Introduction
Microalgae are well recognized for their capacity to produce a wide range of value-added
products and considered as a powerful biotechnological platform to commercially acquire these
products. Various high-value products can be obtained from microalgae and most of these are
having commercial applications in several areas such as pigment industry, animal and human
nutrition, pharmaceuticals, aquaculture, cosmetic products, and fuel industries . All of these
modes provide an appropriate environment for the biosynthesis of various value-added products,
and more recently, interest has been shifted more on carotenoids and lipids . All these advantages
make the algal processing process economically viable and thus made algae as an eligible
candidate for the sustainable production of various high-value products such as carotenoids and
lipids.
The requirement of a huge volume of water for cultivation and nutrient supply converts
commercial-scale algal cultivation into an economical incompetent process . However,
microalgae have an innate capacity to utilize excess nutrients found in wastewater and propagate
readily, consequently, can treat the wastewater and deliver safe water with minimal resources .
Wastewater is a natural pool of organic forms of carbon and nitrogen which includes nonreducing and reducing sugars, nucleic acids, urea, and proteins. Microalgae have been used in
treating wastewater containing higher level of toxic organic compounds and heavy metals .
Microalgae cultivation using wastewater can be a greener and sustainable remediation
technology. Owing to its natural ability to proliferate in open water and utilize its nutrient, these
minuscule organisms also govern the global nutrient cycle comprising C, N, and P pools .
Moreover, microalgae can also sequester the excess carbon dioxide from the atmosphere and flue
gasses released by industries . Among the wide range of products provided by microalgae,
carotenoids are the ones that are gaining importance and have become the focus of significant
biotechnological
commerce
owing
to
its
high
industrial
applications
.
Sensitivity analysis conducted by Rodrigues indicated the view to achieve 107,902.5 kg/year of
total carotenoids on an industrial scale using agroindustrial wastewater . A detailed analysis of
microalgae potential for some industrially important carotenoid along with structure and
biosynthesis is presented. Further, opportunity, benefits, and challenges of using wastewater for
algal cultivation directed towards carotenoid production are discussed.
Potential of microalgae as a source of bioactive metabolites and other applications
The first human consumption of microalgae is reported long back in the 2000s as in China,
Nostoc was used to combat famine . However, lately, microalgae have been emerged as a source
of various high-value beneficial industrial products due to its high nutritional value . On account
of their diverse physiological and biochemical characteristics, microalgae are equipped with a
fairly high concentration of various biochemicals such as 30 % lipids, 50–70 % protein, up to 8–
14 % carotene and vitamins B1, B2, B3, B6, B12, E, K, D, etc. as compared to animals or other
plants . Unlike terrestrial and aquatic animals, microalgae have been projected as cellular
factories for innumerable valuable biomolecules such as sterols , terpenes , fatty acids ,
chlorophylls, phycobiliproteins, alkaloids , etc that can be used in pigments, poultry, aquaculture,
polyunsaturated fatty acid or high-protein supplement in human diet and pharmaceutical
industries
and
also
in
the
biofuel
industry.
A growing range of investigations has also been directed to explore various pharmacological
properties associated with microalgae. Some of the specific genera of microalgae such as
Dunaliella, Spirulina, Chlorella, Botryococcus, Haematococcus, and Nostoc has been well
acknowledged as a source of various bioactive compounds . In addition, as it is obtained from
microalgae based fatty acids, which is renewable, biodegradable, and non-toxic .
Carotenoids are terpenoid pigments having a C40 backbone and lipophilic nature and responsible
for a wide array of pigments present in nature, for instance, orange, red and brilliant yellow in
vegetables, fruits, and invertebrates . Few common carotenoids found in algae and their
biosynthetic pathways are depicted in Fig. Primary carotenoids are structural and functional
constituents of the cellular photosynthetic apparatus. β-carotene in one branch where lycopene is
cyclised at both ends using lycopene β-cyclase and -carotene in the other branch using lycopene
β-cyclase and -cyclases Furthermore chain transformations hydroxylation, epoxidation,
glycosylation, ketolation, and oxygen cleavage mainly catalysed by various enzymes including
cytochrome P450 -hydroxylase, cytochrome P450 β-hydroxylase, carotene β-hydroxylase, βcarotene oxygenase, zeaxanthin epoxidase, violaxanthin de-epoxidase lead to the highly diverse
carotenoids
family.
The major part of the carotenoid biosynthesis is carried out in the chloroplast, with few steps in
the cytoplasm. Phytoene synthase is one of the most significant regulatory enzymes in the
carotenoid biosynthesis as it regulates the rate-limiting step . Keeping this in mind, most of the
genetic engineering approaches employed in enhancing carotenoid content aim its
overexpression by targeting the corresponding gene. Microalgae have been considered as a
natural pool of carotenoids, specifically, strains belong to Chlorophyceae family such as
Dunaliella salina, Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella zofingiensis,
Muriellopsis
sp.
