A comprehensive review of recent reports on cultivation technology for microalgae:

Integrated CO2 bio-sequestration and wastewater treatment

Panneerselvam SundarRajana, Kannappan Panchamoorthy Gopinatha*

a Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering, Kalavakkam, Tamil

Nadu 603110, India

E-mail address: ksundarp@gmail.com; gopinathkp@ssn.edu.in*

* Corresponding author

Highlights

Emphasized the importance of selecting algal species for wastewater treatment.

Biomass productivity is restricted by the cultivation mode and techniques.

Microalgae grown on wastewater under different trophic conditions is reviewed.

Salient features of different cultivation techniques are discussed.

There was synergistic effect while cultivating microalgae in hybrid photobioreactor

Abstract

Microalgae technology has potential to combat the global energy crisis, food security, and

production of various commercial products. On the other hand, biological CO2 fixation and

wastewater treatment by using microalgae have also received much attention by researchers.

Algae being a photosynthetic microorganism their growth and potential can be alleviated

using different cultivation mode, and development of different cultivation techniques, which

generally acts as a major factor that which restricts biomass production and nutrient removal.

There are three major types of cultivation modes: photoautotrophic, heterotrophic and

mixotrophic cultivation; and two major cultivation techniques open systems (raceway ponds)

and controlled closed systems (photobioreactors (PBRs)). The design of cultivation reactors

is usually recommended based on the final use of biomass and quality required. Apart from a

production perspective, other factors, for example, cost effectiveness of the bioreactor, life

span, user-friendly, low maintenance and space convenience should be considered. In this

comprehensive review, we systematically survey the background of microalgae application in

wastewater treatment and CO2 bio-sequestration, relative advantages of various modern

cultivation modes have been emphasized. Recently used cultivation reactors with different

geometry and their uniqueness for biomass production are revised along with the comparative

picture. In this respect, nutrient removal efficiency and biomass productivity were also

compared to cultivation modes and reactors. In addition, some other promising PBRs are

introduced in this paper, hybrid photobioreactor (incorporating different reactors) which can

be utilized for overcoming the bottlenecks of a single PBR.

Keywords: wastewater treatment, CO2 fixation, microalgae cultivation, photobioreactor,

biomass production, nutrient removal

1. Introduction

Rapid industrialization and population explosion has led lead to severe water shortage and

pollution of surface water sources [1-6]. According to World Mapper Project 2007, 990

billion cubic metres of water is utilized for domestic and industrial purpose worldwide each

year and then this freshwater is transformed into wastewater [7, 8]. Many organic and

inorganic pollutants found in wastewater lead to xenobiotic effects in human and other living

beings [9-12].

Eutrophication is mainly due a result of to the enrichment of water by nutrients, especially

inorganic chemicals such as nitrogen in the form of ammonia, urea or organic nitrogen

whereas or phosphorous in the form of phosphate or organic phosphorus [13-15]. These

compounds cause accelerated growth of algae and higher form of vegetation and in so doing

an undesirable disturbance to the balance of organisms and the nature of the water has done

[16]. Hence this global problem has led lead to worldwide severe enforcements of

environmental laws became severe worldwide, to fight against this pollution. This law states

that water quality indices exceeding national discharge standards should be processed prior to

discharge into streams, lakes, seas, and land surfaces using various treatment processes [11,

17].

Normally, the conventional treatment of wastewater utilizes various biological based

treatment process (Table 1) because most of the conventional wastewater treatment

technologies involves chemical and physical methods, which are not economically feasible.

Biological treatment processes involves deals with the use of microorganism for the

transformation of certain substances into others products of greater added value (metabolic

products) [18]. Microalgae are prokaryotic and eukaryotic photosynthetic microorganisms

that which typically live inhabit the in an aquatic environment driven by the same

photosynthetic process similar to terrestrial plants. Exceptionally, uUnlike higher plants,

algae do not have a vascular system to transport nutrients instead of that they are provided

withalgal cells that possess photoautotrophic behavior which can directly absorb dissolved

nutrients [19]. Microalgae do not require arable land and fresh water because they can be

cultivated in non-arable land such as brackish and wastewater which does not compromise

the production of food crops [20, 21]. Microalgae exhibit faster growth rates and

photosynthetic efficiency of microalgae can potentially exceed 10%, which is 10-50 times

greater than that of higher plants because terrestrial plants are relatively inefficient in

capturing light [22 -24]. Microbial cells are sunlight-driven cell factories that have the ability

to convert carbon dioxide (CO2) into raw materials for the production of biofuels (e.g.,

biohydrogen, biodiesel and bioethanol), pharmaceuticals, nutraceutical, biochemical,

pigments, animal feeds, cosmetics, co-products such as carbohydrates, proteins and residual

biomass which may be used as fertilizers (Fig 1 & Fig 2) [25, 26]. A wide range of

metabolites that which are produced from algae has have various bioactive properties and are

yet to be fully exploited. Apart from that, biomass generation of microalgae also has potential

benefits in the cleaning environment, (1) microalgae utilize nutrients from different

wastewater (nitrogen and phosphates), thus providing an alternative for wastewater treatment.

In addition to that, microalgae also have the potential to remove heavy metal ions from

wastewater; (2) microalgae helps in CO2 fixation and storage which can photosynthetically

transform water and CO2 (inorganic carbon) to organic compounds without consumption of

excess energy and secondary pollution. In order to diminish the effect of CO2 on global

warming, the demand has been increased for sequestration of CO2 from the industriesindustry

[19, 21, 27, 28]. In general, there are several CO2 capture methods but they allwhich fall into

three categories: (i) chemical reaction-based methods such as washing emissions with

alkaline solutions, (ii) deep injection of sequestered gas (underground, in the ocean depths)

and (iii) biological methods, by means of autotrophic organisms. Physical and chemical

means of capturing, transporting and storing CO2 methods is an expensive processes, hence

biological means of CO2 capture is chosen as an alternative. Amongst various biological CO2

mitigation processes, microalgae as a bio-agent for CO2 mitigation has have received greater

attention. It is also estimated that 1 kg of dry algal biomass utilizes about 1.83 kg of CO2 [23,

29, 30]. In summary, microalgae cultivation has dual benefits, carbon dioxide mitigation and

wastewater treatment which offersoffer more economical feasibility and environmentally

sustainability (Fig 1).

Enormous efforts have been invested in algae strain selection and development of efficient

cultivation systems because the required amount of biomass is huge and the cost of

production must be very low. Cultivation system will be economically feasible only when the

production cost fall beneath 400$/ton of biomass, which is still long way from the cost that is

currently reported in a full-scale plant: the expenses as of revealed for a medium-scale plant

which is still 173 times higher than the aforementioned target value [29, 31]. Therefore, it

remains a huge challenge in reducing the production cost.

The cultivation technologies currently employed for microalgae farming are conducted at

both laboratory and commercial scales. In general, the cultivation systems selection depends

on the (1) capital cost, (2) desired product, (3) source of nutrients and (4) CO2 sequestration

[19]. Cultivation of microalgae can be done in either open systems (raceway ponds) or

controlled closed systems (photobioreactors) based on their design requirements. Open

systems are preferred in most existing large scale microalgae cultivation plants because they

offer easy operation and are low in investment and maintenance cost. Unfortunately, they are

mostly prone to contamination by other microflora and leads to potential system failure and

also limits the diffusion of CO2 from the atmosphere. Therefore, they can achieve only low

biomass productivities, which causes an impact on biomass harvesting costs [28]. Closed

systems, on the other hand, are more complex in operation, but however, they neglect those

these issues and permits the monoculture growth of microalgae for extended periods under

better-controlled conditions such as pH, temperature, light, CO2 concentration etc. The main

benefits of photobioreactor (PBR) are that they can produce a large quantity of biomass and

prevents water evaporation and CO2 loss [32].

