Developments in Microalgae based Waste Water Treatments : A Review

B. KarpanaiSelvan1, N. Jaya 2 and R. Girija*

1
Dravida Petroleum DMCC, ONGC EPS Bhuvanagiri, Chidambaram 608 001.
2
Department of Petrochemical Technology, Anna University BIT Campus, Tiruchirapalli 620 024
*Department of Zoology, Mercy College, Palakkad – 678 006.

Abstract

In many of the developed and developing countries there is an increased focus to improve the ecological
status of water sources by reducing the content of phosphorus, nitrogen and other nutrient sources in waste waters
generated from various industries. Algal based wastewater treatment systems have emerged as promising
technologies, as they prove to be, a green sustainable alternative to the conventional and existing wastewater
treatments. Many of the algal treatment processes fundamentally rely on breakdown of organic matter and pollutants
by biological breakdown using bacterial consortia. In recent years , mixotrophic micro-algal system is commonly
operated as it is based on the ability of the algae to utilize the organic carbon as well as inorganic nitrogen, phosphorus
in waste water for growth causing the desired result of reduction of concentration of those substances in water. This
technology opened the way to develop the high rate algal pond (HRAP) system for algal biomass production and its
value addition and Algae turf scrubber (ATS) to produce the biomass for the biofuel which later focused on the nutrient
removal from different wastewaters. This review therefore tries to provide a critical account of micro-algae as an
important step in waste water treatment .The review discusses on the components in various types of algal waste water
treatment systems, its pros and cons and introduces to HRAP and ATS systems. Furthermore, the paper highlights on
the mechanism of ammoniacal nitrogen removal in a mixotrophic algal based treatment system. A detailed overview
on the algal based anaerobic digestion of effluents and the use of microalgal system in digestion of alcohol in distillery,
hydrocarbon in petrochemical industry or oil spills, diary wastes from dairy industry with a note on the various
techniques employed and value addition of products generated is also presented. The review is an attempt by the
authors to garner interest in technologists/scientists to integrate micro-algae for enhanced and cost effective treatment
of waste water from industries by overcoming the challenges adopting new strategies.

Keywords: microalgae, waste water treatment, mixotrophic, HRAP

*Corresponding author
Introduction

Disposal of untreated wastes or improper dumping is a major environmental dispute since

time immemorial. The chemicals and toxic compounds released from livestock farming, municipal
and domestic wastes worsen the water quality (Yong et al. 2016 ; De Bhowmick et al. 2018). The
fast developing agro industries across the world as livestock farming itself produces approximately
320 million metric tons of meat. Dumping the large quality of effluent and solid wastes directly to
water source or soil, harmfully alters the natural ecosystem ( Bustillo-Lecompte & Mehrvar, 2017).
Increasing world population and fast urbanization in different regions of the world bring out
rigorous damage to surface and ground water due to massive release of domestic sewage (Nayak
et al. 2016).

India generates around 61754 MLD (million liters per day) of sewage. The total capacity
treatment system installed in the country is 22963 MLD (National status of waste water generation
& treatment updated on 8thMay 2019) nearly around 38791 MLD wastewater was not treated or
not used for any purpose. The sector specific industrial wastewater generation collectively
amounts to around 501 MLD from Chemical (97.8), Distillery (37.0), food, dairy & beverage (6.5),
paper & pulp (201.4), sugar (96.0),textile, bleaching & dyeing (11.4), tannery (22.1), others (28.6)
released out from 764 industries.

Wastewater is a combination of organic, inorganic, and other manmade (artificial) element

mixtures. Proteins, carbohydrates, fasts, amino acids, and volatile acids are the organic carbons
sources inherent in sewage. Potassium, ammonium, sodium, magnesium, calcium, phosphate,
sulfur, chlorine, non-metals and heavy metals are a part of inorganic constituents (Lim et al. 2010).
The heavy metals like mercury, chromium, lead, cadmium, copper, zinc and arsenic are vastly
available in municipal wastewater. In municipal wastewater the nitrogen is present as the source
in the form of nitrite (NO2-), nitrate (NO3-) and ammonia (NH4+), whereas the phosphorus present
as phosphates (PO43+). Wastewater are the sources of macro and microelements. The algal cell
requires macronutrients like carbon (C), Nitrogen (N), Phosphorus (P), Hydrogen (H), Oxygen
(O), Sulphur (S), Magnesium (Mg), Potassium (K), Sodium (Na) and Calcium (Ca) and
micronutrients like Ferrous (Fe), Copper (Cu), Manganese (Mn), Zinc (Zn), Chlorine (Cl),
Vandium (V), Molybdenum (Mo), Boron (B), Cobalt (Co) and Silica (Si) are essential for the
development and biological reactions (Eyster 1964 ; Aron 1961). The microelements like Mn, Zn,
Cu, Ca, Fe are connected with photosynthesis, Cl and Mn are important for O2 evolvement, other
microelements like Si, Mg, Ca, Fe, P, S, Mn, Zn, Cu and Co are associated in algal growth along
with the C, N and P macroelement. As the wastewater are the nutrient source of micro and macro
elements they can used as supplement for algal growth. To produce 1 gram of dry algal biomass,
require the > 1 kg of water (Shen 2014). The rawmaterial source which are readily available at low
cost with suitable pH, dissolved CO2, macronutrients, micronutrients which support the algal
growth (Abdel-Raouf et al. 2012; Ji et al.. 2013; Ajayan et al. 2015; and Ding et al. 2015).

