Journal of CO₂ Utilization 33 (2019) 384–393

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Journal of CO2 Utilization

journal homepage: www.elsevier.com/locate/jcou

Automation of pilot-scale open raceway pond: A case study of CO2-fed pH T

control on Spirulina biomass, protein and phycocyanin production
Jitendra Mehara,b, Ajam Shekha,b, , Nethravathy M. U.a, R. Saradaa,b, Vikas Singh Chauhana,b,
⁎

Sandeep Mudliara,b,
⁎

a
Plant Cell Biotechnology Department, CSIR- Central Food Technological Research Institute, Mysuru, 570020, Karnataka, India
b
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

ARTICLE INFO ABSTRACT

Keywords: Spirulina is known to produce high protein and phycocyanin of commercial importance with concomitant CO2
Spirulina utilisation. The pH is one of the most important factors for cell growth, CO2 utilisation and controlling con-
Automation tamination. In this study, an indigenously developed automated system for Spirulina cultivation was used at
CO2 pilot scale, where CO2 supply was aimed to provide carbon source and to control culture pH. Among all the
Phycocyanin
studied pH (7.5 ± 0.1,8.5 ± 0.1, 9.5 ± 0.1), pH 8.5 was found to be the best where biomass productivity, CO2
Open raceway pond
bio-fixation rate, protein and phycocyanin content were noted as 72 mg L−1 d-1, 0.13 g L-1 d-1, 64%, and 14%,
respectively. Though the culture at pH 9.5 experienced late CO2 feeding, it was healthy and showed almost
similar metabolite content compared to pH 8.5. The pilot scale automation system developed here can be em-
ployed to enhance CO2 fed pH control based performance of Spirulina or any microalgae cultivation.

1. Introduction
Spirulina is considered as one of the best sources of commercially
important renewable feedstock for the production of single cell protein
and other high-value metabolites mainly phycocyanin (C-PC). It is
widely in use for malnourishment eradication owing to its high nu-
tritive value through high quality proteins, carbohydrates, lipids, pig-
ments, vitamins, minerals and antioxidants. Therefore, apart from the
commercial production of Spirulina by various well known industries,
Spirulina cultivation has penetrated into the rural areas of various de-
veloped as well as developing countries [1]. Open cultivation of Spir-
ulina using raceway ponds is most widely adapted cultivation system.
Traditionally, Spirulina is cultivated using chemical source of inorganic
carbon mainly sodium bicarbonate (NaHCO3), and still its use is very
common by various Spirulina cultivator’s [2]. However, the environ-
mental concerns of our period demands research efforts towards de-
veloping new and sustainable advancements for Spirulina production.
Due to the challenges associated with increasing concentrations of CO2
into the atmosphere, Spirulina can be at the centre-stage for replacing
chemical carbon source (like NaHCO3) with gaseous CO2. Since Spir-
ulina are the sunlight-driven cell factories that can convert CO2 into the
biomass rich in high-value metabolites, it remains one of the better
tools for beneficial utilisation of CO2. CO2 utilisation potential of
Spirulina had raised attention around the world owing to its efficient
CO2 removal rate [3,4]. Commercial value of Spirulina biomass and
phycocyanin is quite high as it has wide applications in dietary and
health supplements, pharmaceuticals, cosmeceuticals, natural color-
ants, and diagnosis reagents [5]. The yields of Spirulina biomass in ra-
ceway ponds is influenced by the factors such as temperature, light
intensity, pH, and other on-site weather conditions. The pH is one of the
most important factors for cell growth, CO2 utilisation and prevention
of contamination. The undesirable change in pH of cultivation medium
disturbs the equilibrium between gaseous CO2, and other dissolved
inorganic species (HCO3−, CO32−) in the medium, affects nutrient
availability, and hampers the photosynthesis as well as metabolism in
microalgae. The pH in alkaline range leads to enhanced CO2 mass
transfer resulting in high HCO3− ions, promoting rapid growth of
Spirulina. However, it is also known that the high concentrations of CO2
impedes the accumulation of phycobiliproteins and other pigments
including phycocyanin [6]. Therefore, the Spirulina cultivation needs to
be done at an optimum pH in order to maintain unialgal culture, tackle
predators attack, and obtain high biomass without compromising the
quality. However, the conventional Spirulina cultivation is done
without the control over pH of the cultivation medium. In some cases,
the pH is maintained with the addition of acid (HCl) or alkali (NaOH),
which may not only cause the additional waste of chemicals but may

⁎
Corresponding authors at: Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysuru, 570020, Karnataka, India.
E-mail addresses: azamsheikh24@gmail.com (A. Shekh), sn.mudliar@gmail.com (S. Mudliar).