In this review, we strive to provide a systematic survey on recent developments of

technologies dealing with microalgae culturing for CO2 capture and wastewater treatment.

Table 1. Some of the biological process involved in treating various wastewater.

Sources of wastewater Biological treatment process ReferencesMunicipal wastewater Anaerobic-anoxic sludge blanket reactor [33]Mixed starch wastewater Rhodotorula glutinis (Fungi cultivation) [34]Municipal wastewater Sequencing batch reactor [35]Petrochemical wastewater Ozonation-biological aerated filter [36]

Printing textile wastewater Ozonation-Sequencing batch biofilter granular reactor

[37]

Industrial wastewater Inverse fluidized bed biological reactor [38]Oil refinery wastewater Ultrafiltration hollow fiber membrane

bioreactor[39]

Phenolic wastewater Anaerobic continuous stirred tank reactor [40]Domestic wastewater Anaerobic-aerobic-anoxic biological

reactor[41]

Municipal wastewater Intermittent baffled bioreactor [42]Swine wastewater Integrated constructed wetlands [43]Piggery wastewater Upflow microaerobic sludge reactor [44]Domestic wastewater Flat-panel air-cathode microbial fuel cells [45]Effluent of upflow anaerobic sludge blanket reactor

Downflow rope bed biofilm reactor [46]

Wastewater treatment plant Plug-flow integrated fixed-film activated sludge reactor

[47]

Anaerobically digested wastewater

High rate algae ponds [48]

Bio-industrial wastewater Microalgal airlift Photobioreactor [49]

Fig 1. An integration of microalgae cultivation with wastewater treatment and CO2

sequestration.

Fig 2. Process flow diagram of microalgae product possibilities.

2. Selection of microalgae strains

Since the 1970s, microalgae have been effectively utilized to for the eradication of e the

nutrients in thefrom wastewater in the tertiary treatment phase. In order to produce desired

product and to achieve the maximum microalgae productivity, it is prior importance to pay

attention towards the selection of adequate species or strains is essential (Fig 3). The most

fFavourable characteristics of algae that can be utilized for wastewater treatment includes

higher growth rate, higher productivity, higher tolerance to the possible pollutants such as

metal ions and toxic compounds present in the wastewater, higher NH4+ tolerance, higher O2

generation rates, higher CO2 sinking capacity, and robust growth properties with enhanced

tolerance for different environmental conditions [50, 51]. These criteria are given prime

importance because these are considered as a limiting factor for nutrient and pollutant

removal efficiency of algae. The selection of microalgae for a specific treatment is also based

on the knowledge about the indigenous species present in such wastewater, because they

could also createhave the potential for suppressing a hindrance in algal growth [52]. To date

several microalgae strains have been utilized for various wastewater treatment processes,

Table 2 summarizes some microalgae strains that which have been studied for the removal of

nutrients and pollutants from the wastewater. Among them, chlorella Chlorella and

scenedesmus Scenedesmus species have received major attention inbecome the focus for the

treating treatment of wastewater due to their high growth rate, high environmental tolerance

and high lipid/starch accumulation potential [53].

Lekshmi et al (2014) studied the potential of Chlorella pyrenoidosa and Scenedesmus

abundans in domestic wastewater and observed that both thethese microalgae are capable to

growth and remove removal the nutrients. They also recorded the that the maximum

chlorophyll content as was 11.33 mg/l L and 7.23 mg/l L for C. pyrenoidosa and S.

abundans, respectively [54]. Gupta et al (2016) investigated the role of Chlorella sorokiniana

and Scenedesmus obliquus forin the treatment of domestic sewage. S. obliquus showed

greater potential for removing organic carbon, nutrients and pathogenic removal in when

comparedison to C. sorokiniana. However, C. sorokiniana demonstrated better adaptability

to physiological stresses under similar cultivation conditions in when compared

withcomparison to S. obliquus [55]. Mehrabadi et al (2017) focussed on the five wastewater

colonial algal species that which are found commonly present in high rate algal ponds

(HRAP): Mucidosphaerium pulchellum, Micractinium pusillum, Coleastrum sp.,

Desmodesmus sp. and Pediastrum boryanum and observed that all species showed similar

efficient nutrient removal whereas, under winter conditions, only the M. pulchellum and M.

pusillum cultures showedexhibited efficient nutrient removal. Moreover, M. pulchellum and

M. pusillum cultures had the highest biomass yield, lipid, and energy content under both

summer and winter conditions by the end of the experiment [56]. Mennaa et al (2015)

evaluated the capacity of seven species such as Ankistrodesmus falcatus, Scenedesmus

obliquus, Chlorella kessleri, Chlorella Vulgaris, Chlorella sorokiniana, Botryococcus

braunii, Neochloris oleabundans and a natural bloom of microalgae in urban wastewater.

Results showed that the natural bloom and S. obliquus seem to be the best candidates to grow

in pre-treated wastewater, according to their biomass production, nutrient removal capability

and harvestability [57]. These results highlight the significance of selecting algal species to

extend their applicability for wastewater treatment process.

Table 2. Summary of microalgae species for removal of pollutants.

Microalgae species Wastewater type Research findings ReferencesChlorella zofingiensis

BG-11 medium Adapt the fluctuating outdoor environment

[50]

Mucidosphaerium pulchellum

Primary settled sewage

Higher biomass yields under summer and winter conditions but less settleability

[56]

Micractinium pusillum

Primary settled sewage

High settleability and higher biomass yields under summer and winter

[56]

Coleastrum sp. Primary settled Higher biomass yields under [56]

sewage summerDesmodesmus sp. Primary settled

sewageHigher biomass yields under summer

[56]

Pediastrum boryanum

Primary settled sewage

Higher biomass yields under summer

[56]

Ankistrodesmus falcatus

Urban wastewater Higher biomass concentration [57]

Scenedesmus obliquus

Urban wastewater Maximum biomass productivity, daily phosphorus removal, and short time to harvest

[57]

Chlorella kessleri Urban wastewater Higher specific growth rate [57]Chlorella Vulgaris Urban wastewater Maximum phosphorus removal [57]Chlorella sorokiniana

Urban wastewater Maximum daily nitrogen removal

[57]

Botryococcus braunii

Urban wastewater Maximum daily nitrogen removal

[57]

Neochloris oleabundans

Urban wastewater Maximum daily nitrogen removal

[57]

Natural bloom Urban wastewater Maximum biomass productivity, daily nitrogen and phosphorus removal, and short time to harvest

[57]

Galdieria sulphuraria

Urban wastewater Mixotrophic metabolism yields higher biomass

[58]

Chlorella sorokiniana

Simulated wastewater

Grow under both light and lightless conditions

[59]

Neochloris aquatica

Swine wastewater Carbohydrate-rich biomass production

[60]

Coelastrella sp Swine wastewater Adapt at a high ammonium concentration and pH

[61]

Algal biofilm Municipal wastewater

Lipid and starch-rich biomass production at different HRTs

[62]

Chlorella vulgaris Swine wastewater Carbohydrate-rich biomass production

[63]

Desmodesmus sp. Anaerobic digestion wastewater

Better performance in this type of wastewater

[64]

Scenedesmus obliquus

Municipal wastewater with food wastewater

Enhanced performance under this type of wastewater with flue gas CO2

[65]

Neochloris oleoabundans

Modified soil extract medium

Lipid-rich biomass production [66]

Scenedesmus quadricuada

Herbal pharmaceutical wastewater

Adapt the toxicity and treat this type of wastewater

[67]

Chlorella pyrenoidosa

Piggery wastewater Convert the high organic content of piggery waste

[68]

Chlamydomonas mexicana

Pharmaceutical compound

Tolerant to high toxicity and cellular stresses

[69]

Scenedesmus obliquus

Pharmaceutical compound

Adapt the toxicity and treat this type of wastewater

[69]

Scenedesmus dimorphus

Chlorella medium Adapt the high iron concentration and high salinity

[70]

Haematococcus pluvialis

Inorganic medium Adapt the fluctuating outdoor environment

[71]

Fig 3. Microalgal species selection: A key parameter for product conversion.