In the context of commercial production, few species are highlighted for some specific
carotenoid. This section of the review will try to shed some light on the major player or major
commercial microalgae which are specific carotenoid producers. Such type of information can be
utilized aptly by extrapolating such findings for microalgae cultivation on wastewater aimed at
carotenoid
production.
Carotenoids
obtained
from
D.
.
Along
with
astaxanthin
other
carotenoids
that
are
produced
in
pluvialis are lutein, astaxanthin ester, violaxanthin, zeaxanthin, lutein, and β-carotene .
Canthaxanthin is known to be obtained from microalgae Chlorella zofingiensis in a good amount
. Cyanobacteria, which are prokaryotes commonly known as «bluegreen algae» have also been
identified majorly for carotenoid biosynthesis. β-carotene, zeaxanthin, echineone and
myscoxanthophylls are some of the common carotenoids found in cyanobacteria .
Some of the strains important in carotenoid production are Saccharina japonica and Gelidium
pusillum . The carotenoid production in Cyanobium sp. Total carotenoid content produced from
cyanobacteria Chlorogloeopsis fritschii was 154 μg/g . Similarly, microalga Asterarcys
quadricellulare PUMCC 5.1.1 was found to be prolific producer of carotenoids.
Total amount of carotenoids produced by the organism was 35 1.75 μg carotenoids mg 1 dry
biomass under control culture and 118 μg mg 1 dry biomass under optimized conditions.
Specific carotenoid produced were β-carotene, lutein, astaxanthin and canthaxanthin in 47.0,
28.7, 15.5 and μg/mg dry biomass, respectively, under optimized conditions . The carotenoids
nostoxanthin, canthaxanthin are potent scavenger of peroxyl radical and possess
antiinflammatory activity. Cyanobacteria associated with microalgae not only produce
carotenoids
but
many
active
metabolites
like
lipids
and
polysaccharides.
The major bottleneck associated with the industrial production of carotenoid from microalgae is
the high cost of the entire process and resource utilization. However, the bioremediation based
approach is a novel concept that integrates a wastewater treatment plant with microalgae
cultivation to obtain value-added biomass. Besides, obtaining carotenoid, such type of process
can be made more beneficial by utilizing other by-products generating during the process for
more value-added goods.
Algal cultivation on wastewater. An increase in urban wastewater is one of the major
consequences of the world’s growing population. A huge volume of wastewater is generated by
human activities from agriculture, urbanization, industrialization, and other alterations that are
responsible for various adverse effects associated with the environment . The most common
practices used in different industries for wastewater treatments include primary treatment and
secondary treatment.
Primary treatment aims to remove solid particles, whereas secondary treatments target the
bioremediation of organic matter through microorganism activities. However, such kinds of
treatment require high operational and maintenance costs . Utilization of chemical methods such
as precipitation using iron and aluminum salts produces a huge amount of sludge that needs
further treatment or disposal . Zero drafts of wastewater treatment coupled with microalgal
cultivation were prepared as an approach to minimize the microalgae production cost, however,
nowadays it is exploited as an alternative system for the conventional wastewater treatment
systems.
Considering the huge potential of microalgae in terms of nutrients uptake and biomass
productions, a growing body of research is impending to analyze their competence for the
commercial aspect . The principal nutritional requisite of algal growth comprises carbon ,
nitrogen , phosphorus , and various other micronutrients such as magnesium , iron , and calcium
that are present in abundant amount in wastewater . Large surface areas provided by microalgae
owing to its small size also assist in an efficient nutrient uptake. Microalgal growth and yield are
mainly dependent upon the availability of nutrients such as carbon, phosphorus, and nitrogen.
As most of the algae are mainly autotrophic, so they consume CO2 as carbon source, thus,
microalgae carbon sequestration has been touted as beneficial. Algal cells use this CO2 and fix it
using various biochemical reactions involved in several biosynthetic pathways and produce
carbohydrates, lipids and other value added products . CO2 as a well organic carbon source .
Finally, nitrogen is assimilated in the forms of nitrate-nitrogen , ammonium-nitrogen and nitrite-
nitrogen .
Considering the rich presence of these entire nutrients in wastewater depending upon the source,
decreased the production costs and carbon footprints, recent studies are highlighting the
exploitation of wastewaters for microalgal growth. Various wastewater streams where
microalgae are used for wastewater treatments are municipal , agriculture , and industrial stream
which includes rubber industry textile industry , palm oil mill effluent , sago starch processing
effluent. 8 μg/mL, with 99.97 % of phosphate and 81.10 % of nitrate removal from the
wastewater . Some other micro algae genera which are used to treat wastewater and are
considered as promising microalgae species are Scenedesmus, Botryococcus, and Arthrospira .
