Review

A sustainable perspective of microalgal

biorefinery for co-production and
recovery of high-value carotenoid and
biofuel with CO2 valorization
Ramalingam Dineshkumar , Applied Phycology and Biotechnology Division, CSIR–Central Salt and
Marine Chemicals Research Institute, Bhavnagar, India
Ramkrishna Sen , Department of Biotechnology, Indian Institute of Technology Kharagpur,
Kharagpur, India

Received August 19 2019; Revised April 12 2020; Accepted April 17 2020;

View online at Wiley Online Library (wileyonlinelibrary.com);
DOI: 10.1002/bbb.2107; Biofuels. Bioprod, Bioref. (2020)

Abstract: Process biotechnology can play a very important role in addressing contemporary global
challenges in the areas of energy, the environment, and healthcare. This review discusses the
development of a biorefinery model using sustainable feedstocks such as microalgal biomass, with
multiple benefits. As a case study, it demonstrates the development of a microalgal biorefinery to produce
lipid for biofuel and carotenoids like lutein for healthcare applications, along with CO2 mitigation. However,
there has been a question mark regarding the economic viability of microalgal biorefinery, mainly for low
biomass and product yields and discrete downstream processing steps. To provide sustainable solutions
for these technological challenges, process intensification strategies can be implemented to enhance
the yields of biomass-derived biofuels and value-added products. This article investigates the design
aspects of photo-bioreactors to enhance biomass productivity and CO2 sequestration. Despite efforts
made by researchers to improve product yields, there is ample scope to improve the economic viability
of lutein and lipid production by integrating upstream and downstream operations to reduce the cost
associated with the process. The future of algal biorefineries will rely on the development of rationally
integrated genome and process-scale engineering strategies to improve further the production of lipid and
carotenoids. This review critically analyzes the current state of the art and presents the future prospects
for microalgal biorefinery to address some of the challenges in the areas of healthcare, energy, and the
environment. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

Key words: biorefinery; microalgae; process intensification; downstream processing; carotenoid; lipid

Correspondence to: Ramkrishna Sen, Department of Biotechnology, Indian Institute of Technology Kharagpur,
West Bengal 721 302, India. E-mail: rksen@yahoo.com; rksen@bt.iitkgp.ac.in

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd 1
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization

Introduction: Global energy, Biorefinery

environment, and healthcare
Biorefinery technologies are primarily based on the
challenges
idea of sustainably processing biomass into a spectrum

T
he demand for energy in future decades will primarily
be dictated by a rise in the global population and the
consequent rapid economic growth in the developing
countries. This could lead to an increase in the use of
fossil fuels, which could further harm the global climate.
According to the 2016 International Energy Outlook report,
approximately 10 kg of carbon dioxide (CO2) is emitted
per gallon of diesel fuel burnt. Recently, India’s Central
Electricity Authority1 reported that around 1 ton of CO2 will
be released per MWh electricity from coal-based thermal
power stations. The International Energy Agency2 estimated
that the fuel consumption of developing countries increased
rapidly during the period from 1990 to 2013. Furthermore,
according to the Intergovernmental Panel on Climate
Change,3 the average global atmospheric CO2 concentration
has increased from 280 ppm in 1900 to 403 ppm in 2016.
This is projected to increase further up to 570 ppm by the
year 2100. It is therefore essential to develop green and
feasible solutions to address the current challenges of global
warming, compounded by the scarcity of renewable energy
and fresh water.4
The deleterious impacts of the incessant use of non-
renewable fossil fuels on the environment and health
are well known.5 Several diseases and health disorders,
including different types of cancer, cardiovascular
problems, tuberculosis, and pneumonia are believed
to have been caused or aggravated by emissions from
fossil energy combustion.6 As the nexus between energy,
the environment, and health has well been established,
most of the modern-day R&D challenges thus belong to
the these areas and their interconnected or overlapping
domains.5,6
To derive feasible and sustainable solutions to the
current R&D challenges, the scientific community has
intensified research in attempts to reduce our age-old
dependence on fossil and non-renewable resources
like petroleum, natural gas, coal, and minerals. One of
the current research challenges is therefore to shift the
paradigm of petroleum refining and reforming gradually
towards biomass refining and reforming, thereby
evolving into a new era of the bio-based economy.7 The
development of eco-friendly biorefinery technology is thus
expected to offer sustainable solutions to address some
of the current interconnected challenges of healthcare,
energy, and the environment.
of marketable products, including low-value biofuels,
power, and high-value biochemicals.8 The crucial
steps involved in biorefinery development are given in
Fig. 1.7
The selection of potential feedstock is the key to deriving
multiple products concomitantly in a biorefinery model.
Microalgae are considered to be a potential biomass feedstock
in terms of effective use of land and water and also of their
ability to capture CO2 with 10–50 times more efficiency
than terrestrial energy crops.9,10 The next critical step is the
optimization of biomass conversion processes by adopting
either thermochemical processes or biochemical engineering
strategies.
The commercial potential of thermochemical processes
such as gasification, pyrolysis, and hydrothermal
liquefaction has been recognized increasingly for the
production of bio-oil and other value-added products.
However, these require high temperatures (400–600 °C)
and high pressure (5–20 MPa) operating conditions.11 These
processes must therefore be upgraded by developing more
efficient catalysts to reduce the process energy requirements
significantly to achieve commercial viability.12,13 On the
other hand, biochemical engineering strategies rely on
improving upstream and downstream processes through
a process intensification approach for the production of
biofuels and value-added biochemicals in a biorefinery
platform.14,15
Figure 1. Steps involved in developing a biorefinery model.

2 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid R Dineshkumar, R Senz
and biofuel with CO2 valorization

Microalgae: A sustainable
feedstock for the biorefinery
Microalgae have recently emerged as a potential biomass
feedstock for addressing global sustainable food, feed and
fuel production challenges in a biorefinery. Microalgal strains
are generally considered as miniature biochemical factories
for the production of carotenoids, lipids, and proteins for
potential healthcare, biofuel, and animal feed applications14,16
(Fig. 2). It has also been reported that these microbes can
serve as a sustainable alternative for remediating wastewater
and sequestering flue gas.17,18 Based on the carbon content
(48–53%) of algal biomass, it is estimated that approximately
1.78 to 1.83 kg of CO2 is required to produce 1 kg biomass.19
This multiproduct paradigm with environmental remediation
Figure 2. Microalgal biorefinery for healthcare, energy, and
environment applications.
approach has attracted the attention of the world science
community to envisage microalgae as one of the potential
biomass feedstocks for the biorefinery model. However, it is
essential to address challenges such as producing high-density
microalgal biomass and developing cost-effective downstream
processes, in order to project microalgae as a suitable candidate
for biorefinery. The various products obtained from algal
biomass and their commercial applications are given in Table 1.
The studies reported in the literature mostly emphasize
microalgal cultivation to achieve goals such as the remediation
of waste water or flue gas CO2 and for the production of either
biofuel or carotenoids. However, this hampers the realization
of the full commercial potential of microalgae, so it is essential
to develop an integrated bioprocess – for instance, CO2
mitigation – by microalgae may effectively be integrated with
the concomitant production of high-value and low-value-
added products. This biorefinery strategy is expected to offset
the cost involved in upstream or downstream processing
steps.14,16 Nevertheless, this strategy will be influenced by the
type of algal species employed. It should be noted that most
of the microalgal strains are reported to accumulate one of
the products in major proportion, while others accumulate
negligible quantities. In contrast to this general observation,
green microalgae (belonging to the class Chlorophycea) have
been demonstrated to synthesize significant proportion of two
major products, namely carotenoids (predominantly lutein)
and lipids, besides their CO2 sequestration ability.20,29,30 Hence,
the green microalgal strains (e.g. Chlorella, Scenedesmus,
Nannochloropsis, and Chlorococcum) can be considered as most
suitable for biorefinery development to produce lutein and lipid