3. Overview of cultivation modes for microalgae production

The cultivation conditions mostly influence the growth characteristics and composition of

microalgae which helps in determining the quality and quantity of algale products. There are

three major classifications, i.e., photoautotrophic, heterotrophic, and mixotrophic cultivation,

are discussed in detail in the following sections (Fig 4). Table 3 summarizes the

productivities of several microalgal species using various carbon sources under different

cultivation modes.

3.1. Photoautotrophic cultivation

Photoautotrophic cultivation is the most primitive method for developing microalgae, which

utilizes light (such as sunlight, artificial light), as the energy sources, and inorganic carbon

(e.g., carbon dioxide (CO2), and bicarbonate ion (HCO3-)) as the carbon source to form

chemical energy through photosynthesis, this chemical energy that can later subsequently be

released to fuel thefuelling microalgae activityies [72]. The survey found that under

photoautotrophic mode, there is a huge deviation in the biomass productivity for a different

type of microalgae species (Table 3). Kim et al (2014) demonstrated the that sodium

bicarbonate has the capability as a buffered chemical which canfor maintaining the dissolved

inorganic carbon (DIC) concentration at the desired level in combination with CO2 gas for

enhanced growth of C. vulgaris [73].

Photoautotrophic culture is the most common approach and energy-saving strategy for

microalgae cultivation, usually grown in two photoautotrophic cultivating systems such as

open pond system and closed photobioreactor system. As previously mentioned, open pond

systems are not favourable for microalgae cultivation due to several drawbacks [74]. In order

to overcome the drawbacks, closed culture systems has have been developed. Jacob et al

(2015) reported in the literature that carbon capture efficiencies could be as high as 90% for

closed culture systems such as photobioreactors, with some reports suggesting a range of 45-

70% and lower efficiencies (25-50%) for raceway ponds [75]. Hence, the research has been

become focussed mainly on the design of photobioreactors, which can improve fluid

dynamics, controls the incident irradiance and maximize biomass production [76]. Biomass

efficiency can be enhanced to an extent by the CO2 rich environment to a particular extent;

however, photoautotrophic cultivation experiences issues in accomplishing high biomass

concentration and biomass productivity because light penetration diminishes exponentially

with the elevation of broth turbidity (mainly due to microalgal cells) and dependence on

weather conditions (such as, temperature and light) [75]. Therefore, the choice of preferring

photoautotrophic cultivation has became become limited.

3.2. Heterotrophic cultivation

In contrast to photoautotrophic, heterotrophic cultivation is independent of light and therefore

there is a reduction in could reduce the surface to volume ratio of the bioreactor, thus ease

making thethe design of the bioreactor easier. Heterotrophic cultivation And it also offers

several advantages over photoautotrophic cultivation including the problems associated with

limited light penetration with the elevation of broth turbidity, good control of the cultivation

process, and low harvesting cost due to higher cell density [72]. Moreover, this cultivation

mode can be conjugated combined with plant fermentation technology and facilities, includes

including beverage, pharmaceutical, and nutraceutical industries, makes making the

production costs cheaper. Marudhupandi et al (2016) reported that the heterotrophically

cultivated Nannochloropsis salina with various carbon and nitrogen sources obtained higher

biomass when compared to the a photoautotrophical cultivation [77].

Heterotrophic cultures usually assimilate organic carbon sources for growth and cultivated

cultivation in fermenters. Glucose has been a the main carbon source for heterotrophic

organisms. The growth of microalgae are influenced by the type of organic carbon sources

when chlorella Chlorella vulgaris assimilates glucose, glycerol, and acetate as a carbon

source, higher biomass concentration was acquired achieved at a lower concentration of

glucose and glycerol [74]. However, the cost of glucose is high, in response to that some

researchers have focused on identifying alternative low cost organic carbon sources at low

cost to replace glucose (Table 3). It is has also been reported that sucrose riched wastes and

corn powder hydrolysate (CPH) may effectively reduce the cost of carbon sources for the

heterotrophic culture of microalgae, resulting in high biomass productivities [78, 79]. With

respect to that, Katiyar et al (2017) investigated the use of crude glycerol, a primary by-

product of the biodiesel production for heterotrophic cultivation of microalgae (namely,

Chlorella sp.) in photobioreactor and reported that a two folds increase in biomass

productivity when compared to Bold's Basal media (BBM) used as control [80].

Kishi et al (2015) explored the possibilities of waste ethylene glycol (EG) and propylene

glycol (PG) assimilation for heterotrophic algal treatment and found that among five

Chlorella sp., Chlorella protothecoides seems to assimilated both EG and PG, and with the

results suggested suggesting that glycol has potential as a future application in heterotrophic

cultivation [81]. The heterotrophic system frequently suffers from the problem associated

with contamination of organic carbon source by Escherichia coli and other bacteriuma’s,

which may reduce the quality and quantity of products of interest. Another drawback related

to the heterotrophic system is its cost, which is much higher than photoautotrophic system

because of the large requirement of organic compounds, bioreactors and achieving axenic

microalgae culture [82].

3.3. Mixotrophic cultivation

Mixotrophic cultivation is the mode in which the microalgae utilize both organic compounds

and inorganic carbon (CO2) as a carbon source for growth while whilst undergoing

photosynthesis. This means that they are able to survive under either photoautotrophic or

heterotrophic conditions or both. Microalgae can utilize organic compounds and CO2 as a

carbon source, and the CO2 released by microalgae via respiration will be trapped and

recycled under photoautotrophic cultivation which is affected by illumination conditions,

whereas organic carbon is fed through aerobic respiration which is influenced by the

availability of organic carbon [83].

It is reported that, growth rates under mixotrophic cultivation where higher when compared

to autotrophic cultivation, prompting 6-7 times higher cell density and biomass productivity

[84]. For example, Chodatella sp. in piggery wastewater were investigated by Li et al (2014);

the specific growth rate and biomass production obtained with mixotrophic growth were was

1.74 and 14 times higher than those obtained with autotrophic growth, respectively [85]. Li et

al (2014) evaluated the performance of Chlorella sorokiniana in different cultivation mode

and found that the specific growth rate and maximum dry weight (DW) in mixotrophic

cultivation were 1.8- and 2.4- fold of those in heterotrophic cultivation, and 5.4- and 5.2- fold

of those in photoautotrophic cultivation [86]. Similarly, in Chlorella vulgaris, the biomass

productivity and N and P removal rates were notably increased in the mixotrophic culture

when compared with photoautotrophic and heterotrophic cultures [87].

A few researchers have proposed that the specific growth rate of microalgae under

mixotrophic cultivation is approximately the sum of those under photoautotrophic and

heterotrophic modes, while others trusted have proposed that the specific growth rate in

mixotrophy is not the simple blend of those in photoautotrophy and heterotrophy, but these

two metabolic processes influence each other under mixotrophic culture, which may

contribute to synergistic effects and improve biomass efficiency [76, 82, 88, 89]. Moreover,

mixotrophic culture can able to tolerate at the elevated light intensity. To address it,

Chojnacka and Noworyta (2004) observed the photo-inhibition in of Spirulina sp. at 50Wm-2

under photoautotrophic cultivation, whereas it was not observed under mixotrophic

cultivation (0-65 Wm-2) [90]. With these advantages, mixotrophic cultivation has been

applied to the treatment of organic carbon-rich waste water. The overall differences in the

features of different microalgae cultivation modes are summarized in Table 4.

Fig 4. Various microalgae cultivation modes for wastewater treatment.

Table 3. Productivities of several microalgal species using various carbon sources under different cultivation modes.

Microalgae species Cultivation mediumCultivation condition

Biomass productivity(mg L-1 d-1)

Specific growth rate (d-1)

Ref.