1. Algal flow-way system

Algal based wastewater treatment technologies are the promising emerging technology
than the other treatment technologies. This system is a green sustainable alternative to existing
wastewater treatment technologies. Oswald & his team were the first to engineer the low-cost
wastewater technology based on algal system. This technology opened the way to develop the high
rate algal pond (HRAP) system. HRAP is commonly used for the production of algal biomass and
value-added products. Though the raceway ponds and photobioreactors are used for decades, they
are costlier and energy exhaustive (Christi, 2013). The microalgal cultivation method plays a main
role in biomass harvesting and energy consumption during harvesting creates an impact in
biodiesel production cost (Adeniyi et al. 2018). Hence, adopting to cheap and energy efficient
harvesting methods are the need of the hour. Algae turf scrubber (ATS) is a wastewater treatment
system developed by Dr. Walter Adey (Adey & Loveland 2007). ATS researches primarily used
& focused to produce the biomass for the biofuel , but later also focused on the nutrient removal
from different wastewater from the temperate areas of the United States (Kangas et al. 2017).
Ubeda et al. (2017) and Schulze et al. (2017) utilized urban wastewater as a source for algal
cultivation during treatment and produced the lipid-rich biomass for the biodiesel production.

In India nearly one third of the domestic wastewater is not treated because of insufficient
connectivity of wastewater treatment facilities and lacking the decentralized wastewater system
infrastructure (Williams et al. 2019). Marella et al. 2019 studied the algal biomass and lipid
production and nutrient removal from urban wastewater in a self-seeding algal flow-way (AFW)
system in continuous flow. This proves to be the first attempt in AFW, the result showed above 90
% of sustained community is diatoms in all seasons. The average algal production was 34.83 g dry
weight m2d-1 with 2.52 gm2d-1 of N and 1.25 g m2d-1of P removal. Biomass lipid content was in
14- 22 % range of dry cell weight and in summer season the lipid production was 9.29 g m2d-1.
Biodiesel quality was excellent with high centane number in summer whereas high
eicosapentaenoic acid content was high in winter growth cycles.

1a. High Rate Algal Ponds (HRAP)

HRAP is considered to be an effective treatment method for organic and inorganic

wastewater than the other traditional oxidation ponds (Craggs et al. 2014). Several pilot plants
studies directed that the HRAPs conditions are extremely environmentally variable and can trigger
the changes in microalgal biomass production in actual operating conditions (Monflet & Unc 2017
; Goncalves et al. 2017). Different producer groups, inconstant influent wastewater characters and
vast variations in climatic conditions are referred to describe the microalgal diversity modification
due to variations in nutrient removal (Montemezzani et al. 2016 and Cho et al. 2017). The
microorganism already in wastewater will also grow in HRAPs by utilizing C, N and P.
Association of microalgae and bacteria is suggested as O2 and CO2 source supplementation
respectively and both will compete for nutrients (Lavrinovičs & Juhna 2017).

Galès et al. (2019) studied on ecological interaction in HRAPs wastewater treatment at

various climatic temperature. This study probed the bacterial, microalgal, protozoan and metazoan
community dynamics in HRAPs fed with wastewater continuously. Outdoor HRAPs wastewater
treatment in oceanic and Mediterranean climatic temperature showed similar COD, NH 4+ and
PO43- removal efficiency. Both climatic HRAPs exhibited a similar ecological successions. The
wastewater clarification was due to the organic matter consumption by detritivores, heterotrophic
bacteria, amoebozoa, nematodes which is already present in wastewater and their presence are
essential in spring for microalgae to grow in two weeks. Microalgae and bacteria significantly
contribute in the ammonia utilization. The complete removal of ammonia restricts the complete
removal of dissolved COD by bacteria and phosphate by microalgae in the HRAP utilization.

1.1 Types of culture system

All the algal systems require a carbon source for anabolic activity, an energy source for
catabolic activity, supported by macro-micro nutrients availability for their metabolic activity. The
metabolic selection depends on carbon and energy sources. The algal wastewater treatment
systems are classified in three major groups (Wang 2014).

a. Photoautotrophic culture system

b. Heterotrophic culture system
c. Mixotrophic culture system

1.1a Photoautotrophic culture system

This system utilizes the inorganic carbon as the major carbon source for the algal growth.
This system requires light source energy for the photosynthetic mechanism to convert required
chemical energy of its growth. In this system photoautotrophic algae and heterotrophic bacteria
reduce BOD and remove of available nutrients in municipal wastewater (Oswald, 1963). This
system works with the principle of the photoautotrophic algae produce oxygen needed for the
oxidative assimilation of BOD by heterotrophic bacteria in the wastewater through the
photosynthesis, BOD oxidation produces the carbon dioxide (CO2), this CO2 forms an inorganic
carbon source for photoautotrophic algae. The advantage of this method

(i) energy required for the traditional method of activated sludge process of BOD
removal can be avoided.
(ii) The photoautotrophic algae are subjected to the tertiary process of the activated
sludge process for the nutrient removal. In the activated sludge process the
heterotrophic bacteria cannot remove all the N and P in the primary treatment to
meet the discharge standards, since C:N:P ratios are in mismatch between the
primary effluent treatment and biomass of the activated sludge. Hence, employing
the algae in follow-up end process will utilize the residual N and P to meet the
discharge standards.