https://doi.org/10.1016/j.jcou.2019.07.006
Received 13 March 2019; Received in revised form 10 May 2019; Accepted 10 July 2019
2212-9820/ © 2019 Published by Elsevier Ltd.
J. Mehar, et al.
also damage the cells. Therefore, feeding CO2 into the open ponds for
inorganic carbon supplementation (as a sole source of carbon for en-
ergy) with simultaneous mean of controlling pH remains one of the best
options for Spirulina cultivation. Therefore considering the need of the
time, in this study, we have attempted to address the bottlenecks as-
sociated with the use of chemical carbon species by replacing it with
gaseous CO2, simultaneously optimising the desired pH of the culture
through CO2 supplementation, and automating the Spirulina cultivation
by developing automation system for CO2 feeding and online mon-
itoring of the culture parameters at pilot scale operations.
2. Materials and methods
2.1. Microalgae strain, culture medium and seed culture development
Spirulina (Arthrospira) platensis available at CSIR-CFTRI culture bank
was used for this study. A stock culture of Spirulina was inoculated into
a 1 L autoclaved modified Zarrouk’s medium using a 2 L Erlenmeyer
flask and incubated at room temperature (25 ± 2 °C) under
50 μmol m−2 s−1 continuous cool-white fluorescent light. The slightly
modified Zarrouk's medium (per litre) consisted of 10 g of NaHCO3,
0.5 g of K2HPO4, 2.5 g of NaNO3, 1.0 g of K2SO4, 1.0 g of NaCl, 0.2 g of
MgSO4.7H2O, 0.04 g CaCl2.2H2O, 0.01 g of FeSO4.7H2O, 0.08 g of
Na2EDTA, and 1 ml of trace metal solution (2.8 g of H3BO3, 1.8 g of
MnCl2.4H2O, 0.2 g of ZnSO4.7H2O, 0.074 g of CuSO4.5H2O, and 0.015 g
of MoO3, per litre). For the ease of understanding and data re-
presentation, this modified nutrient medium in the present study is
considered as Zarrouk's medium (ZM). The NaHCO3, NaNO3,
MgSO4.7H2O, and NaCl were of commercial grade and all the other
chemicals used were of analytical grade (Merck, Germany). Following
the exponential growth of the culture and before the development of
stationary growth phase, the seed culture for experiments were se-
quentially developed by scaling up of the culture from 1 L to 4 L. The
culture was then propagated in carboy’s to scale up to 20 L at outdoor
conditions. The culture was acclimatised with supplementation of pure
CO2 (99.99% purity) as a sole source of carbon and scaled up to 360 L
using the seed culture development open raceway ponds available at
the pilot scale Spirulina cultivation facility. Spirulina growth was
monitored daily by measuring the optical density (O.D.) at 560 nm.
2.2. Design, development, and working principle of the automation system
for Spirulina cultivation in pilot scale open raceway pond
As an advancement to the traditional method of Spirulina cultiva-
tion and to address the associated bottlenecks, the automation of
Spirulina cultivation process was done at pilot scale operations in open
raceway pond of capacity 1400 L. An indigenous system for automation
of the Spirulina raceway pond has been conceptualised, designed,
fabricated and installed. The design philosophy of the process auto-
mation includes online measurement and data logging for pH, tem-
perature, light, dissolved oxygen (DO), and agitation. Most of the
Spirulina cultivators record all these measurements manually which is
very laborious and time consuming. In this system, four different sen-
sors were connected to data logger for the measurement of pH, DO,
temperature, and light intensity. The sensors for pH, DO and tem-
perature were submerged in the culture. The main feature of the system
involves control for pH of the cultivation medium via CO2 supple-
mentation at various concentrations and flow rates. The automation
system is also equipped with a CO2 supply unit which operates by
purging CO2 based on the pH set points, a gas mixing system with air
(generated by air compressor) to control/manage the desired CO2
concentration, and the control of gas flow rate (using rotameters). Apart
from CO2 based pH control, conventional acid/alkali based pH control
system was also added as an optional/additional feature. Furthermore,
to conquer the mass transfer limitations associated with gaseous CO2,
the membrane sparger has been provided at the bottom of the culture
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Journal of CO₂ Utilization 33 (2019) 384–393
(10 cm from the pond surface) to enable enhanced CO2 mass transfer in
the growth medium. The membrane sparger helps in better CO2 dis-
solution and avoids CO2 losses. The CO2 feeding system consisted of a
CO2 gas cylinder, a CO2 gas regulator, a gas flow meter (0–20 L/min
range), a solenoid valve, and a membrane sparger. Finally, the data
logger is connected to the automation system that has potential to re-
cord the data of each variable at every one minute’s time interval. It
also has a GSM connectivity through which the pond operators/su-
pervisors mobile phone numbers can be used/connected to receive the
real time data of the set variables, in case the pond operator is not on
the site for any reason.
2.3. Spirulina culture performance at various pH through CO2 fed pH
regulation
The effect of various pH on Spirulina cultivation at pilot scale was
studied using an automated system equipped with CO2 fed pH control.
Spirulina was cultivated at three different pH mainly- pH 7.5 ± 0.1,
pH 8.5 ± 0.1, and pH 9.5 ± 0.1. In this process, the chemical source
of inorganic carbon (NaHCO3) from ZM was completely replaced by
commercially available food grade gaseous CO2 (99.99%). The desired
pH was maintained with CO2 supplementation from CO2 feeding unit of
the automation system. While maintaining the desired pH, when the pH
of the culture rises above the set value, the pH controller opens the
solenoid valve and CO2 is sparged into the culture at a set flow rate
through CO2 sparger till the set pH is achieved. Upon achieving the set
pH value, the solenoid valve of the pH controller gets closed auto-
matically. In these studies, the pH of the culture was monitored and
controlled continuously by the pH control unit of the automation
system. For measurement accuracy, the pH probe of the system was
calibrated every alternate day with standard pH solutions (Sigma
Aldrich) of pH 4, pH 7 and pH 10. In addition, Spirulina culture per-
formance was also studied in other cultivation conditions- (i) ZM (With
NaHCO3; No CO2; No pH control) and (ii) ZM + CO2 (ZM; Without
NaHCO3; With continuous CO2 feeding; No pH control) to compare the
results of Spirulina cultivated at controlled pH via CO2 feeding.
2.4. Experimental conditions
Spirulina cultivation using a pilot scale automated open raceway
pond was carried out to study the effect of various pH (7.5 ± 0.1, pH
8.5 ± 0.1, and pH 9.5 ± 0.1) on overall culture performance. The
study was conducted in the month of March to June, 2018 using open
raceway ponds facility for microalgae cultivation established at CSIR-
CFTRI. Composition of the medium used during these experiments was
similar to the slightly modified ZM mentioned earlier in the literature
except NaHCO3 was replaced by CO2 as a sole source of carbon. The tap
water available at CSIR-CFTRI was used to prepare growth medium
(1400 L). The culture depth was maintained at 20 cm. The CO2 accli-
matised seed culture (in its logarithmic growth phase) developed in
outdoor small scale open raceway pond (used as seed culture devel-
opment pond for this experiment) was used as inoculum. The initial
OD560 of the culture was set at 0.2 in the final working volume of
1400 L. The raceway pond culture was mixed with a paddle wheel at
12 rpm to achieve an average culture flow velocity of about 25–30 cm/