Heavy metals are removed by numerous algal species like Oscillatoria spp. , Chlorella vulgaris,
Chlamydomonas spp. The best growth found is in domestic wastewater using mixotrophic and
heterotrophic growth . Cultivation of microalgae consortia with heterotrophic growth in domestic
wastewater produced 1.0 g/L of biomass within one week .
The algal proficiency and nutrient recovery capacity to treat wastewater and produce biomass is
based upon its capacity to grow in specified conditions, which in turn depends upon several
factors such as solar radiation availability and photosynthetic efficiency. Cultivation of
microalgae using wastewater as a source provides varied ranges of economic and environmental
benefits and is considered as a sustainable approach for the production of high-valued and
marketed products from microalgae which have many health benefits like fatty acids, lipids, and
carotenoids. These products are produced when the microalgae is introduced or cultivated in
wastewater assimilate the organic and inorganic pollutants as a nutrient source. Along with the
growth and production of biomass, the microalgae concurrently treat the wastewater from
different sources such as domestic, industrial, agricultural, sewage effluent .
The biomass produced from microalgae cultivated in wastewater has great potential for
sustainable products such as pigments and fatty acids . The wastewater can be either used as a
medium for cultivation directly or can be supplemented with other medium composition as
required. The other benefit of using wastewater is the availability of convenient water source for
growth and production of higher biomass which in return proves out to be economically feasible.
Pigments generally gets expressed when exposed to stress or supplemented with extra nitrogen
and phosphorus .
The biochemical composition of microalgae is altered under stress condition and presence of
inorganic and organic nutrients along with toxicants stimulate the production of carotenoids and
lipids from microalgae by acting as a natural source of stress to the cell of microalgae . A very
recent study conducted by Ajijah and coworkers investigated the effect of using tofu wastewater
as a cultivation medium for microalgae species Chlorella vulgaris and Arthrospira platensis and
its effect on biomass and carotenoid content of these species. It was observed that after 5 days of
incubation, 5 % tofu wastewater medium provided the highest carotenoid content i. Arthrospira
platensis using 1% fresh palm oil municipal effluent with commercial nutrient in outdoor
cultivation in 7 days .
The utilization of diluted synthetic wastewater for the growth of
Mixotrophic cultivation provided to Nephroselmis sp. with industrial wastewater as a water
source and commercial medium nutrients produced essential fatty acids and carotenoids. Lutein
and β-carotene were produced with μg/g and 188 μg/g maximum productivity to percentage of
wastewater utilized for the cultivation . Microalgae isolated from wastewater produces
carotenoids as bioproducts.
When cultivated in different cultivation systems Kirchneriella sp. , is proved to be a significant
carotenoid producer. Lutein and β-carotene were main carotenoids produced having 0.14 and
0.03 % DW of biomass respectively .
The cost associated with the production of carotenoids, acquiring essential nutrients for the
growth of microalgae, changes in biochemical composition and equipment operation can be
reduced by using wastewater for the mass cultivation of microalgae in systems which combat
these challenges and helps in higher production of market valued biomass and carotenoids which
when generally produced is a costly exercise. Cultivation systems with a focus to produce higher
yields of biomass and carotenoids should be developed with a screening of microalgae species
naturally present in wastewater or can be cultivated in it. It is well known fact that waste water
collected from different sites such as sewage, dairy, municipal, variant industrial, etc. consists of
several microorganisms apart from microalgae depending upon the nature of wastewater. In algal
research, it is often reported that consisting microorganism specially bacteria is regarded as
contaminants, nevertheless recent studies reveals that many bacterial symbionts not only
promotes algal growth but also proves to be advantageous in downstream processing .
To make the algal cultivation and production of metabolites, an economically feasible process it
is very important to study and understand the advantages and risks associated with algal
microbial interactions. Introducing several species of bacterial proves out to increase the algae
growth at large scale and also helps in preventing growth of large algal pathogens. It has also
been reported that few microalgae, live in symbiotic relationship with multiple associated
microorganism throughout their life cycle . It is been reported that removal of these
microorganisms were not proven to be successful and exhibited lesser growth of microalgae
which indicates that the relationship between algae and other microorganism is important for
their existence .
Nitrogen fixed by bacterial species living in close association of microalgae community serves as
nitrogen source for growth and carotenoid production in the form of ammonia . Scenedesmus
dominant municipal wastewater along with Protobacteria helps in nutrient removal. Chlorella
sorokiniana associated with Protobacteria in winery wastewater removes TN = 100 % .
Phosphorus, an essential nutrient required for algal growth, is supplied by bacteria via
decomposing organic phosphorus into organic which was then taken up by algae.