The limitation of this system is that it needs external inorganic carbon source to meet the
standards. The above mentioned process can be carried in open raceway pond operated with paddle
wheels. The main source of energy is sunlight, so the depth pond and the water column are
maintained low to allow the sunlight penetration more. Another limitation is low algal biomass
results in less removal rate of BOD, N and P. whereas the shallow depth pays the way of high
evaporation of water and the growth of predators, competitors and parasites (Richmond, 2004).
The shallow depth is not preferred due to lower densities of biomass production due to
photoinhibition, photolysis, NH3 loss will more than 75% due to volatilization, phosphates
reduction by precipitation (Park et al. 2011). The third limitation is supply of gaseous CO2 through
a sparger results in reduction of the gas-to-liquid transfer efficiency since the bubble detention
time is short in the shallow depths results in loss of CO2, lower growth rate of algae and reduced
pollutant removal rate in volumetric range.

1.1b Heterotrophic culture system

In this culture system the chemoheterotrophic algal cultures get the carbon source and
energy needs from the organic chemicals. Most of algal strains are heterotrophic growth in nature
(Ogawa & Aiba, 1981). This system operates at high algal biomass density to attain the high rate
of pollutant removal. The high rate of algal biomass will be achieved with this system as, the
organic chemicals energy density is higher than that of CO 2. However, the oxygen needs to be
supplied externally. The limitation with heterotrophic system is that there is high chance of
contamination/competition due to enormous amount of the high-energy carbon substrates.
Generally, heterotrophic system in industries are reinstated to fenced reactor configuration to retain
the axenic culture. Highly engineered bioreactors are only employed for heterotrophic mode
operation, and this system is more expensive in the wastewater treatment process because the
volume of liquid is high, and substrate concentration are low (Perez-Garcia et al. 2011). The
heterotrophic wastewater treatment system is restricted to laboratory scale.

1.1c Mixotrophic culture system

Mixotrophic culture system may get the carbon and energy needs either from the organic
or inorganic chemical sources. The energy need is depends on the ratio of the energy available
from organic chemical to that from the light source. In this system, both the photoautotrophic and
chemoheterotrophic process occur independently and results in accumulation/ depletion of CO2
and O2 respectively. The mixotrophic and HRAP systems seem to show similarity among the
reactants and products. A major difference is mixotrophic relies on heterotrophic algae, where the
HARP relies on the heterotrophic bacteria. The variation between the mixotrophic culture system
and photoheterotrophic culture is that the mixotrophs are grow in a single constituent metabolism
i.e. is the algae grow on light photosynthesis when organic carbon is not available, or they grow
in dark with organic carbon availability. The photoheterotrophs need both the light and organic
chemical to grow. Mixotrophs consume organic carbon or inorganic carbon concurrently as the
carbon source, and also utilizes the light and organic carbon also concurrently as the energy source.
This elite metabolic flexibility of mixotrophs builds them a supreme candidate in wastewater
treatment.

Cecchin et al. (2018) grew Chlorella sorokiniana cells by both mixotrophic and autotrophic
methods. In mixotrophic method, acetate was used as the carbon source has increased the biomass
yield, lipid, protein, starch and cell density when compared to the autotrophic growth. Under
mixotrophic conditions the carbon loss was decreased by upregulation of the phosphoenolpyruvate
carboxylase by alternative carbon fixing pathway confirmed by De novo transcriptome assembly
and gene expression analysis. Wan et al. (2011) states that C. sorokiniana with glucose increase
the accD gene (heteromeric acetyl-CoA carboxylase beta subunit) level for fatty acid synthesis,
results with high lipid content in the stationary phase of mixotrophic growth. These findings
recommend that mixotrophic cultures will show excellent performance in maximum carbon atom
recovery lost by organic oxidation process. Several other studies also estimated the mixotrophic
culture system, the algal strains and pure substrates are used a functional element in laboratory
studies. Zhang et al. (2017) reported that more than 60 % of mixotrophic biomass yield more than
the heterotrophic culture when 7.35 fold of light was enhanced in mixotrophic cultivation. Cerón
Garcí et al.. (2000) carried studies on Phaeodactylum tricornutum on glycerol in mixotrophic and
photoautotrophic mode where biomass concentration was 9- fold higher, biomass productivities
was 8-fold higher and eicosapentaenoic acid (EPA) production was 10- fold higher in mixotrophic
than the photoautotrophic mode. Mixotrophic culture growth has resulted high lipid content algal
biomass, higher lipid content reflects in higher lipid productivity transforming to high energy
recovery. Liang et al. (2009) studied Chlorella vulgaris under three modes with acetate, glucose
and glycerol, the mixotrophic mode showed higher biomass and lipid productivities. Mixotrophic
mode showed high lipid production in freshwater Chlorella sp., marine Chlorella sp.,
Nannochloropsis and Cheatoceros sp. (Cheirsilp and Torpee, 2012). The reason for mixotrophic
growth advantage was high photoinhibition tolerance and photooxidative damages. The
mixotrophic modes showed high growth rate, whereas the addition of growth rate which alone
cannot be taken as a valid advantage. In the mixotrophic culture lower sensitivity to wide range of
light intensities in wastewater treatment supports the high cell density and high turbidity levels.
The above mentioned findings are laboratory studies on pure substrates, that provides a solid
promising platform in evaluating the mixotrophic wastewater treatment pathway. Perez-Garcia&
Bashan (2015) stated in review of heterotrophic and mixotrophic culture, the current knowledge
about the mixotrophic systems is a sustainable wastewater treatment (PereZ-Gracia & Bashan,
2015). Nirmalkhandan et al. (2019) studied the algal wastewater treatment with Galdieria
sulphuraria in photoautotrophic and mixotrophic mode in an outdoor pilot and also laboratory.
The both the laboratory and outdoor pilot scale of mixotrophic mode has showed removal of BOD,
nutrients and pathogens in a single step. Mixotrophic mode wastewater treatment in pilot scale
studies showed high volumetric removal rates of BOD, ammonical nitrogen, and phosphates,
which increases the opportunistic potential for a greener and sustainable wastewater treatment.