Chlorella zofingiensis Piggery Wastewater Photoautotrophica 296.16±19.16 0.340 ± 0.001 [91]Chlorella vulgaris Aquaculture Wastewater Photoautotrophica 42.6 0.17 [92]Chlorella saccharophila and Scenedesmus sp.

Treated Dairy Farm Wastewater Photoautotrophica 276±40 NA [93]

Chlorella vulgaris Urban wastewater Photoautotrophica 116 0.48±0.144 [94]Chlorella vulgaris Synthetic wastewater Photoautotrophica 94.1 0.648±0.216 [94]Chlorella kessleri Urban wastewater Photoautotrophica 132.3 0.624±0.168 [94]Chlorella kessleri Synthetic wastewater Photoautotrophica 140.2 0.624±0.144 [94]Scencedesmus obliquus Urban wastewater Photoautotrophica 201.4 0.672±0.168 [94]Scencedesmus obliquus Synthetic wastewater Photoautotrophica 193.2 0.6±0.168 [94]Natural bloom Urban wastewater Photoautotrophica 200.4 0.60±144 [94]Natural bloom Synthetic wastewater Photoautotrophica 201.3 0.6±0.192 [94]Chlorella vulgaris Treated municipal wastewater Photoautotrophicb 39.93±0.73 0.277±0.015 [95]Chlorella ellipsoidea Bold’s basal medium Photoautotrophicb 70 0.161 [96]Chlorella ellipsoidea Wright’s cryptophyte Photoautotrophicb 60 0.138 [96]Chlorella ellipsoidea Closterium medium Photoautotrophicb 65 0.149 [96]Chlorella ellipsoidea Blue green-11 medium Photoautotrophicb 80 0.184 [96]Chlorella protothecoides

Whey permeate from dairy industry

Heterotrophicc,d 9.1±0.2 gL-1 NA [97]

Chlorella protothecoides

Whey permeate from dairy industry

Heterotrophicc,d 17.2±1.3 gL-1 NA [97]

Chlorella pyrenoidosa Blue Green-11 medium Heterotrophice 111.48x106 cells/mL 1.02 [78]Chlorococcum sp. Dairy effluent Heterotrophicf ~2 gL-1 NA [98]Chlorococcum sp. Dairy effluent Mixotrophicf 1.94 gL-1 NA [98]

Nannochloropsis salina Walne medium Heterotrophicg 1.85 gL-1 NA [77]Chlorella protothecoides

Proteose medium Heterotrophich 0.52 gDW gPG-1 NA [81]

Chlorella protothecoides

Proteose medium Heterotrophici 0.081 gDW gEG-1 NA [81]

Chlorella zofingiensis Kuhl growth medium Photoautotrophica 0.44 NA [88]Chlorella zofingiensis Kuhl growth medium Heterotrophicc 1.28 NA [88]Chlorella zofingiensis Kuhl growth medium Mixotrophicc 2.36 NA [88]Chlorella sp. Blue Green-11 medium Photoautotrophicb 0.03 0.4 [99]Chlorella sp. Blue Green-11 medium Photoautotrophicj 0.04 0.3 [99]Chlorella sp. Blue Green-11 medium Photoautotrophica 0.04 0.44 [99]Chlorella sp. Blue Green-11 medium Heterotrophick 0.012 0.017 [99]Chlorella sp. Blue Green-11 medium Heterotrophicl 0.014 0.024 [99]Chlorella sp. Blue Green-11 medium Heterotrophicm 0.022 0.033 [99]Chlorella sp. Blue Green-11 medium Mixotrophicl 0.12 1.22 [99]Chlorella sp. Blue Green-11 medium Mixotrophick 0.10 1.2 [99]Chlorella sp. Blue Green-11 medium Mixotrophicm 0.07 1.47 [99]Chlorella sp. Blue Green-11 medium Photoautotrophica 0.24±0.03 NA [89]Chlorella sp. Blue Green-11 medium Mixotrophicn 0.52±0.02 NA [89]Chlorella sp. Blue Green-11 medium Mixotrophico 0.33±0.04 NA [89]Chlorella sp. Blue Green-11 medium Mixotrophicp 0.35±0.02 NA [89]

NA, not available;a. CO2 b. Air c. Glucose d. Galactosee. Sucrose f. Biodiesel industry waste glycerol g. Glucose + sodium acetate h. Propylene glycoli. Ethylene glycol j. Na-Bicarbonate k. Fructose l. Na-Acetatem. Molasses n. Cheese whey o. Digestate ultrafiltrate + glycerol p. White wine lees

Table 4. The overall differences in the features of different microalgae cultivation modes.

Cultivation system

Carbon Source

Energy Source

Reactor type Pros Cons

Photoautotrophic Inorganic Light Open pond or photobioreactor

(1) High production of pigmentation and phytochemicals

(2) Low cost

(1) Low tolerance towards light intensity(2) Requires special bioreactors(3) Low growth rate and biomass productivity(4) High harvesting cost

Heterotrophic Organic Organic Conventional fermenter

(1) Bioreactors design with little limitation

(2) Higher growth rate and biomass productivity

(3) Low harvesting cost

(1) Dark conditions weakens the pigmentation and production of phytochemicals

(2) Higher cost(3) More CO2 emission(4) Prone to contamination hence requires

sterile media (5) Requires large amount of organic

compounds(6) High substrate cost

Mixotrophic Inorganic and Organic

Light and Organic

Closed photobioreactor

(1) High tolerance towards light intensity

(2) High production of pigmentation and phytochemicals

(3) Higher growth rate and

(1) Higher cost(2) Prone to contamination hence requires

sterile media(3) High substrate cost(4) High equipment cost

biomass productivity(4) Less CO2 emission

4. Microalgal cultivation technique-Photobioreactor (PBR) technology

Over 70 years, a wide range of the microalgae cultivation systems has have been investigated

and reported in the scientific literature by scholars and industrialists. The suitable cultivation

conditions such as adequate illumination, appropriate algae strains, ideal climate condition,

overall year-round production and least land utilize, are still challenges and have a long way

to go in the futuregoing forward. In recent times, large scale algae cultivation for commercial

purpose has turned into an intriguing issue, in order to diminish culture cost, improve

biomass efficiency and stay away from other harmful microscopic organisms or parasites as

much as possible, hence it is important to pay more endeavors on the cultivation procedure.

Enthusiasm for large-scale algae cultures of algae was invigorated through the construction of

a Chlorella pilot plant and supplemental laboratory studies by Arthur D. Little, Inc for the

Carnegie Institution of Washington during in 1951 [100]. This valuable source of information

is summarized in a report which is more useful even today for algae cultivation. In the 1960s,

a Japanese research group experimented in “open circulation system”, which utilizes utilized

shallow open pond to culture microalgae [101]. Later on, the Japan Nutrition Association

developed a pilot scale plant to further study the industrial cultivation of algae further. As

time went on, iIn the late 1970s, commercial cultivation of algae was started by Japanese,

Europeans, and Israelis; during this period, algae, cultures were commercially developed as

healthy foods from the view of public health. Eventually, microalgae cultivation became

significant in biofuels (e.g., biohydrogen, biodiesel, and bioethanol), aquaculture, as well as

the production of pharmaceuticals, nutraceutical, biochemical, pigments, animal feeds,

cosmetics and biofertilizers [25, 26]. Likewise, algae growth techniques turned out to be

more sophisticated and, with the advent of technology, the utilization of PBRs turns out to

bebecame more common. Currently, two cultivation systems were are widely used either

open systems or controlled closed systems based on their design requirements. Table 5

indicates the general understanding of pros and cons of the both two methods simultaneously

so that the appropriate methods can be preferred basedchosen on the requirement of final

products and the operating conditions.