s. The real time culture temperature, DO, and pH were measured by
submerged sensors of automation system and the data was recorded in a
data logger. The regulation and maintenance of the set pH of the culture
was done via submerged pH probe, pH controller through the real-time
monitoring of pH and control of the electromagnetic valves. The pH of
the culture was regulated and maintained at the set pH (either pH
7.5 ± 0.1, pH 8.5 ± 0.1, or pH 9.5 ± 0.1) by feeding pure food grade
gaseous CO2 only. The CO2 was injected through the membrane sparger
(length- 2.2 m, diameter- 0.8 cm, pore size- 0.05 mm) with flow rates
measured and regulated to 5 lpm by gas flow meters connected with an
electromagnetic valve. The electromagnetic valves were turned on
J. Mehar, et al.
automatically when the pH values reached the upper limit of the set
range. Once the pH value of the system was reduced to the lower limit
of the range under the action of injected CO2, the electromagnetic valve
was turned off. The culture depth/level of 20 cm was maintained by the
addition of water to compensate for evaporative losses. Samples were
collected daily at 10.00 a.m. throughout a period of an experiment for
monitoring the culture performance through various analysis such as
growth, nitrate, phosphate and alkalinity. To ensure the representative
sampling, the samples were collected from the well mixed zones of the
pond. The experiments were continued till the culture OD560 reached 1,
since most of the commercial cultivators are known to (partially/
completely) harvest the biomass at this OD. Spirulina culture was
checked for bacterial load after every 48 h throughout the cultivation
period. The standard plate count was performed to estimate the number
of viable bacteria and the results being reported as colony forming units
per ml (CFU/ml).
2.5. Cell growth, biomass quantification and ash content dermination
Cell growth was monitored by measuring the optical density (OD) at
560 nm using UV–vis spectrophotometer (Shimadzu, UV-1800). The
standard calibration curve of OD560 verses concentration of cells
(mg L−1) was plotted. For this, Spirulina culture suspensions (in tri-
plicates) of varying OD have been prepared. Then, same volume of each
suspension was filtered through pre-weighed 0.45 μm, GF/ C glass fibre
filters (Whatman, USA) and dried overnight at 60 °C. Subsequently, the
biomass concentration of the corresponding samples of varying OD was
gravimetrically calculated based on the change in filters weight, and the
dry weight (mg L−1) was noted. To analyse the ash in Spirulina bio-
mass, about 3 g of the lyophilized biomass was dried and corrected for
the amount of moisture present in the sample using an automated
moisture analyser (Sartorious, MA160). Further, the total ash content
was determined by incineration of the sample received from the
moisture analyser using a muffle furnace at 525 °C for 6–8 h [7].
2.6. Biomass harvesting, lyophilisation, and storage
After the completion of the experiment, the biomass was harvested
by filtration. The 1 mm opening size sieve was used as a first step to
take out any possible debris or foreign material from the culture. The
pre-filtered Spirulina culture was then filtered in continuation using a
nylon cloth (100 μm mesh size) to get the wet biomass. The harvested
wet biomass was washed with mild HCl to remove the salts. Further, the
biomass was freeze-dried over the time period of 18 h using a lyo-
philizer operated with vacuumed pressure and condenser temperature
of 0.34 mbar and −55 °C, respectively. The dried and powdered bio-
mass was stored at 4 °C prior to its use for further analysis.
2.7. Biomass productivity, proteins, carbohydrates, lipids and phycocyanin
2.7.1. Biomass productivity
(P , mg L−1 d-1) of Spirulina was calculated from the change in
biomass concentration ( X , mg L-1) according to the following equation:
X
P= t
X0
(t t0 )
Where, X0 is the initial biomass concentration at time t 0 and Xt is the
biomass concentration at any time t .
2.7.2. Total protein
The lyophilised biomass (30 mg) was used for the estimation of total
nitrogen content using the protein analyzer (Thermo Flash 2000).
Further, the total protein content of the biomass was determined using
the following formula [8]:
Total protein (%) = Total nitrogen (%) × 6.25
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Journal of CO₂ Utilization 33 (2019) 384–393
2.7.3. Total carbohydrate
Total carbohydrate content was determined using the Phenol sul-
furic acid method with glucose as a standard. The known amount of
lyophilised biomass was dissolved in distilled water (w/v) and digested
with 5 ml of 2.5 M HCl in boiling water bath for 20 min. The hydrolysed
biomass was cooled and neutralized with anhydrous Na2CO3 to remove
effervescence and diluted for final quantification (10 ml). To the diluted
solution (0.2–1.0 ml), 1 ml of 5% phenol and 5 ml of 96% H2SO4 was
added and the developed colour was measured at 490 nm [9].
2.7.4. Total lipid
Slightly modified Bligh and Dyer method was used for total lipid
extraction [10]. 100 mg of lyophilized Spirulina biomass was dissolved
in chloroform: methanol: water in the ratio of 10:10:9. The samples
were vortexed and kept overnight on the shaker followed by cen-
trifugation at 5000 g for 5 min. The bottom layer of lipids dissolved in
chloroform was then removed using syringe and filtered through 0.2 μm
Whatmann PVDF filter in a pre-weighed glass vial (W1). To quantify
lipids, chloroform was then evaporated and the glass vials were re-
weighed (W2). The difference in weight of the glass vial (W3 = W2−
W1) was used to calculate the percent lipid content considering the
amount of biomass used for the extraction.
2.7.5. Phycocyanin
For the quantitative estimation of crude phycocyanin (Cp), 20 mg of
lyophilised biomass was added in 20 ml of 100 mM phosphate buffer
(w/v) of pH 7. It was then mixed properly and stored overnight in a
refrigerator at 4 °C. Next day, the suspension was centrifuged and the
absorbance of a supernatant was measured at 615 nm and 652 nm [5].
The phycocyanin extract was quantified using the equation defined by
Bennett and Bogorad [11] as follows:
Cp = A615 − (0.474 × A652) / 5.34
Where; Cp = Crude phycocyanin concentration (g L−1);
A615=Absorbance of the extract at 615 nm; and A652=Absorbance of
the extract at 652 nm.
2.8. Elemental composition (C, H, N, S and O content) analysis of biomass
The lyophilised biomass was weighed in the range of 5–10 mg and
analysed for the quantification of carbon (C), hydrogen (H), nitrogen
(N), and sulphur (S) content using a CHNSO analyser (ELIII, Vario,
Germany).
2.9. Analysis of carbon dioxide (CO2) bio-fixation rate
The inorganic carbon (IC) utilization (as equivalent CO2 bio-fixa-
tion) rate RCO2 (g L−1 d-1) was calculated using the following equation
[12].
MCO2
RCO2 = Cc P ( )
Mc (1)
Where, Cc denotes carbon content (% dry cell weight) of Spirulina
biomass, P symbolizes biomass productivity. MCO2 and Mc . Where, the
molecular weights of CO2 and carbon, respectively.
2.10. Measurement of alkalinity, NO3- and PO42−
To determine the concentration of Alkalinity, NO3- and PO42-; the
representative sample of the culture medium collected and centrifuged
at 5000 rpm for 10 min and the supernatant was used for analysis of
Alkalinity, NO3- and PO42-. The alkalinity was determined by titrating
the sample against standard acid as per the standard method protocols
[13].
J. Mehar, et al.
The alkalinity of the culture medium was measured daily to de-
termine the concentration of dissolved inorganic carbon (HCO3−, CO32-
). Also, the concentration of NO3- and PO42- were analysed daily