1.2 Removal Ammoniacal Nitrogen

The anthropogenic activities leading to release of more amount of N & P in water bodies
causes unwanted phytoplankton bloom growth affecting the water quality and inequity of rivers
and lakes ecology. Ammonium (NH4+-N) discharged to waters bodies exist in reduced inorganic
form of N and it is the source for eutrophication (Wang et al. 2019). Formerly NH4+-N was
removed by various physicochemical and biological methods. The biological method involving
Activated sludge process technology utilizes the various types of microorganisms at optimized
dissolved oxygen to remove NH4+-N by nitrification tailed denitrification to nitrogen gas (N 2)
(Gardner-Dale et al. 2017). Reduction of NH4+-N in wastewater is huge success technology with
limitations like exhaustive energy cost, operation conditions are complex and the production of
enormous quantity of activated sludge development with sustainable ecology is a massive
challenge. Another problem is aerobic bacteria oxidize the organic matter with carbon dioxide
(CO2) production. CO2 is main donor for climate change and global warming. Therefore, a new
method is essential to reduce the N discharge from wastewater at low energy and material
consumption cost. There are few new technologies like ion echange resin (Jorgensen and
Weatherley, 2003), aerobic granular sludge (Ni et al. 2009), anammox process (Kartal et al. 2010),
and capacitive deionization (Feng et al. 2017) are drawn consideration but failed in N resource
recycling. NH4+-N rich wastewater is a perfect medium for algal growth since majority of
microalgal species will uptake this form of N source, to achieve the better wastewater recycling
and reduction of energy crisis the microalgae are believed as a rapid green bio bridge (Chen et al.
2017).
Scenedesmus species is omnipresent in water ecosystem, it shows prominent NH 4+-N
tolerance. Countless efforts have been made to compare it with other algal species to convert the
nutrients into commercial products. It shows high photosynthetic efficiency and counted as eco-
friendly strain (Yang et al. 2016). Heterotrophic and mixotrophic modes are recommended as
viable options for wastewater treatment because of concurrent removal of C, N and P in a single
reactor system by avoiding the regular complex recycling process of conventional method (Shen
et al. 2017). Predominantly mixotrophic cultivation increase the algal biomass considerably. The
heterotrophic growth system is different. The CO2 and organic carbon are integrated concurrently
in which both the photosynthetic and respiratory metabolism function simultaneously (Ji et al.
2014). The main problem limiting the microalgae-based treatment is the cell harvesting. There are
enormous researches focusing on microalgae harvesting like giving importance to flocculation,
filtration, gravity sedimentation and centrifugation (Lei et al. 2015). Among the cultivation method
immobilization is considered as feasible cultivation method for harvesting. The microalgae
cultivation in entrapped matric will restrict the cell freedom and is easy to separate the algae cell
from the water (Lam & Lee 2012). Generally, sodium alginate (SA) is preferred as polymer matrix
for microalgae immobilization, as it has the property like high transparency, high diffusivity and
it is low toxicity to immobilize (Zamani et al. 2012). The immobilized beads are larger in size
than the algal cell and hence can be separated by simple sieving without any additional energy
requirement. After separating, the beads dissolve in sodium citrate to collect the biomass. Thus,
the municipal wastewater treatment by microalgal immobilization method is a practical and an
economical method.
Fig 1: Simplified representation of the NH4+-N assimilation pathways by microalgae during the
mixotrophic cultivation (adopted from Liu et al. 2019)

Liu et al. (2019) has studied the immobilization with sodium alginate of Scenedesmus
obliquus. The free and immobilized cells are studied in heterotrophic and mixotrophic modes to
compare the NH4+-N removal. They observed a positive C dependent effect in mixotrophic mode
and a negative N dependent effect in heterotrophic modes in both the free and immobilized cells.
The immobilized cells exhibited a better tolerance to high concentration of NH4+-N. Nearly 96 %
of removal was observed at 50 ppm concentration of NH 4+-N in mixotrophic mode. Amino acids
like Arginine (Arg), Cystine (Cys), Glutamic acid (Glu), and Proline (Pro) dominated in protein
synthesis pathway of NH4+-N removal (Fig.1).

