Bioresource Technology 289 (2019) 121625

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Towards protein production and application by using Chlorella species as T

circular economy
⁎
Yu-Cheng Laia, Chien-Hsiang Changa, Chun-Yen Chenb, Jo-Shu Changa,c,d,e, I-Son Nga,
a
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
b
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
c
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan
d
Research Center for Circular Economy, National Cheng Kung University, Tainan 701, Taiwan
e
College of Engineering, Tunghai University, Taichung 407, Taiwan

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, productions of microalgal proteins were explored via a circular economy concept. First, production
Microalgal proteins of proteins from Chlorella vulgaris FSP-E (CV) and Chlorella sorokiniana (CS) was optimized by using favorable
Chlorella cultivation conditions and strategies. The optimal CO2 concentration for the growth of both microalgae was 5%
Antibacterial activity (v/v), while the optimal nitrogen source for CV and CS were 12 mM of NaNO3 and NH4Cl, respectively. Addition
Prebiotic properties
of 12 mg/L ammonium iron (III) citrate enhanced protein production. Next, semi-batch cultivation strategy was
employed to achieve a protein production of 793.3 and 812.8 mg/L for CV and C S, representing a 4.86 and 2.77
fold increase, respectively, in protein productivity. The obtained microalgal proteins consist of 40% essential
amino acids. The CV and CS proteins possess prebiotic activities as they enhanced the growth of Lactobacillus
rhamnosus ZY by 48 and 74%, respectively, with a good antibacterial activity against predominant pathogens.

1. Introduction
With global industrial development and urbanization, excess use of
fossil fuels led to severe environmental issues like climate change and
global warming. Hence there is a need for alternative sustainable bio-
fuel feedstocks with a lower carbon footprint. The consumption of food,
water, and natural resources have significantly increased in the past
decades (Steinfeld et al., 2006). Along with anthropogenic behaviors
like deforestation, the carbon emission (i.e. greenhouse gas) was esti-
mated to account in 20% of global CO2 emission (Solomon et al., 2007;
Van der Werf et al., 2009). As agricultural activities also directly in-
fluence waste generation and land use significantly (Gasparri et al.,

⁎
Corresponding author.
E-mail address: yswu@mail.ncku.edu.tw (I.-S. Ng).

https://doi.org/10.1016/j.biortech.2019.121625
Received 18 May 2019; Received in revised form 6 June 2019; Accepted 7 June 2019
Available online 08 June 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Y.-C. Lai, et al. Bioresource Technology 289 (2019) 121625

Fig. 1. Nitrogen effect in time course of dry cell weight, pH and protein concentration in the cultivation of (a, c) CV and (b, d) CS. The pH is indicated by line, biomass
is represented by symbols. Nitrate (red bar), ammonium (green bar), urea (blue bar). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

Table 1
The effect of different nitrogen sources, nitrogen and carbon dioxide concentration in BG11 medium on the dry cell weight, productivity of biomass and protein by C.
vulgaris (CV) and C. sorokiniana (CS).
Nitrogen and CO2 Dry cell weight Biomass productivity Protein concentration Protein productivity Protein content (%, Protein content (%, w/
conditions (g/L) (mg/L/d) (mg/L) (mg/L/d) w/w) at day 3 w) at day 7

Nitrogen sources
CV-NO3− 2.96 423.1 715.0 102.1 41.1 24.1
CV-NH4+ 1.32 188.3 220.6 31.5 39.4 16.7
CV-urea 2.11 301.8 536.3 76.6 41.3 25.4
CS-NO3− 1.68 240.0 489.4 69.9 46.0 29.1
CS-NH4+ 3.98 568.6 645.6 92.2 27.7 16.2
CS-urea 2.95 420.7 588.0 84.0 25.7 20.0

Nitrogen concentration
2 mM NO3− 1.37 195.2 115.0 16.4 6.5 8.4
12 mM NO3− 4.55 649.8 658.1 94.0 15.2 14.5
36 mM NO3− 4.13 589.3 581.1 83.0 16.1 14.1
72 mM NO3− 3.93 562.0 648.3 92.6 18.1 16.5
2 mM NH4+ 2.21 315.4 495.6 70.8 15.3 22.5
12 mM NH4+ 3.65 522.1 594.2 84.9 13.7 16.3
36 mM NH4+ 4.06 579.5 558.3 79.8 16.0 13.8
72 mM NH4+ 3.95 563.8 548.3 78.3 17.2 13.9

CO2 concentration
CV-2% 2.96 423.1 715.0 102.1 41.1 24.1
CV-5% 4.55 649.8 708.2 101.2 24.9 15.6
CV-10% 3.56 508.7 618.8 88.4 23.5 17.4
CV-17% 2.58 368.2 537.2 76.7 31.4 20.8
CS-2% 3.98 568.6 645.6 92.2 27.7 16.2
CS-5% 4.24 605.8 712.6 101.8 17.5 16.8
CS-10% 3.97 566.5 586.6 83.8 14.6 14.8
CS-17% 2.64 376.5 259.4 37.1 15.3 9.8

The highest amounts were shown in bold number.