throughout the cultivation period to determine the NO3- and PO42-
utilization pattern of Spirulina and nutrient sufficiency during the
cultivation period. The concentration of NO3--N was determined by
measuring the absorbance at 220 nm and 275 nm using UV–vis spec-
trometry [14]. The concentration of PO42--P was determined by the
Vanadomolybdophosphoric acid colorimetric method [14].
3. Results and discussion
3.1. Effect of various pH regulated by CO2 feeding on Spirulina biomass
productivity
The pH of culture medium plays a significant role in defining the
microalgae cell behaviour during cultivation. Dearth in understanding
the uptake and secretion of chemical species has confounded under-
standing of the stoichiometry of photosynthetic growth of algae [15].
Therefore, it is challenging to achieve high growth of Spirulina which
otherwise can improve biomass productivity and economic feasibility of
commercial systems like open raceway ponds. In this study, Spirulina
has shown growth and produced biomass at all three different pH (pH-
7.5; 8.5; 9.5) studied for cultivation. The biomass productivity of
Spirulina was found to be in the range of 49–76 mg L−1 d-1 (Table 1).
The biomass production at these CO2 fed conditions was also compared
with ZM without CO2 supplementation and pH control. The biomass
productivity at ZM was noted to be 76 mg L−1 d-1 and was maximum
when compared to all cultivation conditions. Among all three studied
pH, the maximum biomass productivity of 72 mg L−1 d−1 was achieved
at pH 8.5, which was 14% higher than pH 9.5 and 5.5% lesser than ZM
(Table 1). The biomass productivity is also a function of carbon utili-
sation from the nutrient media. HCO3- is the most favoured inorganic
carbon species that are utilised by Spirulina. In media, the dissolved
CO2 remains in equilibrium with H2CO3, HCO3– and CO32–. Con-
centration of these species mainly depends on pH. Due to rapid trans-
formation/interchange between these species, the use of any of these
inorganic carbon species by microalgae does not affect their equili-
brium. In fact, Spirulina cells prefer to utilise HCO3– rather than CO2 in
its native form. The maximum biomass productivity obtained with ZM
as compared to the tested pH conditions can be attributed to the highest
HCO3- alkalinity observed at this condition. The high HCO3- alkalinity
with ZM can be attributed to easy dissociation of NaHCO3 into HCO3-
compared to the hydration of CO2 when purged to maintain defined pH
conditions (at pH-7.5, 8.5 and 9.5). The trend of biomass productivity
obtained is directly correlated to the trend of available HCO3- alkali-
nity. The increase in biomass productivity is characterised by the
availability of HCO3- which is discussed in detail under Section 2.1.
However, in case of pH 9.5 (62 mg L−1 d-1), it took 6 days for the
culture to attain pH above 9.5 so that the feeding of CO2 could be in-
itiated. The slow rise in pH in this case was an indication of the slow-
ness in the growth and photosynthetic activity which can be attributed
to the non-availability of externally added chemical carbon (NaHCO3)
source. The culture was solely dependent on the ambient CO2 for the
Table 1
Biomass productivity (mg L−1 d−1), carbon content (%) and CO2 bio-fixation rate (g L-1 d-1) of Spirulina at various cultivation conditions.
Cultivation conditions Biomass productivity
(mg L−1 d-1)
pH 7.5 ± 0.1 (+ CO2) 49 ± 2.45
pH 8.5 ± 0.1 (+ CO2) 72 ± 2.88
pH 9.5 ± 0.1 (+ CO2) 62 ± 1.86
ZM (- CO2; No pH control) 76 ± 2.66
ZM (+ CO2; No pH control) 61 ± 2.44
(All pH conditions were maintained with automated CO2 injection; “+” indicates CO2 supply and “–” indicates no CO2 supply).
387
Journal of CO₂ Utilization 33 (2019) 384–393
growth till it reached above the set pH of 9.5. Further, once the CO2
feeding started to maintain the pH at 9.5 as well as to supplement the
carbon source, increase in biomass productivity was observed. On the
other hand, when compared to pH 8.5, the biomass productivity at pH
7.5 was marked with remarkable decrease of 32%. The least biomass
productivity at pH 7.5 was because of the poor culture health due to
contamination by undesirable local microalgae species (2–8 μm in
diameters) and by organisms that graze upon both foreign algae and
Spirulina. At this pH, the culture appeared extremely susceptible to
contamination and was unable to maintain its unialgal status. The
grazers & contaminants generally reported during cultivation in the
open ponds include protozoans (ciliates, amoeba, etc.), mosquito
larvae, viruses, fungi, and bacteria. In large-scale culture, especially in
open ponds, there is a greater susceptibility to bacterial and other algal
contamination in cultures maintained at near neutral pH. The results of
the standard plate count showed the higher bacterial load in Spirulina
culture at pH 7.5 (4.6 × 105 CFU/ml). However, the bacterial load at
pH 8.5 and pH 9.5 was noted to be in the range of 4–6 × 103 CFU/ml,
which is generally considered as satisfactory in food products.
The pH 6.5 and 10.5 were omitted for this study considering the fact
that Spirulina does not survive at pH 6.5 due to acidic conditions.
Whereas, at pH 10.5, CO2 feeding might not have been possible re-
sulting in poor productivities due to limited CO2 availability. Various
researchers have used Spirulina for CO2 utilisation and biomass pro-
duction in varied cultivation systems which includes close PBRs and
open raceway ponds. In fact, recently Chen et al have studied Spirulina
biomass production at three different pH of 8, 9 and 10 with CO2 fed pH
control in a laboratory scale 1 L closed PBR. At these pH, with CO2
supplementation in a closed PBR, the biomass productivities were
above 0.8 g L−1 d-1 [6]. As such these results could not be compared
with the current study considering the varied cultivation system and the
scale of operation used in that study. However, their trend of biomass
productivity which was found almost similar at pH 9 and 9.5 whereas
decreased at pH 10 can be fairly extrapolated to the current study. On
the other hand, our biomass productivities were higher than the report
of Lu et al. (41.35–58.38 mg L−1 d-1) who used aerobic composting
exhaust as a carbon source [16]. In addition, our findings were also in
corroboration with the results of Duarte et al who reported the com-
parable biomass productivities (80 mg L−1 d-1) for CO2 fed culture and
standard Zarrouk’s medium [17]. Similar results of biomass pro-
ductivity were also reported by Radmann et al. [18].
Varied biomass productivities at different pH can be attributed to
the fact that changes in the pH of the medium are also known to alter
the ionisation of nutrient molecule as well as chemical species, which
may limit their intracellular availability. On the other hand, in case of
Spirulina or in general for microalgae, when the external pH is acidic;
maintaining an alkaline cytosolic pH is the major challenge to these
organisms. This results in membrane disruption, inhibition of the ac-
tivities of various enzymes and transport proteins involved in carbon
capture and concentration. In the present investigation, Spirulina cul-
ture behaviour was studied at three different pH (pH 7.5 ± 0.1, pH
8.5 ± 0.1, and pH 9.5 ± 0.1) maintained through CO2 supplementa-
tion. Since CO2 was the sole source of carbon, it is worth understanding
that the equilibrium amongst various species of inorganic carbon like
Carbon content (%) CO2 bio-fixation rate (g L−1 d-1)
43.50 ± 0.42 0.08 ± 0.003
50.82 ± 0.58 0.13 ± 0.006
49.71 ± 0.61 0.11 ± 0.004
51.20 ± 0.32 0.14 ± 0.006
46.75 ± 0.88 0.10 ± 0.004
J. Mehar, et al. Journal of CO₂ Utilization 33 (2019) 384–393