Table 1. Microalgal products and their potential applications.

Products Examples
Carotenoids β-Carotene Lutein Astaxanthin
Zeaxanthin Lycopene
Fucoxanthin neoxanthin
Pigments Chlorophyll Phycocyanin
Phycoerythrin
Lipids Eicosapentaenoic acid (EPA),
Docosahexaenoic acid (DHA),
Triacyl glycerides (TAGs)
Exo- Glucose, galactose, mannose,
polysaccharides ribose, arabinose, xylose,
(EPS) fucose, rhamnose, glucuronic
and galacturonic acid
Nutritional Protein, carbohydrate,
supplement essential vitamins and trace
minerals.
Microalgal strains
Dunaliella Haematococcus
Chlorococcum Spirulina
Chlorella Scenedesmus
Spirulina Chlorella Nostoc
Oscillatoria Porphyridium
Chlorella Crypthecodinium
Botryococcus Isochrysis
Nannochloropsis Tetraselmis
Porphyridium cruentum,
Nostoc calcicola
Chlorella Spirulina Nitzchia
Navicula Chaetoceros
Scenedesmus
Applications References
20–22
Antioxidants, Prevention and
treatment of cancers and age-
related blindness, Enhances
immune system, Colorants in food,
drugs and cosmetic industries.
23,24
Colorants, Antioxidant and
anticancer activities.
25,26
TAGs–Biodiesel; EPA and
DHA–therapeutic purposes such
as atherosclerosis, cholesterol,
coronary heart diseases.
22,27
Emulsifiers, stabilizers or thickening
agents in industries, Anticancer
agents, Antiviral immunostimulants
24,28
Aquaculture/ animal feed

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107 3
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization

concomitantly with flue gas CO2 sequestration for healthcare, CO2 to be compressed to high pressures and, hence,
energy, and environmental applications, respectively. additional energy needs to be derived by burning more coal
One of the major challenges in the production of lipid in thermal power plants.9 On the other hand, microalgae
and lutein is the availability of nitrogen sources in the have been reported to utilize CO2 (present in atmospheric
culture medium at the time of harvesting biomass. In or flue gas) as the carbon source, when it is grown in photo-
general, nitrogen-enriched conditions favor maximal lutein autotrophic mode with the required macro- and micro-
biosynthesis,31,32 whereas nitrogen-deficient conditions nutrients. Moreover, the biomass obtained from microalgae
improve lipid production.33,34 It is consequently challenging can be used for the production of valuable chemicals and
to attain maximum levels of both lutein and lipid over biofuels and microalgal strains therefore represent attractive
a similar cultivation period. In this context, the optimal candidates for carbon sequestration.35
medium or process conditions and the harvest time can be
prioritized for a product that is growth associated. In this Overview of carbon flow in an algal cell
review, the carotenoid lutein is considered as a primary
In eukaryotic microalgae, carbonic anhydrase enzyme
product, and hence the conditions and cultivation time are
catalyzes the reversible reaction of conversion of bicarbonate
focused primarily on enhancing lutein biosynthesis, while
and carbonate ions into CO2. Then, ribulose 1,5-bisphosphate
lipid can be extracted as a co-product. Another challenge in
carboxylase (Rubisco) enzyme assimilates CO2 into the
developing this integrated process is the concomitant recovery
first organic compound 3-phosphoglycerate (3-PGA).36
of both products from biomass, and this is discussed below.
Subsequently, 3-PGA will be utilized for the conversion
As some review articles investigated the potential significance
of different cellular molecules. According to Becker37 and
and applications of all the pigments and carotenoids produced
Polle et al.,38 most of the carbon flows into protein synthesis
from microalgae, this paper will give special emphasis to the
(50%), followed by the synthesis of carbohydrates (15%),
co-production and recovery of lutein along with biodiesel.
triglycerides (15%), nucleic acids (5%) and isoprenoids (1%)
This review will give insights about recent developments and
in a green microalgal cell depending upon the cultivation
future trends associated with the algal biorefinery model
conditions (Fig. 3). This biochemical composition can be
with regard to the following topics: (i) microalgae-mediated
varied depending upon different growth conditions and
CO2 sequestration; (ii) critical photobioreactor design
species. It should be noted that glycerolipids and isoprenoids
considerations for improved algal biomass cultivation; (iii)
are precursors for lipids and lutein, respectively.
process intensification strategies for the co-production of lutein
and lipid; (iv) downstream processing strategies for biomass
harvesting, cell disruption, and concomitant recovery of lutein
Flue gas CO2 bio-sequestration
and lipid; and (v) preliminary techno-economic implications The typical composition of coal-fired flue gas is as follows:
of the algal biorefinery model. CO2, 10–15% (v/v); N2, 72–77% (v/v); O2, 3–4%(v/v); SOx,
541–657 mg m−3; NOx, 449–516 mg m−3; CO, 30–40 mg m−3;
Microalgae mediated carbon heavy metals and particulate matter.39 The effect of flue gas
components on microalgal growth has been investigated
sequestration
comprehensively in the literature. For instance, the ability to
The main sectors involved in CO2 emissions are power utilize flue gas as the carbon source for algal growth depends
generation and transport.1 According to the International
Energy Agency2, power and heat generation accounts for 42%
of global CO2 emissions, transport accounts for 24%, followed
by industrial (19%), residential (6%), and other sources
(10%) including public /commercial services, agriculture /
forestry, fishing, and emissions not specified. Hence, coal-
based thermal power stations are prime targets for CO2
sequestration studies. Various carbon sequestration strategies
have received widespread attention over the past few years
to mitigate the harmful effects of CO2. Non-biological CO2
mitigation methods including geological storage, ocean
storage, mineral carbonation, and industrial consumption Figure 3. The percentage of carbon accumulation into
have been surveyed.2,3 However, these techniques require various biochemicals in a green microalgal cell.38

