Tolerance Comparison Among Selected Spirulina Strains Cultured Under High Carbon Dioxide and Coal Power Plant Flue Gas Supplements

ZHAO Qianqian, JIN Guiyong, LIU Qiuke, PAN Kehou, 2), ZHU Baohua, and LI Yun, *

Tolerance Comparison Among SelectedStrains Cultured Under High Carbon Dioxide and Coal Power Plant Flue Gas Supplements

ZHAO Qianqian1), JIN Guiyong1), LIU Qiuke1), PAN Kehou1), 2), ZHU Baohua1), and LI Yun1), *

1),,266003,2),,266037,

In order to explore the changes in the growth and protein contents ofand obtain a proper strain for the fixation of carbon dioxide (CO2) from flue gas, the strains isolated from thefarms and the strain 208 were cultured under different aeration conditions including no CO2, 10% CO2and coal power plant flue gas supplements. The physiological indexes including filament length, biomass yield and chlorophyll, soluble protein and phycocyanin contents were determined, respectively. When cultured without CO2supplement, the strain 4-5 exhibited the highest biomass yield (1.880gL?1) and a specific growth rate (0.367d?1). However, the specific growth rate of all strains decreased significantly when they were cultured under 10% CO2and unfiltered coal power plant flue gas supplements. Considerable differences were noted in the performance of the experimental microalgal strains under different contemporaneous conditions. The strain 7-8 achieved the highest biomass yield (1.603gL?1) and relatively high phy- cocyanin content (7.1%) under 10% CO2supplement. We noted that strain 4-5 had the highest specific growth rate (0.182d?1) and biomass yield (0.43gL?1) under coal power plant flue gas supplement. Strain 6-10 displayed the highest soluble protein content (66.02%), and strain 7-8 showed the highest phycocyanin content (9.28%) under coal power plant flue gas supplement.

; CO2; coal power plant flue gas; physiological index

1 Introduction

Global warming, one of the world’s most unsolved en- vironmental issues, was mainly caused by excessive CO2production (Chiaramonti, 2013; Yi, 2015; Pérez-López, 2017). Except for reducing CO2emissions fromthe production source, effective capturing of CO2has been becoming a solution which is a researching hotspot cur- rently. At present, the harness of CO2mainly focused on the physical, chemical and biological aspects (Kumar, 2011). When compared with the physical and chemical methods, biological carbon sequestration through photo- synthesis showed more efficient, clean and sustainable ad- vantages (Zhao, 2015).

Previous researches have shown that nearly 40% of the CO2on earth could be absorbed through photosynthesis, in which microalgae and cyanobacteria have a huge con- tribution (Jacob-Lopes., 2008). Microalgae are super- excellent, which assimilate CO2owing to their short growth cycle, fast reproduction rate, high photosynthetic efficien- cy and strong environment adaptation capacity (Wang, 2008; Zhao, 2015; Khan, 2018). In addition, when CO2is absorbed and utilized by microalgae, high value-added products can be synthesized (McGinn, 2011). As a prokaryotic algal genus,species are spirally formed through multi-cell aggregation, which can exist in theenvironment with high pH and high temperature (Ogbonda., 2007). Moreover,species can produce plentiful protein, pigment and polysaccharide which can be used in food processing and pharmaceutical industries (George, 2014). Particularly, phycocyanin, a type of functional protein for human health, exhibits some characteristics such as antioxidant, anti-in?ammatory and anti-cancer (Begum, 2016; Wu, 2016). To date,species have been cultured on large-scales, bring- ing the possibility of usingto fix CO2.