gaseous CO2, HCO3− and CO32− gets affected with the change in pH of
the medium. It is understood that the HCO3− ions dominate in the pH
range of 8–9 and CO32- remains in a minimum presence. On the other
hand, CO32− dominates above pH 10 and the HCO3− are known to be
negligible. HCO3− are the most favoured inorganic carbon species for
the transport across the cell membrane. Since it is dominated at the
near alkaline pH range (8–9) and infact being the maximum at pH 8.5,
the biomass productivity was maximum at this pH. It is also corrobo-
rated by the fact that the intracellular and transmembraneous carbonic
anhydrases work most efficiently at this pH. These enzymes are known
to be the fastest enzymes in the universe responsible for the reversible
conversion of bicarbonates to CO2 with the Kcat value of 1.4 × 104 M/
sec [19]. The bicarbonate ions are converted into the CO2 molecules
and are made available to RuBiSCo to fix it into the energy compounds
of various classes [20]. The reported pH range for the optimum CA
activity is 8–9 [20]. Altogether, these facts support the maximum bio-
mass productivity obtained at pH 8.5 and the decreased biomass pro-
duction at pH 7.5 in this and other reports across the literature. The ash
content of Spirulina biomass cultivated at different cultivation condi-
tion was observed in the range of 7.6–13.4%. The ash content of bio-
mass was found significantly lower when CO2 was used as a sole carbon
source as a replacement of NaHCO3. However, a contamination prone
unhealthy culture at pH 7.5 (ash, 13.40%) was noted with a remarkable
increase of 73.12% and 34.02% ash when compared to the ash content
of biomass at pH 8.5 and ZM, respectively (Table 2). The high per-
centage of ash can be related to the presence of salts on the surface of
the dry biomass [21], which may be due to poor health, contamination
and inadequate assimilation of inorganic salts. High ash content in
Spirulina is generally undesirable because safe limits of trace and heavy
metals should not exceeded. However, algal ash can contribute to
meeting the recommended daily intake of minerals in human nutrition
[22].
3.2. Protein, carbohydrate, lipid, phycocyanin content of Spirulina biomass
The concentration of metabolites in microalgae biomass is influ-
enced by environmental and cultivation factors including pH.
Microalgae store their chemical energy in the form of carbohydrates,
lipids, and proteins, according to the metabolic strategy used by the
microalgae to acclimatize to the conditions [23]. Spirulina is reported
to produce 40–70% of high quality protein that may contain all the
essential amino acids. Spirulina proteins due to the presence of essential
amino acids at the quantities reported by FAO/WHO, are being used to
feed children to tackle malnourishment. Protein, carbohydrate, lipid,
and phycocyanin content of Spirulina biomass obtained in this study is
provided in Fig. 6. The total protein content of Spirulina biomass at all
cultivation conditions was found to be in the range of 52–65% (Fig. 6).
The maximum protein content of 65% was found at the standard cul-
tivation condition (ZM) which was comparable with the protein content
(64%) observed at pH 8.5. The protein content of 64% at pH 8.5 was
maximum amongst all the studied pH conditions. Also, no significant
difference in protein content was observed at pH 9.5 when compared to
pH 8.5. This may be attributed to quick adaptation of culture (at pH
9.5) to its normal metabolism/photosynthetic behaviour post CO2
purging. In contrast, a contamination prone unhealthy culture at pH 7.5
was noted with remarkable decrease of 18.75% protein when compared
to the yield at pH 8.5. Our results are substantiated by reported protein
content in the range of 55–65% by earlier studies [16,24]. Similarly, the
total carbohydrate content has also showed the comparable trend like
protein accumulation in biomass at these pH conditions. The total
carbohydrate content was ranged in 13–18%, with a maximum of 18%
observed at both pH 8.5 and pH 9.5. This was comparable to the car-
bohydrate content obtained with ZM. The noted decrease in carbohy-
drate accumulation at pH 7.5 was 27.77%, with total carbohydrate
content of 13%. The carbohydrate contents in this study were com-
parable with the earlier report [25]. The total lipid content of Spirulina
biomass was found to be in the range of 6.9–9%. The lipid contents at
pH 8.5 and standard cultivation condition (with ZM) were comparable.
The other two pH conditions were noted to accumulate 8.3% (pH 9.5)
and 6.9% (pH 7.5) of lipid. The lipids of Spirulina are known to have
nutraceutical importance due to the presence of gamma-linoleic acid.
Most importantly, Spirulina is widely cultivated to produce C-phyco-
cyanin (C-PC) which it accumulates in the range of 10–15% [26]. C-PC
plays a major role as a light harvesting pigment, exhibit various
bioactive functions, and has extensive commercial importance in nu-
traceutical and pharmaceutical industry [27]. In this study, the C-PC
content of Spirulina cultivated at various cultivation conditions was
observed in the range of 10–14% (Fig. 6). The maximum C-PC content
was noted at standard cultivation condition (with ZM) (Fig. 6). When
compared with the C-PC content at the various pH conditions, the
maximum of 14% C-PC was obtained at pH 8.5, followed by pH 9.5
which was characterised with 13% C-PC. Again, the minimum C-PC
content of 10% was observed at pH 7.5. These results were in agree-
ment with the findings of Chen et al who reported 14% C-PC under CO2
supplementation. In another study, the C-PC content of 17.5% was re-
ported at pH 9–10, which is 34.61% more than the current findings
which may be due to the controlled cultivation in lab scale closed PBR
[6]. With the objective to study the and compare the Spirulina perfor-
mance at various pH, the better performance of Spirulina culture with
respect to biomass and metabolites production at pH 8.5 and 9.5 can be
explained by the fact that the higher pH is associated with higher the
CO2 gradient under a given alkalinity as well as the possible con-
tribution of free CO2 from the atmosphere. Compare to pH 7.5; the
other pH on the higher alkaline side had advantage of cultivation
performance without affecting the algal growth. In addition, at these
pH, the culture medium exhibited the self-defence mechanism to pre-
vent the possible contamination, and maintained its monoalgal status. It
is reported that Spirulina can be cultivated at a wide range of pH be-
tween pH 9–10.5 [6]. However, on contrary to the current study where
the CO2 feeding strategy was used, other studies were carried out with
NaHCO3 as a carbon source. The pH range of 8.5–9.5 where the Spir-
ulina performance was almost similar (except biomass production) and

