is widely applied to obtain optimized
conditions for pretreatment methods, which hold the key for enhancing
the yield of bioethanol. It is also possible to interpret the interaction
among the factors considered for the study. DOE includes various de-
signs such as Factorial design, Taguchi design, Central Composite
Design (CCD) and Box Behnken Design (BBD). A summary of various
optimization studies citing the optimum pretreatment parameters for
bioethanol production have been enlisted in Table 5.
2.2.3. Fermentation with yeasts
The selection of appropriate yeast strains and operational conditions
are the crucial parameters in fermentation process. In addition, the
Crabtree effect also decides the yeast cell growth pattern and utilization
of carbon sources [51,52]. In general, alcoholic fermentation is carried
out under anaerobic conditions. Recently, various engineered strains of
bacteria and yeast have been developed using genetic engineering to
increase the bioethanol yield from pretreated macroalgal biomass [53].
But, depending on the type of strain used in the process and growth
kinetics, it can switch between semi-anaerobic and aerobic conditions
[54]. Nowadays, apart from conventional SHF process, new methods
such as SSF and SSCF have been employed to improve the bioconver-
sion of macroalgal biomass. A study on bioethanol conversion using the
algal strain Gelidium amansii revealed that autoclaving can increase
bioethanol yield up to 76.9% [55]. The process of macroalgal bioe-
thanol production involves two steps: preparation of macroalgal hy-
drolysate followed by yeast fermentation. Post-pretreatment, the mac-
roalgal hydrolysate is sterilized. Thereafter, the selected yeast strain is
inoculated into macroalgal hydrolysate and fermented in a batch mode,
N. Dave, et al. Algal Research 42 (2019) 101606

Table 4
A comprehensive review of various pretreatment processes for macroalgal biomass.
Macroalgae strain Pre-treatment method Pre-treatment steps Reducing sugar yield Reference

Mechanical Chemical Enzyme Conditions for

Enzymatic
digestion

Taxonomic division – I: Chlorophyta (green algae)

Enteromorpha Hydrothermal Drying and milling (140 – Viscozyme L and 170 °C, 20.1 g/L [77]
intestinalis treatment followed by mesh sieve), Cellic CTec2 170 rpm
enzymatic hydrolysis S/L ratio (1:10), (1:1 v/v) 30 min
170 °C, 60 min [Novozymes]
Enteromorpha sp. Enzymatic hydrolysis Air-drying and milling – Cellulase from Aspergillus 50 °C, pH 5, 70.48% [89]
niger 96 h
(0.8 U/mg biomass)
[FLUKA:22178,
Germany]
Ulva rigida Enzymatic hydrolysis Drying (70 °C, 48 h), – Amylo-glucosidase 37 °C, 3 h 196.0 ± 2.5 mg/g dw [85]
under mild sonication milling and sonication: (300 U/mL),
37 °C, 3 h [A7095; Sigma],
(40 kHz frequency, alpha-amylase
power:120 W) (250 U/mL)
[A7595; Sigma]
and cellulase
(0.3 U/mg) [C1184;
Sigma]
Ulva rigida Thermal acid Drying at 60 °C for 10% (w/v) – – 34 ± 0.25 mg/mL [78]
hydrolysis 24 h followed by grinding biomass,
4% (v/v) H2SO4,
121 °C, 1 h
(pH 7)
Ulva lactuca liquid hot water Air-drying and milling – Celluclast 1.5 L 50 °C, 900 rpm, 97.5 mg/g dried [90]
pretreatment followed followed by (10 U/g biomass) 120 h biomass
by enzymatic liquefaction at [Novozymes]
hydrolysis 10% (w/v) solid content,
121 °C, 1 h
Ulva sp. Alkali treatment Drying and grinding 3% NaOH, CMCase 45 °C, 135 rpm, 345 mg/g substrate [91]
followed by enzymatic 120 °C, 20 min from Aspergillus fumigatus 40 h
hydrolysis (pH 5) SL1
(13 U/mg dw)
Ulva lactuca Mechano-enzymatic Air-drying, size reduction – Haliatase 37 °C, 500 rpm, 0.131 g/g total solid [92]
hydrolysis using (30 g/L) 72 h
centrifugal and vibratory [KURA BIOTECH SPA,
ball mill Puerto Varas, Chile]
(screen size: 0.25 mm,
12,000 rpm;
frequency: 15/s., for
5 min at ambient
temperature)

Taxonomic division – II: Phaeophyta (brown algae)

Sargassum Hydrothermal Drying followed by – – – 30.1% (w/w) [93]
sagamianum liquefaction liquefaction at
200 °C, 15 MPa,
15 min
Saccharina japonica Thermal acid – 10% w/v Termamyl- 30 °C, 200 rpm, 45.60 ± 5.0 g/L [94]
hydrolysis followed biomass, 120 L 7.5 days
biological 40 mM H2SO4, (1.5 KNU/mL)
saccharification 121 °C, 60 min [Novozymes],
Bacillus sp. JS-1 [Dry
powder]
(1 g dcw/L)
Sargassum sp. Combined acid Drying (70 °C) 3 g biomass, Cellulase 50 ± 1 °C, 120.0 mg/g dw [34]
hydrolysis and enzyme and milling 3.4–4.6% w/v (10 FPU/g substrate) 100 rpm, 96 h
digestion H2SO4, 115 °C, [Sigma] and cellobiase
1.5 h (250 CBU/g substrate)
[Sigma]
Ascophyllum Microwave Assisted Drying and grinding 3.13% (w/v) – – 127 mg/g [95]
nodosum Acid Hydrolysis biomass, biomass
0.4 M H2SO4,
150 °C, 1 min
Ascophyllum Microwave Drying by microwave 0.4 M H2SO4, – – 44.97 mg/g biomass [79]
nodosum assisted heating under vacuum 150 °C, 1 min
thermo-chemical (80–94 °C, 600 W, (pH 7)
treatment 24 min), grinding and
sieving (1–2 mm particle
size)
(continued on next page)

5
N. Dave, et al. Algal Research 42 (2019) 101606

Table 4 (continued)

Macroalgae strain Pre-treatment method Pre-treatment steps Reducing sugar yield Reference

Mechanical Chemical Enzyme Conditions for

Enzymatic
digestion

Laminaria digitata Combined acid Drying (80 °C, 10% w/v Novozymes Biomass Kit 50 °C, 150 rpm, 29.30 g/L [38]
hydrolysis and enzyme 48 h) and milling biomass, 0.2 M 18 h
digestion H2SO4,
121 °C, 20 min
(Final pH 5.5)
Sargassum Combined acid Homogenization 10 g biomass, Cellulase 30 °C, pH 5.5, 68.32 g/L [96]
crassifolium hydrolysis and enzyme (25 °C, 10 min) 0.2 M H2SO4, (30 U/g) [Sigma] 1500 rpm,
digestion 121 °C (15 psi), (0–0.5 g/mL) 10 min
15 min
Sargassum latifolium Acid treatment Drying at room 10 g biomass, Trichoderma 30 °C, pH 5.5, 510 mg/g algal [80]
followed temperature for 1% HCl, asperellum RM1 21 days biomass
by fungal 72 h, milling and 121 °C, 1.5 bar, [Petroleum
saccharification sieving (1.5–2 mm) 60 min Biotechnology Lab, EPRI,
Egypt]

Taxonomic division – III: Rhodophyta (red algae)