Microalgae demonstrated different growth rates with va- rious concentrations of CO2aerated into the culture sub- strate (Thomas, 2016). Moreover, microalgae show- ed different performances when cultured under pure CO2and power plant flue gas supplements. Shurair(2016) estimated the CO2capture capacity of aspecies exposed to 0%, 5% and 10% CO2, finding that thespecies exhibited the highest growth rate at 10% of CO2. However, only a few works have explored the growth of microalgae exposed to unfiltered flue gas. For instance, Singh and Dhar (2019) deemed that microalgae are impro- vable for debasing and utilizing the carbon source. How- ever, the flue gas containing 10%–15% CO2inhibits micro- algal growth because of some NOx, SOx, and dust in flue gas. Therefore, it is quite necessary to screen a good strain with strong tolerance to high concentrations of CO2and flue gas (Hauck., 1996;Pavlik, 2017; Camargo and Lombardi, 2018).

In this study, we separated and purified thesamples collected from large-scale cultivation farms in In- ner Mongolia and selected ideal strains after preliminary screening. The selected strains and strain 208 were used todetermine the effects of CO2and flue gas onspe- cies with the excellent ones utilized to fix CO2from coal power plant flue gas. The evaluation was based on the bio- mass accumulation rate and the chlorophyll, soluble pro- tein and phycocyanin contents when these strains were cul- tured without CO2supplement and with 10% CO2supple- ment and unfiltered coal power plant flue gas supplement.is a genus of cyanobacteria. In this experiment, the selected strains and strain 208 were identified as. We call these isolatesstrains in this study.

2 Materials and Methods

2.1 Experimental Spirulina Strains

Thestrains used in this study included select- edstrains and strain 208. The selected strainswere isolated from seven samples collected fromfarms in Inner Mongolia, China (38?18??40?11?N, 106? 41??108?54?E). The strain 208 was stored at Applied Microalgae Biology Laboratory, Ocean University of Chi- na. The strain 208 had been cultured in large-scale open raceway ponds in Inner Mongolia, which achieved consi- derable biomass production.

2.2 Purification and Morphological Observation of Spirulina Strains

The selected strains were separated with micropipette se- paration method based on the trichome length, helix width and spiral number. The single algal filament selected from the same sample was marked with sample labels. For ex- ample, the isolated strains fromsample 1 were tagged as 1-1, 1-2, 1-3 among others.

All selected microalgal strains were cultured with mo- dified Zarrouk medium (Zarrouk, 1966) in 24-well plates placed in light incubator at 25℃ and in a 12h:12h light: dark cycle. Two weeks later, the strains were observed un-der a microscope to analyze their singleness. The pure strains were transferred into 50-mL triangle bottles. At exponen- tial growth phase, the isolated and purified strains were inoculated to 100mL triangle bottles with an initial opti- cal density at 750 nm of 0.1, respectively. The strains were cultured in the light incubator set at a 12h:12h light:dark cycle, 100μmol photonsm?2s?1on bottle surface, 28℃ and 100rmin?1. Five mL of microalgal culture was filtered to measure dry cell weight.

The morphological parameters including trichome length, helix width and spiral number were measured for the se- lected strains. These parameters were counted using a mi- croscope equipped with a micrometer. The length, pitch and spiral number were averages of 30 filaments.

2.3 Growth of Spirulina Strains Under Different Aeration Conditions

Three aeration conditions included no CO2supplement and 10% CO2and coal power plant flue gas supplements were used to culture the isolated strains at 28℃ and in a 12h:12h light:dark cycle.

2.3.1 Growth of the strains without CO2supplement

Without CO2supplement, the strains were cultured in triangle flasks with 300mL volume. The strains were in- oculated with a biomass of 0.1gL?1and cultured in a light incubator and at 100rmin?1and 100μmolphotonsm?2s?1on the surface of triangle flask for 8d with 10mL of cul- ture collected every 24h for biomass determination.

2.3.2 Growth of the strains with 10% CO2supplement

The strains were cultured in column photobioreactors (inner diameter 49mm, outer diameter 53mm). The me- dium was inoculated with 0.1gL?1as initial biomass con- centration. The culturing volume was 800mL. The strains were cultured continuously. At 100mLmin?1, the culture medium was aerated continuously with 10% CO2for 12h, approximately 72Ld?1. In dark, air was aerated into cul- tures at the same rate. The irradiation was 100μmol pho- tonsm?2s?1on the surface of reactor. In this experiment, thestrains were cultured for 8d, and 10mL of culture was taken every 24h for biomass determination.