Table 2
Ash content of Spirulina biomass obtained from various cultivation conditions and its comparison with reported literature.
Sr. No. Cultivation Condition and Working Volume Carbon Source Biomass Ash (%) References

1 Outdoor; open raceway tank; 240 L 16.8 g NaHCO3 19.35 ± 0.06 [11]
2 Outdoor; open raceway pond; 500–5000 L 9 g NaHCO3 10.0 [33]
3 Indoor; vertical tubular PBR; 2 L 16.8 g NaHCO3 22.7 ± 0.97 [34]
10% (v/v) CO2; No NaHCO3 13.8 ± 0.92
4 Indoor; vertical glass column; 9 L 1.2 % CO2 (No NaHCO3; pH maintained at 8.5–9.5) 8.52 ± 0.27 [35]
Outdoor; open raceway tank; > 5000 L 1.2% CO2; (No NaHCO3; pH maintained at 8.5–9.5) 9.83 ± 0.06
5 Outdoor; automated open raceway pond; 1400 L pH 7.5 ± 0.1 (+ CO2; No NaHCO3) 13.40 ± 0.66 Current study
pH 8.5 ± 0.1 (+ CO2; No NaHCO3) 7.74 ± 0.39
pH 9.5 ± 0.1 (+ CO2; No NaHCO3) 7.61 ± 0.38
ZM (10 g NaHCO3; No pH control) 9.98 ± 0.5
ZM + CO2 (Continuous CO2; No pH control) 7.26 ± 0.36