Gelidium amansii Acidified Desalting, NaClO2 r-cellulase 37 °C, pH 5, 661.8 g/kg biomass [97]
salt solution treatment homogenization followed (1 g/g biomass) (200 U/g dw), 24 h
followed by by freeze-drying at and CH3COOH r-beta-glucosidase
enzymatic hydrolysis −50 °C (0.2 mL/g (100 U/g dw) and
biomass), r-xylanase
70 °C, 0.5–4 h (20 U/g dw)
Gelidium elegans Combined acid Drying (60 °C, 1.5 g biomass, Meicelase 50 °C, 100 rpm, 123.0 g/L [84]
Kuetzing hydrolysis and enzyme 2 days) and milling 2% H2SO4, (5 g/L) 120 h
digestion 121 °C, 30 min [CEPB5394, Meiji Seika
(Final pH 5.5) Kaisya Ltd., Japan]
Gracilaria salicornia Dilute-acid treatment Drying with paper towel 2% H2SO4, Cellulase 40 °C, pH 5, 13.8 g/kg fresh algal [98]
followed by enzymatic followed by 120 °C, 30 min (5 g/L buffered 4h biomass
hydrolysis homogenization (pH 7) hydrolysate)
[MP Biomedicals, LLC]
Gelidium amansii Acid treatment Drying and crushing 1 g dry biomass, Viscozyme-L and 50 °C, 150 rpm, 55% (w/w) [73]
followed by enzymatic (< 0.5 cm particle size) 0.10 N HCl, Celluclast-1.5 L (1:1) 24 h
digestion 121 °C, 15 min (1 mL/100gdw)
(pH 5.5) [Novozymes]
Gelidium corneum Weak acid treatment Drying and sieving (200 1% C2H2O4, – – 2.2% [99]
mesh screen) 121 °C, 30 min
(pH 7)
Kappaphycus alvarezii Acid hydrolysis Sun-drying and crushing 6.25% (w/v) – – 72 g/L [56]
biomass,
0.9 N H2SO4,
100 °C, 60 min
(pH 6.4–6.8)
Kappaphycus alvarezii Thermal acid Chopping and grinding 0.4 M H2SO4, – – 4.08 mg/g biomass [100]
hydrolysis (10 min) 100 °C, 3 h
(pH 7)
Kappaphycus alvarezii Dilute acid treatment Drying (60 °C) 10 g biomass, – – 30.50 g/L [101]
and milling (5 min) 0.2 M H2SO4,
130 °C, 15 min
(Final pH 5.0)
Gracilaria verrucosa Alkali and acid Drying (25 °C, 24 h) and 50 g biomass, Cellulase (20 FPU/g) 50 °C, 150 rpm, 38.93 ± 0.76 g/L [75]
treatment with milling 5.0% NaOH, [Sigma] 48 h
enzymatic hydrolysis 80 °C, 2 h and beta-glucosidase
and (60 U/g) [Sigma]
1.5% H2SO4, 2 h
(Final pH 5.0)
Gracilaria sp. Combined acid and Drying (60 °C), 2% (w/v) Cellulase 50 °C, 100 rpm, 592 mg/g biomass [82]
enzymatic hydrolysis homogenization followed biomass, (0.01 g/g biomass) 6h
by desiccation at room 0.1 N H2SO4, [Challenge- Bioproducts
temperature 121 °C, 60 min Co. Ltd., Touliu, Taiwan]
(pH 4.8)
Kappaphycus alvarezii Enzymatic Soaking in water – Cellulase 50 °C, 100 rpm, 8.04 g/L [87]
Hydrolysis (2 h) and size reduction (1 g/mL) 12 h
[Endsany Jaya
Engineering, Surakarta]
Gelidium amansii Dicationic acidic ionic Drying (40 °C), 5 wt% biomass, – – 45.07 ± 2.46 wt% [102]
liquid (IL) grinding and sieving 0.5 mmoL [Tri-
treatment (−80 mesh fractions) EG-(MIm)2]
2HSO4 DAIL,
120 °C,
1 atm, 3 min
(continued on next page)

6
N. Dave, et al. Algal Research 42 (2019) 101606

Table 4 (continued)

Macroalgae strain Pre-treatment method Pre-treatment steps Reducing sugar yield Reference

Mechanical Chemical Enzyme Conditions for

Enzymatic
digestion

Kappaphycus alvarezii Alkali treatment Drying (25 °C) and 8 g biomass, Cellic CTec II 45 °C, pH 4.8, 13.7 g/L [45]
followed by enzymatic sieving (1 mm) 6% KOH (w/v), (10 FPU/g dw) 120 rpm,
hydrolysis 25 °C, 24 h [Novozymes] 72 h
Ahnfeltia plicata Hot water treatment Drying (50 °C) and – Xylanase from Bacillus sp. 30 °C, pH 7, 233 ± 5.3 [103]
followed by enzymatic grinding followed by BT21 140 rpm, μg/mg biomass
digestion thermal hydrolysis (50 IU/mg dw) 6h
(121 °C, 45 min)
Gracilaria sp. Acid treatment Air drying and chopping 2 g biomass, Cellulase (53 FPU/g dry 50 °C, pH 5.0, 140.6 ± 1.7 mg/g [83]
followed by enzymatic followed by desalination 4% H2SO4, substrate) and 150 rpm, 4 h biomass
digestion 121 °C, beta-glucosidase
30 min (30 U/g dry substrate)
(pH 6.5–7) [Sigma]
Gracilaria verrucosa Combined acid and Drying at 60 °C for 48 h 2% biomass, r-agarases 37 °C, 200 rpm 47.4 ± 1.0% [49]
enzymatic hydrolysis followed by grinding and N HCl, (aga50D 0.5 U/mg plus for 48 h
sieving (< 200 mesh) 121 °C, 15 min NABH
(pH 7) 0.5 U/mg)
Gracilaria verrucosa Acid treatment Freeze-drying, grinding 7.5% biomass, Viscozyme L, Cellic 50 °C, 180 rpm, 69.1% [104]
followed by enzymatic and milling (< 100 μm 100 mM CTec2 and Cellic HTec2 72 h
digestion particle size) H2NSO3H, (1:1:0.1 v/v/v ratio per
130 °C, 90 min dried biomass)
(pH 4.8) [Novozymes]
Gracilaria fisheri Thermal acid Drying, grinding 6% (w/v) – – 16.69 g/L [88]
hydrolysis and sieving biomass,
1 M H2SO4,
95 °C, 150 min
(pH 6.5)
Gracilaria manilaensis Dilute-acid Drying (80 °C, 2.5% (w/v) – – 0.34 g/gdw [105]
treatment overnight) and milling H2SO4,
(1 mm) 121 °C, 40 min
(pH 5)
Gracilaria fisheri Acid hydrolysis Air-drying, 2 g biomass, – – 11.39 g/L [106]
grinding and sieving 1 M H2SO4,
95 °C, 150 min
(pH 6.5)

Macroalgal residual biomass

Marine macroalgal Dilute acid Drying (40 °C) 0.1% w/v Cellulase 50 °C, 150 rpm, 277.5 mg/g [107]
processing waste- pretreatment followed and milling H2SO4, (45 FPU/g) 48 h dw
Floating residue by enzymatic 121 °C, 1 h [Jienuo Enzyme
(FR) hydrolysis Co., China] and
cellobiase
(55 CBU/g)
[Sigma, USA]
Marine macroalgal Mild acid hydrolysis Drying under sunlight 1% H2SO4, – – 13.07 ± 0.00 [108]
industrial spent and milling 121 °C, 15 min mg/g dw
biomass (BS) (Final pH 5.3)

Fig. 2. Schematic representation of pretreatment process for macroalgae.

7
N. Dave, et al. Algal Research 42 (2019) 101606

Table 5
Examples of statistical optimization strategies for the bioethanol production using macroalgae.
Source/Strain of macroalgae Optimization method Optimum conditions Bioethanol yield References
(g/g substrate)

Taxonomic division – I: Chlorophyta (green algae)

Ulva sp. Taguchi orthogonal array design (a) Solid load: 15% (w/v) 0.22 [109]
(Beach of Ramat Aviv, Israel) (b) H2SO4 concentration: 2% (v/v)
(c) Pretreatment time: 30 min
(d) Process temperature: 121 °C
Chaetomorpha linum BBD (a) Liquid to solid (L/S) ratio: 100 mL/g 0.28 [110]
(Coastal region of Monastir, Tunisia) (b) Enzyme concentration: 52 U/g substrate
(c) Hydrolysis time: 44 h

Taxonomic division – II: Phaeophyta (brown algae)

Sargassum sp. CCD (a) Solid loading: 10% (w/w) 0.13 [34]
(Coastal region of Bolinao, Pangasinan, Philippines) (b) H2SO4 concentration: 4.0% (w/v)
(c) Pretreatment time: 1.5 h
(d) Process temperature: 115 °C
Undaria pinnatifida CCD (a) Slurry content: 13% (w/v) 0.33 [111]
(Gijang Local-Products Co. Ltd., Busan, Korea) (b) H2SO4 concentration: 75 mM
(c) Pretreatment time: 60 min
(d) Process temperature: 121 °C
Saccharina japonica CCD (a) Slurry content: 10% (w/v), 0.34 [112]
(Gijang Local- Products Co. Ltd., Busan, Korea) (b) H2O2 concentration: 1% (v/v)
(c) Pretreatment time: 60 min
(d) Process temperature: 121 °C

Taxonomic division – III: Rhodophyta (red algae)