2
Y.-C. Lai, et al.
Fig. 2. Time course of dry cell weight, pH and protein concentration in the cultivation of (a, c) CV with different nitrate concentration, and (b, d) CS with different
ammonium concentration. The pH is indicated by line, biomass is represented by symbols. Nitrogen at 2 mM (red bar), 12 mM (green bar), 36 mM (blue bar) and
72 mM (dark bar). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2013), several conventional products need alternative sustainable and
emerging materials as feedstock (Brodie and Lewis, 2007; Tilman and
Clark, 2014).
The concept of circular economy is being promoted recently
(Korhonen et al., 2018; Stiles et al., 2018), which entails an essentially
free process of transformation from excess waste to renewable energy
sources in order to design an economic, natural and circular model,
different from the consumption of finite resources. Algal biomass shows
potential benefits in maximal energy recovery via recycling as a bio
fertilizer to enhance soil quality and crop nutrition in a sustainable
manner (Alobwede et al., 2019). However, very few studies report
microalgae based circular economy designs. With increase in global
food demand and critical climate change events, microalgal biomass
has recently become significant as an alternative source for food, feed,
fertilizers, nutraceuticals, cosmetics, chemical compounds and energy.
The use of microalgae as a feedstock for renewable bioenergy produc-
tion has considerably increased in the past few years (Abo et al., 2019;
Barreiro et al., 2014; Jazrawi et al., 2015). On the other hand, the
utilization of microalgae is converting it into food, chemical feed and
pharmaceuticals due to their abundant protein content presents as an
attractive option to be explored.
Despite current research in investigating the importance of micro-
algal biofuels, cost of competitive biofuel production from microalgae is
challenging. Therefore, exploitation of the complete algal biomass with
high value co-products, e.g., proteins, lipids, carbohydrates, pigments,
vitamins and antioxidants, with applications in health supplements,
fertilizers, cosmetics, pharmaceuticals and industrial chemicals is pi-
votal (Chew et al., 2017). Especially, protein is one of the essential
products in a microalgal biorefinery, as microalgae are naturally rich in
proteins (Grossmann et al., 2018; Unterlander et al., 2017; Waghmare
et al., 2016). Due to the nutritional and environmental advantages of
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Bioresource Technology 289 (2019) 121625
algal protein, it is critical to produce algal protein in a sustainable
equilibrium (Henchion et al., 2017). Some studies reported that
Chlorella is a protein-rich microalgae and serves as a potential feedstock
for animal feed (Bleakley and Hayes, 2017). Nevertheless, the compo-
sition and functional aspects of proteins from Chlorella was rarely
evaluated. Even though the annual production of microalgal biomass is
comparatively lower than seaweed, the operation of microalgal bior-
efineries to produce algal biomass and value added compounds is
economically valuable (Pulz and Gross, 2004). The goal of this study is
to develop feasible cultivation strategies to enhance microalgal protein
productivity in photoautotrophic cultivation, and to evaluate the ap-
plication of proteins from Chlorella as an alternative to applicable bio-
products in various fields.
2. Materials and methods
2.1. Materials
All chemicals, salts, solvents and reagents used in this study were of
analytical grade and were purchased from Sigma-Aldrich, USA. The
microalgae strains CV and CS were isolated from a freshwater area
located in southern Taiwan. To compare antibacterial and prebiotic
activities of the algal proteins, one of the functional proteins, phyco-
cyanin, was bought from Febico Bio-Tec, Taiwan.
2.2. Culture mediums and conditions
BG-11 medium was used as the base medium for algal growth. It
consists of (mg/L): NaNO3, 1000; K2HPO4, 40; MgSO4·7H2O, 75; Citric
acid, 6.0; Na2CO3, 20; CaCl2·2H2O, 36; Ferric ammonium citrate, 6.0;
EDTA·2Na, 1.0; H3BO3, 2.86; MnCl2·4H2O, 1.81; ZnSO4·7H2O, 0.222;
Y.-C. Lai, et al. Bioresource Technology 289 (2019) 121625

Fig. 3. Carbon dioxide concentration effect in time course of dry cell weight, pH and protein concentration in the cultivation of (a, c) CV and (b, d) CS. The pH is
indicated by line, biomass is represented by symbols. CO2 (v/v air) at 2% (red bar), 5% (green bar), 10% (blue bar) and 17% (dark bar). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 2
The effect of different nitrogen sources, nitrogen and carbon dioxide concentration in BG11 medium on the content and productivity of protein by C. vulgaris (CV) and
C. sorokiniana (CS) at day 3.
Nitrogen and CO2 conditions Protein concentration (mg/L) Protein content (%, w/w) Protein productivity (mg/L/d)

CV
2 mM NO3−, 2% CO2 69.8 6.5 23.3
12 mM NO3−, 2% CO2 289.8 15.2 96.6
12 mM NO3−, 5% CO2 473.3 24.9 157.8
12 mM NO3−, 5% CO2, 12 mg/L iron 312.8 15.4 104.3
12 mM NO3−, 5% CO2, 12 mg/L iron (day 7) 793.3 19.4 113.3

CS
2 mM NH4+, 2% CO2 125.7 15.3 41.9
12 mM NH4+, 2% CO2 414.6 13.7 138.2
12 mM NH4+, 5% CO2 528.9 17.5 176.3
12 mM NH4+, 5% CO2, 12 mg/L iron 342.2 17.5 114.1
12 mM NH4+, 5% CO2, 12 mg/L iron (day 7) 812.8 19.2 116.1

The highest amounts were shown in bold number.