Aromatic polycyclic hydrocarbons are types of pollutants present in wastewater and possess high
risk to human and animal health, these can be degraded co-culturing Chlorella sorokiniana and
Peuodmonas migulae . Microalgal-bacterial consortia not only play significant role in treating
wastewater but are proven to be potential for industrial production of highly valuable metabolites
like lipids, proteins and pigments. Wastewater generated from modern world industrialization,
urbanization, and agriculture operations has a strong detrimental effect on the usable water pool
and the aquatic environment. Extensive research has been carried out in recent times on the
utilization of microalgae for wastewater treatment.
Similarly, compared to common nitrogen removal treatments such as denitrification/nitrification,
most of the nitrogen is released as nitrogen gas, on the other hand, algal-based treatment convert
nitrogen into biomass. Thus algal-based treatment is considered an ecologically safer and less
expensive way to remove nitrogen . As an end product, microalgal treated wastewater provides
oxygenated water for environments and biomass provided by it can be used in various
applications such as pigment production, biofuel, feed, nutraceuticals, and various high-value
bioproducts . Algal harvesting also assists in carbon dioxide sequestration release from flue gases
and consequently helps in mitigating greenhouse gas emission.
Compared to terrestrial plants having photosynthetic efficiency of 1–2 % only, photosynthetic
efficiency of the microalgae system is far better and ranging around 10–20 % and thus
microalgae-based systems have reported to quench up to 53 % of CO2 from simulated flue gases
. For better utilization of the algal capacity of CO2 sequestration, an appropriate selection of
algal species and a comprehensive understanding of various environmental factors governing
algal growth is a basic need. Few algal species specifically, Scenedesmus sp. The faster growth
rate of the microalgae system is also a factor responsible for higher carbon sequestration rates as
compared to terrestrial plants .
Upstreaming process in carotenoid production
Considering the high commercial potential of algae-based wastewater treatment and associated
bio-based products, a growing body of research is conducted for the upstreaming and
downstreaming process by various industries and companies. This part of the review will briefly
describe some of the cultivation techniques and strategies used to enhance carotenoid production.
Microalgal cultivation in wastewater can be classified into open or closed systems, broadly.
These include raceway ponds and turf scrubbers which are mainly used in wastewater treatment
along with algal cultivation .
Open ponds consist of an oval pond impermeable in nature, combined with a series of plates
divided where the circulation of water occurs through tanks for uniform mixing and maintaining
the algal growth . Due to simple design with minimum requirements for maintenance, an open
pond system proves out to be a low cost and most commercially accepted operating system for
algal cultivation. It has been found that cultivation of Scenedesmus sp. LX1 in an open pond fed
by secondary wastewater, yield maximal microalgal biomass production rate about 20 gm/m2/d
biomass under optimized condition.
Constructing raceway pond is inexpensive than many types of closed systems nevertheless it has
drawbacks such as no proper mixing, uneven light intensity, poor mass transfer, carbon dioxide
underutilization , and microbial contamination . production uses an open pond system . This
technology is designed to stimulate natural wastewater treatment processes. It uses filamentous
algal species which improves the quality of wastewater from agriculture and domestic sludge.
It contains sloping surfaces with attached, naturally seeded filamentous algae coupled with
polluted water flows, which provides treatment through the uptake of organic and inorganic
compounds from wastewater during photosynthesis . Photobioreactors are designed to reduce the
complications associated with large open microalgae cultivation system. When compared to open
an pond cultivation system, photobioreactors helps in the growth of monoculture microalgae
species, with controlled parameters like pH, temperature, light, and CO2 concentration. Chlorella
protothecoides cultivated in wastewater from two different treatment plants in a photobioreactor
with continuous batch achieved a specific growth rate and sufficient nutrient uptake from the
wastewater .
Batch cultivation is a closed system in which the fresh nutrients are added to the cultivation
system, under aseptic conditions, at the beginning of cultivation and the volume of the culture
medium in the cultivation system remains constant during cultivation. It is one of widely used
cultivation regime in bench- scale processing. Most industrial cultivation systems are processed
in batch mode because of the simple steps of the process and lower risk in contamination. This
process has its own disadvantages as cost of harvesting is high, sterilization and washing of the
cultivation system is also an expensive exercise .
Fed-batch cultivation refers to a semi-open system where in additional nutrients are added
gradually in aseptic conditions to the bioreactor. In this case, the culture broth in the cultivation
system rises as the product is discharged only at the end of cycle. This mode is exploited for the
microalgal cultivation in wastewater in different cultivation systems for the production of various
value added compounds like carotenoids. Many advantages have been reported of this mode for
microalgal cultivation such as controlled progressive addition of nutrients enhances the capacity
to yield higher biomass.