1.3 Removal of N and P

N and P discharge cause eutrophication and deterioration of water ecosystems in water

bodies (Garrido Cardenal et al. 2018). N and P in wastewater streams can be removed at high rate
(Rinna et al. 2017). Effluents of anaerobic membrane bioreactor (AnMBR) contains high amounts
of nutrients and are considered as sources of nutrients for microalgal growth (Giménez 2011).
AnMBR effluents shows several advantages than the other conventional treatments in nutrients
recovery by microalgae (Romero villegas et al. 2018) as listed.

(i) N and P removal from AnMBR effluents does not require any additional organic
carbon source or addition of extra chemical reagents (Tan et al. 2016).
 (ii) The effluent discharged are oxygenated and
(iii) The biomass production by this process can be processed for biogas production.
The processed sludge enhanced fertilizer property with nutrient enriched (Guldhe
et al. 2017).

The reduction of carbon footprint has been attempted by combining the microalgal cultivation with
AnMBR effluents to recover nutrients and energy from sewage (Seco et al. 2018) . Though the
microalgae cultivation can be carried in open or closed photobioreactors, the open photobioreactor
systems are only preferred since the operating cost is lesser than the closed photobioreactor system.
The control of biological process is difficult because the open reactors are oddly affected by the
ambient factors than the closed systems. Loss of nitrogen and CO 2 occurs due to strip off ammonia
and CO2 in open systems whereas in closed photobioreactor there is increase of photosynthetic
efficiency, biomass productivity and nutrient recovery (Huang et al. 2017; Behera et al. 2018; Vo
et al. 2019 and Nwoba et al. 2019). Microalgal productivity and nutrient removal was influenced
by the light intensity, light frequency and photoperiods (Binnal & Babu, 2017). Growth of
microalgae is proportional to the light intensity till the saturation point to achieve the maximum
photosynthetic activity (Raeisossadati et al. 2019). Microalgal growth is limited when intensity is
below the optimum valve. On other hand when the light intensity exceeds the optimum value the
photosystem I (PSI) and photosystem II (PSII) will be injured and initiate the photoinhibition
(Ramanna et al. 2017 ; Martinez et al. 2018). Photoinhibition reduction can be achieved by merging
the high light irradiance with darkness period. According to the reports the algae will respond to
light intensity immediately and the dark reactions photosynthesis is followed in light lack
conditions temporarily, the dark reactions are slower than the light reactions. Energy stored from
the light reactions cannot be used without the addition of extra photons for photosynthesis
(Barcelo-villalobos et al. 2019). The adsorption of excess photons is released as heat or
fluorescence and it reduces the efficiency of photosynthesis. The suitable light-dark (L:D)
photoperiods decrease the light energy demand with comparable or higher productivity. However,
mass productivity is less in the longer optimum dark periods (Ferro et al. 2018 ; Lehmuskero et al.
2018). There are three major groups on photoperiods are

(i) Long term photoperiods (L:D cycles in hours)

(ii) Frequency photoperiods (several L:D cycles per day)
 (iii) Short photoperiods or Flashing light effect (FLE) (L:D cycles of seconds or even in
miliseconds)

Solar light is the major light source for outdoor algal cultivation. However, the weather
variations in day: night cycles and seasonal changes will disturb the light intensity and spectrum,
which destructively effect the microalgae (Castrillo et al. 2018). If microalgae are dispersed with
high density the light distribution will not be in uniform manner. The algal cells which is near to
the reactor surface will get high light intensity upto 1800 µmol m-2S-1 at mid of the day as the
photoinhibition occurs. The algal cell close to the surface will absorb the maximum light and
initiate the dark zone, this is called as show effect or self-shading (Jebali et al. 2018). Dark zone
volume is depending on the factors like microalgal biomass concentration, microalgae pigments,
light intensity, light path, culture turbidity and reactor opacity (Wagner et al. 2018). The self-
shadow will affect the chlorophyll, because in dark cycle chlorophyll is not synthesized. The only
way to reduce the shadow effect is proper mixing and high illumination, whereas, in open system
the mixing is improper. The artificial illumination should have high energy efficiency and
reliability, low heat dissipation and cost, the emission should be in the microalgae spectrum range.
Among all the artificial light sources LED lamps are considered as best artificial light source for
the microalgal growth.

Gonzalez-Camejo et al. 2019 studied on the light intensity effect, light duration,
photoperiods, N and P removal from AnMBR effluent. The outdoor photobioreactor is operated
with the AnMBR effluent with high photon flux showed high biomass productivity and nutrient
recovery. The maximum nitrogen recovery rate is around 7.7 ± 1.6 mg of N/L/d, the maximum
phosphorus recovery rate is around 1.03 ± 0.21 mg fg P/L/d and biomass productivity is around
100 ± 32 mg VSS/L/d respectively. Nearly 300 µmol of m-2S-1was supplied under artificial
illumination. They have not observed any photoinhibition due to high net photo flux. Biomass
productivity: light irradiance ratios is observed higher when photon flux is less. Limitation in
phosphorus results in increasing microalgal biomas.