Na2MoO4·2H2O, 0.39; CuSO4·5H2O, 0.079; Co(NO3)2·6H2O, 0.049. The
BG-11 medium was modified as required for optimizing algal growth.
Sodium nitrate (NaNO3), ammonium chloride (NH4Cl) and urea were
evaluated as nitrogen source (at 2 mM, 12 mM, 36 mM, 72 mM con-
centration) for growth and protein productivity of Chlorella sp., while a
4.065 g/L of Tris was used to stabilize the pH of cultures when NH4Cl
and urea were used as nitrogen source (Nguyen et al., 2016). Modified
BG-11 medium (MBG-11) with an additional supplementation of ferric
ammonium citrate was employed based on the previous study (Kona
et al., 2017). Algae were cultured in a 1 L laboratory-scale airlift pho-
tobioreactor (PBR) at ambient temperature under continuous white
light at 100 μmol photons/m2/s with shaking at 300 rpm. The PBR has a
working volume of 500 mL and was aerated with CO2 and air mixture to
obtain a final CO2 concentration of 2%, 5%, 10%, 17% (v/v).
2.3. Measurement of growth and biomass
Cell growth was monitored by determining the optical density of the
culture at 680 nm (OD680) with a spectrophotometer (SpectraMax 340,
Molecular Devices, USA). Wet biomass (g/L) were collected from 1 mL
cultures by centrifugation at 2000×g for 10 min and dried at 110 °C for
20 min in an Infrared Moisture Analyzer (FD-660, Kett electric