With reference to wastewater treatment, Desmodesmus sp. removed higher concentration of
harmful metals when grown in fed batch mode as compared to batch mode with biomass
production of 25 mg/L/day. By utilizing the fed batch mode, the excess nutrient in the
wastewater can be limited and helps in avoiding high COD level which limits the light
penetration in the wastewater medium . High astaxanthin productivity was reported in Chlorella
zofingenesis when cultivated under semi-continuous cultivation and with nitrogen deprivation.
Researchers have employed this mode to increase certain high valued metabolites like lutein
yield with mixotrophic growth conditions . Phycocyanin content of Galderia sulphuraria was
found to be enhanced in continuous cultivation regime . The biomass productivity in continuous
batch was found to be 5.8 fold higher than fed batch cultivation in Chlorella vulgaris . In general,
Heterotrophic cultivation of microalgae has been reported as the best and promising pathway for
increased biomass as well as the biochemical composition of microalgae Mainly there are three
metabolic pathways found in microalgae-based on their nutritional requirements to grow and
produce valuable biochemical.
These are autotrophic mode, heterotrophic mode, and mixotrophic mode . The autotrophic mode
is also known as photoautotrophic in the context of photosynthetic microalgae. In contrast,
heterotrophic mode refers to the acquisition of energy and carbon from organic compounds
available from the environment. For large scale production, a heterotrophic mode has been
successful to only a few microalgae species however, based upon cell production per unit energy,
heterotrophic mode provides it better i.
Astaxanthin content in Haematococcus pluvialis in heterotrophic mode with acetate as carbon
source comes out to be 10.5 mg/g cells .
Chlorella zofingiensis using sucrose as a carbon source produces
Mixotrophic mode refers to the combination of autotrophic and heterotrophic mode which results
in increased growth and utilization of resources by microalgae . In this, conventional
photoautotroph supplemented with organic carbon source is used to cultivate microalgae. In this
mode, wastewater from industries is utilized as carbon source which is coupled with the light
source to cultivate microalgae in an efficient and cost effective way. It has been reported that this
mode of cultivation resulted in higher cell yields per unit of energy, for example, mixotropic
mode provided 0.00749 g cells k/J as compared to autotrophic mode which yields 0.00177 g
cells k/J .
The valuable carotenoids like β-carotene, zeaxanthin, astaxanthin, lutein can be produced in
substantial amount in mixotrophic and autotrophic mode . In mixotrophic mode, where glucose
was used as an organic carbon source, Chlorella pyrenoidosa was found to produce 218 mg/L
lutein. Oxidative stress plays an important inducer for carotenogenesis in microalgae. In
Chlorella zofingensis cultivation, oxidative stress produced by reactive oxygen species present in
wastewater can enhance carotenoid accumulation.
The oxidative stress provided by certain chemicals present in wastewater is effective in
accumulating lutein and astaxanthin in microalgae species . Wastewater collected from different
sites like sewage, dairy municipal, industrial, etc. consists of several microorganisms apart from
microalgae. Although presence of microorganism specially bacteria is regarded as contaminants,
yet recent studies reveals that many algal symbionts promotes algal growth and proves to be
advantageous for cultivation and downstream processing. Compounds like growth stimulants,
micronutrients which are essential for microalgae growth and biomass production are
synthesised .
Bacterial communities present in wastewater along with algae not only enhances the production
of growth but this association also leads to perform more complex task with diversified
applications as in wastewater treatment. Nitrogen is fixed by several bacterial species living with
microalgae community and serves as nitrogen source for growth and carotenoid production in the
form of ammonia .
Downstream process in carotenoid production
Downstream processing of algal pigments is a very important aspect, considering the commercial
aspect of the process. Despite having a higher yield of carotenoids as compared to terrestrial
plants, the commercialization of algal pigments is not reached to the projected level due to the
high cost associated with multiple steps needed in the downstreaming process. There is a dire
need for an integrated process that amalgamates multiple steps including harvesting, extraction,
and isolation of selected pigment. Since most of the present methods use conventional
approaches which may impact the overall efficiency, thus, major efforts are needed to develop
more efficient techniques.
Nevertheless, the wastewater industry had wealth of experience for eliminating low
concentration small particles from huge volumes of water, thus, such can be extrapolated for the
harvesting of biomass from algal biomass for carotenoid recovery. Centrifugation is the efficient,
rapid, and most widely acceptable method for microalgal harvesting . This method is mostly used
in both lab-scale and pilot-scale industries and found to be appropriate for all the algal strains.
This method has the potential to provide improved recovery and a higher concentration of
microalgal biomass in a short time .
A low-cost filtration method is a process that works mainly based on particle size . There are
different types of filtration methods, for instance, microfiltration, dead-end filtration,
ultrafiltration, vacuum filtration, pressure filtration, and tangential flow filtration . Tangential
flow filtration is found to be more feasible specifically for smaller suspended algae than deadend filtration, though membrane fouling is a major issue . A method which is commonly
employed and preferred over sedimentation methods in wastewater treatment sludge removal is
dissolved air flotation .