4 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid R Dineshkumar, R Senz
and biofuel with CO2 valorization

on pH, temperature, light, nutrients, biomass density, and
type of microalgal species.40 NOx and SOx gas components
are considered as alternative sources of nitrogen and sulfur
for microalgal growth. However, the presence of NOx and
SOx in flue gas will reduce the medium pH, due to the
formation of respective acids such as HNO2 or HNO3 and
sulfite or sulfate ions.10,40 The microalgal growth was found
to be inhibited when the concentration of SO2 was more
than 100 ppm, mainly due to the drastic reduction of pH
to 2.5–3.5.41 Hence, it is suggested that either controlling
the pH of culture medium or diluting the flue gas with air
could effectively sequester flue gas CO2 by algae and improve
biomass yields. Another concern in handling the flue gas is its
high temperature (around 120 °C), as this prevents its direct
introduction into the algal reactors.
To overcome this issue, researchers have considered the
use of temperature tolerant species.17 However, the growth
rate of most of the thermotolerant species was found to be
relatively less than mesotolerant species. Hence, an efficient
strategy is to cool the incoming flue gas using appropriate
heat exchangers that can reduce the temperature to as
low as 40 °C.41 Very low temperatures (<15 °C) decrease
the activity of Rubisco enzyme, whereas exceedingly high
temperatures (>40 °C) affect microalgal metabolic behavior
and also decrease CO2 solubility.10,36,40 Hence, it is essential
to maintain critical physicochemical parameters such as
pH and temperature at optimal levels to improve carbon
sequestration and algal biomass production.
Microalgal strains studied for CO2
bio-mitigation
Strain selection is one of the most important criteria for
optimal biomass production and carbon sequestration. An
increase in CO2 concentration from a limiting level (air,
0.04% CO2) to an optimal level (4–6% CO2) was found
to influence the biochemical composition of microalgae
positively.42 Some algal species have more tolerance to
CO2 than others. Hence, one of the essential steps towards
biorefinery development is to find potential algal strains
that possess high carbon capture ability with improved
productivity of biomass and value-added products. In
general, green microalgae are reported to be the most suitable
species for CO2 capture studies. Some examples of green
microalgae-based carbon sequestration studies are mentioned
in Table 2.
Photobioreactor design
considerations for microalgal
biomass cultivation
Microalgae are generally cultivated in two primary reactor
types, namely open ponds and closed photobioreactor
systems. Open-pond cultivation systems are usually favored
for their low capital and operational costs, as compared with
closed photobioreactors. However, the major drawbacks
are contamination issues and poor control over cultivation
conditions, and so the open raceway ponds result in very
low biomass productivity.53 The microalgal strains that could
adapt to certain environmental conditions such as high
salinity (e.g. Dunaliella salina), high alkalinity (e.g. Spirulina)
and high nutrient (e.g. Chlorella, Scenedesmus) could be
grown effectively in open ponds at a large-scale level.54,55
On the other hand, closed photobioreactors provide
precise control over operating conditions with maximum
photosynthetic efficiency and high biomass productivity.
Closed photobioreactor systems are essential for the

Table 2. Carbon dioxide sequestration rate and biomass productivity of some microalgal isolates with
different photobioreactor configurations.
Microalgal strains CO2 (%, v/v) Photo-bioreactor Biomass productivity CO2 fixation rate References
(mg L−1 d−1) (mg L−1 d−1)
43
Nannochloropsis salina 10–11 Raceway pond 32 60
44
Chlorella sp. AG10002 5 Bubble column 335 630
45
Botryococcus braunii 5 Fermenter 263 496
46
Chlorella emersonii 15 Airlift column 67 113
47
Scenedesmus dimorphus 15 Bubble column 485 889
48
Chlorella sorokiniana 4 Airlift reactor 150 251
49
Chlorella variabilis 0.03 Raceway pond 120 219
50
Chlorella sp. MTF-15 23–27 Bubble column 197 370
51
Chlorella vulgaris 5 Flat panel 830 1510
52
Scenedesmus sp 2.5 Raceway pond 97 182

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107 5
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization

production of high-value pigments and carotenoids,53,56
as these reactors ensure good manufacturing practice for
therapeutic, food, and cosmetic applications. The main
photobioreactor configurations are vertical tubular (bubble
column and airlift), horizontal tubular, flat panel and hybrid
reactors.42,57 The working principle, advantages, and drawbacks
of each reactor configurations are discussed in Table 3.58,59
The important design considerations of a photobioreactor
are as follows:57,60 (i) it provides a higher illumination
surface area to volume ratio for better light utilization;
(ii) it minimizes non-illuminated reactor surface area
and contamination; (iii) it provides precise control over
operational parameters and optimal mixing and mass transfer
characteristics; (iv) it minimizes capital costs and operational
energy consumption, and (v) it reduces fouling, and increases
ease of cleaning and sterilization. The critical design aspects
that significantly affect the performance of a photobioreactor
are light utilization, mixing, and mass transfer characteristics,
and this is discussed in the following sections.
Light distribution and utilization efficiency
Reactor geometry can greatly influence the distribution of
light to the microalgal cells. One of the main challenges
associated with light utilization in the reactor is the self-
shading effect of cells at a higher biomass concentration.
As light intensity critically influences the performance of
a photobioreactor, different reactor configurations that
improve light distribution inside the photobioreactor have
been reported. For instance, an internally illuminated
bioreactor was designed and tested to maximize the light-
utilization efficiency of Synechococcus sp.61 The flashing light
phenomenon (i.e. the frequency of the light/dark cycle) to
enhance the light utilization efficiency by algal cells was
reported in conventional airlift reactors.62,63 Other design
strategies that were attempted to improve light distribution
inside the reactor are as follows: (i) decreasing the light path
of a reactor,64,65 and (ii) developing some hybrid reactors
such as transparent glass sponge-based flat panel reactors,66
hydraulically integrated serial turbidostats,67 multi-
compartment reactors,68 hybrid flat-panel airlift bioreactors69
and Taylor vortex-flow photobioreactors.70 Although these
reactor configurations improved the light regime to achieve
high-density biomass production, scaling-up of these
photobioreactors may be cumbersome, considering capital or
operating costs and long-term performance.71
Despite significant advances in the development of
efficient photobioreactors, the installation and electricity

Table 3. Types of photobioreactors reported for microalgal biomass cultivation.