4
Y.-C. Lai, et al.
Fig. 4. Effect of ammonium iron citrate in time course of dry cell weight and pH; (c, d) protein concentration in the cultivation of (a, c) CV and (b, d) CS. pH is
indicated by line, biomass is represented by symbols. Ammonium iron citrate at 6 mg/L (red bar), 12 mg/L (green bar), and 18 mg/L (blue bar). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
laboratory, Japan) until the weight was constant. The calibration be-
tween OD680 and dry cell biomass (g/L) were calculated by the equation
Y (g/L) = 0.4839 × (OD680) for CV, and Y (g/L) = 0.4884 × (OD680)
for CS, respectively. All experiments were performed in triplicate in-
dependently.
2.4. Batch and semi-batch fermentation
To enhance the algal growth and the protein productivity of CV and
CS, engineering strategies were applied for enhancing the fermentation
performance. CV and CS were grown semi-continuously in a 1 L PBR
with 500 mL MBG-11 medium. Eighty percent replacement of sterilized
MBG-11 medium was conducted for four repeated cycles in PBR with
some modifications (Chen et al., 2018; Chia et al., 2013). The optimized
cultivation conditions were 12 mM of nitrate for CV and 12 mM of
ammonium for CS, CO2 aeration at 5% (v/v air), and other physical
parameters were applied as described in Section 2.2.
2.5. Protein extraction and quantification
Microalgal protein was prepared as previous reports with mod-
ifications (Ling et al., 2015). The cells were harvested by centrifugation
at 2000×g for 10 min, and washed with deionized water for 2 times.
The whole cell (WC) was concentrated to OD680 at 5. Then, the cells
were disrupted by a high-pressure homogenizer with a pressure of 30
kpsi (Constant system, One-Shot Model, UK). Cell extract and pellet
were separated by centrifugation at 8000×g for 15 min. The protein
concentration was determined by BioRad Protein Assay using bovine
serum albumin (BSA) as standard. The calibration curve was converted
between absorption of OD595 (X) and concentration (mg/L) (Y) by
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Bioresource Technology 289 (2019) 121625
equation Y = 0.0012 X + 0.0038. The protein content (%, w/w) is de-
fined by protein concentration (mg/L) × volume of solution (L)/dry
cell weight (g/L), while the productivity (mg/L/d) were obtained from
protein concentration (mg/L) divided by culture time (days). The data
shown are the average of three replicates ( ± SD standard deviation).
2.6. Protein identification via SDS-PAGE and tandem MS/MS
The microalgal proteins were prepared at a standard concentration
of 0.5 g/L and were further analyzed by SDS-PAGE through 10% (w/v)
and 4% (w/v) as separating and stacking gels, respectively. Tris–glycine
buffer (pH 8.3) containing 0.1% SDS was used as electrophoresis buffer.
Samples at same concentration were suspended in water and heated at
100 °C for 10 min before loading onto the gel. Electrophoresis was
conducted at a constant current of 20 mA per slab at room temperature
in a Biorad mini gel electrophoresis unit. After separation, proteins on
SDS-PAGE were visualized by staining with Coomassie blue R-250 and
followed by Image scanner. The target bands on gel were selected and
further analyzed by tandem mass (MS/MS).
2.7. Amino acid analysis
Microalgal proteins were concentrated to 1 g/L by centrifugation as
described in Section 2.5 and were further analyzed by Amino Acid
Analysis System (Waters PICO-TAG, US) containing hydrolysis and
HPLC (White et al., 1986).
2.8. Antibacterial and prebiotic activity
Bacillus cereus, Pseudomonas putida and Staphylococcus aureus were
Y.-C. Lai, et al.
Fig. 5. Semi-batch fermentation for CV and CS. (a) Time course of dry cell
weight (dark circle for CV and open square for CS), and pH (up triangle for CV
and down triangle for CS). (b) Time course of protein concentration (dark bar
for CV and white bar for CS) and nitrogen consumption rate (dark circle for CV
and open square for CS) in the cultivation of CV and CS. The red arrows in-
dicated the medium replaced point. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this
article.)
inoculated in 20 mL of LB agar at 2% (v/v). To test the antibacterial
activity of algal proteins, 1 g/L of phycocyanin and 5 g/L of crude
proteins extracted from CV and CS were added on the filter paper in a
cooled agar plate. The sizes of the bacterial inhibition zone were
measured after culturing at 37 °C for 6 h. All experiments were per-
formed in triplicate independently. On the other hand, Lactobacillus
rhamnosus ZY was inoculated in 100 mL of Erlenmeyer flask containing
20 mL of MRS medium at 5% (v/v) and cultured at 37 °C with shaking at
200 rpm. To test the prebiotic activity of algal proteins, phycocyanin
and crude proteins extracted from CV and CS were added into the
cultures. After culturing for 12 h, samples were diluted by deionized
water for 107 times. The colony forming units (CFU) of probiotics were
counted on MRS agar plates after culturing at 37 °C for 24 h.
3. Results and discussion
Herein, microalgae biomass and protein productivity of C. vulgaris
(CV) and C. sorokiniana (CS) were investigated by using 1 L photo-
bioreactor (PBR), which was illuminated by cold cathode fluorescent
lamps at the light intensity of 100 μmol photons/m2/s, and was aerated
by CO2 as the sole carbon source to grow the protein-rich microalgae
photo-autotrophically. To ensure the effect of protein production by
mixotrophic cultivation, glycerol, glucose and acetic acid were also
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Bioresource Technology 289 (2019) 121625
used as different carbon sources to culture CV and CS. However, the
protein productivities of CV and CS in mixotrophic cultivation were
lower than that in photoautotrophic cultivation. Therefore, photo-
autotrophic cultivation was the optimal strategy to obtain the highest
protein productivity of CV and CS in this study. The composition of the
nutritional medium including nitrogen, carbon and ferric ion (Fe2+)
concentration were optimized for maximal biomass and protein pro-
ductivity. To establish a stable platform for producing functional pro-
tein from microalgae with high protein content, the PBR was operated
in semi-batch mode for a prolonged period under the optimal operation
conditions. Crude proteins from both algal biomass were analyzed by
electrophoresis and tandem mass (MS/MS) spectrometry. The amino
acid composition of the microalgal protein extract was determined.
Finally, to access the possibility of practical applications of the algal
proteins, probiotic and antibacterial activities of the crude algal protein
extracts were studied.
3.1. Effect of nitrogen sources on microalgal growth and protein production
Nitrogen is an essential macro-nutrient for microalgal cultivation
and nitrogen repletion is crucial for maximal protein production. The
effect of three nitrogen sources, including sodium nitrate (NaNO3),
ammonium chloride (NH4Cl) and urea (CO(NH2)2) were investigated at
same nitrogen concentration (12 mM) and the results are shown in
Fig. 1. CV cultured in nitrate had a higher biomass of 2.96 g/L, and the
pH inclined towards the neutral range (Fig. 1a). The initial pH of the
culture was 7.4, followed by a sharp decrease in pH when nitrates were
assimilated by algae after 3 days. In media supplemented with nitrate,
microalgae transported nitrate through NO3−/H+ symporter (Perez-
Garcia et al., 2011; Xie et al., 2017). Therefore, the hydrogen ions
would be counteracted and the pH of the medium stabilized at the
neutral range during the culture time. On the other hand, to maintain
the proton concentration outside the cellular membranes, Tris was
added in the medium with ammonium and urea to adjust the pH value
to 7–8 initially. Compared to CV, CS could more easily acclimate to
ammonia medium and had a higher biomass of 3.98 g/L (Fig. 1b). A
previous study reported the use of ammonium chloride as nitrogen
source for the mixotrophic cultivation of C. sorokiniana (León-Vaz et al.,
2019). The pH value of medium ranged from 6.5 to 9, which was sui-
table for the cultivation of CV and CS (Fig. 1a and b). The accumulated
protein concentration of CV cultured with nitrate was consequently
higher than other nitrogen sources (Fig. 1c), while the CS cultured with
ammonium had higher protein content than with nitrate and urea
(Fig. 1d). Compared with both CV and CS at different culture time,
higher biomass led to higher protein concentration of those cultured at
day 7. Due to their much higher biomass obtained at day 7, the protein
content would decrease. Another reason is that when carbon sources
were assimilated by algae for a long period, the lipid synthesis was
promoted in the cell machinery, which was not beneficial to protein
synthesis (Lin et al., 2018). The biomass and protein content for CV and
CS with different nitrogen sources are summarized in Table 1. After 7-
days culture, the highest biomass productivity was 423.1 mg/L/d for
CV and 568.6 mg/L/d for CS with adequate nitrogen sources (i.e., ni-
trate for CV and ammonium for CS), and the protein productivity was
102.1 mg/L/d for CV and 92.2 mg/L/d for CS. As a result, nitrate and
ammonium was selected as the best nitrogen source for culturing CV
and CS, respectively.
3.2. Effect of nitrogen concentration on microalgal growth and protein
production
The effect of different concentrations of nitrate and ammonium on
the biomass and protein production of CV and CS are shown in Fig. 2.
When CV and CS were cultured with 2 mM nitrate and ammonium, the
nitrogen concentration was limiting leading to poor biomass accumu-
lation (Fig. 2a and b). Among the different nitrogen concentrations
Y.-C. Lai, et al. Bioresource Technology 289 (2019) 121625