2.3.3 Growth of the strains with unfiltered power plant flue gas supplement

The strains were cultured at Hairong Microalgae Co., Ltd., Penglai County, Shandong, China. Owning to the highlevel of hardness of water used in the experiment, calcium chloride and magnesium sulfate were not added to the me-dium. The strains were cultured in 5-L spherical bottles (in- ner diameter 224mm, outer diameter 232mm) with 3.5L of medium. The initial concentration was 0.1gL?1and the irradiation was 100μmol photonsm?2s?1on the surface of reactor. During the light phase, the unfiltered power plant flue gas was continuously aerated into the cultures for 8h at 400mLmin?1(192Ld?1). The power plant flue gas con- tained 13% (v/v) CO2, 6.7% O2, 26×10?6CO, 115×10?6NO, 129×10?6NOx, 14×10?6NO2and 30×10?6SO2(Zhu, 2014). Similarly, the strains were cultured for 8d and 10mL of culture was collected every 24h for biomass de- termination.

2.4 Measurement of pH

Two microliters of microalgal culture was collected every24h to determine the pH with a pH meter (Sartorius PB-10).

2.5 Determination of Biomass, Specific Growth Rate and Biomass Production

The microalgal cells in culture were filtered onto a What- man GF/C filter paper, 47mm in diameter, with the aid of a vacuum pump. The cells were washed thrice with distil- led water and dried at 70℃ overnight.

The specific growth rate (μ, d?1) and biomass produc- tion (P, gL?1d?1) were calculated with Da Silva Vaz. (2016):

=(lnX–ln0)/(?0),

P=(X?0)/(?0),

whereXrepresents the biomass at time(day) and0represented the biomass at time0(day).

2.6 Measurement of Harvesting Efficiency

strains were harvested with 400-mesh silk screens (aperture 37–38μm) at the end of culture. The har- vesting efficiency was calculated with

Harvesting efficiency(%)=((0?1)/0)×100%,

where0represents the dry weight of strain on Whatman GF/C membrane without filtration, and1represents the dry weight of strain on Whatman GF/C membrane after filtration.

2.7 Measurement of Chlorophyll a Content

Chlorophyllcontent was measured with method de- scribed by Lichtenthaler in 1987 with modifications. First, 10mL of culture was collected and mixed with 10mL of 90% methanol. Then, the mixture was incubated at 55℃ in a water bath for 15min, centrifuged at 8000rmin?1for 15min. The absorbance of the supernatant was measured at 665nm and 652nm wavelengths on a spectrophotome- ter (UV-8000; Metash, Shanghai, China). Chlorophyllcontent was calculated with Xiong(2016):

Chlorophyll(mgL?1)=16.82A665?9.28A652.

2.8 Determination of Protein Content

2.8.1 Determination of soluble protein content

The soluble protein was extracted with BCA protein as- say kit and the method enclosed (Wiechelman., 1988). The processed samples were placed in an incubator set at 37℃ for 25min and assayed at 562nm on a microplate reader (Synnergy HT; BioTek, USA). The soluble protein content was converted according to the standard bovine se-rum albumin curve.=0.8633+0.0588 (2=0.9967, whererepresents OD562value whilerepresents protein content).

2.8.2 Determination of phycocyanin content

The dry algal powder (30mg) was fully ground and 5mL of phosphate buffer (0.15molL?1PBS, pH=7.0) was add- ed. All treated samples were placed at 4℃ for 36h and then centrifuged at 8000rmin?1for 5min. The absorption of supernatant at 620nm and 652nm wavelengths were measured using a spectrophotometer (UV-8000; Metash, Shanghai, China). The concentration and content of phyco- cyanin were calculated with following equations (Bennett and Bogorad 1973; Rathnasamy and Debora, 2014):

Phycocyanin (gL?1)=(OD620?0.474OD652)/5.34,

Phycocyanin (%)=(CV/M)×100%,

where C, V and M represent concentration, volume of PBS (0.005L), and weight of algal powder, respectively.