Reactor types Working principle Advantages Limitations
Raceway ponds Open cultivation system with a depth • Low capital, maintenance and • Limited control of operational
of 15 to 25 cm receives sunlight and operating costs parameters
CO2 directly from the atmosphere. • Contamination issues,

• No oxygen accumulation • High CO2 and water losses

Vertical tubular Cylindrical vessels that are aerated from
(bubble column/ the bottom by suitable sparger and
airlift) illuminated through translucent reactor
surface
Horizontal tubular An array of the parallel set of transparent
(horizontal/ tubes (of <0.1 m diameter) oriented
inclined) towards sunlight for improved light
utilization efficiency by algae
Flat panel Rectangular shaped reactors with high
illumination surface area
Hybrid (modified/ E.g. helical type and α-shaped reactors.
improved Designed to overcome the limitations of
designs) the conventional reactor configurations.
• Efficient mixing and mass transfer
rate
• Better control of process
parameters
• Higher biomass productivity and
photosynthetic efficiency
• Higher illumination surface area
• Moderate biomass productivity
• High biomass productivity
• Low oxygen buildup
• High biomass productivity and
photosynthetic efficiency
• Efficient mass transfer
• Low biomass productivity and
requires more land area
• High capital and operating costs
• Decreased S/V upon scale up
• Modest scalability
• High power consumption
• Difficult to scale up
• Expensive temperature control
• Fouling / settling of biomass
• Difficult to scale up
• Wall growth
• Difficult temperature control
• High power consumption
• Difficult to scale up

6 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
and biofuel with CO2 valorization
costs associated with artificial illumination remain a major
obstacle, while designing a novel photobioreactor. To
reduce the power consumption due to artificial lighting,
Chen et al.72 proposed a solar-energy-excited optical
fiber photobioreactor to supply electricity for providing
illumination to microalgal cultures. As the solar energy
supply varies with time and season, Chen et al.72 also
developed a light-dependent resistor that instinctively
switches to the artificial light sources when the intensity
of sunlight drops to a set value. This was demonstrated to
ensure a continuous and stable illumination supply to the
photobioreactor. Chen et al.73 further postulated that solar
panels and wind power generators (with solar energy excited
optical fiber) can be used to meet electricity requirements
adequately for the entire cultivation period. Recent
developments and future research trends of optical fiber
technology can contribute effectively to reducing the costs of
the artificial illumination supply for photobioreactors.
Mixing and mass transfer characteristics
The important hydrodynamic parameters of a photobioreactor
are mixing and gas hold up and they depend on the flow
regime of the reactor. In microalgal cultivation systems,
mixing is generally achieved by either aeration or mechanical
agitation. As mechanical agitation creates hydrodynamic
stress to some microalgal cells, aeration by indigenously
designed spargers is usually a preferred method of mixing.42
Mixing or aeration of a photobioreactor significantly affects
the performance of microalgal cultivation for the following
reasons:57 (i) it ensures uniform light and nutrient utilization
by algal cells; (ii) it avoids fouling or sedimentation of the
cells; (iii) it improves heat and mass transfer effects; and (iv) it
influences the gas hold up of the reactor.
Gas–liquid mass transfer is one of the important design
considerations of a photobioreactor, in terms of increasing
CO2 fixation by the microalgae, and O2 evolution from the
reactor. The solubility of CO2 in water is around 1.46 g L−1
(at 25 °C and 1 atm), which is very high compared with O2
solubility. In general, the solubility of CO2 increases with
increasing pH and pressure, and decreases with increasing
temperature.36,40 The gas–liquid mass transfer rate of CO2 is
given by Eqn (1):
N CO2  K La C *  C  (1)
where KL (ms−1) is the liquid-phase mass transfer
coefficient, a (m2 m−3) is the specific area available for mass
transfer, C* and C (mg L−1) are dissolved CO2 concentration
at saturation and in the liquid medium, respectively. When
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
R Dineshkumar, R Senz
the culture medium is sparged with CO2 or flue gas CO2,
the inorganic carbon exists in four different forms such as
free CO2, bicarbonate ion (HCO3−), carbonate ion (CO32−),
and true carbonic acid (H2CO3).74 The set of chemical
equilibrium reaction states of these carbonate species is
given below:
Carbonic acid formation:
CO2 + H2O  H2 CO3 (2)
Carbonic acid equilibrium:
 
H2CO3  HCO3  H (3)
 2 
HCO3  CO3  H (4)
The predominant inorganic carbon species at pH values
2–6, 6.5–10, and 10.5–12 are H2CO3, HCO3−, and CO32−,
respectively. In most of the studies reported in the literature,
the pH level of the microalgal culture ranges between 6.5
and 10 and, hence, bicarbonate ions are the major inorganic
carbon species available for microalgal growth.40,74 The
challenges associated with microalgae-mediated CO2
sequestration with regard to mass transfer characteristics
could be addressed by (i) minimizing CO2 loss from the
reactor by adopting a reactor-in-series strategy;48,75 (ii)
improving CO2 retention along the photobioreactor by
installing hollow fiber membrane systems;76 and (iii)
maintaining an adequate CO2 concentration in the culture
medium to avoid carbon excess or limitation conditions by
using advanced process controllers.71
An illustrative case study
Process intensification strategies
for microalgae-based lutein and lipid
production
Lutein: Properties and applications
Lutein is one of the commercially important carotenoids
and it is an essential component of the macular region of
the eyes. Conventionally, it has been produced from the
petals of marigold flowers through intensive labor and land
processes. The increasing demand for lutein has driven
the researchers to search for an alternative source like
microalgae, so as to reduce dependency on agricultural
land.21,77 The blue-light filtering efficiency of lutein is
very high, compared with other carotenoids, due to its
electron-rich conjugated structure e.g. lutein > zeaxanthin
7
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization

> β-carotene > lycopene.78 In plants and algae, the major
role of lutein is to protect the photosynthetic components
and acts as an accessory pigment for chlorophyll. Due to
various health benefits, the demand for lutein has steadily
increased, which is evident from its market potential. It
shall be noted that the global market potential of lutein is
expected to increase drastically with an annual growth of
3.6%.79,80 In addition to its antioxidant characteristics, some
of the classic applications of lutein are the prevention and
treatment of age-related blindness and cataract formation.21
Recently, lutein has been reported for its ability to prevent
and ameliorate breast and colon cancers,81,82 and it also
helps in stimulating cell-mediated and humoral immune
responses.78
Biosynthesis of lutein and lipid
Figure 4 shows the biosynthesis pathway that illustrates the
flow of carbon from CO2 to lipid and lutein.83,84 The enzyme
ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo)
fixes the inorganic CO2 into the first organic carbon
compound, namely, 3-phosphoglycerate (3-PGA), which is
then converted into pyruvate and acetyl-CoA.
In lutein biosynthesis, as in that of any other carotenoids,
isopentyl pyrophosphate produced through the MEP
(2-C-methyl-d-erythritol-4-phosphate) pathway, acts as
the precursor molecule. It is then converted to phytoene
and lutein through a series of enzymatic reactions. While
in lipid synthesis, acetyl-CoA is converted to malonyl-
CoA by a critical enzyme, acetyl-CoA carboxylase,
and finally, the storage lipid triacylglycerol (TAG) will
be produced by fatty acid synthase and other critical
enzymes. The neutral lipid TAGs are then extracted
from the cells and transesterified to produce fatty acid
alkyl esters – that is, biodiesel. It has been reported that
medium and process parameters could either up-regulate
the key enzyme levels or act as cofactors for the enzymes
involved in the lutein and lipid metabolic pathways.
Consequently, the production of lutein and lipid could be