388
J. Mehar, et al.
Fig. 1. Sketch of the automated open raceway pond with details of control panel and CO2 feeding system.
better than pH 7.5 is characterised by the ample availability of in-
organic carbon species to assimilate by the cells. On the other hand, the
culture at pH 7.5 was more prone to contamination by the other uni-
cellular green microalgae. This cross-contamination could have invited
predators attack. This culture was also characterised with slow growth,
unnatural colour and typical odour. The overall decrease in photo-
synthetic and metabolic activity at this pH has led the under-perfor-
mance of this culture with respect to biomass and other metabolites
production. Since, the time required for the culture devoid of any
carbon source to reach pH 9.5 was more before the CO2 could be
purged, the studies with initial supplementation of NaHCO3 to quickly
attend pH 9.5 and then maintain with CO2 can be done to further im-
prove the culture performance.
3.3. CO2 bio-fixation rate
The CO2 bio-fixation rate was theoretically calculated using Eq. (1).
The estimated carbon content in the biomass obtained at various cul-
tivation conditions varied in the range of 43.5–51.20%. This was higher
than the assumptions of Braga et al who assumed 40% carbon in
Spirulina biomass composition for the calculations of CO2 bio-fixation
rate [28]. Though the molecular formula of microalgae biomass
(CO0.48H1.83N0.11P0.01) allows the calculation of the bio-fixation rate
(RCO2) using equation RCO2 = 1.88×biomass productivity [28], cal-
culations done in this study with actual carbon content leads to more
precise results. The CO2 bio-fixation rates for various cultivation con-
ditions were noted to be 0.08–0.14 g L−1 d-1 which are in agreement
with the previous study [28]. In the current study, following the trends
of biomass production, the best CO2 fixation rate among all cultivation
conditions was observed at ZM (No CO2, No pH control) which could be
corroborated to the highest carbon content at this condition (51.20%).
When compared to the various pH conditions, the best CO2 fixation was
observed at pH 8.5 which was calculated to be 0.13 g L−1 d−1
(Table 1). The CO2 fixation rates at ZM and pH 8.5 were almost com-
parable. The better CO2 fixation rates at these conditions may be at-
tributed to the availability of sufficient amount of inorganic carbon for
assimilation into biomass.
389
Journal of CO₂ Utilization 33 (2019) 384–393
3.4. Alkalinity, NO3− and PO42−
The alkalinity of the culture medium, which is a function of pH,
plays an important role in biomass production and defining the overall
biochemical composition and productivity of microalgae. In the present
study, due to the absence of any other inorganic carbon source except
CO2, the alkalinity in the culture medium was purely due to the con-
tribution of dissolved HCO3− and CO32- species. The maximum HCO3−
alkalinity was noted at ZM (Fig. 3C). The HCO3− alkalinity at ZM can
be attributed to easy dissociation of NaHCO3 into media to form
HCO3−. At pH 8.5, the HCO3− alkalinity was found to be in the range
of 1040–1100 ppm, (Fig. 5B). Similarly, pH 7.5 was noted to have the
alkalinity in the range of 590–605 ppm (Fig. 5A). However, the alka-
linity gradually decreased from1425 ppm to around 850 ppm, and
maintained in the range of 840–890 ppm at pH 9.5 (Fig. 5C). This de-
crease was due to shift in the equilibrium of dissolved inorganic carbon
species with the gradual increase in pH to 9.5 during the initial days of
cultivation at this condition. The contribution of CO32- to total alkali-
nity gradually increased (340 ppm to 890 ppm) with an increase in pH
from 8.5 to 9.5. On the other hand, the HCO3− alkalinity decreased
gradually for the cultures grown with ZM (5100 ppm to 0 ppm) and the
CO32- alkalinity increased from 1900 ppm to 3950 ppm (Fig. 3C). This
was also marked to the change in slightly alkaline condition to more
alkaline conditions with increase in pH of the culture. The contribution
of CO32- towards alkalinity at pH 7.5 and in continuous CO2 purging
cultivation was nil. The comparatively better performance of the Spir-
ulina culture at pH 8.5 can be attributed to the conducive physiolo-
gical/nutritional environment due to the interdependence of pH and
alkalinity. This pH condition was marked with sufficient and constant
availability (more than other conditions) of HCO3− ions which are
preferred over CO32- and are readily taken up by the cells through CCM
components and various transport channels. However, under un-
controlled conditions of pH, the OH− are released into the medium as
an exchange for HCO3− and the pH gradually increases, which results
in the decrease in HCO3− and increase in CO32-. This shift also hampers
the growth, and towards higher pH the Spirulina biomass productivity
decreases. The initial alkalinity at various cultivation condition has
significantly affected the biomass production, and was found to be in
J. Mehar, et al.
positive correlation with it.
Phosphates contribute by providing phosphorous, which is an es-
sential element for synthesis of nucleic acid and high energy com-
pounds. Interestingly, the percent phosphate utilisation among all the
cultures at various cultivation conditions was almost similar and ranged
between 67.35–71.14% of initially supplemented phosphate (Fig. 3A,
Fig. 4). The slightly more phosphate utilisation was recorded for the
culture grown using ZM (71.14%) (Fig. 3A). Nitrogen is another ele-
ment which is required for growth and biomass production by micro-
algae. The nitrate utilisation at pH 8.5 (Fig. 4B) and pH 9.5 (Fig. 4C)
was almost same and was recorded to be 32.36% and 32.73%, re-
spectively. When compared with ZM (Fig. 3A), the pH 8.5 has utilised
9.36% less nitrate. The nitrate utilisation when CO2 was purged con-
tinuously (Fig. 4D) was lesser by 48.23% compared to pH 8.5. Further,
optimisation with respect to initial nitrate concentrations can be at-
tempted to minimise unspent nitrate in the culture media. The better
performance of the culture with respect to nitrate, phosphate and
HCO3− utilisation at pH 8.5 could be because of ease of maintenance of
physiological state where nitrate reductase, carbonic anhydrase and
other permeases remains at most active state.
3.5. Automation system: temperature, pH, DO, light intensity, and culture
behaviour
Although the use of CO2 fed pH control strategy is practised com-
monly for commercial cultivation of microalgae, the detail studies
Fig. 2. Light intensity (LI) pattern during the experimentation period [Automation system + ZM + No CO2+No pH Control]: A- LI on the surface of the culture; B- LI
at 5 cm culture depth; C- LI at 15 cm culture depth; D- Reduction in LI from surface to 15 cm culture depth.
pertaining to Spirulina cultivation at the pilot scale are rare. Infact, the
diverse strategies apart from using the CO2 have been employed to
control the culture pH. The pH control studies carried out earlier differs
significantly with the current study with respect to the operating scale,
means of pH control, the type of reactor employed, and the species for
which it was used. Chen et al (2016) have used the indoor photo-
bioreactor (1 L) to study the effect of pH-stat CO2 feeding strategy to
enhance the cell growth and C-phycocyanin production from Spirulina
390
Journal of CO₂ Utilization 33 (2019) 384–393
platensis [6]. The cost-effective CO2 fed bicarbonate/phosphate buffer
system was developed to control the pH and enhance growth and as-
taxanthin production by H. pluvialis [29]. Zhang et al. (2016) have
developed and studied a CO2 spraying absorption tower coupled with
an outdoor open runway pond for cultivation of C. pyrenoidosa through
controlled pH in the range of 6.5–8.5 [30]. As an advancements in the
automation system, Cao et al have developed the Algal Station system
for online monitoring of microalgae and have studied Nannochloropsis
oceanica IMTE1 and Isochrysis zhangjiangensis under pH-steady modes
through CO2 supplementation [31]. Further, CO2 fed online pH mon-
itoring and maintenance system was also employed to study the effects
of various pH on the biomass and lipid production potential of Grae-
siella sp. WBG1 cultivated in 10 L circular ponds and 5 m2 open ra-
ceway reactors [32]. Even though the various groups have used the pH
control strategies for diverse applications, not all of these were through
automation system, and only Chen et al. (2016) have studied it for
Spirulina performance that too using a 1 L indoor photo-bioreactor [6].
Design sketch of the automated open raceway pond with details of
control panel and CO2 feeding system used in the current study is
provided as Fig. 1. Temperature and light intensity individually and in
combination plays an important role in defining the culture behaviour
and overall biomass productivities of the closed PBRs as well as open
raceway ponds. The light intensity pattern recorded during the various
cultivation experiments carried out in this study is shown in Fig. 2 (ZM,
No CO2, No pH control), Supplementary Figs. 1–3 (All CO2 fed pH
controlled conditions), Supplementary Fig. 4 (ZM + CO2, No pH con-
trol). The temperature and DO profiles of the culture during cultivation
with ZM without CO2 feeding are shown in Fig. 3B and D. The data
collected by the automation system clearly indicated that there were
three major temperature zones. The temperature, pH, DO and light
intensity were recorded from 8 a.m. to 6 pm and the data presented is of
every 2 h interval. The temperature and DO profiles of the cultivation
days during all experiments are provided as Supplementary Figs. 5 and
6. The mornings at the cultivation location were cooler compared to the
J. Mehar, et al.
Fig. 4. Change in biomass concentration, and nitrate and phosphate utilisation pattern during Spirulina cultivation at [A] - (pH-7.5 controlled with CO2 feeding); [B]
(pH-8.5 controlled with CO2 feeding); [C] (pH-9.5 controlled with CO2 feeding); [D] (ZM + CO2+No pH control).
afternoons which were bright and sunny, and the evenings were again
cooler with the breeze. The culture temperatures were recorded below
30 °C till the mid-afternoon, and rose to 33 °C by 4 pm and then a sharp
decline to 28 °C by 6 pm. This pattern was observed for almost all the
days of cultivation. Interestingly, even though the culture temperature
was still rising from mid-afternoon to late-afternoon, light intensity
started decreasing (from 2 pm to 6 pm). We have also manually re-
corded the light intensity at various depths of the culture using a
spherical LI-COR underwater light sensor (SPQA-5574, LI-1500, USA)
to understand light penetration and its availability to the cells. The
maximum light intensity of 1000–1300 μmol m−2 s-1 was recorded in
the afternoon (during 12 pm to 2 pm). The fluctuations in the light
391
Journal of CO₂ Utilization 33 (2019) 384–393
Fig. 3. Culture monitoring during experimenta-
tion period [Automation system + ZM + No
CO2+No pH Control]: (A) - Nitrate and phos-
phate utilisation pattern with change in biomass
concentration; (B)- Observed temperature pat-
tern during the experimentation; (C)- Change in
alkalinity as a function of pH; (D)- Observed DO
pattern during the experimentation.
intensity and the temperature on a daily basis were observed and can be
attributed to the weather conditions. When compared to surface light
intensity during the initial days of cultivation, a 70% and 97% decrease
in light intensity was observed at depths of 5 cm and 15 cm, respec-
tively. At 5 cm depth, the light intensity has reduced from around ˜400
to below 100 μmol m−2 s-1 from the initial day of cultivation to the final
day. At 15 cm depth, light penetration was further affected and was
noticed to be 30 μmol m−2 s-1 and reached below 5 with the increasing
biomass concentrations. The pH of the culture was maintained with CO2
feeding. The cultures with ZM only showed a gradual increasing in pH
over the cultivation period, which is attributed to the release of OH- in
exchange of HCO3- by the cells undergoing photosynthesis. The DO was
J. Mehar, et al. Journal of CO₂ Utilization 33 (2019) 384–393