Fig. 6. (a) 10% (w/v) SDS-PAGE Analysis of algal protein harvested in C. vulgaris (CV) and C. sorokiniana (CS) under different culture conditions. Standard molecular
weight of protein with sizes (kDa) are indicated in the left. The arrows indicated proteins are further analyzed by MS/MS. (b) Amino acid composition of CV and (c)
amino acid composition of CS.

Table 3
Mascot protein identifications of C. vulgaris and C. sorokiniana from selected proteins.
No. Protein name Accession no. Score Mw (Da) pI Coverage (%)

CV-55 ATP synthase CF1 alpha subunit YP_003058335 495 54.7 5.70 13
CV-27 light-harvesting complex II chlorophyll a-b binding protein M3 XP_001703699 570 27.4 5.68 52
CV-24 ribosomal protein S4 YP_004347822 93 23.8 10.56 24
CS-75 heat shock protein 70 EFN51789 645 70.7 5.20 14
CS-60 ATP synthase CF1 alpha subunit NP_045781 642 54.6 5.9 38
CS-50 elongation factor thermo unstable YP_004347788 652 44.8 5.37 44
CS-20 40S ribosomal protein S5 EFN51870 194 21.6 9.91 19

Table 4 producing algal proteins (Hülsen et al., 2018; Lourenço et al., 2004). In
The antibacterial and prebiotic activities of the algal proteins. summary, 12 mM of nitrogen source concentration was optimal for CV
Added protein CV CS
and CS to produce maximal amount of protein at day 7. Table 1 shows
that CV and CS had the highest amount of protein concentration (i.e.,
Pathogen strain Diameter of inhibition zone (cm) 658.1 mg/L in CV and 594.2 mg/L in CS), and productivity (i.e.,
Bacillus cereus 1.43 ± 0.06 1.53 ± 0.06 94.0 mg/L/d in CV and 84.9 mg/L/d in CS) at 12 mM nitrogen sources.
Pseudomonas putida 1.30 ± 0.10 1.37 ± 0.06
Staphylococcus aureus 1.40 ± 0.10 1.60 ± 0.10
The maximal protein concentration decreased (i.e., 581.1 mg/L in CV
and 558.3 mg/L in CS) at 36 mM nitrogen sources due to growth in-
Protein amount (mg) CFU of L. rhamnosus (×108)
hibition. Nitrogen concentration at 12 mM was chosen for further ex-
0 5.0 5.0 periments for maximal protein content and productivity.
10 5.2 6.0
20 6.5 7.6
30 7.4 8.7 3.3. Effect of CO2 concentration on microalgal growth and protein
production