2.9 Statistical Analysis

All data were expressed as mean±SD and processed using Origin Pro 9.0. Difference amongstrains was calculated with the same method with the significance tested with Tukey’s test using SPSS Statistical Package (v 22.0). The difference was significant if<0.05.

3 Results and Discussion

3.1 Purification and Screening of Spirulina Strains

All the 7samples were mixtures of strains; they contained individuals with different filament length and helix pitch. A total of 30 strains were isolated (Table 1). These strains were amplified from 24-well plates, to 50-mL triangle bottles and finally to 100-mL triangle bottles, which adapted to the culture condition, and were ge- netically stable in a few months.

The trichome length, helix width and spiral number of the selected strains showed significant differences. For exam- ple, among six strains (7-2, 7-3, 7-4, 7-5, 7-8 and 7-9) pu- rified from sample 7, the filament length of strain 7-2 was the longest (532.95μm) and the filament length of strain 7-5 was the shortest (53.46μm). The helix numbers ranged from 3 to 11 while the helix pitch varied between 58.91μm and 14.85μm. The strains isolated from other samples also showed obvious difference in their morphology. Be- causesp. grew by cell division, its trichome length could increase in the culture cycle (Cheng, 2018). The filament length and helix number of the microalga in- creased during the process of growth while the helix width of the strains exhibited no change when the culture envi- ronment remained the same. Eachhas its unique helix width, which indicates the difference in the microal- gal morphology. In addition, the trichome length and helix pitch ofsp. were associated with harvesting ef- ficiency (Cheng, 2018). The helix width and trichome length are important characteristics ofrelevant to actual production. A long filament length could improve the biomass harvesting efficiency (Zhu, 2020). The strains performing short lengths of filament could be knock- ed out in large-scale culture. Thus, it was necessary to ob- tain the filament length and helix width of the strains.

The 30 isolated strains were cultured in 100-mL triangle bottles for preliminary screening. The biomass of strains were measured, and strain 208 was used as control. Fig.1 shows the biomass accumulation of strains in 7 days. The strain 7-8 showed the highest biomass (1.63gL?1), follow- ed by strain 7-5 (1.43gL?1), strain 7-2 (1.42gL?1), strain 7-3 (1.41gL?1), strain 6-10 (1.40gL?1), strain 2-6 (1.40g L?1), strain 1-3 (1.40gL?1), strain 7-4 (1.38gL?1), strain 6-5 (1.31gL?1) and strain 4-5 (1.29gL?1). These strains hadbiomass higher than strain 208 (1.17gL?1). The strains showing the highest biomass of each mixture samples in- cluded strain 1-3 (1.4gL?1), strain 2-6 (1.4gL?1), strain 3-2(1.19gL?1), strain 4-5 (1.29gL?1), strain 5-1 (1.22gL?1), strain 6-10 (1.4gL?1) and strain 7-8 (1.63gL?1). The top five strains from the seven samples were selected, which included strain 1-3, strain 2-6, strain 4-5, strain 6-10 and strain 7-8. All these strains had been cultured in large-scale open raceway ponds, which provided an application base to use them to explore their growth under different aera- tion conditions.