Pre-treatment of the algal cells with chemical flocculants is another approach that is used to
enhance the algal cell size prior to use any other harvesting method. This method is extensively
used in wastewater treatment at the industrial scale because of its lower cost . These flocculants
work on the principle of neutralization of algal surface charge and consequently flocculate the
cells . The nature of flocculants applied in the process can vary from inorganic salts, principally
ferric, or aluminium-based polymer-based or magnetic and nanoparticles particles .
Considering the environmental burden, metal salt-based flocculants are not preferable as
compared to biopolymer-based flocculants which are potentially non-toxic and do not cause
secondary pollution. Natural blooms of microalgae sometimes underwent natural flocculation in
lakes or rivers . This is usually associated with high energy consumption needed for various
operations involved in extraction and majorly involves biomass drying, the use of organic
solvent and their recovery, maintaining necessary operational conditions, etc. Besides all these
necessary procedures, one of the major challenges with microalgae biomass extraction is the
rigid and thick glycoproteins layer of glycoproteins and carbohydrates around algal cells which
need to be disrupted to enhance the extraction yield . Accordingly, based upon the requirement,
microalgae extraction is conducted as a two or one-step procedure involving extraction following
cell disruption or direct extraction.
Some algae species having high carotenoid content yield higher recovery rate with hardly any
need for cell disruption e. Isochrysis galbana released approximately 95 % fucoxanthin by single
solvent extraction . Although to increase the extraction yield several folds, it is always preferable
to include cell-disruption as a pre-treatment step. In a study conducted by Chan and co-workers,
it was found that cell disruption enables the highest lutein extraction efficiency as compared to
non-treated microalgal biomass.
The selection of an appropriate disruption method is mainly species-specific . These cell
disruption methods are classified into three types based upon the process involved. Mechanical
methods use physical force to break the cells and include various techniques such as manual
grinding, ultrasonication, microwave, high-pressure homogenization , bead beating, and
electroporation. The advantage of these methods is that these can be applied to all species.
Manual grinding with wet biomass and cryogenic grinding
The high-pressure homogenizer is a continuous type of cell disruption process that performs its
functions on algal cell suspension. Hydraulic shear force and cavitation are generated, when cell
slurry is sprayed through a narrow tube under high pressure. Carotenoids extracted in this
process were neoxanthin, violaxanthin, lutein, -carotene, and β-carotene . The heat generated in
this process is comparatively very low thus reducing the chances of thermal degradation of
carotenoids.
N a similar study, analysing the impact of chemical and mechanical disruption methods,
ultrasonic bath method employed on unfrozen biomass provided optimum results and exhibited
193.5 25.8 and 2.34 μg/g carotenoids from R. Algal cell disruption by chemical means involves
the usage of alkalis, acids, surfactants, and ionic liquid. The permeability of cells can be
enhanced by using these chemicals as chemical linkages on the cell envelope are degraded by
applying these chemicals. This interrupts the osmotic balance between the exterior and the
interior of the cells and thereby disrupts the cell wall by creating pressure. Though, osmotic
shock does not work on the algal cell with a rigid cell wall and is incapable to extract pigments
from these cells .
Biological methods apply to procedures that destroy the cell wall with the help of enzymes. The
other biological method suggested was co-cultivation of algae with lead to lysis of cell walls .
Electroporation utilizes a pulsed electric field approach to disintegrate the cell envelope’s
molecules using dipole moments, therefore, cause cell disruption. High intensity intermittent
electric fields applied on cell for the micro-seconds duration enhance the cell membrane
permeability which can be regulated as reversible or irreversible by controlling the intensity of
the electric field.
This method does not cause degradation of carotenoids thus found to be an efficient method for
their extraction . Carotenoid extraction is a vitally important step in the entire downstreaming
process. Several procedures for the extraction of carotenoids from microalgae have been applied.
The conventional extraction method involves soxhlet extraction involving simple solid-liquid
extraction.
Owing to the non-polar nature of carotenoids, their efficient extraction necessitates the use of
non-polar solvents such as chloroform, dichloromethane, chloroform/methanol,
hexane/isopropanol, etc . Usage of binary solvent extraction systems have been reported to have
better extraction efficiency and yield . Pressurized liquid extraction , subcritical and supercritical
solvent extraction , microwave-assisted extraction , and ultrasonication assisted extraction are
some of the extraction techniques which are used for extraction biomolecules from algal
biomass. PLE is also recognized as accelerated solvent extraction , has been considered as a
green alternative method for the extraction of bio metabolites from any biological matrices.
It is a very recent technique and has emerged as an innovative technique as compared to
conventional extraction. It involves exhaustive extraction from biomass using elevated pressure
and temperature and reducing solvent consumption.