Figure 4. A biochemical pathway leading to lipid and lutein synthesis in microalgae. RuBisCO, ribulose-1,5-bisphosphate
carboxylase/oxygenase; PDH, pyruvate dehydrogenase complex; G-3P, glyceraldehyde-3-phosphate; MEP, 2-C-methyl-
d-erythritol-4-phosphate; ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; MAT, malonyl-
CoA:ACP transacylase; FAT, fatty acyl-ACP thioesterase; DHAP, dihydroxyacetone phosphate; G3PDH, glycerol-3-phosphate
dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lyso-phosphatidic acid acyltransferase; PAP, lyso-
phosphatidylcholine acyltransferase; DGAT, diacylglycerol acyltransferase.

8 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid R Dineshkumar, R Senz
and biofuel with CO2 valorization

strategically improved through the optimization of critical
physicochemical parameters.
Influence of physicochemical parameters on
lutein and lipid production
One of the most essential steps in bioprocess and bioproduct
development is to identify and optimize the critical
physicochemical parameters that significantly influence the
production of desired products. It has been reported that the
optimal levels of medium components along with process
parameters could effectively improve the production of lutein
and lipid.15,21 The main physicochemical parameters that have
non-synergistic effects towards lutein and lipid synthesis are
nitrogen and light intensity.
In an attempt to improve lutein synthesis in microalgae,
most of the studies suggested the use of a higher concentration
level of nitrogen and some metal ions (e.g. Cu, Mn, and Fe)
in the medium along with the optimal level of light intensity
and CO2 (Table 4). For example, the lutein productivity of
Desmodesmus sp. F51 increased from 1.71 to 2.31 mg L−1 d−1,
upon increasing the nitrate concentration from 4 to 8 mM.31
Besides the role of nitrogen as a key factor in synthesizing
enzymes for lutein synthesis, supplementation of the copper
ion from 0.06 mM to 2 mM positively influenced the lutein
production by 20% via oxidative stress.87 In another study, the
optimal light intensity level (from 150 to 300 μmol m−2 s−1)
enhanced the lutein productivity of Scenedesmus obliquus
FSP-3 by 133%.32 However, a further increase in light
intensity level decreased the accumulation of lutein. This
might be attributed to the rapid up-regulation of carotenoid
synthesis at optimal light intensity levels and the reduction
of light-harvesting receptors (where lutein is predominantly
accumulated) at high light intensity.32,80
On the other hand, lipid production has been reported
to increase under low nitrogen concentration and at
high light intensity levels (Table 5). For instance, upon
reducing the nitrogen in the medium by 75%, Converti
et al.94 reported twofold improvement in lipid content in
the tested microalgal species, namely Chlorella vulgaris
and Nannochloropsis oculata. Under nitrogen limitation
or starvation conditions, the flow of carbon shifts from
protein to lipid synthesis, and hence it leads to increased
lipid production at the expense of biomass concentration.
However, the synthesis of primary carotenoid lutein will be

Table 4. Influence of physicochemical parameters on biomass and lutein productivities.

Microalgae Critical nutrient Process conditions Biomass productivity Lutein productivity References
studied (g L−1 d−1) (mg L−1 d−1)
Coccomyxa acidophila 0.67 g L−1 urea 150 μmol m−2 s−1 5% CO2 0.25 2.0 85

86
Chlorella sorokiniana 40 mM nitrate; 690 μmol m−2 s−1 1% CO2 0.84 2.5
2 mM NaCl
Coccomyxa onubensis 0.2 mM Cu2+ 160 μmol m−2 s−1 5% CO2 0.42 2.12 87

31
Desmodesmus sp. F51 8.8 mM nitrate 600 μmol m−2 s−1 2.5% CO2 0.494 2.31
88
Dunaliella salina 36 mM nitrogen; 170 μmol m−2 s−1 2.5% CO2 0.5 3.68
1.7 M NaCl
Scenedesmus 0.5 g L−1 nitrate 300 μmol m−2 s−1 2.5% CO2 0.918 4.15 32

obliquus FSP-3

Table 5. Influence of critical physicochemical parameters on lipid production.

Microalgae Critical nutrient Process conditions Lipid content (%) Lipid productivity References
studied (mg L−1 d−1)
Scenedesmus obliquus 120 mg L−1 nitrate 140 μmol m−2 s−1 2.5% CO2 19 139 89

CNW-N
Botryococcus braunii 86 mg L−1 nitrogen; 200 μmol m−2 s−1 0.04% 43 29 90

27 mg L−1 iron CO2

Chlorella vulgaris 0.766 g L−1 nitrate 60 μmol m−2 s−1 2% CO2 25% 56.2 91

−2 −1 92
Chlorella sorokiniana Deep sea water; 150 μmol m s 5% CO2 57.7 140.8
0.04 g L−1 PO4
93
Chlamydomonas sp. High salt medium; 250 μmol m−2 s−1 4% CO2 33.1 169.1
JSC4 3.5% sea salt