Table 1 The filament length, helix width andhelix number for Spirulina strains

Fig.1 The final biomass (at 7th day of growth) for Spirulina strains. Error bars represent the mean±SD and different superscripts indicate signi?cant difference (Tukey’s test; P<0.05). 1, Spirulina strain 1-1; 2, Spirulina strain 1-3; 3, Spirulina strain 1-5; 4, Spirulina strain 1-8; 5, Spirulina strain 2-1; 6, Spirulina strain 2-2; 7, Spirulina strain 2-6; 8, Spirulina strain 2-7; 9, Spirulina strain 2-8; 10, Spirulina strain 3-2; 11, Spirulina strain 3-8; 12, Spirulina strain 4-3; 13, Spirulina strain 4-4; 14, Spirulina strain 4-5; 15, Spirulina strain 4-10; 16, Spirulina strain 5-1; 17, Spirulina strain 5-2; 18, Spirulina strain 5-5; 19, Spirulina strain 5-7; 20, Spirulina strain 6-1; 21, Spirulina strain 6-5; 22, Spirulina strain 6-7; 23, Spirulina strain 6-8; 24, Spirulina strain 6-10; 25, Spirulina strain7-2; 26, Spirulina strain 7-3; 27, Spirulina strain 7-4; 28, Spirulina strain 7-5; 29, Spirulina strain 7-8; 30, Spirulina strain 7-9; 31, Spirulina strain 208.

3.2 Growth of Spirulina Strains Without CO2 Supplement

When the strains were cultured without CO2supplement, the prime pH value of six strains was 9.30±0.01 (Fig.2A). During the breeding cycle, the pH of cultures kept increas- ing, which contributed to the conversion of CO2into HCO3?,providing inorganic carbon for the growth of microalgal strains (Chen, 2016). Finally, the pH arrived at 10.05±0.01 on the 8th day. The growth curve of six strains, based on the biomass, is indicated in Fig.2B. The biomass of six strains significantly increased with time. The strain 4-5 ex-hibited the highest biomass (1.880gL?1) and the strain 7-8 presented with the lowest biomass (1.660gL?1) (Table 2); however, the biomass of six strains showed no significant difference. Different collection times and experimental con- ditions, such as light, temperature and gas composition, ledto differences in different biomass accumulation (Cheah and Show, 2015). Rafiqul(2003) reported thatachieved a biomass at 2.3gL?1when it was cultivated in Zarrouk media, higher than that of our strains. Moreover, when no carbon dioxide ?lter-sterilized air was aerated into the photobioreactors, the maximum biomass concentration was 0.85gL?1in 21 days (de Morais and Costa, 2007), which was lower than ours. A previous research also reported that thesp. LEB 18 show- ed a biomass of 1.05±0.08gL?1when cultured with Zar- rouk synthetic medium in a photobioreactor (Cardoso,2020), which was also lower than that of our strains. Inanother research,sp. LEB 18 obtained the bio- mass at 1.25±0.01gL?1, cultured with Zarrouk medium inoutdoor cultivation (Mata, 2020). In this study, sixstrains exhibited similar biomass production, which show- ed no significant difference. As shown in Fig.2C, thestrain 7-8 showed the highest harvesting efficiency (94.37%), while the strain 1-3 showed the lowest harvest- ing efficiency (80.61%). These results indicated that the harvesting efficiency was basically consistent with the fila- ment length ofstrain. The strain 7-8 had a rela- tively long filament length (409.86±61.95μm), while the filament length of strain 1-3 was only 266±95.21μm (Table 1).

Fig.2 The change in the pH (A), biomass (B), harvesting efficiency (C), the final pigment content (D), and protein content (based on dry biomass (DW)) (E) of six Spirulina strains cultivated without CO2 supplementation.Error bars represent the mean±SD calculated from three replicates. Different superscripts indicate signi?cant difference in the six Spirulina strains (Tukey’s test; P<0.05).

Table 2 Growth parameters of Spirulina strains cultured without CO2 supplement, with 10% CO2 supplement and with unfiltered coal-fired power plant flue gas supplement

Notes: Data are shown as mean±SD (=3). Different superscripts indicate signi?cant difference (Tukey’s test;<0.05).