4.1. Open system

Cultivation of algae in an open system has been used for large scale microalgae cultivation

because they it offers a simpler construction, and relatively easy operation and for economic

reasons. These open systems permit microalgae to take-up CO2 from the air directly from the

ambient atmosphere. They mostly preferred for economical reasons. Such cultivation systems

can be categorized into natural water such as lakes, lagoon, ponds and artificial water systems

such as artificial ponds, tank, and containers [26]. Depending on the requirement of treatment

facilities, different shapes, sizes and types of open systems (agitated, inclined and others)

have been investigated. These pond systems are normally kept shallow so that the algae are

exposed to sunlight for photosynthesis, and that sunlight can reach a limited depth [102]. For

commercial cultivation of algae, unstirred open system, circular ponds, and raceway ponds

are usually normally used (Fig 5).

Fig 5. Open cultivation technique: (a) Paddle wheel raceway pond. (b) Circular pond.

4.1.1. Unstirred open pond

Amongst commercial culture methods, unstirred, open ponds are the most economical and

simple, open ponds have the with least technical but also most ineffective method due to poor

mixing or contaminants such as protozoa, other microalgae, viruses, and bacteria. Unstirred

open ponds are simply natural lakes or constructed from natural water ponds with a depth of

less than 50 cm deep [103]. Borowitzka et al (1999) reported that large shallow open-air

ponds (unmixed other than by wind and convection) have been used for culturing Dunaliella

salina for β-carotene production in Australia, Betatene Ltd [104]. It is has aslo also reported

that more than 30 tons year-1 of microalgae biomass have been harvested from natural lakes in

SoutheEast Asia [105].

4.1.2. Circular open pond

Circular open ponds are provided with a centrally pivoted rotating agitator (a clock dial with

the second rotating hand running around). However, the pivoting arm is a restricting

component in circular ponds because of the poor mixing efficiency when it gets too long,

(e.g. >50 m in diameter) the poor mixing efficiency increases due to the strain of water

resistance on the rotating motor. In such cases, circular ponds are is normally build with a

diameter of <45 m and a depth ranging from 30-70 cm [103, 105]. Lee (2001) reported that

algal biomass is cultivated widely in Japan, Taiwan, and Indonesia using large-scale circular

ponds [106]. Rao et al (2012) focused on cultivation and seasonal variation in growth of

Botrycoccus braunii in raceway and circular ponds under outdoor conditions and suggested

that B. braunii can be exploited for mass cultivation under outdoor conditions [107].

4.1.3. Raceway pond

Since the 1950s, the raceway pond is an extensively used as a typical open system for algae

cultivation. This type is normally shallow with a depth ranging from 15 and 30 cm. Raceway

ponds are available in either oval channel (single channel) or race track channel (joining

individual raceways together). They are normally constructed with concrete materials or they

are just dug into the earth and fixed with a plastic liner (such as thick silpauline sheets) to

keep the ground from leachate [108]. Raceways are encouraged to conduct in extremely

selective environment or covered by building a greenhouse over, in order to avoid species

contamination and other contaminants [109]. Amongst various raceway ponds designs of

raceway ponds, this type (i.e., paddle wheel type) has have been used commercially over the

past 30 years. This paddle type promotes the vertical mixing by generating turbulent eddies in

the raceway ponds (average water velocity typically from 0.15 to 0.30 m s-1) so that the

deposition of settling cells or the accumulation of cells via flocculation can be prevented

[110]. This system is frequently operated in a continuous mode, where the influent

(containing nutrients including nitrogen phosphorus and inorganic salts) is supplied at the

front of the paddle wheel, and when the algal culture has circulated through the loop, it is

segregated behind the paddle wheel [48].

An aerator can be utilized to enhance the air flow rate and the CO2 usage. Thus the algae

culture is mixed and circulated around raceway ponds with continuous CO2 and nutrients

supply supplied [111]. In order to support that, Posadas et al (2015) evaluated the influence

of pH and CO2 source on the microalgae growth in secondary domestic wastewater and found

that the supply of CO2 enhanced the chemical oxygen demand (COD), total organic carbon

(TOC) and total phosphorus (TP) removals and also recorded the maximum biomass

productivity as 17 ± 1 gm-2 d-1 in the an outdoor pilot raceway pond [03]. It was reported that

Chlorella sp., Spiriluna platensis, Hematococcus sp. and D. salina are commercially

cultivated using raceways ponds. Compared with other open ponds, raceway pond shows

display a high superiority in convenient and efficient operation, therefore, it has become is

preferred a lot when it comes to thefor large-scale outdoor large-scale cultivation of

microalgae [112].

4.1.4. Limitations of open pond systems

Open ponds face various technical barriers that which prevent them from being

commercialized: (1) light diffusion into the pond diminishes with depth and therefore pomds

have to be shallow and have a . Subsequently, they require shallow depth for ponds and have

low volume to area ratio, (2) limited utilization of CO2 from the atmosphere, (3) requirements

for of large areas of land areas, (4) monoculture of the desired microalgae is least possible

due to consistent airborne contamination, with the exception of extremophile species, (5)

cultivation are is mostly influenced by the environmental growth parameters such as local

weather conditions, which may cannot be controlled and thus makinges production seasonal,

(6) due to inefficient stirring mechanisms, the mass transfer rates becomes very poor resulting

in low biomass productivity (7) considerable amount of water dissipates due to increasing in

temperatures which results in reduction of the water being treated (8) harvesting is laborious,

costly to separate algae from water and sometimes constrained by low cell density and (9)

production of pharmaceutical or food supplements is not achievable because the desired

product is not quite pure [105, 113].

4.2. Closed system

In order to overcome the problems associated with the open system and to attain a better yield

of microalgae biomass, much attention is nowhas become focused on the development of

suitable closed systems, which does not allow direct mass transfer between culture media and

atmosphere and consumes a large amount of energy, which seems uneconomical. Therefore,

promoting closed PBRs to pilot or large scale is normally considered to be problematic. In

contrast, it is of great value when value-added products are produced such as

biopharmaceuticals, top grade cosmetics, human health foods and biofuels. These fine

chemicals are required to meet sufficient purity grade, therefore, the development of suitable

and sustainable closed PBRs becames very essential [101]. This type of system is normally

classified as a flat panel, horizontal tube, bubble column, airlift PBR and their modified

configurations (Fig 6).

Fig 6. Closed cultivation technique: (a) Flat panel PBR, (b) Horizontal tubular PBR (c) Bubble column PBR (d) Internal loop Airlit PBR (e) External loop Airlit PBR (f) Large-scale plastic bag PBRs.

4.2.1. Flat panel

The flat plate reactor is a kind of common PBR with cuboidal shape appearance which can be

used for algae cultivation. It can be operated with either indoor exposed to artificial light or

outdoor exposed to sunlight [114]. These types of photobioreactors are made with transparent

materials or semi-transparent materials like glass, plexiglass, plastic bags and polycarbonate

etc., for maximum utilization of solar light energy (i.e., oriented into the direct light path of

light to receive maximum exposure to solar energy) [115]. Moreover, flat panels have a

minimal light path which permits light to penetrate easily through the culture medium. The

key characteristic of this type is high surface area to volume ratio, vertical or tilted inclination

to obtain the best intensity of light and absence of mechanical devices for cell suspension.

Generally, agitation, gas exchange and degassing are promoted by bubbling air from the

bottom of each channel through the perforated tube. In comparison, with horizontal tubular

PBRs, the accumulation of dissolved O2 concentrations in flat panel PBRs is relatively low

[116].