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107 9
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization
drastically affected, as sufficient nitrogen is required for the
continued synthesis of light harvesting proteins and other
functional xanthophylls.31,95 Similarly, in response to excess
light conditions (>300 μmol m−2 s−1), the concentration of
lipid was found to increase as a result of stress stimulus.89,96
Conversely, the synthesis of lutein was found to decrease
substantially, because lutein accumulation takes place in
close proximity to photosynthetic antenna complexes.80
These research findings clearly demonstrate the underlying
practical issues for enhancing the production of both lutein
and lipid simultaneously. Hence the optimal conditions can
be prioritized for the production of lutein, as it is a growth-
associated and high-value product. In this context, lutein can
be considered as a primary target product, and lipid (as a
co-product) can be concurrently extracted from the biomass.
The existence of no review reports on the co-production of
lutein and biodiesel along with CO2 mitigation served as one
of the main motivations for this review article.
Traditionally, the optimization of the parameters mentioned
above for increasing product yields is usually performed
with a one-factor-at-a-time strategy (Table 4; Table 5). It
should be noted that response surface methodology-based
optimization may not completely describe the complex non-
linear biological processes by a second-order polynomial
equation. As this conventional method of optimization
is either inefficient or laborious, the advanced modeling
approaches such as artificial neural network modeling and
the particle swarm optimization technique have emerged to
solve non-linear and multivariate bioprocesses.97,98 Recently,
this modeling technique was used for the optimization of
environmental parameters to improve lipopeptide production
by Bacillus megaterium.99 The same tool was implemented
to optimize essential medium and process parameters for
enhancing lutein and lipid productivities in a microalga,
Chlorella minutissima.100,101 It was observed that the
nutrient components such as nitrate, phosphate, manganese,
and copper were found to influence lutein production
significantly, by around 90%. Moreover, the optimization of
critical medium and process parameters through an advanced
Table 6. Performance evaluation of different photobioreactor modes of operation on lutein content and
productivity.
Microalgae Modes of operation
Scenedesmus almeriensis Continuous
Desmodesmus sp. Fed-batch
Desmodesmus sp. Repeated fed-batch
Scenedesmus obliquus FSP-3 Two-stage (light
intensity shift)
Scenedesmus obliquus FSP-3 Semi-continuous
10 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
optimization technique enhanced the production of lutein
and lipid by 260% and 164%, respectively, as compared with
conditions that were not optimized.100,101
Role of bioreactor strategies on lutein and
lipid production
In general, fed-batch, semi-continuous, and continuous
reactor strategies can be used for long-term operation for
the synthesis of the required products. Critical nutrients
like nitrates or ammonium have been strategically fed to
the photobioreactor to further improve microalgal lutein
production (Table 6). For instance, Sánchez et al.102 observed
that the continuous reactor mode involving the addition of
fresh medium at a dilution rate of 0.03 h−1 resulted in higher
lutein productivity of 4.77 mg L−1 d−1 with lutein content
of 5.5 mg g−1. In another study, Xie et al.31 assessed that the
pulse feeding of 2.2 mM nitrate was found to increase the
lutein productivity and content by 16% and 27% respectively,
in comparison with the batch process.
As high light intensity shrinks the photosynthetic antenna
complexes, lutein accumulation is usually reduced at high
illumination levels. Accordingly, Ho et al.103 developed a
two-stage cultivation strategy involving shifting of light
intensity from a high level (300 μmol m−2 s−1) to a low
level (75 μmol m−2 s−1). It was observed that lutein content
(mg g−1) of Scenedesmus obliquus FSP-3 increased by 13%,
but there is a slight decrease in lutein productivity because
of lower biomass yield in the second stage. However, the
use of semi-continuous reactor mode with 10% medium
replacement resulted in maximum lutein productivity of
6.01 mg L −1 d−1.103 Although this strategy improved the
lutein productivity by 36% in comparison with the batch
operation, the lutein content did not increase (Table 6).
Hence, it is essential to develop bioreactor strategies that will
facilitate higher lutein productivity and lutein content for the
cost-effective lutein production process. One of the effective
bioprocess strategies to enhance both lutein productivity
and content is to reduce photo-inhibition during the initial
growth stages and also avoid photo-limitation at later growth
Lutein productivity (mg L−1 d−1 Lutein content (mg g−1) References
102
4.9 5.5
31
3.56 5.05
79
4.61 4.42
103
4.2 4.7
103
6.0 4.6
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
and biofuel with CO2 valorization
phase. In this context, Dineshkumar et al.104 employed
an optimal light-feeding approach followed by a semi-
continuous mode of photobioreactor operation for improved
lutein content and productivity of Chlorella minutissima. The
combination of a linear light-feeding strategy with 20% fresh
medium replenishment showed consistent lutein production
for about five consecutive reactor operations. Moreover,
this optimized light-feeding and semi-continuous strategy
resulted in comparatively higher lutein productivity and
content than other studies reported in the relevant literature.
This incremental light-feeding approach can be employed
with the photobioreactors using microcontroller devices to
vary light intensity with time. These microcontrollers can also
be coupled with solar-energy excited optical fiber system to
reduce the dependence on artificial lighting.
For lipid production, a two-stage reactor operation involving
sufficient nitrogen in the first stage and nitrogen starvation
in the second stage has been considered to be an efficient
reactor strategy for enhancing lipid productivity without
affecting biomass yields significantly (Table 7). For instance,
Rodolfi et al.105 demonstrated this two-stage reactor strategy
by cultivating Nannochloropsis sp. in a 110 L photobioreactor
under outdoor conditions. It was observed that the lipid
production increased from 117 mg L−1 d−1 in a nitrogen-
rich phase with 32% lipid content to 204 mg L−1 d−1 in the
nitrogen-deprived stage with 60% lipid content. Similarly,
Mujtaba et al.33 cultivated Chlorella vulgaris in two-stage
reactor operation and the lipid productivity was found to
be improved by around 125%, as compared with the batch
process. However, this strategy may not be suitable while
enhancing lutein and lipid simultaneously. Chen et al.92
studied the use of semi-continuous operation involving
medium replenishment to assess the stability of microalgal
biomass and lipid production for long-term operation. A slight
decrease in lipid productivity was observed in their study,
but the growth and biomass production remain unaffected
(Table 7). Hence, the development of semi-continuous
or appropriate bioreactor strategies can be strategically
performed for enhancing both lutein and lipid production.
Table 7. Performance comparison of various photobioreactor modes of operation on lipid content and
productivity.
Microalgae Modes of operation
Nannochloropsis sp Two-stage; nitrogen starvation
Chlorella sp. Semi-continuous (25% replacement)
Nannochloropsis oculata Two-stage; nitrogen starvation
Chlorella sorokiniana Semi-continuous (50% replacement)
Chlorella sp., BUM11008 Two-stage; nitrogen starvation
Desmodesmus sp. F2 Semi-continuous (90% replacement)
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
R Dineshkumar, R Senz
Downstream processing of microalgal
biomass
To achieve an economically viable production process,
it is essential to develop cost-effective downstream
processing techniques along with improved photobioreactor
strategies.110,111 The steps involved in the downstream
processing of microalgal biomass are (i) harvesting biomass
from culture media; (ii) dewatering or drying the harvested
biomass; (iii) disrupting the cells to release intracellular
products; and (iv) extracting and recovering the desired
products simultaneously.
Biomass harvesting
Harvesting biomass may significantly impact the economics
of the process, as the cost of biomass separation contributes to
around 20–30% of the total production costs.17,112 The major
challenges associated with harvesting biomass are: (i) the
small size (2–10 μm) of the cells; (ii) relatively lower biomass
concentration (1–3 g L−1) compared to bacterial and yeast
biomass; and (iii) negative surface charge of cells that prevents
settling of biomass by gravity. The conventional harvesting
techniques including centrifugation and filtration are not
recommended for the large-scale processing of microalgal
culture due to their high capital and operational costs.14,53
Flocculation using cationic chemicals like aluminum and
iron-based metal salts also has a negative impact when
recycling the medium and also affects the quality of the value-
added products.113
On the other hand, flocculation using biopolymers like
chitosan, cationic starch, cassia gum, and guar gum showed
higher biomass recovery with lower dosages and negligible
negative impact on the environment.113,114 Rashid et al.115
reported the highest harvesting efficiency of 99% to recover
around 1 g L−1 algal concentration at pH 6 with a chitosan
concentration of 120 mg L−1. Similarly, Farid et al.116
employed nano-chitosan and the dosage requirements
were found to decrease by 40% with improved harvesting
efficiency, as compared with chitosan. In another study,
Lipid content (%) Lipid productivity (mg L−1 d−1) References
105
60 204
106
45.1 139
107
44.5 170.1
92
37 73.5
108
42.8 53.96
109
45.6 302
11
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization
Vandamme et al.117 observed that the use of cationic starch
(10 mg L−1 dose) showed 80% flocculation efficiency for an
initial biomass concentration of 0.15 g L−1. Despite higher
harvesting efficiency and lower environmental impacts of
these flocculation methods, non-recycling of biopolymers
impedes their applicability at a large-scale level. Hence,
reusability of the polymers by conjugation with magnetite
(Fe3O4) particles is suggested as a viable option for cost-
effective biomass harvesting and long-term use.118,119
Recently, magnetite nanoparticles conjugated with chitosan,