used, 12 mM was the optimal nitrogen concentration to culture CV and

The biomass, culture medium pH and protein concentration of CV
CS. As the nitrogen concentration increased from 12, 36 to 72 mM, the
and CS under different concentration of CO2 (2%, 5%, 10% and 17%) is
growth of CV and CS gradually decreased, leading to lower biomass and
shown in Fig. 3. The maximum biomass of CV and CS with CO2 aeration
protein productivity. Likewise, CS grew more rapidly at 12 mM am-
at 2% was 2.96 g/L and 3.98 g/L, respectively (Fig. 3a and b). The dry
monium. The pH value of the medium was around 9 when 72 mM ni-
cell weight of CV and CS was 4.55 g/L and 4.24 g/L when the CO2
trogen was added initially, followed by a sharp decrease of pH when
concentration increased to 5% CO2 (Table 1). The carbon assimilation
nitrogen was assimilated by algae on day 1 (Fig. 2a and b). In the ex-
of CV and CS enhanced with increasing CO2 aeration at low CO2 con-
ponential growth phase of CV and CS, the pH of the medium increased
centration. However, the biomass of CV and CS decreased with 10%
at first and then stabilized at the neutral range on day 7. The results
and 17% CO2 concentration. The results indicate that CS and CV are not
confirmed that algal strains exchanged hydrogen ion with other ions
high CO2 tolerant microalgae, and lower levels of CO2 would suffice
from nutrients in vivo for surviving. The protein concentration in algae
their growth needs. In the aspect of pH, Fig. 3a shows that pH of the
was directly related to the amount of nitrogen consumed by algae. As a
medium with CV remained stable from 6.4 to 7.8. And with an increase
result, the algal protein productivity of CV and CS cultured at 2 mM
in CO2 concentration, the pH of the medium decreased
were the lowest, and the highest was at 12 mM and 72 mM (Fig. 2c and
(2% > 5% > 10% > 17%). Similarly, Fig. 3b reveals that the pH of
d). Several reports emphasized the importance of nitrogen sources in
medium with CS also remained stable from 7.2 to 9.1. The pH decreased

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Y.-C. Lai, et al. Bioresource Technology 289 (2019) 121625

sharply for both the cultures at day 1 due to carbon and nitrogen as-

Animal Feed, Anti-antibacterial, Prebiotic Additives

similation by microalgae. Also, at higher CO2 concentrations, protein
concentration decreased, with the lowest values at 17% CO2. Since
biomass growth was also limited at this higher CO2 concentration, it
reflected in protein productivity as well, with a decrease after day 6
(Fig. 3c and d). As shown in Table 2, it is worth noting that by in-
creasing CO2 from 2% to 5%, and protein concentration of CV and CS
increased by 6.6% and 17.2% (i.e., 762 and 757 mg/L at day 6).
Carbon fixation/photosynthesis is essential for effective biomass
Proteins by CV & CS

production (Li et al., 2019). On the other hand, metabolic pathways of

carbon assimilation in microalgae routes the fixed carbon into proteins,
In this study

lipids and other components (Lin et al., 2018). It has been shown that
batch and semi-continuous strategies were optimal for C. protothecoides
in autotrophic mode with 20% CO2 (Liu et al., 2019) while C. sor-
50

okiniana exposed to high CO2 levels (5–15%) showed an increase in

cellular carbohydrate content (> 50% by dry weight) in stationary
phase (Varshney et al., 2018). Exponential growth of microalgal cells
Food, Prebiotic Additives

always show a higher protein content, since most of the cellular func-
tions are carried out by protein based enzymatic machinery. Carbon
partitioning between lipids and carbohydrates vary with different
algae, and oleaginous (lipid accumulating) and carbohydrate accumu-
ProBiotein

lating algae differ even on strain basis. The protein content in algal
Amazon
75–80

biomass increased with increasing pH, while the addition amount of

CO2 decreased (Qiu et al., 2017). Therefore, the effect of aeration at
different CO2 on algal growth and protein production are critical. In this
section, it is clearly seen that for both CV and CS, 5% CO2 is optimal for
Anti-inflammation, Antioxidation

maximal protein content and productivity.

3.4. Effect of ferric ion addition on microalgal growth and protein

production
C-Phycocyanin

Ammonium iron citrate was used as the source of ferric ion and was
added in BG-11 medium at three different concentrations (6, 12,
135–190
Alibaba

18 mg/L) as modified BG-11 medium (MBG-11) for enhancing the

growth of CV and CS (Fig. 4). As illustrated in Fig. 4a, CV in MBG-11
with 12 mg/L and 18 mg/L ammonium iron citrate had a higher bio-
mass than with 6 mg/L. To maximize functional protein production
from microalgae, the composition of medium should be investigated,
Spirulina Powder

such as nitrogen, carbon sources and other trace elements. As far as

Animal Feed

trace elements are concerned, several studies proposed that iron is an

Alibaba

essential micronutrient for algal growth (Choi et al., 2016;

1–6

Sivaramakrishnan and Incharoensakdi, 2017; Zhao et al., 2018), but

very few studied the correlation between iron and protein production
from microalgae. It was revealed that iron plays an important role of
Chlorella Powder

improving energy molecules (i.e., ATP and NADPH), photosynthetic

Animal Feed

efficiency and further enhancing biomass production in the cultivation

media (Briat et al., 2015). However, the growth rate of CV with 18 mg/
Alibaba
10–50

L ammonium iron citrate was slightly higher than with 12 mg/L during
The prices of various algal proteins used in different fields.

the early stage of growth curve (day 1–3), but was lower for the next
4 days (day 4–7). It was suggested that excessive iron in the medium
had a negative effect on growth of Chlorella due to oxidative stress
Corn Gluten Meal

(Estevez et al., 2001). For the same reason, the effect of ferric ion on the
* Data sourced from Alibaba in May 2019.