1.4 Algal based anaerobic digestion effluent treatment

 Anaerobic digestion (AD) is defined as a sequential biological process where the
biodegradable materials are broken down in the absence of oxygen by the microbes. In other term
organic wastes are digested to produce the methane biogas. AD is a well-known technology
involving various hurdles in field application. The main environmental problem in field application
is the AD effluent management, malodor and eutrophication, when discharged without treatment.
The environmental risk is because of high nitrogen content. The AD effluent can be used as
fertilizer, but direct application will cause risk to both groundwater contamination and pathogen
transmission (Xie et al. 2018). Nitrogen removal from wastewater by conventional two stage
nitrification-denitrification process has limitations as requirement of high quantity of energy for
the aeration and externally the carbon source must be added. Hence, the conventional method is
not appropriate for AD effluent treatment, because it has nitrogen in bulk quantity and less amount
of available organic carbon (Zhang et al. 2011). There are several substitute methods have been
developed such as bypass the nitrification and denitrification process, anammox,
bioelectrochemical denitrification (Pandit et al. 2014 ; Fernandes et al. 2018). Microalgae has
showed promising nutrient uptake in high rate and maximum growth rate in wastewater
enviornments, stains like Chlorella, Neochloris and Scenedesmus maximum nitrogen removal
from various AD effluents (Franchino et al. 2013). Carbon dioxide from biogas is utilized by
microalgae as carbon source for growth and simultaneously remove the effluent nutrients (Marin
et al. 2018). Microalgae cultivation in AD effluent is a feasible solution. Since AD could be a
mixotrophic cultivation medium for microalgae, where the residual organic matter from effluent
can be carried. Wastewater based microalgae cultivation was studied with commercially available
monoculture strains, several research studies also stated that exogenous monocultures face
complications in adaption of wastewater as a media and further contamination risk by other
organisms in AD effluent (Johnson & Admassu, 2013 ; Chen et al. 2015).Mixed culture
environment microalgae consortia have enhanced adaptation in wastewater, short lag phase,
maximum biomass productivity and maximum nutrient removal (Cho et al. 2017). Microalgae
(inoculum) and wastewater (source) both will affect the nutrient removal and growth of microalgae
in mixed-culture process. Yu et al. (2019) studied the microalgae capability with various inocula
and AD effluents for ammoniacal nitrogen removal and biomass production. The mixed-culture
microalgal consortia enriched form aerobic sludge (AeS) and Anerobic sludge (AnS) were
examined for the maximum possible NH4+-N from four different AD effluents. Both AeS and AnS
inoculum removed the NH4+-N completely in 14 days. Further, the Nitrogen starvation studies
showed the changes in microbial community structures, influenced in lipid metabolism and growth
of microalgae. Both the AeS and AnS inoculum grown well on the all the AD effluents even under
non-sterile conditions, where in mixed culture conditions with various bacteria also coexisted.

1.5 Algal based distillery wastewater treatment

Ethanol production rate is increasing in India in recent years by the Indian government
policy since 2010 related to blending of 5-10% of ethanol with petrol. To achieve the goal the
distilleries face two problems.

(i) The continuous supply and availability of molasses to the distillery industry due to
climatic change in rainfall.
(ii) There is operational difficulty in wastewater treatment to meet the standards.

Generation of wastewater after distilling the alcohol was enormous with high load (BOD
40,000-50,000 ppm & COD 80,000-100,000 ppm) of rich organic and inorganic contaminants
(Sankaran & Premalatha, 2014). The distillery wastewater needs 1000 time of dilution to suppress
the harshness effect. Since, Central Pollution Control Board (CPCB), Government of India
enforced all distilleries to achieve the Zero Liquid Discharge (ZLD) (Tewari et al. 2007). After
this policy came in act the distilleries followed the effluent treatment process (ETP) sequence as
evaporation tailed by incineration/anaerobic digestion tailed by reverse osmosis (RO) process
(Sankaran et al. 2015). Evaporation tailed by incineration is costlier and energy exhaustive process
because wastewater has only 9-11% of solids. Hence, the anaerobic digestion process is broadly
followed in all distilleries because it removes 80-90% BOD, 65-70 % COD and simultaneously it
produces the biogas. Approximately 1 m3 of distillery wastewater produces 38-40 m3 biogas.
Anaerobic digestion tailed by RO process achieves to obtain the clean water after the treatment of
wastewater and the clean water is recycled for the production process. But, the RO process have
the disadvantages like

(i) Influent wastewater has the high pollution load

(ii) This process of operation requires high pressure
(iii) Power requirement
 (iv) Maintenance service is essential
(v) Membrane replacement at a certain duration.

These disadvantages created an opportunity for an alternative suitable unit operation in

ETP process before tailing the RO system. The new alternative system is to reduce the pollution
load to increase the RO efficiency. Numerous physico-chemical treatment techniques are not
suitable to treat the distillery wastewater because of,

(i) the high pollution load;

(ii) unit operation process requires high cost and
(iii) secondary pollutant generation (Sankaran et al. 2015).