polyacrylamide, polyethylenimine, and amino clays have
been tested for biomass harvesting and consequent reuse of
the polymer.120–122
The detachment of magnetic particles from biomass
(while recycling) involves the use of acid or base, so Lee
et al.112 developed magnetic nanocomposites containing
octyltriethoxysilane, which helps in the immediate
detachment of magnetic flocculants at the water-nonpolar
solvent interface by an external magnetic force. Similarly,
Ge et al.121 prepared a steric acid-coated ZnO magnetic
nanocomposite to achieve separation of magnetic particles
from algae through a hydrophobicity shift under ultraviolet
(UV) illumination. Hence, the following design strategies
can be implemented to develop sustainable harvesting
methods: (i) avoiding chemicals and reducing fossil energy
consumption; (ii) employing green methods / biopolymers
for harvesting; (iii) recycling the used biopolymers for
subsequent batches; and (iv) developing an integrated process
that concomitantly addresses subsequent downstream steps.
Cell disruption
This downstream unit operation facilitates the release
of intracellular products from the cells. The three main
approaches to disrupt microalgal cells are (i) mechanical
operations such as ultrasonication, high-pressure
homogenization, bead-beating, and microwave radiation;
(ii) chemical methods like cationic detergents and alkali;
and (iii) biological methods such as the use of enzymes.
Among the disruption techniques, mechanical methods
reported in the literature exhibit higher biomass disruption
efficiency.71,123 However, the energy required by different
physicochemical methods to disrupt 1 kg of biomass was
calculated to be 33–132 MJ.123 Although detergents or alkali
can effectively disrupt the algal cells, these chemical agents
will hamper the recyclability of culture medium and also pose
ecological problems when it is released to the environment.113
On the other hand, the use of enzymes such as cellulase,
hemicellulase, and pectinase was found to disrupt around
80% of Chlorella sp. cells.124 In order to reduce the cost of
enzymes, Fu et al.125 performed enzyme immobilization
12 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
and upon recycling it for five batches, the cell disruption
efficiency was found to have decreased by 40%, indicating the
inefficiency of this process. This shows that the conventional
cell disruption methods mentioned above are either energy
/cost intensive or environmentally hazardous. Hence, it is
essential to develop an integrated downstream process like
the simultaneous separation and disruption of biomass, with
a focus on reducing the dependency on chemicals or any
energy-intensive equipment.
Recently, aminoclay-conjugated-TiO2 nanoparticles
have been reported to simultaneously harvest and disrupt
the cells of Chlorella sp. after UV-irradiation at 365 nm
for 3 h.126 In another study, the chemical surfactant cetyl
trimethyl ammonium bromide was utilized to separate
and disrupt the microalgal biomass.127,128 Despite these
attempts to develop an integrated downstream process,
the use of chemicals and UV-irradiation could not be
employed for processing microalgal biomass at a large scale.
In this context, Dineshkumar et al.129 developed multi-
functionalized magnetic nanocomposites (MNCs) that
consist of cationic charged chitosan biopolymer, which was
functionalized with TiO2–Fe3O4 nanoparticles to achieve
concomitant biomass separation and cell disruption along
with subsequent reuse of the prepared nanocomposites. The
MNCs employed in this study could harvest over 98% of
cells and also effectively disrupt the cell walls through visible
light-driven photocatalysis. The harvesting efficiency and
photocatalytic cell disruption efficiency were not significantly
affected by recycling the MNCs for five consecutive
downstream operations. Moreover, the preliminary economic
analysis indicated that this integrated downstream process
approach could reduce the cost by 73%, in comparison with
conventional methods of biomass harvesting, drying, and
disruption.129 However, a detailed life-cycle assessment
must be performed to ensure the economic viability of the
developed integrated downstream process.
Concurrent extraction and recovery of lutein
and lipid
One of the critical issues in developing a biorefinery is the
concomitant extraction of two or more products from the
biomass. The extraction of carotenoids and lipids is mostly
carried out by solvents such as chloroform, methanol,
ethanol, hexane, dichloromethane, diethyl ether, and
isopropanol.80,114 There are many reports that investigated
the extraction of lipids from microalgal biomass using binary
solvents involving chloroform and methanol.130,131 However,
this binary solvent system can also extract significant
amounts of pigments along with lipids in both aqueous
and organic phases, and hence it cannot be employed for
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
and biofuel with CO2 valorization
the concomitant separation of the products. Moreover,
hazardous solvents like chloroform must be avoided, as
they cannot be used at an industrial scale. Bai et al.132 found
that a methanol-hexane solvent system could recover 90%
of chlorophyll pigment and 98% of lipids from Chlorella
pyrenoidosa biomass simultaneously. Although supercritical
CO2 extraction has been tested to recover lipid and pigments
concurrently, the lower pigment recovery (70%) and higher
capital and operating costs hamper its commercial viability
compared with traditional solvent extraction.133 Recently,
Prommuak et al.134 reported the use of an alkali catalyst for
simultaneous saponification and transesterification reactions
to recover lutein and biodiesel concurrently under optimal
process conditions. Despite the advantages of this extraction
method, possible azeotrope formation during extraction
complicates the unit operations associated with evaporation
and the recovery of used solvents. Moreover, the yields of
fatty acid methyl esters (biodiesel) may be affected due to
partial saponification of lipids by the alkali catalyst.134
Alternatively, lutein and lipid by virtue of their preferential
partitioning towards dissimilar phases can also be extracted
concomitantly with the use of a polar and a non-polar
solvent. This method is preferable only if the solvents used
are non-hazardous and recycled back to the process. The
carotenoid lutein has been found to be more soluble in polar
solvents such as methanol, ethanol, and isopropanol than in
non-polar solvents such as hexane.135 It is well documented
that a non-polar solvent like hexane can extract neutral lipid
fraction efficiently from the disrupted biomass.136,137 In this
context, Dineshkumar et al.101 employed binary solvent
systems involving methanol or ethanol with the non-polar
solvent hexane to extract lutein and lipid effectively in
corresponding aqueous and organic phases. The lutein-
rich aqueous methanol or ethanol phase was saponified to
obtain pure lutein, whereas the lipid-rich hexane portion was
transesterified to obtain biodiesel. It was observed that this
binary solvent system recovered 94.3% of lutein and 92.4% of
biodiesel concomitantly from the algal biomass. This proof of
concept study indicates the feasibility of developing an algal
biorefinery, which can contribute to the demand for bioenergy
cost effectively by the simultaneous production of a high-
value carotenoid product. While separating the carotenoid
lutein fraction after saponification, the major photosynthetic
pigment such as chlorophylls could be concurrently recovered
and used as an antioxidant and food-coloring agent.
Preliminary techno-economic implications
The process flow sheet of the algal biorefinery model that
is proposed in this review is shown in Fig. 5. As most of
the studies concluded that microalgae-based biodiesel is
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107
R Dineshkumar, R Senz
uneconomical, Prommauk et al.134 investigated the economic
viability of concurrent production of lutein and biodiesel
from algal biomass. The preliminary economic assessment
was calculated based on 20-year annuities with 7% of capital
costs per year (for maintenance, taxes, insurance), operating
costs (utilities, labor and miscellaneous at 0.9% of capital),
and fixed costs (depreciable capital cost, spare parts, initial
expenses, process startup, and license fee). Considering
0.15–0.35% of lutein content and 3–5% of biodiesel yield by
dry weight biomass, the sensitivity and economic analyses
studied by Prommauk et al.134 suggested that a maximum
of $95 value of lutein could be obtained per kg biodiesel.
Hence, this preliminary economic analysis indicated that
the concurrent production of lutein and biodiesel may be
economically viable. However, a detailed life-cycle assessment
(LCA) involving cost and energy analyses for the entire
upstream and downstream process should be performed
to understand the economic feasibility of this proposed
biorefinery model. Moreover, the systematic scaling up of
this process to pilot-scale level could be helpful for LCA
studies. The results from such an LCA report could reveal the
commercialization potential of microalgae-based lutein and
biodiesel production.
The proposed algal biorefinery is in the research and
development phase, so the possibility of leveraging other
value-added products from the de-oiled and de-pigmented
algal biomass can also be envisioned to strengthen further
the microalgal biorefinery concept. For instance, the de-oiled
biomass containing proteins and carbohydrates of low carbon
to nitrogen-rich material can be used for biofertilizer or
animal-feed applications.138,139 Alternatively, the hydrolysate
part of de-oiled biomass may be employed as a nutrient
source for microbial fermentation processes like bioethanol,
biomethane, or biohydrogen production. Further, it is also
suggested that the residual defatted biomass may be used as
a biomaterial for the preparation of nanoparticles or carbon
dots for biomedical applications.138
Conclusions and future perspective
In view of current energy and environment requirements,
more research must be aligned to transforming the paradigm
of producing fuels, power, and value-added chemicals
systematically from a fossil-based economy to a biomass-
based economy. In this context, the microalgal biorefinery
has emerged as one of the sustainable solutions for the
production of biofuels and high-value carotenoids along with
concomitant CO2 mitigation.
As a case study, this review article focused on the
co-production and recovery of high-value carotenoids
13
 R Dineshkumar, R Sen Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid
 and biofuel with CO2 valorization