growth of CV resembled which on the growth of CS (Fig. 4b). Chen et al

Animal Feed

verified that protein productivity of Chlorella vulgaris was on the same

Alibaba
0.4–0.6

level at iron concentration ranging from 22.5 to 90 μM, but significantly

decreased at a concentration of 180 μM (Chen et al., 2015). The
medium pH curves of CV and CS with different ferric concentration
were similar because of the same cultivation conditions, especially the
Prices per kilogram (USD)

nitrogen and carbon sources (Fig. 4a and b). After 7-days of culturing,
the maximal protein concentration of CV and CS (793.3 and 812.8 mg/
L) were obtained by culturing with 12 mM ammonium iron citrate
(Fig. 4c and d).
Applications

Providers*
Chemicals

3.5. Semi-continuous cultivation of CV and CS strains

Table 5

Optimization of cultivation parameters like nitrogen source,

8
Y.-C. Lai, et al.
nitrogen concentration, iron concentration and CO2 concentration were
carried out in batch cultures. However, other cultivation strategies like
semi-batch or continuous needs to be applied for higher biomass pro-
duction without the hassles of the repeated batch operations. Hence,
semi-continuous cultivation mode was adapted for both CV and CS with
80% medium replacement. Time courses of growth curve, medium pH
and protein concentration of CV and CS in semi-continuous culture are
shown in Fig. 5. The dry cell weight of CV and CS were 3.6 and 3.9 g/L
in the MBG-11 medium at day 7. The initial medium pH of CV and CS
were 7.6 and 8.7 and it dropped quickly at the first day (Fig. 5a). After
the first replacement cycle, replacing 80% of modified medium in the
PBR at day 14, the dry cell weight of CV and CS slightly decreased,
while the average medium pH increased from 7.4–7.6 to 7.7–7.8. On
the other hand, Fig. 5b reveals that the protein concentration of CV and
CS increased by 25.6% and 7.9% (632 and 628 mg/L), respectively. The
rational explanation could be that the inhibitory compounds/metabolic
wastes accumulated in the first cycle of growth carried along with the
20% inoculum could result in the slight increase of pH (Chen et al.,
2018). In Fig. 5c, the nitrogen consumption rate (%) refers to milli-
grams of nitrogen amount (i.e., nitrate and ammonium) consumed in
one liter of CV or CS solution containing a milligram of nitrogen source.
With the enhancement of nitrogen assimilation (86.3% for CV and
81.2% for CS) as shown in Fig. 5c, the first cycle of semi-continuous
cultivation did not result in improved biomass growth but showed
improvement in protein content. In the third and fourth cycle of cul-
tivation (indicated by red arrows), the biomass of CV and CS succes-
sively increased and finally increased up 23.1–27.8% (4.6 and 4.8 g/L)
after 28-days cultivation. During the cultivation, the medium pH of CV
at 7.59–7.94 was higher than that of CS at 7.01–7.81, and both of them
started to decline. More importantly, the protein concentration of CV
and CS finally increased up to 794 and 852 mg/L as the biomass in-
creased. The nitrogen consumption rate of CV was higher than that of
CS in the fourth cycle of cultivation (90.2%, 85.8% at day 28). There-
fore, the continuous operation proved effective for growth and protein
production of CV and CS. Actually, previous study of different algae
produced 1 g/L of biomass from semi-continuous culture (Chae et al.,
2006) while scale-up cultivation of microalgae in outdoor are favorable
to obtain higher productivity and yield (Schoepp et al., 2014). To the
best of our knowledge, we report for the first time the increase in
protein content and productivity by semi-continuous mode of cultiva-
tion.
3.6. Characterization of microalgal proteins
The microalgal proteins were extracted by cell disruptions and
analyzed by electrophoresis. The protein patterns and distributions as
seen in SDS-PAGE analysis is shown in Fig. 6a. The major proteins in CV
and CS were different, even though both were cultured in BG-11
medium. The dominating proteins designed as at molecular weights of
20, 24, 27, 50, 55, 60 and 75 (kDa) were further analyzed by tandem
mass (MS/MS). The detailed peptide sequencing information and
Mascot results are summarized in Table 3. The best matches from the
database are ribosomal protein, light-harvesting complex II chlorophyll
a-b binding protein, elongation factor thermo-unstable, ATP synthase
CF1 alpha subunit and heat shock protein 70. The eukaryotic ribosomal
subunit (40S) is the smaller subunit of 80S ribosomes containing de-
coding center which complement tRNA and mRNA in protein transla-
tion (Götz and Arnold, 1980; Manuell et al., 2005). The light-harvesting
complex consists of chlorophylls and the chlorophyll a-b binding pro-
tein, which functions as a light receptor that captures and delivers ex-
citation energy to photosystems I and II. The light harvesting chlor-
ophyll a-b binding proteins are located in the thylakoid membrane of
plants and function in balancing the excitation energy between the two
photosystems by reversible phosphorylation under changing light
condition (Liu and Shen, 2004). ATP synthase subunit is integrated into
thylakoid membrane and participates in the dark reactions of
9
Bioresource Technology 289 (2019) 121625
photosynthesis in Calvin cycle where ATP synthesis takes place (Walker