Anaerobically digested distillery wastewater (ADDW) offers the acceptable conditions for
microalgal cultivation (Travieso et al. 1999) and it doesn’t generate any secondary pollution
(Rawat el al 2011). The microalgae exhibiting the mixotrophic or heterotrophic machinery in
wastewater treatment integrate the organic pollutants into its cellular components as
carbohydrates, lipids, proteins and inorganic pollutants as essential components for growth and
multiplication ( Lowrey et al. 2015; Wang et al. 2010). After the ADDW treatment the microalgal
biomass can be used as feedstock for biofuel production, feed for animal and aquaculture, fertilizer
and high value chemicals rawmaterial source (Mata et al. 2012). Microalgal strains like Chlorella,
Scenedesmus, Phormidium, Oscillatoria, Botryococcus, Chlamydomonas and Spirulina has been
effectively applied in industrial effluent wastewater treatment (Sankaran & Premalatha et al., 2014;
Solovchenko et al., 2014; Ruiz-Marin et al.., 2010; Travieso et al., 2008).

Krishnamoorthy et al. (2019) studied the ADDW treatment with microalgae in 50 L

laboratory and 500 L open raceway ponds (outdoor) reactor. The ADDW has bicarbonate around
12,800-12,850 ppm, ammoniacal nitrogen around 1000-1050 ppm, nitrates around 350-400 ppm,
dissolved phosphates around 350- 360 ppm as the source of C, N, P for Spirulina sp. cultivation
(Krishnamoorthy et al. 2017). Krishnamoorthy et al. observed that Spirulina sp. has exhibited the
better adaptability to ADDW environment, and the stain showed high sustainability in 500 L
capacity photobioreactor. The pollution load of wastewater was reduced considerably 60-70 % of
inorganic compounds as its nutrients. After anaerobic digestion, tailing the algal reactor to reduce
the pollution load considerably minimize the operational cost in ETP and minimize the salt
concentration in consequent RO process. Thus, the possible option for the ADDW in distillery
ETP was algae-based wastewater treatment.

1.6 Algal based Hydrocarbon wastewater treatment

Water is an essential resource for human life and modern society industrialization. The
enormous usage of petroleum and fossil fuels products creates the water contamination by oil spill
and cargo accidents. The water contamination by oil spill pose threat the water ecosystem and
human life (Al-majed et al. 2012). Oil refinery industries are working on to reduce the water
uptake and wastewater treatment system development. The conventional method of wastewater
treatment still leaves the residues of hydrocarbons and recalcitrant organics in high quantity (de
Abreu Domingos and da Fonseca 2018). The remediation of petroleum hydrocarbon is essential as
their presence in water bodies are high toxic, mutagenic and cancer-causing (Van der oost et al.
2003). The conventional methods like adsorption, agglomeration, dispersion, dissolution,
evaporation, microbial degradation and photochemical oxidation were generally adopted to
remove hydrocarbons whereas, the involvement of high cost, production secondary pollution and
non-eco-friendliness are the major restrictions (Banerjee et al. 2006).Dispersing agents are highly
flammable and hazardous in nature effects marine mammals and humans health. The adsorbents
and petroleum wastewater contact alter the fatality and hydrocarbon transport (Suganthi et al.
2018). Hydrocarbons in wastewater can be removed economically by biosorption technique.
Microalgae based wastewater treatment is preferred since recovery of resource of algal biomass.
can be achieved. The biofuel, cosmetics, fertilizer, pharmaceuticals, protein rich supplement and
products like pigment, paraffin, glycerol, olefin, plastic, antioxidant are produced from the algal
biomass (Sundarajan et al. 2018). There are several research reports that algal biomass has the high
sorption capacity to phenolic compounds, dyes, aromatic hydrocarbons and heavy metals
(Gunasundari & Kumar 2016). Hence, microalgae can be applied as low-cost adsorbents since it
will grow mixotrophically by utilizing the atmospheric CO2, inorganic carbon (CO32-, HCO3- and
H2CO3) and nutrients like N and P from wastewater for the growth. Microalgae can be used as
biosorbent for hydrocarbon removal from wastewater and the spent biosorbent after wastewater
treatment can be subjected to biofuel production. The biofuels are widely accepted because of the
low nitrogen and sulfur content, discharge of CO2 to environment is less which results in reduction
in global warming phenomenon. The non-renewable energy will create unnatural weather change
that can bring in destructive effect on environment (Agarwal et al. 2013). Recent method used to
extract the oil from algal biomass was the hydrothermal liquefaction (HTL) (Arun et al. 2017 ;
Biswas et al. 2018). HTL method is preferred since this method does not need high temperature
and energy intensive dewatering of feedstock is not needed (Wang et al. 2018). Fossil fuels
compete with the biofuels produced from algal source in economic limitations. In HTL method,
the aqueous phase can be used in medium liquefaction reaction for the energy production without
discharging (Hu et al. 2017). The biochar produced from this method can also be used as adsorbent
to remove the contaminants of wastewater.

Rajan et al. (2019) has studied the petroleum wastewater hydrocarbon removal by
Scenedesmus abundans biomass as the sorbent. The S. abundans biomass has sorbed nearly 92 %
of hydrocarbon from petroleum wastewater. The spent biosorbent was exposed to hydrothermal
liquefaction the bio oil yield was around 43 % at 300 ̊C. The bio oil yield was further increased
upto 47 % for two recycle process with HTL wastewater. The liquid hydrocarbons expressed
presence of C8 to C21 range of higher molecular weight hydrocarbon. The HTL biochar was
adsorbing the Pb (ii), Ni (II) and Cr (VI) in wastewater as a sorbent and around 86 % of adsorption
was observed in Ni (II) ions.