A flat plate was built up by Ruiz et al (2013) from methacrylate sheets having which

possessed an illuminated area of 1022 cm2. The system was aerated at with an airflow of 2.81

min-1 and enriched in CO2 (5%) to grow Scenedesmus obliquus in urban wastewater. They

also recorded the biomass productivity and nutrient concentration under different hydraulic

retention time (HRTs) and suggested that biomass concentration and CO2 bio-fixation would

be higher at longer HRT of 2 µ-1 whereas efficient nutrient removal would be higher at

shorter HRT of µ-1 [117]. In, 2007, Feng et al (2011) evaluated the feasibility of culturing

Chlorella zofingiensis in a 60 L flat plate PBR using natural sunlight to reduce the cost of

microalgae culturing and demonstrated that it was possible to culture under an outdoor

conditions with biomass productivity of 58.4 mg L-1 d-1 [50]. Issarapayup et al (2011)

examined the cultivation of Haematococcus pluvialis in a flat panel airlift photobioreactor

based on its economic performance and found that reactor size appeared to be important for

the profitability of the system. The results revealed that, even though the small scale 17 L

exhibited higher performance, it was operated operating at a higher cost (197 US$ per year),

on the other handhowever, 200 L reduces reduced the costs per unit production (121 US$ per

year). Thus, these are fully scalable photobioreactor units [118].

Meanwhile, the major drawbacks of the flat panel included: (1) possibility of hydrodynamic

stress to algal strains due to aeration, (2) algae growth near wall region is was limited, (3)

facing difficulty in controlling the temperature during cultivation and (4) biofouling near the

internal surface [116].

4.2.2. Horizontal tube

Horizontal tube PBRs, are normally referred to as a tubular reactor, they possess has a high

surface to volume ratio, which is favorable to maximize exposure of the algae to incident

light (i.e., photosynthesis process). This type can be arranged in multiple possible

orientations, such as horizontal, inclined, α shape, spiral, helicoidal and their modifications,

resulting in a high light conversion efficiency. All these types are made with transparent

materials and have the similar working operations. The size of PBRs usually depends on the

desired product requirement, the diameter of tubes normally ranges from 10-60 mm but the

length can extend several hundred meters. The key parameters for this type of PBR

isparameters for this type of PBR are a flow velocity and mixing efficiency in the radial

direction [101]. Other than tubes, it is providedpossesses a with gas exchange chamber,

which helps to introduce the fresh culture, circulate cooling water in a loop for temperature

control and utilization of CO2. In addition, exhaust gases such as O2 is are removed from this

chamber because during photosynthesis oxygen will build up and causes photo bleaching and

thus reducesing photosynthetic efficiency. A mechanical pump is usually employed for the

circulation of liquid between gas exchange chamber and irradiated tubes which help in

suspension of cells without settling [115]. Lastly, the algae culture can be harvested

continuously after the completion of traveling cyclically.

The major drawbacks of this technology are the accumulation of excessive dissolved oxygen

and excessive power consumption about 2000 Wm-3 compared with 50 Wm-3 for bubble

column and flat plate PBR. This high energy input is necessary to attain liquid velocities of

about around 20-50 ms-1 for promoting turbulent conditions with sufficient short light/dark

cycles [119]. Arbib et al (2013) and Nwoba et al (2016) compared the nutrient removal and

biomass production between tubular PBR and high rate algal pond (HRAP) in tertiary and

piggery wastewater. The results revealed that the photosynthetic activity in tubular PBR was

higher when compared with HRAP, similarly, total nitrogen (TN) and total phosphorus (TP)

removal and biomass productivity were also higher in tubular PBR rather than that in HRAP

[120, 121]. However, there are some limitations in tubular PBR such as biofouling on the

inner wall. Michels et al (2014) continuously operated a tubular PBR for culturing

Tetraselmis suecica in fish farm wastewater. This pilot scale experiment was carried out

indoor turbidostat and continued for 65 d. This type of tubular PBR can handle the high

organic load of wastewater treatment [122].

4.2.3. Vertical tube

Vertical tube PBRs which are also described as a cylindrical reactor which isare available in

two types of configurations; , the bubbling, and airlift. Two of them are transparent in nature

to allow the penetration of light for improved photosynthesis and provided with air sparger at

the base of the reactor, which transforms spared gas into far smaller and more numerous tiny

bubbles to ensure suspension of algae without settling and also to improve gas/liquid mass

transfer, CO2 sequestration and O2 discharge [114, 115]. When cycling of the medium

between the irradiated and dark zone is high, bubble and airlift column can achieve a

considerabely increase ind radial movement of fluid. These reactor designs have a low

surface/volume ratio but they can give a significantly improved gas hold-up than horizontal

tube reactors with a conceivably more prominent gas-liquid flow [32].

4.2.3.1. Bubble columns

Bubble column reactor is a cylindrical vessel with no internal structure, thus the fluid flow is

done through bubbling the gas mixture from an air sparger provided positioned at the base of

the reactor, which will enhance the mixing and CO2 mass transfer. Generally, these sorts of

cylindrical vessels will have a diameter which is twice that of the height and get receive

illumination from an external light source. Bubble column reactor has have characteristics

advantages in of high surface area to volume ratio, low capital cost, lack of moving parts,

favourable heat and mass transfer, relatively homogenous culture environment, proficient

discharge of O2 and residual gas mixture. Perforated plates are installed during scale-up of

bubble column PBR, in order to disrupt and redistribute coalesced bubbles [123].

Photosynthetic efficiency incredibly relies upon gas flow rate, as the gas flow rate increases

increased (≥ 0.05 ms-1) significantly, fluid is circulated from central dark zone to outer

illuminated zone leading to shorter light and dark cycles. If the gas flow rate seems to beis

less than 0.01 ms-1, back mixing does not take place which will result in absence of

circulation flow pattern [124].

Lopez-Rosales et al (2015) investigated the growth of dinoflagellate Larlodinium veneficum

in bubble column photobioreactor under different hydrodynamic shear stress and evaluated

revealed that the cell growth is was optimum optimal at gas flow rates as of 0.26 L min-1

[125]. Zhu et al (2013) studied the growth and the productivity of Chlorella zofingiensis in a

1.37 L bubble column PBR containing piggery wastewater which was later utilized for the

production of biodiesel [91]. A bubble column bioreactor was built up by Maroneze et al

(2014) using borosilicate glass with height/diameter (h/D) ratio of 1.33 having no

illumination facility, for cattle-slaughterhouse wastewater treatment. This configuration in is

fact utilized for heterotrophic bioreactors. Then the eExperiments were performed using

Phormidium sp. microalgae, which revealed thatshowed COD, TN and TP removal was

90%, 57%, and 52%, respectively [126]. These results indicated that choosing choice of

material is was important to avoid the erosion of the wastewater and to allow the light

penetration of light. Hende et al (2011) investigated the potential of microalgae bacterial flocs

for the secondary sewage treatment in a working volume of 4L bubble column PBR

supplemented with flue gas mimicking a coal burning process. At a gas flow rate of 0.6 L h -1

(0.0025 vvm) with an HRT of 0.67 d, the reactor showed thehad a removal efficiencyies of

48 ± 7 % CO2, 87 ± 5 % NOx and 99 ± 1 % SO2 and by achieved attaining European

discharge standards. In the meantime, under a low intensity of light, the biomass productivity

is wasobtained as 180 mg L-1 d-1.

4.2.3.2. Airlift PBR

The An airlift reactor can be considered as an upgraded version of the bubble column but it is

divided into two interconnecting zones (called as, riser and downcomer) using a baffle or a

draft tube. In the riser zone, similar to bubble column, gas mixture is sparged to drive liquid

upward which is assisted by the gas hold up of riser, whereas in the downcomer zone, the gas

leaves the liquid and the left over gas which does not disengage will get trapped by the liquid,

illuminated and recycled back into riser zone [127]. The difference in mean density between

the downcomer and the riser generates the pressure gradient which acts as a driving force for

the recirculation to happen. Thus the large circulatory currents were produced in the

heterogeneous flow regime [115].

Generally it wasthey are classified into two main type on the basis of their morphology: (1)

internal loop (baffled) vessels, in which the riser is set as a concentric tube or split-cylinder,

while air is sparged inside the concentric tube and exit to the outer zone (downcomer zone)

where the liquid cycle backs to riser zone again; (2) another form is external loop vessels, in

which circulation takes place through separate and distinct conduits (i.e., downcomer is set

outside the vertical tube) [128] .