et al., 1982). The elongation factor and 70 kDa heat shock proteins only
exist in CS and play a crucial role in translation and protein folding, and
help to protect cells from stress. It is supposed that the components of
algal protein participating in energy conservation in Calvin cycle, and
translational regulation are essential cellular machinery.
The amino acid profiles of algal protein produced by CV and CS are
shown in Fig. 6b and c. The microalgal protein consisted of 40% es-
sential amino acids, including alanine (Ala), glycine (Gly), leucine
(Leu), proline (Pro), valine (Val), isoleucine (Ile), methionine (Met),
phenylalanine (Phe), tyrosine (Tyr), serine (Ser), threonine (Thr), cy-
steine (Cys), lysine (Lys), arginine (Arg), histidine (His), glutamate
(Glu) and asparagine (Asp). Algal proteins produced by CV and CS re-
vealed a similar composition of total protein content at 1 g/L, which
mostly contained aliphatic amino acids. Related studies demonstrated
that the quality of the proteins extracted from microalgae is compara-
tive to protein from other organisms (Waghmare et al., 2016). Because
of high protein content and productivity of algae, it is suggested that
algal protein obtained in this work is a practically high-value product
which is already available in emerging markets. Further applications
for the extracted proteins are shown in the next section.
3.7. Antibacterial and prebiotic activities of the microalgal proteins
Proteins extracted from CV and CS were evaluated for their anti-
bacterial activity against three highly pathogenic bacteria Bacillus
cereus, Pseudomonas putida and Staphylococcus aureus, using the cyano-
bacterial protein phycocyanin as control. The diameter of inhibition
zones produced in LB agar with three kinds of pathogen strains is shown
in Table 4. The diameter of inhibition zones for phycocyanin varied
from 1.07 to 1.32 cm, which was similar to that of a cell extract of
filamentous freshwater cyanobacterium (Sabarinathan and Ganesan,
2008). The results of our study indicate that the microalgal proteins had
a higher antibacterial activity than phycocyanin. The diameter of in-
hibition zones for microalgal proteins varied from 1.30 to 1.60 cm.
Maximal zone of inhibition (1.6 cm) was obtained against S. aureus
when using CS proteins.
Table 4 summarizes the probiotic effect of algal proteins based on
the CFU of L. rhamnosus spread on MRS agar after culturing with pro-
tein supplement at 37 °C for 24 h. Compared to the cultivation of pro-
biotics without protein addition, the CFU of probiotics was higher when
cultured with algal proteins. The growth rate of L. rhamnosus was di-
rectly proportional to the amount of algal proteins. The CFU of L.
rhamnosus was the highest while adding phycocyanin. As a result, the
prebiotic activity of phycocyanin (4 mg) on L. rhamnosus effectively
increased the biomass to 1.75 × 109 CFU in this study. The prebiotic
activities of proteins from CV and CS on L. rhamnosus effectively in-
creased the biomass to 7.4 × 108 and 8.7 × 108 CFU, respectively.
Several studies verified that phycocyanin is a biliprotein found in blue
green algae such as Spirulina and Arthospira with efficient antimicrobial
activity (Mohite et al., 2015; Najdenski et al., 2013). Although the
prebiotic activities of protein from CV and CS were lower than that of
phycocyanin, it is conceivable that the algal protein extracted from CV
and CS has potential as a dietary supplement. The prices of various algal
proteins used in animal feed, anti-inflammation, antioxidation, anti-
antibacterial, and prebiotic additives are listed in Table 5 (data sourced
from Alibaba in May 2019). For algal proteins used in animal feed, the
price is under $50 USD/Kg while anti-inflammation and antioxidation
proteins were higher than $80 USD/Kg. Our product showed anti-
bacterial and prebiotic activity, but the cost would be approximately
$50 USD/Kg which implies that high protein production of CV and CS
achieves the goal of circular economy.
4. Conclusions
Biomass and protein productivity of CV and CS were enhanced by
Y.-C. Lai, et al.
optimizing CO2 concentration, nitrogen source concentration and ferric
ion addition. The algal protein production was further maximized by
semi-continuous mode of fermentation. Finally, the protein pro-
ductivity is up to 157.8 mg/L/h for CV and 176.3 mg/L/h for CS. Both
microalgae proteins consisted of over 40% of essential amino acids and
could be used as food/feed additive. Antibacterial and prebiotic prop-
erties of the algal proteins were also evaluated. This study provides a
potential method for producing high value products in circular
economy concept with enhanced protein titer, content and pro-
ductivity.
Acknowledgements
The authors are grateful for the financial support received from the
Ministry of Science and Technology (MOST 108-2218-E-006-006,
MOST 107-2218-E-006-016, MOST 106-3114-E-006-008, MOST 105-
2221-E-006-225-MY3 and MOST 105-2621-M-006-012-MY3) in
Taiwan.
Ethics approval and consent to participate
All the authors have read and agreed the ethics for publishing the
manuscript.
Consent for publication
The authors approved the consent for publishing the manuscript.
Declaration of Competing Interest
The authors declare that they have no competing interests.
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