Kappaphycus alvarezii CCD (a) Solid (CR) concentration: 18% (w/v) 0.29 [11]
(Itaguaí, Rio de Janeiro, Brazil) (b) Enzyme load: 45 FPU/g substrate
(c) Hydrolysis time: 24 h
Kappaphycus alvarezii Taguchi orthogonal array design (a) Substrate Concentration: 0.5% 0.27 [113]
(Qingdao, People's Republic of China) (b) HCl-Methanol ratio: 1.0 mol/L
(c) Pretreatment time: 20 h
(d) Process temperature: 80 °C
Gracilaria verrucosa Taguchi orthogonal array design (a) Substrate consistency: 15% (w/v) 0.49 [114]
(Coasts of Orissa and Tamil Nadu, India) (b) Enzyme dosage: 30 U/mL
(c) Hydrolysis time: 36 h
Gelidium amansii CCD (a) Slurry content: 12% (w/v) 0.50 [115]
(Morocco) (b) H2SO4 concentration: 358.3 mM
(c) Pretreatment time: 11 min
(d) Process temperature: 142.6 °C

Macroalgal residual biomass

Macroalgal waste CCD (a) Slurry content: 8% (w/v) 0.45 [116]
(Gwangalli beach, Busan, Korea) (b) H2SO4 concentration: 286 mM
(c) Pretreatment time: 90 min
(d) Process temperature: 121 °C

aerobically or anaerobically (e.g., capped serum bottle and Duran glass
bottle) in the operating range of 30–37 °C, pH 4–7, 100–200 rpm for
2–8 days (Table 6). Post-fermentation, the biomass is separated by
centrifugation and centrate containing bioethanol is analyzed using gas
chromatography [48].
For bioethanol fermentation, yeast strains such as, Saccharomyces
cerevisiae, Pichia angophorae and Pichia stipitis, obtained from terrestrial
resources, are commonly used (Table 6). However, the current research
emphasizes on the utilization of indigenous halotolerant marine yeast
strains to improve the fermentation process by eliminating additional
desalination step for bioethanol production [56]. For example, when
the hydrolysate of Kappaphycus alvarezii was fermented using haloto-
lerant marine isolate Candida sp., it was observed that the yeast could
tolerate high concentration of salt in fermentation medium and result in
a bioethanol yield up to 1.76% [57].
Additionally, various studies have been carried out in order to iso-
late wild-type strains or to develop genetically modified yeast strains
with capability of fermenting wide range of monomeric sugars from
macroalgal hydrolysate into bioethanol. Fermentation of the hydro-
lysate of brown algal strain Laminaria hyperborea using Pichia ango-
phorae revealed that both the monomeric sugars (laminarin and man-
nitol) in the hydrolysate can be fermented simultaneously to recover
bioethanol yield of 0.43 g/g substrate [54]. Similarly, the engineered
Saccharomyces cerevisiae strain has also been developed with conversion
yield up to 83% from macroalgal hydrolysate containing mannitol and
alginate monomers for bioethanol production [13]. To maximize the
bioconversion yield from maroalgal hydrolysate, yeast metabolic ana-
lysis is also used. In this context, metabolic profiling of 24 different
yeast strains were performed on macroalgal strains and a bioethanol
yield of 13 g/L was achieved from Chondrus crispus using selected yeast
strain [58].
Till now, various fermentation processes have been studied for the
macroalgal bioethanol production (Table 6). The table shows the
comparative analysis of different yeast strains, fermentation conditions
and bioethanol yield using pretreated macroalgal biomass. In the pre-
sent scenario, bioethanol is produced at laboratory scale by fermenting
various carbohydrate-rich macroalgae. The most commonly used strain
is Gelidium amansii, which contains approximately 71% of the total
carbohydrate per dry cell biomass [59]. Apart from this, several species
of Laminaria and Ulva genus are also widely used for bioethanol pro-
duction. Usually, the carbohydrate profile of macroalgal biomass de-
termines the conversion routes and their metabolic fates for bioethanol
synthesis. For example, macroalgal genera, such as, Laminaria and
Saccharina belonging to brown algal group are known to contain large
amounts of alginate, laminarin and mannitol rather than structural
polysaccharide (cellulose) [60]. The red and green macroalgal genera
like Gelidium, Gracilaria, Kappaphycus and Ulva mainly comprise cellu-
lose, glucan and galactan [43]. Of these, majorly the polymers of D-

8
N. Dave, et al. Algal Research 42 (2019) 101606

Table 6
A comparative study of various fermentation processes for bioethanol production using macroalgae.
Macroalgae Fermentation agent Fermentation mode Nutrient source Fermentation Bioethanol yield Reference
strain conditions

Taxonomic division – I: Chlorophyta (green algae)

Chaetomorpha linum Saccharomyces Semi- anaerobic SSF Bacto-peptone: 20 g/L, 32 °C, pH 4.8, 44.0 ± 3.0 g/g raw- [117]
cerevisiae ATCC96581 yeast extract: 10 g/L, glucose: 100 rpm, glucan
20 g/L. 200 h
[additive: 2 mL/L urea)]
Ulva rigida Saccharomyces Anaerobic SSF under 200 μM sodium acetate (2.1% w/ 37 °C, 333.30 ± 4.7 mg/g [85]
cerevisiae mild sonication v) pH 5.0, glucose
3h
Ulva prolifera Saccharomyces Anaerobic Glucose: 20 g/L, 28–36 °C, 13.20 g/g UPR (substrate) [86]
cerevisiae YSC2 yeast extract: 0.5 g/L, 150 rpm,
(NH4)2SO4: 6 g/L, MgSO4.7H20: 24–72 h
0.4 g/L,
FeSO4.7H20: 0.4 g/L
KH2PO4: 1 g/L,
urea: 1 g/L.
Ulva fasciata Delile Saccharomyces Anaerobic Peptone: 5 g/L, 28 ± 2 °C, 120 rpm, 0.45 g/g reducing sugar [48]
cerevisiae MTCC180 yeast extract: 3 g/L. 12 h
Ulva lactuca Saccharomyces Semi- anaerobic SSF Yeast extract: 50 g/L, urea: 4 g/L, 37 °C, pH 5.5, 0.06 g/g [92]
cerevisiae chloramphenicol: 0.5 g/L, 50 mM 500 rpm, total solid
acetate buffer 72 h (substrate)
(pH 5).
Ulva rigida Pachysolen tannophilus Anaerobic Yeast extract: 10 g/L, peptone: 30 °C, pH 6, 120 rpm, 0.12 g/g dw [78]
10 g/L, 4 days
dextrose: 20 g/L.

Taxonomic division – II: Phaeophyta (brown algae)

Laminaria hyperborea Pichia angophorae Aerobic Mannitol: 40 g/L, 30 °C, pH 4.5, 0.43 g/g reducing sugar [54]
CBS5830 yeast extract: 0.3 g/L, OTR (5.9 mM/Lh),
malt extract: 0.3 g/L, 40 h
(NH4)2SO4: 5.0 g/L,
KH2PO4: 0.2 g/L
MgSO4.7H2O: 0.4 g/L.
Saccharina latissima Saccharomyces Semi-anaerobic SSF CH3COONa buffer: 32 °C, pH 6, 0.45% (v/v) [118]
cerevisiae 50 mM 60 h
Sargassum sagamianum Pichia stipitis CBS7126 Aerobic Yeast extract: 10 g/L, peptone: 30 °C, pH 5, Aeration 0.386 g/g reducing sugar [93]
20 g/L. rate: 100 mL/min,
200 rpm,
200 h
Laminaria japonica Escherichia coli KO11 Semi-anaerobic SSF Yeast extract: 5 g/L, 30 °C, pH 5.5, 0.4 g/g substrate [73]
peptone: 10 g/L, 150 rpm,
NaCl: 10 g/L. 120 h
Laminaria digitata Pichia angophorae Semi-anaerobic SSF Yeast extract: 10 g/L, peptone: 24 °C, pH 4, 8.86 ± 0.05 μL/mL slurry [119]
CBS5830 20 g/L. 69 h
Laminaria japonica Saccharomyces Anaerobic Yeast extract: 10 g/L, peptone: 30 °C, pH 7, 150 rpm, 0.269 g/g substrate [120]
cerevisiae KCCM50550 20 g/L, 96 h
dextrose: 20 g/L
(NH4)2SO4: 10.8 g/L, KH2PO4:
5.0 g/L, MgSO4.7H2O: 1.1 g/L.
Saccharina japonica Pichia angophorae Anaerobic SSF Yeast extract: 10 g/L, peptone: 30 °C, 7.70 g/L [94]
KCTC17574 20 g/L, 200 rpm,
dextrose: 20 g/L. 136 h
Sargassum sp. Saccharomyces Semi- anaerobic (NH4)2SO4: 0.9 g/L, 40 °C, 2.79 ± 0.09 g/L [34]
cerevisiae yeast extract: 0.375 g/L. pH 4.5,
48 h
Ascophyllum nodosum Saccharomyces Anaerobic Yeast extract: 10 g/L, bacto- 37 °C, 5.57 g/L [95]
cerevisiae peptone: 20 g/L, 130 rpm,
ATCC200062 dextrose: 20 g/L. 72 h
Sargassum crassifolium Saccharomyces Anaerobic Casein hydrolysate:5 g/L, yeast 30 °C, 43.92 g/L [96]
cerevisiae JCM3012 extract: 5 g/L, pH 5.5,
glucose: 5 g/L 200 rpm,
12 h
Laminaria digitata Kluyveromyces Anaerobic Malt extract: 20 g/L 30 °C, 6.0 g/L [38]
marxianus NCYC1424 100 rpm,
3–150 h
Ascophylum nodosum Scheffersomyces stipitis Anaerobic Malt extract: 20 g/L 30 °C, 2.40 g/L
NCYC1542 100 rpm,
3–150 h
Laminaria digitata Ambrosiozyma Anaerobic Yeast extract: 5 g/L, 30 °C, pH 4.0, 42.51 ± 9.14 μL/g DS [121]
(Hudson) JV angophorae CBS5830 glucose: 20 g/L. 72 h
Lamouroux
Padina tetrastromatica Saccharomyces Anaerobic Trypton: 10 g/L, 30 °C, pH 4.0, 161.0 mg/g algal biomass [122]
cerevisiae yeast extract: 5 g/L, 200 rpm,
NaCl: 5 g/L. 7 days
(continued on next page)