The residence time of gas has a significant influence in on the gas–liquid mass transfer, heat

transfer, mixing and turbulence. The attractive features of airlift PBR is the circular mixing

pattern which causes flashing light effect to algal cells, where liquid culture is exposed

continuously to dark and light phase and also the high photosynthetic efficiency but the

drawbacks is are its complexity and difficulty while scaling-up, higher manufacturing, and

maintenance cost, higher shear stress on algal cultures and smaller irradiation per unit area

[124, 129].

Miron et al (2002) suggested that airlift PBR has the capability to yield higher dry biomass

weight compared to bubble column PBR when Artrospira platensis (Spirulina platensis) was

applied to assess biomass production in Zarrouk’s medium [130]. Similarly, Oncel and Sukan

(2008), Chiu et al (2009) compared the bubble column and airlift PBR with respect to their

performance and concluded the same observation made by Miron et al (2002) [127, 131].

Chu et al (2015) demonstrated the possibility of large scale cultivation of Chlorella

pyrenoidosa adapted to anaerobically digested starch processing wastewater using 820 L

airlift photoautotrophic-heterotrophic PBR. This liquid culture exhibited enhanced tolerance

towards variation in the environmental conditions without a temperature-control device

(except in the winter). Thus the seasonal variations significantly influenced microalgae

production [132].

4.2.4. Plastic bag PBR

Plastic bag PBRs has have received greater attentions for large scale production when

compared with the other PBRs, due to their low cost and good sterility at start up - because of

high film-extrusion temperatures. It has three components such as plastic bags, a frame that

support the plastic bags and aeration system for suspending algal cells. The key design

parameters of this type of PBR are materials, size, aeration type (insertion of aeration tube or

provision for aeration outlet at the bottom), mixing behavior (airlift or mixing by sway) and

structure of the frame [133].

Wang et al (2013) built up 20 L transparent plastic bags PBR to treat pig effluent by using

Spirulina platensis. Results showed that this type of reactor can obviously promote the

nutrient removal: ammonia nitrogen, nitrate, and phosphorus removal reached 91.8%, 54.0%

and 65.4%, respectively, and the COD and suspended solids (SS) reached toachieved the

drainage standards [134]. Menke et al (2012) designed two sorts of plastic bags for screened

screening microalgae cultivation adapted to hypersaline wastewater. These experiments were

carried out to study the salt stress tolerance of ten different microalgae cultures. Results

indicated that Dunaliella salina, Tetraselmis tetrathele, and Nanochloropsis salina could

grow in hypersaline wastewater. Amongst two types of plastic bags, vertical airlift is suitable

for T. tetrathele, and N. salina cultivation, while D. salina was more suitable to cultivate in a

shaking see-saw PBR [135]. Chinnasamy et al (2010) compared the area ofl biomass

productivity of Chlamydomonas globosa and Chlorella minutissima using the 20 L

suspended polybags, 100 L vertical tank reactors and 2000 L raceway ponds containing

carpet manufacturing wastewater. Results revealed that the area ofl biomass productivity is

was higher in the plastic bags with the value of 21.1 g m-2 d-1 and the estimated biomass

production is was 51-77 tons ha-1 year-1 [136]. Altogether, in anFrom an economical point of

viewconsideration this type is more beneficial, however, scaling-up is a huge task because of

complexity in installing and maintenance of too much connection and structure structural

units. In addition, they also suffer from inadequate mixing, frequent culture crashing, disposal

of large quantities of used plastic bags, and are inherently fragile.

4.2.5. Hybrid PBR

In addition to the traditional PBRs, there is some hybrid type forms of innovative PBRs

which have been designed by researchers to for the treatment of wastewater, which is

broadlythese are utilized because it they exploits the benefits of two different types of

reactors only after integrating these reactors with one another (Fig 7). This is the only best

way to overcome the drawbacks associated with other. Rocha et al (2016) developed a novel

integrated bioreactor combined with recyclable iron oxide nano/micro-particle adsorption

interfaces, to eradicate CO2, and undesired organic air pollutants using natural particles while

generating oxygen. And further discussed the implication of usage of this system to existing

industrial setups [137]. Fernandez et al (2001) presented a methodology for designing

photobioreactors integrated with tubular loop solar receivers assisted by airlift device for

circulating fluid which was later used to model and develop outdoor PBR. This outdoor 200

L airlift-driven external-loop tubular photobioreactor was tested with the a continuous culture

of Phaeodactylum tricornutum and it was observed that biomass productivity is was higher at

velocities between 0.5-0.35 ms-1 [138]. A two-stage hybrid system was designed and

constructed by Narala et al (2016) in which a portion of rapidly growing cells from the PBR

was transferred to open raceway ponds where the nutrients diminished and algae were

harvested. This result was compared with the separate open pond and closed PBR system,

suggests suggesting that the hybrid system is was superior for the production of lipid-rich

microalgae [139]. However, it is not economically feasible because of the costs associated

with artificial illumination and land area requirementsd.

Fig 7. An outlook of hybrid PBR cultivation system

Table 5. Comparative study of major prospects and challenges of various microalgal cultivation system.

Cultivation technique

Temperature control

S/V ratio

MixingGas exchange

Prospects Challenges

Open ponds

None High Paddle wheel Poor, only achieved through surface aeration

(1) Easy to construct(2) Relatively economical(3) Low energy input(4) Easy maintenance and

cleaning(5) Suitable for mass cultivation

(1) Poor biomass productivity(2) Requires huge areas of land(3) Less control over mixing, light

and CO2 capture(4) Prone to contamination(5) High evaporative losses(6) Limited to few strains of algae(7) Difficulty in cultivating algae for

long periods(8) Lower mass transfer capacity(9) Sensitive to seasonal changes in

temperature and humidityFlat panel PBR

Heat exchange coils

High Airlift/bubble provided at bottom or side

Open gas exchange at head space

(1) Smaller light path(2) Less energy consumption(3) Low operating cost(4) Good for immobilization of

algae(5) Easy to sterilize(6) Less prone to contamination(7) Low dissolved O2

accumulation(8) Suitable for outdoor mass

culture

(1) Scale-up requires many support materials and huge areas of land

(2) Difficult temperature regulation(3) Frequent fouling(4) Clean up issues(5) Small degree of hydrodynamic

stress(6) Some degree of wall growth

(9) Low space requirement(10)High photosynthetic

efficiency Horizontal tube PBR

Water spraying, shading, overlapping

High Recirculation via pumps

Injection intofeed, dedicateddegassingunit

(1) Good biomass yield(2) Suitable for outdoor mass

culture(3) Less prone to contamination(4) Low hydrodynamic stress

(1) Fouling due to algal growth(2) Requires huge areas of land(3) Some degree of wall growth(4) High dissolved O2, and CO2 build-

up(5) High capital and operating costs(6) Susceptible to photoinhibition

Vertical column PBR

None Low Airlift/bubble provided at bottom

Open gas exchange at head space

(1) High mass transfer capacity(2) Relatively homogeneous

culture environment(3) Efficient release of O2 and

residual gas mixture(4) Compact(5) Lak of moving parts(6) Low fouling(7) Good mixing with low shear

stress(8) Easy to sterilize(9) Reduced

photoinhibition/photo-oxidation

(1) High energy consumption(2) Cells are exposed to prolonged

high or low light intensities for a long time

(3) Greater air throughput and high pressure needed

(4) Insufficient in breaking the foam(5) Sophisticated materials are

required for construction(6) High cleaning cost(7) Scale-up will lead to decreases in

illumination surface area(8) Expensive compared to open

pondsPlastic bag PBR

None High Airlift/bubble provided at bottom

Injection intofeed

(1) Low capital cost in the short term

(2) Pump free operation(3) Simple technology and

(1) Photolimitation(2) Bad mixing(3) Prone to contamination during the

escape of excess CO2

easy to handle (4) Clean up issues(5) Frailty to leakage(6) Short life span(7) Increasing formation of biofilm

Table 6. Biomass productivity and nutrients removal reported in some outdoor PBRs

PBRs Culture strainsSource of wastewater

Volume (L)

Light source

Nutrients removal (%) Biomass productivity (mg L-1 d-1)

RefTN DIN NO3

- TP DIP PO43-

Raceway pond

Chlorella saccharophila, Scenedesmus sp.