PLE include the extraction at elevated pressure and temperature, normally in the ranges
35–200 bar and
The main benefit of employing PLE is quick extraction and less solvent consumption. A growing
trend of using supercritical extraction as a greener extraction technique was found during the last
decade . It is a state-of-the-art extraction technique utilizing the capacity of liquefied carbon
dioxide gas also known as supercritical fluids as the extraction fluid for the extraction of
bioactive molecules from solid matrices . Supercritical fluid possesses low viscosity and high
diffusivity thus provides better solvating and transport properties than liquids and resulted in
better extraction efficiency.
Though, supercritical fluids assist in the extraction of non-polar compounds because of its
hydrophobic nature but the polarity of the solvent can possibility customized when used in
combination with different co-solvent such as ethylene for extraction of carotenoids and ethanol
for the extraction of moderately polar compounds as xanthophylls . CO2 pressure and
temperature is a critical step for the better extraction efficiency with respect to carotenoid of
interest. Since both the parameters plays important role for the efficient extraction of carotenoid,
so it is necessary to observe the combined effect of both the parameters. In a study, investigating
the effects of pressure and temperature on lutein extraction, pressure range was selected between
and temperature range was selected between .
It has been observed that intermediate pressures lead to maximum lutein extraction, with the
exception of extraction done at 46⁰C where pressure of 600 bar worked best for maximum
recovery. Apart from temperature and pressure, other factors which play significant role in
influencing the extraction efficiency along with selectivity of compounds of interest for
extraction are flow rate, co-solvents time, etc. Accordingly, optimization of these parameters also
needs to be considered carefully for a selective and efficient extraction of target analytes. Similar
to SFE, subcritical fluid extraction is a technique employing subcritical fluids as an extraction
solvent. Due to its recent advent, a limited amount of studies are available defining carotenoids
extraction from microalgae and seaweeds using subcritical fluid .
It works at relatively lower pressure and temperature as compared to
The most common carotenoids studied with respect to sub critical extraction are lutein,
astaxanthin and β-carotene. This result was comparatively much better, when compared to
supercritical CO2 extraction, subcritical water extraction and soxhlet extraction .
Microwave radiations are the frequencies extending from
This technique is a well-known green extraction technique known for its effective extraction
yield with lower extraction time and reduced solvent consumption . Several factors play an
important role in optimizing extraction efficiency and some of these are the nature of substrate,
solvents used for extraction, temperature, pressure, solid– liquid ratio, and particle size . MAE
offers a tremendous opportunity for the extraction of all the carotenoids, however, extraction of
astaxanthin from microalgae employing MAE received much attention. Ultrasound-assisted
extraction is considered an environmentally safe and efficient extraction method to recover the
number of range of metabolites from biomass .
Considering the thermolabile nature of carotenoids, the possibility of their degradation through
thermal extraction is extremely high. Heat exchange system used in this process assists in
regulate the extraction temperatures and thus marked valuable in extracting thermolabile
metabolites specifically carotenoids. Numerous studies have reported the UAE as a suitable
technique for the extraction of various carotenoids such as lutein, neoxanthin, violaxanthin,
astaxanthin, β-carotene from various algae species . These findings leads to conclude that
optimization of extraction parameters that includes duty cycles, microwave intensity extraction
temperature, solvent employed, extraction time, solid to solvent ratio, and specific pre-treatment
is a pivotal step for the optimum recovery of carotenoid components.
Specific pre-treatments followed by UAE extraction helps in complete recovery of metabolites.
Similarly, UAE combined with other techniques was found to be proficient in the extraction of
carotenoids . The implementation of hyphenated and multi-phases extraction techniques using
variable extraction techniques, solvents combinations and parameters helps to recover maximum
metabolites based upon the selection of metabolites. For example, employment of PEF
disintegration techniques when combined with single solvent extraction and/or extraction with
biphasic mixture of organic solvents lead to recovery of low water solubility carotenoids with
optimum yields from Nannochloropsis spp.
Technology selection criteria for commercialization process
Careful selection of algal strain, innovative upscaling and downstreaming approaches applied at
commercial scale might direct towards extensive intensification benefits. The first and foremost
step for algal cultivation at large scale is the selection of suitable strain for up-scaling process.
For instance, in a pilot scale study, three strains of Dunaliella salina were investigated for βcarotene production in an open pond. G was found to be suitable for β-carotene accumulation in
outdoor cultivation under specific circumstances .
Another principal step in algal processes is large-scale cultivation, which includes both open
ponds and their variations or in closed systems as discussed above. However the most recognized
system in commercial microalgae biomass cultivation are open raceways, shallow ponds, and
tubular photobioreactors. Moreover, 90 % of industrial cultivation around the world prefers open
systems, specifically raceway ponds owing to its less construction and maintenance cost and easy
operation. Some of the industries which are using open raceway ponds for commercial
production of β-carotene are Cyanotech , Nature Beta Technologies, Tianjin Lantai
Biotechnologies .