9
N. Dave, et al. Algal Research 42 (2019) 101606

Table 6 (continued)

Macroalgae Fermentation agent Fermentation mode Nutrient source Fermentation Bioethanol yield Reference
strain conditions

Ecklonia kurome Saccharomyces Anaerobic Yeast extract: 10 g/L, peptone: 37 °C, pH 6, 2.10 ± 0.70 g/L [123]
cerevisiae 20 g/L, 60 rpm,
AM1 and dextrose: 20 g/L, 120 h
Saccharomyces MES buffer: 50 mM.
cerevisiae
CDY
Sargassum latifolium Saccharomyces Semi-anaerobic Yeast extract: 10 g/L, 30 °C, pH 6, 150 rpm, 0.29 g/g reducing sugar [80]
cerevisiae KH2PO4: 2 g/L and 96 h
ATCC76621 and MgSO4.7H2O: 1 g/L
Saccharomyces
cerevisiae
RM2

Taxonomic division – III: Rhodophyta (red algae)

Gracilaria salicornia Escherichia coli KO11 Anaerobic MOPS buffer: 10× 30 °C, 79.1 g/kg dw [98]
K2HPO4: 100×, pH 7.2,
ACGU solution: 40 X, 50 h
supplement EZ: 20 X,
chloramphenicol solution:
1000×.
Kappaphycus alvarezii Saccharomyces Semi-anaerobic Yeast extract: 6 g/L, peptone: 5 g/ 30 ± 2 °C, 2.06% [56]
cerevisiae L, pH 6.4–6.8,
NCIM3523 dextrose: 10 g/L. 150 rpm,
72 h
Gelidium corneum Saccharomyces Semi-anaerobic Yeast extract: 2 g/L, peptone: 30 °C, pH 5.6, 10% (w/w) [99]
cerevisiae 10 g/L, 120 rpm,
(NH4)2HPO4: 1.5 g/L. 96 h
Kappaphycus alvarezii Saccharomyces Anaerobic Yeast extract: 10 g/L, peptone: 30 °C, pH 7, 200 rpm, 9.6% (v/v) [100]
cerevisiae 10 g/L, 72 h
dextrose: 20 g/L.
Gelidium amansii Brettanomyces custersii Semi- anaerobic Yeast extract: 10 g/L, 30 °C, pH 5.5, 27.60 g/L [124]
KCTC18154P soy peptone: 20 g/L, 150 rpm,
galactose: 120 g/L 39 h
Kappaphycus alvarezii Saccharomyces Anaerobic (NH4)2SO4: 0.02% 30 °C, 0.21 g/g reducing sugar [101]
cerevisiae NaH2PO4: 0.006% 120 rpm,
72 h
Gracilaria verrucosa Saccharomyces Anaerobic Glucose: 30 g/L, 30 °C, 20 h 0.43 g/g reducing sugar [75]
cerevisiae HAU yeast extract: 3.0 g/L, peptone:
5.0 g/L, (NH4)2HPO4: 0.25 g/L
Kappaphycus alvarezii Saccharomyces Anaerobic SSCF Urea: 1.25 g/L, 30 °C, 64.30 g/L [11]
cerevisiae CBS1782 yeast extract: 2 g/L, 150 rpm,
KH2PO4: 1.1 g/L, galactose: 20 g/ 144 h
L
Gracilaria sp. Saccharomyces Semi-anaerobic Yeast extract: 10 g/L, peptone: 30 °C, pH 4.5, 0.47 g/g reducing sugar [82]
cerevisiae 10 g/L, 120 rpm,
Wu-Y2 dextrose: 20 g/L 48 h
Gelidium amansii Saccharomyces Anaerobic SSF Yeast extract: 0.1% w/v, peptone: 37 °C, pH 4.8, 25.70 g/L [55]
cerevisiae KCTC7906 0.2% w/v 200 rpm,
48 h
Chondrus crispus Saccharomyces Anaerobic Yeast extract: 10 g/L, 30 °C, 13.0 g/L [58]
cerevisiae YPS128 peptone: 20 g/L, 120 rpm,
glucose: 20 g/L. 120 h
Gracilaria manilaensis Saccharomyces Anaerobic Yeast extract: 6 g/L 30 °C, pH 5.0, 0.32 g/g reducing sugar [105]
cerevisiae 150 rpm,
4 days
Gracilaria sp. Saccharomyces Semi-anaerobic Yeast extract: 50 g/L, (NH4)2SO4: 30 °C, pH 5, 125 rpm, 28.7 ± 0.4 g/L [83]
cerevisiae 50 g/L, K2HPO4: 1.25 g/L, 96 h
MTCC174 KH2PO4: 8.75 g/L,
NaCl: 1 g/L,
MgSO4·7H2O: 5 g/L, CaCl2·2H2O:
1 g/L, CuSO4.5H2O: 1 g/L.
Gracilaria tenuistipitata Saccharomyces Semi-anaerobic Yeast extract: 3 g/L, peptone: 5 g/ 30 °C, pH 6.5, 0.19 g/g glucose [88]
cerevisiae L 120 rpm,
TISTR5339 96 h
Gracilaria fisheri Candida glabrata Anaerobic Yeast extract: 3 g/L, peptone: 5 g/ 37 °C, pH 6.5, 0.025 g/g reducing sugar [106]
L, 120 rpm,
96 h

Macroalgal residual biomass

Marine macroalgal Saccharomyces Anaerobic (NH4)2SO4: 0.9 g/L, 30 °C, pH 6.5, 48 h 23.3 g/L [107]
processing waste- cerevisiae yeast extract: 0.375 g/L
Floating residue (FR)
(continued on next page)

10
N. Dave, et al. Algal Research 42 (2019) 101606

Table 6 (continued)

Macroalgae Fermentation agent Fermentation mode Nutrient source Fermentation Bioethanol yield Reference
strain conditions

Marine macroalgal Saccharomyces Anaerobic yeast extract 10 g/L, 30–34 °C, 0.02 ± 0.003 g/g [108]
industrial spent cerevisiae peptone: 20 g/L, (pH 5.3), reducing sugar
biomass (BS) dextrose: 20 g/L, 50 rpm,
(NH4)2SO4: 1 g/L, 5 days
Na2HPO4: 0.5 g/L,
KH2PO4: 2.5 g/L,
FeSO4: 0.001 g/L
MgSO4: 1 g/L and
Urea: 1 g/L
Mixed marine macoalgal Saccharomyces Anaerobic Yeast extract: 10 g/L, 30 °C, 13.5 g/L [116]
waste cerevisiae peptone: 20 g/L, 150 rpm,
KCTC1126 and galactose: 20–120 g/L, mannitol: 144 h
Pichia angophorae 20–120 g/L.
KCTC17574

Fig. 3. Pathways for carbohydrate metabolism derived from macroalgal biomass.