Fig. 5. Alkalinity contributors and change/shift in alkalinity pattern of Spirulina culture medium during cultivation at [A] - (pH-7.5 controlled with CO2 feeding); [B]
(pH-8.5 controlled with CO2 feeding); [C] (pH-9.5 controlled with CO2 feeding); [D] (ZM + CO2+No pH control).

reducing the carbon footprint, dependence on the chemical carbon

source for energy, significant reduction in ash content, reducing the
excess CO2 loss when pumped continuously into the pond. The pH 8.5
was found to be the best cultivation conditions among all three tested
pH conditions where biomass productivity, protein, phycocyanin and
ash content were noted to be 72 mg L−1 d-1, 64%, 14% and 7.74%,
respectively. A slightly reduced biomass productivity and metabolic
content at pH 9.5 can be attributed to the initial time taken by culture
to rich the pH just above 9.5 where the CO2 feeding initiated. The
performance of the culture at pH 8.5 was in near comparison with
standard ZM. In terms of commercial scale cultivation of microalgae,
pH control must be achieved to enhance culture performance of the
microalgae that may experience highly fluctuating day-to-day growth
conditions. Therefore, the automation system developed and studied
here can be utilised for cultivation of any microalgae specie with the
same control and effect.

Fig. 6. Concentrations of protein, lipid, carbohydrate and phycocyanin at Authors contribution

various cultivation conditions (all pH conditions were controlled via CO2
feeding). JM has performed the major experiments with the help from NMU
and AS. SR, SM, and VC have designed and installed the automation
found to increase with increase in temperature and light intensity and system. SM and AS have conceptualised the study. JM and AS have
was noted to rise from 6 mg L-1 to 11 mg L-1. Interestingly, it showed the analysed the data and written the manuscript.
similar pattern of decrease with decrease in light intensity. This may
clearly be attributed to the lowered photosynthetic activity of the cells
Acknowledgements
due to decrease in light intensity which has led to decreased O2 emis-
sion as a by-product of photosynthesis.
The automation system was developed under the major lab project
(MLP187) of CSIR-CFTRI, Mysore. The experimental study was funded
4. Conclusion
by DST-INSPIRE Faculty Project grant (DST/INSPIRE/04/2016/
000742). JM is thankful to CSIR-UGC for providing fellowship for
The scope of this work relates to understanding the algal culture
doctoral studies. AS is thankful to DST for DST-INSPIRE Faculty award.
behaviour at various pH maintained through automatic CO2 feeding at
a defined gas concentration and flow rate. We have successfully de-
signed, fabricated, installed and operated the pilot scale automation Appendix A. Supplementary data
system for Spirulina cultivation in the open raceway pond of capacity
1400 L. In this study, to test the automation system, Spirulina was Supplementary material related to this article can be found, in the
cultivated by CO2 as NaHCO3 replacement, a strategy which helps in online version, at doi:https://doi.org/10.1016/j.jcou.2019.07.006.

392
J. Mehar, et al.
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