Dairy farm wastewater

600 Sunlight NA NA 99.4 NA NA 98.8 276±40 [93]

Raceway pond

Scenedesmus sp. Activated sludge wastewater

700 Sunlight 97 NA NA 62 NA NA 7 g m-2 d-1 [3]

Raceway pond

Scenedesmus sp. Activated sludge wastewater

800 Sunlight 98 NA NA 61 NA NA 5 g m-2 d-1 [3]

Raceway pond

Scenedesmus sp. Activated sludge wastewater

850 Sunlight 97 NA NA 56 NA NA 6 g m-2 d-1 [3]

Raceway pond

Scenedesmus obliquus

Urban wastewater

530 Sunlight 65.12

NA NA 58.78

NA NA 8.26 g m-2 d-1 [120]

Raceway pond

Chlorella sp.,Scenedesmus sp.

Piggery wastewater

160 Sunlight NA NA NA NA NA NA 25.03 [121]

Raceway pond

Tetraselmis sp. F/2 Medium NA Sunlight NA NA NA NA NA NA 36±2 [150]

Raceway Chlamydomonas Carpet 500 Sunlight NA NA NA NA NA NA 5.9 g m-2 d-1 [136]

pond globosa, Chlorella minutissima and Scenedesmus bijuga

industry wastewater

Flat panel PBR

Chlorella vulgaris Municipal wastewater

20.3 LED lamps

NA 52.6 NA NA 66.6 NA 10.54±0.77 [140]

Flat panel PBR

Chlorella vulgaris Domestic wastewater

37 LED Lamps

83.91

NA NA 80.98

NA NA 2300±400 [144]

Flat Panel PBR

Chlorella sorokiniana

Internal circulation reactor effluent

0.4 LED lamps

NA NA >99.9 NA NA >95 5.87 [147]

Flat Panel PBR

Chlorella sorokiniana

Dilute human urine + Mg

1 Sodium lamps

87 NA NA 76 NA NA 14800 [148]

Horizontal Tube PBR

Chlorella sp., Scenedesmus sp.

Piggery wastewater

40 Sunlight NA NA NA NA NA NA 11.7 [121]

Horizontal Tube PBR

Tetraselmis sp. F/2 Medium 40 Sunlight NA NA NA NA NA NA 67±5 [150]

Horizontal Tube PBR

Scenedesmus obliquus

Urban wastewater

380 Sunlight 89.68

NA NA 86.71

NA NA 21.7 g m-2 d-1 [120]

Horizontal Tube PBR

Tetraselmis suecica Fish farm 40 Sunlight NA 49.4 NA NA 99 NA 350±30 [122]

Bubble column PBR

Chlorella zofingiensis

Piggery wastewater

1.37 FLUOR lamp

82.7 NA NA 98.17

NA NA 296.16±19.16 [91]

Bubble column PBR

Scenedesmus sp. Domestic wastewater

0.5 FLUOR lamp

NA NA 70.2 NA NA 78.9 61.4±1.8 [141]

Bubble Nannochloropsis Synthetic 1.8 FLUOR NA NA 87 NA NA 18.9 88 [143]

column PBR

oculata wastewater lamp

Bubble column PBR

Chlorella sp. 40% Sludge liquor + Municipal wastewater

NA FLUOR lamp

46.8 NA NA 94.16

NA NA 450 [149]

Bubble column PBR

Chlorella ellipsoidea Horticulture fertlizer

NA FLUOR lamp

36.52

NA NA 88.21

NA NA 470 [149]

Bubble column PBR

Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga

Carpet industry wastewater

100 Sunlight NA NA NA NA NA NA 8.1 g m-2 d-1 [136]

Airlift PBR

Chlorella sorokiniana

Tris Acetate Phosphate medium

1.4 FLUOR lamp

NA NA NA NA NA NA 230 [142]

Airlift PBR

Chlorella pyrenoidosa

Starch processing wastewater

820 Sunlight 57.9 NA NA 89.9 NA NA 342.6±12.8 [132]

Airlift PBR

Chlorella pyrenoidosa

Starch processing wastewater

820 Sunlight 83.06

NA NA 96.97

NA NA 370±50 [146]

Plastic bag PBR

Scenedesmus obliquus

Piggery wastewater

400 FLUOR lamp

74.63

NFA NA 81.73

NA NA 311.2±12.3 [145]

Plastic bag PBR

Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga

Carpet industry wastewater

20 Sunlight NA NA NA NA NA NA 21.1 g m-2 d-1 [136]

FLUOR, fluorescent; TN, total nitrogen; DIN, dissolved inorganic nitrogen; TP, total phosphorus; DIP, dissolved inorganic phosphorus; NA, not available.

5. Conclusion and perspectives

Over the past 60 years, our insight has been enhanced significantly in the area of

environmental, biological and technological aspects of microalgae cultivation. Some of them

are geographical location of cultivation system, solar radiation, investment and operating

cost, contamination, CO2 delivery system, O2 build-up, mixing techniques, energy

consumption, illumination, salinity etc. This has lead to the development of different

cultivation modes and techniques for microalgae cultivation. In cultivation modes,

mixotrophic microalgae cannot just retain CO2 and discharge O2 by photosynthesis, yet

additionally grow with organic carbon, which gives it a promising application in the

environmental treatment of wastewater. Contrasted with the high cost of heterotrophic

cultivation and lower biomass yield of autotrophic cultivation, the mixotrophic cultivation

might be an optimal culture method for microalgae large-scale culture and application.

Amongst cultivation technique, open systems are cheaper to build and operate, but it is

mostly prone to contamination by other microflora and this leads to potential system failures

and also limits the diffusion of CO2 from the atmosphere and requires large area of lands,

whereas closed PBR systems neglect those issues and permits the monoculture growth of

microalgae for extended periods under better-controlled conditions such as pH, temperature,

light, CO2 concentration etc. Even though a great progress is has been made in developing

PBRs, still more efforts are required to advance the PBR techniques. The major concerns

associated with the designing of efficient PBRs is scalingscale-up to pilot scale, with low

energy input and land area, high mass transfer rates, and utilizing maximum solar irradiance.

Moreover, the operating costs, which includescosts, which includes temperature controlling,

periodic cleaning and maintenance are commonly underestimated, and the lifespan of newly

designed PBRs is ignored by many researchers. Hence further research is necessary to

intensify the algal biomass production by optimizing those aforementioned parameters to

prevent eutrophication of water bodies.

Four types of PBRs, including flat panel PBRs, horizontal tube PBRs, vertical PBRs and

plastic bag PBRs, are have been suggested for commercial utilization because of their unique

features. Despite those aforementioned problems, these reactors are mostly preferred for

producing sensitive and highly valuable strains used in the production of nutraceutical,

pharmaceutical and feeds. To date, no perfect type of cultivation technique for the mass

cultivation of microalgae, due to practical difficulties.

It is noteworthy that a new technique called the hybrid PBR, have has proved to be superior

in mass production of algae when compared with the separate open ponds and closed PBR

systems. This type of PBR, exploits the benefits of different reactors and also overcomes the

drawbacks associated with each other, and has been developed in recent years [139]. It is not

possible to assess whether hybrid PBR is preferable because this depends on technical

progress and on the market.

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