glucose and D-galactose (e.g., cellulose, starch, laminarin and agar) can
be utilized by various yeast strains in its monomeric form for alcoholic
fermentation via glycolysis and/or Leloir pathway (Fig. 3) [61–63].
Besides, oleaginous macroalgal residue, after oil extraction, results in
some amount of fermentable sugars that can be utilized for other value-
added product synthesis using yeast [64]. However, the research work
concerning the breakdown of complex polysaccharides (e.g., fucoidan,
carrageenan, ulvan, etc.) from macroalgal feedstocks into bioethanol is
in the preliminary phase [43,65]. Hence, future research needs to focus
on technical problems associated with macroalgae bioconversion pro-
cess for its commercialization.
Macroalgal biomass has gained huge attention as the indelible
sustainable solution for biomass-derived energy. As per the current
industrial applications, macroalgae are widely used as a source of
natural pigments [66]. In addition, the industrial processing of mac-
roalgae produces organic waste, which mainly contains cellulose and
some amount of algal colloids, such as carrageenan and alginate. These
wastes can be a potential substrate for bioethanol production and serves
as an eco-friendly platform for the renewable energy generation [67].
Akin to macroalgal biomass, the bioethanol production from industrial
spent residues also requires various pretreatment processes such as
chemical and enzymatic hydrolysis followed by fermentation using
appropriate microbial strain for efficient bioconversion (Tables 4 and
5). Recently, various studies have been conducted to optimize pre-
treatment methods to improve saccharification and the fermentation
process for bioethanol production [68]. A study on bioconversion of
cellulose from organic waste of marine macroalgae showed that liquid
hot water pretreatment followed by SSF using Clostridium thermocellum
under optimum conditions can produce upto 2% of bioethanol [68].
Hence, the industrial waste of marine macroalgae may be used as a
cost-effective source for bioethanol production to meet future energy
requirements.