Parry Nutraceuticals use open raceway ponds for C-phycocyanin production . Some of the
industrial examples which are using closed tubular photobioreactors in conjugation with open
race ponds are Cyanotech and Mera Pharmaceuticals for the production of astaxanthin. Further
selection of intensified downstreaming process principally pigments extraction, is another vital
step that needs to be considered carefully. Considering the expansion of microalgae market,
availability of these pigments at competitive price is a need of hour.
There is no universal system to make microalgae cultivation and downstreaming process fitted
and suitable to obtain these pigments at attractive price, however, vigilant process optimizations
can make these processes economically feasible.
Carotenoids market value, industrial application and cost economy
European market leads in the carotenoids based cosmetics and feed industries. These are
preferred as food colouring agents in the European market. European carotenoids market size has
increased up to 579.90 million in 2018. According to a recent report by «Fiormarkets»
worldwide carotenoids market value is expected to reach US$ 3.59 Billion by 2025 at a
compound annual growth rate of 5.5 % considering the forecast period from 2018 to 2025 .
The increasing market trend of pigments obtained from microalgal origin has been observed due
to the consumer preference over organic and natural food ingredients.
β-carotene a preferred choice by the health market . In terms of the nutrition industry,
astaxanthin is always highlighted as one of the most potent antioxidants obtained from nature
and attracts a large proportion of attention with respect to the antioxidant market. Astaxanthin is
immensely used by the animal feed and nutraceutical industry and thus annual market value of
this pigment is rising. The present production of carotenoid pigments including synthetic and
natural is ruled by two main chemical manufacturers i.
Most of the lutein present in the market nowadays is generally obtained from the marigold
flower. The lutein market is exceeded US $250 million per year and also approved by the EU as
E161b for the colorant to the eye health care products and food additives. Having an annual
growth rate of 3.6 %, the lutein market is expanded . Algal biomass is another nutrition-rich algal
product attracting special attention in various food applications and having increasing market
trends .
Although algae have been explored for various applications includes metabolites production for
over 50 years now, still, only few examples of commercial exploitation for such applications
have been reported. Taking into consideration the negligible cost of feedstock utilized and the
price of oleoresin in market, the net profit estimated in the study was 70.6 %. This study
highlighted the fact that implementation of waste feedstock might resulted in less production cost
and resulted in wide economic margin to explore industrial application . In an another study, to
assess the economic feasibility of astaxanthin production by large scale cultivation of
Haematococcus pluvialis in a pilot plant having large scale outdoor photobioreactors and a
raceway pond was studied for 2 years.
Further establish processes were scaled up with a theoretical production capacity of 900 kg
astaxanthin per year. Using this process, the production cost of astaxanthin and microalgae
biomass was reduced up to $718/kg and $18/kg respectively. The results obtained were found to
be very promising for commercial application and much lower than commercial astaxanthin
production.
Challenges for wastewater algal cultivation
Although the cultivation of algae in wastewater has been considered as a sustainable option for
wastewater management and biomass production yet most of the studies are mainly published
from lab-scale observations. Some of the challenges are varying composition of wastewater, flow
rate, nutrient concentration, microbial interactions, presence of heavy metals, total suspended
solids, and environmental condition such as pH, light, temperature, etc. which probably are not a
clear expression of critical interventions experienced at full-scale level. Different type of
wastewater sludge has differential composition so strategy to make process productive should
consider the type of wastewater that is required to be treated. Wastewater obtained from agroindustries and other industries contain a large amount of suspended solid which may lessen the
light penetration into wastewater thus interfere with microbial growth.
Pre-treatment of wastewater with flocculants and add turbulence to it might assist in overcoming
the problem . Another factor which may affect the carotenoid production from algae grown on
wastewater is the variation in content and molecular form of various metal ions and other basic
nutrients present in specific wastewater. Numerous studies have highlighted the effect on media
composition and metal content on the biomass and carotenoids production. The astaxanthin
content was maximum in Fe2+ EDTA selection of appropriate strain according to wastewater .
Conclusion and recommendations
Various applications of carotenoid pigments and consumer preference towards natural-based
solutions have encouraged exhaustive cultivation of microalgae for the production of these
compounds. Some of the key challenges associated with bio-based products industry from
microalgae impede its commercial viability and sustainability. However, considering the highvalue of carotenoid, associated products such as lipids along with others by-product and
wastewater treatment efficiency of grown microalgae make this concept a promising approach.
There is a critical requirement for a more economic, effective, and efficient harvesting method
that can be employed in various microalgae industrial processes.