1.7 Algal based Dairy wastewater treatment

Industrial pollution and CO2 emission are the world environmental threat that creates
impact on climate change (Ho et al. 2013). The dairy industry occupies the top position among
the most polluting industries because almost approximately 2 – 10 L of wastewater is generated
for processing a liter of milk. The wastewater characteristics of Dairy industry depends on the
quantity of milk and the type of milk product although it contains biodegradable organic
constituents. Apart from this dairy waste, the water also contains sanitizers, detergents, casein and
inorganic salts with high sodium content as caustic soda is used for cleaning (Raghunath et al.
2016). High concentration of dairy wastewater discharged into free environments causes
eutrophication which threatens life. Microalgae has high rate of CO2 fixation, rapid cell growth,
high photosynthetic efficiency, carbohydrates and lipid accumulation that leads to generation of
biofuels, nutraceuticals, food additives and feed for livestock (Venkata mohan et al. 2016 ;
Sudhadra & Edwards 2010). Microalgae has high carbon rich compounds that is counted as major
feedstock in many processes like hydrolysis, anaerobic digestion and yeast fermentation to produce
the reduced sugars, short chain carboxylic acids and alcohols. Microalgae has rapid growth, energy
rich compounds and nutrient utilization hence it is used as alternative replacement feedstock (John
et al. 2011). Microalgae carbohydrates are complex in nature because it is obtained from
chloroplast starch and cell wall cellulosic polysaccharide (Domozych et al. 2012). This complex
nature carbohydrates are not readily fermentable it needs to hydrolyze to fermentable simple sugars
before to prior use (Hahn-Hagerdal et al. 2007). Lipid extracted residual biomass rich in carbon
can be used in fermentation (Naresh Kumar et al. 2018). There are various methods like
ultrasonication, freeze-fracture, acid/base treatment, thermo acid/alkaline chemical and enzymatic
pretreatment methods followed for hydrolysis of lipid extracted microalgal biomass (de Farias
Silva & Bertucco 2016). In these methods’ acid hydrolysis is followed to hydrolyse the lipid
extracted biomass to convert them to a complex carbohydrates and then into simple monomer
sugars (Girio et al. 2010).

Hemalatha et al. (2019) studied the possibility of integration of photosynthesis and

fermentation in dairy wastewater treatment using microalgae. Cultivation of microalgae in dairy
wastewater expressed high fraction of carbohydrates (38%) and lipids (22%) than proteins (15%)
compositions. 90 % of organic carbon removal was achieved along nutrients removal. Nearly 1.4
g/L of biomass was obtained at end of each cycle of dairy wastewater treatment. Hydrolysis of the
lipid extracted biomass has exhibited 54 % of glucose recovery than other monomers. The reducing
sugar produced from the hydrolysis process was able to produce 116 g/L bioethanol using
Saccharomyces cerevisiae. The microalgae based dairy wastewater treatment produce the potential
biomass feedstock for bio products production by tailing the close loop recycling to avoid the
waste streams.

1.8 Value-added products production by microalgae during wastewater treatment

Microalgal biomass production for biofuel is not economically feasible because of

expensively high CAPEX (capital expenditure) and OPEX (operational expenditure) and zero net
energy gain (Yong et al. 2016). Lutein is identified as highly commercial important carotenoids
with different healthcare applications among the other high value products like carotenoids, poly
unsaturated fatty acids (PUFA), exopolysaccharides, antioxidants and vitamins (Dineshkumar et
al. 2015). Lutein express the prevention and improvement in age related blindness, cataracts,
atherosclerosis, it has antioxidant, anti-inflammatory and colorant properties. The health beneficial
properties of lutein world market in 2010 is around 233 million US $ and in 2018 it has grown up
to 308 million US $ with 3.6 % estimated growth rate (Lin et al. 2015). Instead of concentrating
on low value – large volume biofuel one can concentrate on high value-low volume product like
lutein.

De Bhowmick et al. (2019) studied the open raceway pond cultivation performance of
Chlorella minutissima with wastewater and flue gas CO2. Kitchen wastewater (KW), poultry litter
wastewater (PLW), ethanol distillery wastewater (EDW) and dairy wastewater (DW) were used
individually and in combination to cultivate the C. minutissima. The formulation of mixture with
20% of KW, 10g L-1 of PLW and 5 % of flue gas was used to cultivate the C. minutissima in 60 L
open raceway pond. C. minutissima has showed better result in using the formulated mixture
wastewater to produce the lipid, lutein and biomass. The Lutein production was around 1.2 ± 0.08
mg/L/day by consuming 80.74 ± 0.07 mg/L/ day of CO2. C. minutissima completely removed the
nitrate, nitrite and ammonium.

Conclusion

Microalgal cultivation is believed as alternative method for remediation of harmful waste.

Microalgal cultivation in various wastewater showed that microalgae-based wastewater treatment
is a cost effective and a sustainable method. The listed methods are having enhancement in
treatment process whereas the large-scale studies need to be carried to optimize the conditions for
effluent treatment. Adaptation of microalgae in existing treatment system will enhance the nutrient
removal at low cost and pave the way to zero liquid discharge. The microalgal biomass recovery
with value additions can be carried out for the benefit of mankind.

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