11
N. Dave, et al.
3. Future perspective
The demand for bioethanol is on the surge over a few decades. This
is necessitated because of the fact that bioethanol is utilized as an ad-
ditive to gasoline. The supplementation reduces the load on the re-
quirement for non-renewable fuels. However, the first and second
generation feedstocks may not be sufficient to bridge the gap between
the demand and supply of bioethanol. Hence, recent energy research
has driven the hunt for potential feedstocks for bioethanol production,
with special attention towards macroalgal biomass. Since various
strains of macroalgae are known to contain huge amounts of pentose
and hexose sugars, it acts as a suitable candidate for bioethanol pro-
duction. However, the development and utilization of macroalgal
bioethanol on a large scale is still in the early stage due to the re-
quirement of costly and high energy-intensive pretreatment methods.
Henceforth, the future work should emphasize on development of an
economical and scalable method for bioethanol production from mac-
roalgae.
4. Conclusion
To combat the present environmental concerns and increasing fuel
prices, the macroalgal biomass has shown greater potential as feedstock
for biofuel production. In particular, carbohydrate-rich macroalgae are
widely used for bioethanol production. For macroalgal biomass to be
used as a substrate for bioethanol production, it has to be pretreated
using various methods. However, the major limitation lies in the pre-
treatment process due to formation of fermentation inhibitors, which
affect further downstream processing. Apart from this, fermentation
also plays a critical role in determining the bioethanol yield. In the
present review, we have critically summarized the pretreatment and
fermentation strategies of bioethanol production from various strains of
macroalgae.
Author contributions
The original idea was suggested by RV and the concept of this study
was developed in discussion with all the authors. ND and TV drafted the
article. RS and RV were involved in the critical revision of the article for
important intellectual content. All the authors read and approved the
final manuscript.
Informed consent, human or animal rights
No conflicts, informed consent, human or animal rights applicable.
Declaration of author agreement
The authors agree to authorship and submission of the manuscript
for peer review.
Declaration of Competing Interest
The authors have declared no conflict of interest.
Acknowledgement
The authors are thankful to the financial support obtained from Dr.
T.M.A. Pai Ph.D. scholarship programme provided by Manipal
Academy of Higher Education (MAHE).
References
[1] L.S. Khuong, et al., Effect of gasoline–bioethanol blends on the properties and
lubrication characteristics of commercial engine oil, RSC Adv. 7 (25) (2017)
15005–15019.
12
Algal Research 42 (2019) 101606
[2] A. Mohr, S. Raman, Lessons from first generation biofuels and implications for the
sustainability appraisal of second generation biofuels, Energy Policy 63
(Supplement C) (2013) 114–122.
[3] G. Markou, I. Angelidaki, E. Nerantzis, D. Georgakakis, Bioethanol production by
carbohydrate-enriched biomass of Arthrospira (Spirulina) platensis, Energies 6 (8)
(2013) 3937–3950.
[4] C.S. Jones, S.P. Mayfield, Algae biofuels: versatility for the future of bioenergy,
Curr. Opin. Biotechnol. 23 (3) (2012) 346–351.
[5] C. Formighieri, Bioethanol from algae polysaccharides, Solar-to-fuel Conversion in
Algae and Cyanobacteria, Springer International Publishing, Cham, 2015, pp.
13–17.
[6] M. Lackner, 3rd-generation biofuels: bacteria and algae as sustainable producers
and converters, in: W.-Y. Chen, T. Suzuki, M. Lackner (Eds.), Handbook of Climate
Change Mitigation and Adaptation, Springer International Publishing, Cham,
2017, pp. 3173–3210.
[7] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal
biomass: a renewable source for bioethanol, Bioresour. Technol. 102 (1) (2011)
186–193.
[8] E. Hernández-Garibay, J.A. Zertuche-González, I. Pacheco-Ruíz, Isolation and
chemical characterization of algal polysaccharides from the green seaweed Ulva
clathrata (Roth) C. Agardh, J. Appl. Phycol. 23 (3) (2011) 537–542.
[9] D. Hebbale, M.D.S. Chandran, N.V. Joshi, T.V. Ramachandra, Energy and food
security from macroalgae, J. Biodivers 8 (1) (2017) 1–11.
[10] K. Li, S. Liu, X. Liu, An overview of algae bioethanol production, Int. J. Energy Res.
38 (8) (2014) 965–977.
[11] P.I. Hargreaves, C.A. Barcelos, A.C.A. da Costa, N. Pereira, Production of ethanol
3G from Kappaphycus alvarezii: evaluation of different process strategies,
Bioresour. Technol. 134 (2013) 257–263.
[12] S.H.M. Azhar, et al., Yeasts in sustainable bioethanol production: a review,
Biochem. Biophys. Reports 10 (2017) 52–61.
[13] M. Enquist-Newman, et al., Efficient ethanol production from brown macroalgae
sugars by a synthetic yeast platform, Nature 505 (7482) (2014) 239–243 Jan.
[14] Z. Fan, W. Wu, A. Hildebrand, T. Kasuga, R. Zhang, X. Xiong, A novel biochemical
route for fuels and chemicals production from cellulosic biomass, PLoS One 7 (2)
(2012) 1–8.
[15] M. Ciani, et al., Non-conventional yeast species for lowering ethanol content of
wines, Front. Microbiol. 7 (2016) 642.
[16] K. Kumar, et al., Recent developments on biofuels production from microalgae and
macroalgae, Renew. Sust. Energ. Rev. 65 (2016) 235–249.
[17] D. Pimentel, T.W. Patzek, Ethanol production using corn, switchgrass, and wood;
biodiesel production using soybean and sunflower, Nat. Resour. Res. 14 (1) (2005)
65–76 Mar.
[18] S. Kraan, Algal polysaccharides, novel applications and outlook, in: C.-F. Chang
(Ed.), Carbohydrates, IntechOpen, Rijeka, 2012.
[19] M. Yanagisawa, S. Kawai, K. Murata, Strategies for the production of high con-
centrations of bioethanol from seaweeds: production of high concentrations of
bioethanol from seaweeds, Bioengineered 4 (4) (2013) 224–235.
[20] S. Vandendriessche, M. Vincx, S. Degraer, Floating seaweed in the neustonic en-
vironment: a case study from Belgian coastal waters, J. Sea Res. 55 (2) (2006)
103–112.
[21] J. Chen, J. Bai, H. Li, S. Fang, Prospects for bioethanol production from macro-
algae, Trends Renew. Energy 1 (3) (2015) 185–197.
[22] G.-S. Kim, Manufacturing Technology of Bioenergy Using Algae, Handb. Mar.
Macroalgae Biotechnol. Appl. Phycol, 2011, pp. 451–460.
[23] K. Balina, A. Lika, F. Romagnoli, D. Blumberga, Seaweed cultivation laboratory
testing: effects of nutrients on growth rate of Ulva intestinalis, Energy Procedia
113 (2017) 454–459.
[24] F. Fernand, A. Israel, J. Skjermo, T. Wichard, K.R. Timmermans, A. Golberg,
Offshore macroalgae biomass for bioenergy production: environmental aspects,
technological achievements and challenges, Renew. Sust. Energ. Rev. 75 (2017)
35–45.
[25] N. Neveux, M. Magnusson, T. Maschmeyer, R. de Nys, N.A. Paul, Comparing the
potential production and value of high-energy liquid fuels and protein from
marine and freshwater macroalgae, GCB Bioenergy 7 (4) (2015) 673–689.
[26] K. Balina, F. Romagnoli, L. Pastare, D. Blumberga, Use of macroalgae for bioe-
nergy production in Latvia: review on potential availability of marine coastline
species, Energy Procedia 113 (2017) 403–410.
[27] I. Smolina, S. Kollias, A. Jueterbock, J.A. Coyer, G. Hoarau, Variation in thermal
stress response in two populations of the brown seaweed, Fucus distichus, from the
Arctic and subarctic intertidal, R. Soc. Open Sci. 3 (1) (2016) 150429.
[28] R.A. Andersen, Algal culturing techniques, Elsevier, 2005.
[29] A. Mhatre, M. Navale, N. Trivedi, R. Pandit, A.M. Lali, Pilot scale flat panel
photobioreactor system for mass production of Ulva lactuca (Chlorophyta),
Bioresour. Technol. 249 (2018) 582–591.
[30] A. Falace, S. Kaleb, G. De La Fuente, V. Asnaghi, M. Chiantore, Ex situ cultivation
protocol for Cystoseira amentacea var. stricta (Fucales, Phaeophyceae) from a
restoration perspective, PLoS One 13 (2) (2018) 1–16.
[31] J.K. Kim, C. Yarish, E.K. Hwang, M. Park, Y. Kim, Seaweed aquaculture: cultiva-
tion technologies, challenges and its ecosystem services, ALGAE 32 (1) (2017)
1–13 https://www.e-algae.org/journal/view.php?number=2819.
[32] P. Kaladharan, P.U. Zacharia, K. Vijayakumaran, Coastal and marine floral bio-
diversity along the Karnataka coast, J. Mar. Biol. Assoc. India 53 (1) (2011)
121–129.
[33] B. Jha, C.R.K. Reddy, M.C. Thakur, M.U. Rao, Seaweeds of India: The Diversity and
Distribution of Seaweeds of Gujarat Coast, vol. 3, Springer Science & Business
Media, 2009.
N. Dave, et al.
[34] M.G. Borines, R.L. de Leon, J.L. Cuello, Bioethanol production from the macro-
algae Sargassum spp, Bioresource Technology, vol. 138, Elsevier Ltd, 2013, pp.
22–29.
[35] E.T. Kostas, D.A. White, D.J. Cook, Development of a bio-refinery process for the
production of speciality chemical, biofuel and bioactive compounds from
Laminaria digitata, Algal Res. 28 (2017) 211–219.
[36] N. Wei, J. Quarterman, Y.-S. Jin, Marine macroalgae: an untapped resource for
producing fuels and chemicals, Trends Biotechnol. 31 (2) (2013) 70–77.