Figure 5. Process flow diagram of proposed microalgal biorefinery model demonstrating the proof of concept for the
co-production and recovery of biodiesel and high-value nutraceuticals like lutein with simultaneous CO2 capture.

like lutein for healthcare applications and lipids for biofuel
purposes, along with CO2 sequestration. The major
underlying technological challenges associated with
microalgae-based lutein and biodiesel production are low
product yields and high cost involved in cultivation and
downstream processing of biomass. Hence, the strategies
to develop an efficient algal biorefinery model should
be aimed towards (i) improving the bio-manufacturing
process by employing process intensification strategies, and
(ii) developing an energy-efficient downstream process.
Economically feasible lutein and lipid production can be
achieved by integrating the upstream and downstream
operations to reduce the cost and energy associated with the
process. A comprehensive cost and energy analysis should
also be conducted to explain the economic viability of the
proposed algal biorefinery.
The future prospects of the algal biorefinery also include
the construction of metabolically engineered strain(s)
that are capable of hyper-producing lipid and carotenoid
simultaneously. The systematic metabolic engineering
coupled with bioprocess strategies would be useful for
developing genetically engineered microalgal strains with
improved productivities of lipid and carotenoid for biodiesel
and nutraceutical applications. In addition to leveraging
carotenoid and lipid production from algal biomass it is also
essential to focus on deriving other value-added products
like carbohydrates for bioethanol and protein for animal feed
or bio-fertilizer applications. Thus, this review on microalgal
biorefinery provides valuable insights which are required to
address some current healthcare, energy, and environmental
challenges.
Acknowledgements
The authors acknowledge their institute, IIT Kharagpur
and CSIR–Central Salt and Marine Chemicals Research
Institute (CSIR-CSMCRI), for providing Internet facilities.
CSIR-CSMCRI Registration No. PRIS 108/2019 has been
assigned to this manuscript. R.D. gratefully acknowledges
the valuable technical suggestions and language editing of
Dr Vivek Rangarajan.
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Dineshkumar Ramalingam
Dr Dineshkumar Ramalingam is a senior
scientist in the CSIR–Central Salt and
Marine Chemicals Research Institute,
Bhavnagar, India. He obtained his PhD
(in Bioprocess Engineering) from Indian
Institute of Technology (IIT) Kharagpur.
His research interests involve an
interfacial role between chemical engineering and biology.
His work focuses on developing optimal upstream and
downstream processes for bio-products that benefit society
in the sectors of healthcare, energy, and the environment.
 Review: A sustainable perspective of microalgal biorefinery for co-production and recovery of high-value carotenoid R Dineshkumar, R Senz
and biofuel with CO2 valorization

Ramkrishna Sen
Dr Ramkrishna Sen, professor and
head, Department of Biotechnology,
Indian Institute of Technology (IIT)
Kharagpur, served as Fulbright Visiting
Professor at Columbia University, USA.
He works in the areas of energy and
the environment. Professor Sen has
so far supervised 24 PhD students, has authored about
150 international publications; has produced 14 patent
applications, and has completed 21 sponsored projects.
Professor Sen has been honored with a Jadavpur
University Distinguished Alumni award; a Fulbright
Fellowship; a UK-India Education and Research
Initiative (UKIERI) Visit Award, and a National Award for
Technology Innovation.

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2107 19