[37] R. S. Baghel, C. R. K. Reddy, and B. Jha, “Characterization of agarophytic sea-
weeds from the biorefinery context,” Bioresour. Technol., vol. 159, no. March, pp.
280–285, 2014.
[38] O. Obata, J. Akunna, H. Bockhorn, G. Walker, Ethanol production from brown
seaweed using non-conventional yeasts, Bioethanol 2 (1) (2016) 134–145.
[39] P. Schiener, K.D. Black, M.S. Stanley, D.H. Green, The seasonal variation in the
chemical composition of the kelp species Laminaria digitata, Laminaria hy-
perborea, Saccharina latissima and Alaria esculenta, J. Appl. Phycol. 27 (1) (2015)
363–373 Feb.
[40] J.M.M. Adams, et al., Seasonal variation in the chemical composition of the
bioenergy feedstock Laminaria digitata for thermochemical conversion, Bioresour.
Technol. 102 (1) (2011) 226–234.
[41] Z.A. Popper, M.-C. Ralet, D.S. Domozych, Plant and algal cell walls: diversity and
functionality, Ann. Bot. 114 (6) (2014) 1043–1048.
[42] D.P. Maurya, A. Singla, S. Negi, An overview of key pretreatment processes for
biological conversion of lignocellulosic biomass to bioethanol, 3 Biotech 5 (5)
(2015) 597–609 Oct.
[43] S. Maneein, J.J. Milledge, B.V. Nielsen, P.J. Harvey, A review of seaweed pre-
treatment methods for enhanced biofuel production by anaerobic digestion or
fermentation, Fermentation 4 (4) (2018).
[44] S.W. Kim, C.H. Hong, S.W. Jeon, H.J. Shin, High-yield production of biosugars
from Gracilaria verrucosa by acid and enzymatic hydrolysis processes, Bioresour.
Technol. 196 (2015) 634–641.
[45] F. Masarin, F.R.P. Cedeno, E.G.S. Chavez, L.E. de Oliveira, V.C. Gelli, R. Monti,
Chemical analysis and biorefinery of red algae Kappaphycus alvarezii for efficient
production of glucose from residue of carrageenan extraction process, Biotechnol.
Biofuels 9 (1) (2016) 122 Jun.
[46] E.T. Kostas, S.J. Wilkinson, D.A. White, C.Y. Du, D.J. Cook, Optimization of a total
acid hydrolysis based protocol for the quantification of carbohydrate in macro-
algae, J. Algal Biomass Util 7 (1) (2016) 21–36.
[47] E.T. Kostas, S.J. Wilkinson, D.A. White, D.J. Cook, Optimisation of carbohydrate
quantification in seaweed optimization of a total acid hydrolysis based protocol
for the quantification of carbohydrate in macroalgae, J. Algal Biomass Utln 7 (71)
(2016) 21–36.
[48] N. Trivedi, et al., An integrated process for the extraction of fuel and chemicals
from marine macroalgal biomass, Sci. Rep. 6 (1) (2016) 30728.
[49] S.W. Kim, et al., Recombinant agarase increases the production of reducing sugars
from HCl-treated Gracilaria verrucosa, a red algae, Algal Res. 31 (2018) 517–524.
[50] Q. Al Abdallah, B.T. Nixon, J.R. Fortwendel, The enzymatic conversion of major
algal and cyanobacterial carbohydrates to bioethanol, Front. Energy Res 4
(2016) 36.
[51] H.G. Crabtree, Observations on the carbohydrate metabolism of tumours,
Biochem. J. 23 (3) (1929) 536.
[52] G. Perez-Samper, et al., The crabtree effect shapes the Saccharomyces cerevisiae
lag phase during the switch between different carbon sources, MBio 9 (5) (2018).
[53] M. Daroch, S. Geng, G. Wang, Recent advances in liquid biofuel production from
algal feedstocks, Appl. Energy 102 (2013) 1371–1381.
[54] S.J. Horn, I.M. Aasen, K. Østgaard, Ethanol production from seaweed extract, J.
Ind. Microbiol. Biotechnol. 25 (5) (2000) 249–254 Nov.
[55] H.M. Kim, S.G. Wi, S. Jung, Y. Song, H.J. Bae, Efficient approach for bioethanol
production from red seaweed Gelidium amansii, Bioresour. Technol. 175 (2015)
128–134.
[56] Y. Khambhaty, et al., Kappaphycus alvarezii as a source of bioethanol, Bioresour.
Technol. 103 (1) (2012) 180–185.
[57] Y. Khambhaty, D. Upadhyay, Y. Kriplani, N. Joshi, K. Mody, M.R. Gandhi,
Bioethanol from macroalgal biomass: utilization of marine yeast for production of
the same, BioEnergy Res 6 (1) (2013) 188–195.
[58] E.T. Kostas, D.A. White, C. Du, D.J. Cook, Selection of yeast strains for bioethanol
production from UK seaweeds, J. Appl. Phycol. 28 (2) (2016) 1427–1441.
[59] S. Jang, Y. Shirai, M. Uchida, M. Wakisaka, Production of mono sugar from acid
hydrolysis of seaweed, African J. Biotechnol 11 (8) (2012) 1953–1963.
[60] M.M. Nielsen, et al., Variation in biochemical composition of Saccharina latissima
and Laminaria digitata along an estuarine salinity gradient in inner Danish waters,
Algal Res. 13 (2016) 235–245.
[61] L. Pasteur, Experiences et vues nouvelles sur la nature des fermentations, C. R.
Acad. Sci. 53 (1861) 1260–1264.
[62] O. Meyerhof, On the influence of oxygen on the alcoholic fermentation of yeast,
Nat. Sci. 13 (49) (1925) 980–984.
[63] R. Caputto, L.F. Leloir, R.E. Trucco, C.E. Cardini, A.C. Paladini, The enzymatic
transformation of galactose into glucose derivatives, J. Biol. Chem. 179 (1) (1949)
497–498.
[64] E.T. Kostas, M. Cooper, B.J. Shepherd, J.P. Robinson, Identification of bio-oil
compound utilizing yeasts through phenotypic microarray screening, Waste and
Biomass Valorization (2019), https://link.springer.com/article/10.1007/s12649-
019-00636-7#aboutconten Feb article in press.
[65] J.J. Milledge, P.J. Harvey, Potential process ‘hurdles’ in the use of macroalgae as
feedstock for biofuel production in the British Isles, J. Chem. Technol. Biotechnol.
91 (8) (2016) 2221–2234.
13
Algal Research 42 (2019) 101606
[66] M.P. Sudhakar, A. Jegatheesan, C. Poonam, K. Perumal, K. Arunkumar,
Biosaccharification and ethanol production from spent seaweed biomass using
marine bacteria and yeast, Renew. Energy 105 (2017) 133–139.
[67] Uju, A.T. Wijayanta, M. Goto, N. Kamiya, Great potency of seaweed waste biomass
from the carrageenan industry for bioethanol production by peracetic acid–ionic
liquid pretreatment, Biomass and Bioenergy, vol. 81, 2015, pp. 63–69.
[68] O.S.R. Pasanda, A. Azis, H.S. Kusuma, Utilization of waste seaweed through pre-
treatment with liquid hot water method and simultaneous fermentation using
bacteria Clostridium thermocellum, J. Mater. Environ. Sci 7 (7) (2016)
2526–2533.
[69] L. Mojović, S. Nikolić, M. Rakin, M. Vukasinović, Production of bioethanol from
corn meal hydrolyzates, Fuel 85 (12) (2006) 1750–1755.
[70] D.A. Salvi, G.M. Aita, D. Robert, V. Bazan, Ethanol production from sorghum by a
dilute ammonia pretreatment, J. Ind. Microbiol. Biotechnol. 37 (1) (2009) 27 Oct.
[71] F. Wang, et al., An environmentally friendly and productive process for bioethanol
production from potato waste, Biotechnol. Biofuels 9 (1) (Mar. 2016) 50.
[72] B. Buaban, et al., Bioethanol production from ball milled bagasse using an on-site
produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis, J. Biosci.
Bioeng. 110 (1) (2010) 18–25.
[73] N.-J. Kim, H. Li, K. Jung, H.N. Chang, P.C. Lee, Ethanol production from marine
algal hydrolysates using Escherichia coli KO11, Bioresour. Technol. 102 (16)
(2011) 7466–7469.
[74] S.-H. Ho, S.-W. Huang, C.-Y. Chen, T. Hasunuma, A. Kondo, J.-S. Chang,
Bioethanol production using carbohydrate-rich microalgae biomass as feedstock,
Bioresour. Technol. 135 (2013) 191–198.
[75] S. Kumar, R. Gupta, G. Kumar, D. Sahoo, R.C. Kuhad, Bioethanol production from
Gracilaria verrucosa, a red alga, in a biorefinery approach, Bioresour. Technol.
135 (2013) 150–156.
[76] N. Trivedi, V. Gupta, C.R.K. Reddy, B. Jha, Enzymatic hydrolysis and production of
bioethanol from common macrophytic green alga Ulva fasciata Delile, Bioresour.
Technol. 150 (2013) 106–112, 2013.
[77] D.-H. Kim, S.-B. Lee, G.-T. Jeong, Production of reducing sugar from
Enteromorpha intestinalis by hydrothermal and enzymatic hydrolysis, Bioresour.
Technol. 161 (2014) 348–353.
[78] M. El Harchi, F.Z.F. Kachkach, N. El Mtili, Optimization of thermal acid hydrolysis
for bioethanol production from Ulva rigida with yeast Pachysolen tannophilus,
South African J. Bot 115 (2018) 161–169.
[79] Y. Yuan, D.J. Macquarrie, Microwave assisted step-by-step process for the pro-
duction of fucoidan, alginate sodium, sugars and biochar from Ascophyllum no-
dosum through a biorefinery concept, Bioresour. Technol. 198 (2015) 819–827.
[80] R.M. Soliman, S.A. Younis, N.S. El-Gendy, S.S.M. Mostafa, S.A. El-Temtamy,
A.I. Hashim, Batch bioethanol production via the biological and chemical sac-
charification of some Egyptian marine macroalgae, J. Appl. Microbiol. 125 (2)
(2018) 422–440.
[81] F. Abd-Rahim, et al., Production of high yield sugars from Kappaphycus alvarezii
using combined methods of chemical and enzymatic hydrolysis, Food Hydrocoll.
42 (P2) (2014) 309–315.
[82] F.-C. Wu, J.-Y. Wu, Y.-J. Liao, M.-Y. Wang, I.-L. Shih, Sequential acid and enzy-
matic hydrolysis in situ and bioethanol production from Gracilaria biomass,
Bioresour. Technol. 156 (2014) 123–131.
[83] K. Saravanan, S. Duraisamy, G. Ramasamy, A. Kumarasamy, S. Balakrishnan,
Evaluation of the saccharification and fermentation process of two different sea-
weeds for an ecofriendly bioethanol production, Biocatal. Agric. Biotechnol 14
(2018) 444–449.
[84] M. Yanagisawa, K. Nakamura, O. Ariga, K. Nakasaki, Production of high con-
centrations of bioethanol from seaweeds that contain easily hydrolyzable poly-
saccharides, Process Biochem. 46 (11) (2011) 2111–2116.
[85] L. Korzen, I.N. Pulidindi, A. Israel, A. Abelson, A. Gedanken, Single step produc-
tion of bioethanol from the seaweed Ulva rigida using sonication, RSC Adv. 5 (21)
(2015) 16223–16229.
[86] Y. Li, et al., Optimization study on the hydrogen peroxide pretreatment and pro-
duction of bioethanol from seaweed Ulva prolifera biomass, Bioresour. Technol.
214 (2016) 144–149 Elsevier Ltd.
[87] S. Puspawati, Wagiman, M. Ainuri, D. A. Nugraha, and Haslianti, “The production
of bioethanol fermentation substrate from Eucheuma cottonii seaweed through
hydrolysis by cellulose enzyme,” Agric. Agric. Sci. Procedia, vol. 3, pp. 200–205,
2015.
[88] N. Nunraksa, S. Rattanasansri, J. Praiboon, A. Chirapart, Proximate composition
and the production of fermentable sugars, levulinic acid, and HMF from Gracilaria
fisheri and Gracilaria tenuistipitata cultivated in earthen ponds, J. Appl. Phycol.
31 (1) (2018) 683–690 https://link.springer.com/article/10.1007/s10811-018-
1552-9 Jun February 2019.
[89] M.A. Jmel, G. Ben Messaoud, M.N. Marzouki, M. Mathlouthi, I. Smaali, Physico-
chemical characterization and enzymatic functionalization of Enteromorpha sp.
cellulose, Carbohydr. Polym. 135 (2016) 274–279.
[90] M.A. Jmel, et al., Variations in physicochemical properties and bioconversion ef-
ficiency of Ulva lactuca polysaccharides after different biomass pretreatment
techniques, Appl. Biochem. Biotechnol. 184 (3) (2018) 777–793 Mar.
[91] N. Ben Yahmed, N. Berrejeb, M.A. Jmel, S. Jazzar, M.N. Marzouki, I. Smaali,
Efficient biocatalytic conversion of stranded green macroalgal biomass using a
specific cellulases-based cocktail, Waste and Biomass Valorization (2018), https://
link.springer.com/article/10.1007/s12649-018-0397-4 Jun article in press.
[92] S. Amamou, C. Sambusiti, F. Monlau, E. Dubreucq, A. Barakat, Mechano-enzy-
matic deconstruction with a new enzymatic cocktail to enhance enzymatic hy-
drolysis and bioethanol fermentation of two macroalgae species, Molecules 23 (1)
(2018).
N. Dave, et al.
[93] H.Y. Lee, S.E. Lee, K.H. Jung, J.H. Yeon, W.Y. Choi, et al., Repeated-batch op-
eration of surface-aerated fermentor for bioethanol production from the hydro-
lysate of seaweed Sargassum sagamianum, J. Microbiol. Biotechnol. 21 (3) (2011)
323–331.
[94] J.S. Jang, Y.K. Cho, G.T. Jeong, S.K. Kim, Optimization of saccharification and
ethanol production by simultaneous saccharification and fermentation (SSF) from
seaweed, Saccharina japonica, Bioprocess Biosyst. Eng. 35 (1–2) (2012) 11–18.
[95] Y. Yuan, D.J. Macquarrie, Microwave assisted acid hydrolysis of brown seaweed
Ascophyllum nodosum for bioethanol production and characterization of alga
residue, ACS Sustain. Chem. Eng. 3 (7) (2015) 1359–1365.
[96] T. Widyaningrum, I. Prastowo, M. Parahadi, A.D. Prasetyo, Production of bioe-
thanol from the hydrolysate of brown seaweed (Sargassum crassifolium) using a
naturally β-glucosidase producing yeast Saccharomyces cereviceae JCM 3012,
Biosci. Biotechnol. Res. Asia 13 (3) (2016) 1333–1340.
[97] S.G. Wi, H.J. Kim, S.A. Mahadevan, D.-J. Yang, H.-J. Bae, The potential value of
the seaweed Ceylon moss (Gelidium amansii) as an alternative bioenergy resource,
Bioresour. Technol. 100 (24) (2009) 6658–6660.
[98] X. Wang, X. Liu, G. Wang, Two-stage hydrolysis of invasive algal feedstock for
ethanol fermentation F, J. Integr. Plant Biol. 53 (3) (2011) 246–252.
[99] M.H. Yoon, Y.W. Lee, C.H. Lee, Y.B. Seo, Simultaneous production of bio-ethanol
and bleached pulp from red algae, Bioresour. Technol. 126 (2012) 198–201.
[100] R.F. Mansa, W.-F. Chen, S.-J. Yeo, Y.-Y. Farm, H.A. Bakar, C.S. Sipaut,
Fermentation study on macroalgae Eucheuma cottonii for bioethanol production
via varying acid hydrolysis, in: R. Pogaku, R.H. Sarbatly (Eds.), Advances in
Biofuels, Springer US, Boston, MA, 2013, pp. 219–240.
[101] M.D.N. Meinita, J.Y. Kang, G.T. Jeong, H.M. Koo, S.M. Park, Y.K. Hong,
Bioethanol production from the acid hydrolysate of the carrageenophyte
Kappaphycus alvarezii (cottonii), J. Appl. Phycol. 24 (4) (2012) 857–862.
[102] L.B. Malihan, N. Mittal, G.M. Nisola, T.G. Weldemhret, H. Kim, W.-J. Chung,
Macroalgal biomass hydrolysis using dicationic acidic ionic liquids, J. Chem.
Technol. Biotechnol. 92 (6) (2016) 1290–1297.
[103] P. Parab, R. Khandeparker, U. Amberkar, V. Khodse, Enzymatic saccharification of
seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain
BT21, 3 Biotech 7 (5) (Aug. 2017) 296.
[104] M.-R. Park, S.-K. Kim, G.-T. Jeong, Biosugar production from Gracilaria verrucosa
with sulfamic acid pretreatment and subsequent enzymatic hydrolysis, Biotechnol.
Bioprocess Eng. 23 (3) (2018) 302–310 Jun.
[105] M.J. Hessami, S.-M. Phang, A. Salleh, R. Rabiei, Evaluation of tropical seaweeds as
feedstock for bioethanol production, Int. J. Environ. Sci. Technol. 15 (5) (2018)
977–992 May.
[106] S. Rattanasaensri, N. Nunraksa, N. Muangmai, J. Praiboon, A. Chirapart, Ethanol
production from Gracilaria fisheri using three marine epiphytic yeast species, J.
Appl. Phycol. 30 (6) (2018) 3311–3317 https://link.springer.com/article/10.
1007/s10811-018-1527-x Jun December 2018.
[107] L. Ge, P. Wang, H. Mou, Study on saccharification techniques of seaweed wastes
for the transformation of ethanol, Renew. Energy 36 (1) (2011) 84–89.
[108] M.P. Sudhakar, R. Merlyn, K. Arunkumar, K. Perumal, Characterization, pre-
treatment and saccharification of spent seaweed biomass for bioethanol produc-
tion using baker's yeast, Biomass Bioenergy 90 (2016) 148–154.
[109] R. Jiang, Y. Linzon, E. Vitkin, Z. Yakhini, A. Chudnovsky, A. Golberg,
14
Algal Research 42 (2019) 101606
Thermochemical hydrolysis of macroalgae Ulva for biorefinery: Taguchi robust
design method, Sci. Rep. 6 (June) (2016) 27761.
[110] M. Neifar, et al., Optimization of enzymatic saccharification of Chaetomorpha
linum biomass for the production of macroalgae-based third generation bioe-
thanol, AIMS Bioengineering 3 (2016) 400–411.
[111] Y. Cho, H. Kim, S.-K. Kim, Bioethanol production from brown seaweed, Undaria
pinnatifida, using NaCl acclimated yeast, Bioprocess Biosyst. Eng. 36 (6) (2013)
713–719 Jun.
[112] C.H. Ra, S.-K. Kim, Optimization of pretreatment conditions and use of a two-stage
fermentation process for the production of ethanol from seaweed, Saccharina ja-
ponica, Biotechnol. Bioprocess Eng. 18 (4) (2013) 715–720. Aug.
[113] X. Li, Y.-P. Li, H. Mou, H.-M. Hwang, P. Wang, Red seaweed derived poly-
saccharides, a novel marine resource for bio-ethanol production, J. Renew.
Sustain. Energy 6 (3) (2014) 33122.
[114] R. Shukla, et al., Process development for the production of bioethanol from waste
algal biomass of Gracilaria verrucosa, Bioresour. Technol. 220 (2016) 584–589.
[115] P. Sukwong, C.H. Ra, I.Y. Sunwoo, S. Tantratian, G.-T. Jeong, S.-K. Kim, Improved
fermentation performance to produce bioethanol from Gelidium amansii using
Pichia stipitis adapted to galactose, Bioprocess Biosyst. Eng. 41 (7) (2018)
953–960 Jul.
[116] I.Y. Sunwoo, J.E. Kwon, T.H. Nguyen, C.H. Ra, G.-T. Jeong, S.-K. Kim, Bioethanol
production using waste seaweed obtained from Gwangalli Beach, Busan, Korea by
co-culture of yeasts with adaptive evolution, Appl. Biochem. Biotechnol. 183 (3)
(2017) 966–979 https://link.springer.com/article/10.1007%2Fs12010-017-2476-
6 November 2017.
[117] N. Schultz-Jensen, et al., Pretreatment of the macroalgae Chaetomorpha linum for
the production of bioethanol - comparison of five pretreatment technologies,
Bioresour. Technol. 140 (2013) 36–42.
[118] J.M. Adams, J.A. Gallagher, I.S. Donnison, Fermentation study on Saccharina la-
tissima for bioethanol production considering variable pre-treatments, J. Appl.
Phycol. 21 (5) (2009) 569.
[119] J.M.M. Adams, T.A. Toop, I.S. Donnison, J.A. Gallagher, Seasonal variation in
Laminaria digitata and its impact on biochemical conversion routes to biofuels,
Bioresour. Technol. 102 (21) (2011) 9976–9984.
[120] S.-M. Lee, J.-H. Lee, Ethanol fermentation for main sugar components of brown-
algae using various yeasts, J. Ind. Eng. Chem. 18 (1) (2012) 16–18.
[121] J.M.M. Adams, G. Bleathman, D. Thomas, J.A. Gallagher, The effect of mechanical
pre-processing and different drying methodologies on bioethanol production using
the brown macroalga Laminaria digitata (Hudson) JV Lamouroux, J. Appl. Phycol.
(2017) 1–7.
[122] V. Ashokkumar, et al., Production of liquid biofuels (biodiesel and bioethanol)
from brown marine macroalgae Padina tetrastromatica, Energy Conversion and
Management, vol. 135, Elsevier Ltd, 2017, pp. 351–361.
[123] Y. Sasaki, T. Takagi, K. Motone, T. Shibata, K. Kuroda, M. Ueda, Direct bioethanol
production from brown macroalgae by co-culture of two engineered
Saccharomyces cerevisiae strains, Biosci. Biotechnol. Biochem. 82 (8) (2018)
1459–1462.
[124] J.H. Park, et al., Use of Gelidium amansii as a promising resource for bioethanol: a
practical approach for continuous dilute-acid hydrolysis and fermentation,
Bioresour. Technol. 108 (2012) 83–88.