The chlorophyllcontent of five strains is displayed in Fig.2D. The strain 7-8 obtained the highest chlorophyllcontent at 35.55mgL?1, followed by that of strain 208 (31.67mgL?1) and strain 6-10 (31.23mgL?1). However, the strain 2-6 showed the lowest chlorophyllcontent at 25.60mgL?1, which manifested significant difference with strain 7-8. Rafiqul(2003) reported that the chlorophyllcon- tent ofcultured in Zarrouk medium was only 12.5mgL?1, which was significantly lower than our result. In addition, our result was similar to the report of Mata, (2020), who reported that thesp. LEB 18 showed the highest chlorophyllcontent at 37.64μgmL?1when cultured with Zarrouk medium.

not only showed a high biomass but also con- tained affluent nutritive material; it was the prime micro- organism used to produce phycocyanin (Hsieh-Lo, 2019). Phycocyanin, a protein with antioxidant properties, showed the application value ofto a greater ex- tent. The contents of the soluble protein and phycocyanin of six strains were analyzed at the end of the cultivation period. As shown in Fig.2E, there were significant differ- ences in the extent of protein accumulation among the six strains. These results indicated that strain 208 showed the highest soluble protein content (51.59%), while strain 2-6 showed the lowest soluble protein content (35.06%). Simi- larly, the highest phycocyanin content was 11.33%, which was obtained from strain 208. The strain 2-6 showed the lowest phycocyanin content (3.48%), exhibiting a signifi- cant difference with strain 208.

Six strains showed no significant difference in the amountof biomass when they were cultured in the absence of CO2. In this experiment, strain 4-5 exhibited the maximum bio- mass content (1.880gL?1), while strain 7-8 showed a high chlorophyllcontent at 35.55mgL?1. It was found that strain 208 had the highest soluble protein content (51.59%)and phycocyanin content (11.33%) (Fig.2E). Based on these results, we plan to eliminate strain 1-3 from the next ex- periment because its harvesting efficiency was the lowest and the extent of biomass accumulation, content of the so- luble protein, and phycocyanin content were not high when compared with those of other strains.

3.3 The Growth of Spirulina Strains with 10% CO2 Gas Supplement

A previous research referred the ?ue gas from the coal power plant usually contained 10%–15% CO2(Lee, 2002). Wang(2014) also suggested that the CO2con-centration of flue gas in the power plants was usually 10%–20%. Therefore, the concentration of 10% CO2was se- lected to explore the growth of the strain 208, strain 2-6, strain 4-5, strain 6-10, and strain 7-8 in column photobio- reactors.

Once CO2enters the nutrient medium, it gets transfer- red into various forms, for example, CO32?, HCO3?and H2CO3, whichcan induce a change in the pH of medium. In our study, the pH value of five strains showed a similar change trend during the breeding cycle (Fig.3A). On the first day, the pH value descended from 9.64 to 8.59. From the third day onward, the pH ?uctuated and was stabilized at 8.48 (observed in 5 strains). It has been reported that the pH value ofexposed to 12% CO2was 7.08– 8.76 in 21 days (de Morais and Costa, 2007). Whenwas cultured with 3% CO2and 6% CO2, Yong and Lee (2018) found that the medium pH decreased from 9.5 to 8.7–8.8 and 8.4–8.5, respectively.Cheng(2017) also found that the pH decreased from 8.65 to 8.25 and thebiomass yield ofmutant decreased by 84.9% whenthe CO2concentration increased from 5% to 10%. It could be seen thatthe influence of CO2on the pH of the me- dium was obvious and that the adaptation to CO2may be the adaptation to the pH.

The dried biomass weight is depicted by a growth cur- ve during the culture period (Fig.3B). Our result showed that the growth trends demonstrated no significant differ- ence among them. The highest final biomass (1.603gL?1) was measured from the strain 7-8, and the strain 4-5 show- ed the lowest biomass (1.327gL?1). The specific growth rate and biomass productivity are indicated in Table 2. Therange of the biomass productivity in strains was from 0.188gL?1d?1to 0.153gL?1d?1. Our result was consistent with those of previous researches. For example, de Morais and Costa (2007) reported the maximum speci?c growth rate and maximum productivity were0.33d?1and 0.17gL?1d?1when thesp. was cultivated with 12% CO2in a 3-stage serial tubular photobioreactor for 21 days. Duarte. (2020) reported that thesp. LEB 18 obtain- ed the highest specific growth rate at 0.20±0.01d?1when it was aerated into 10% CO2in tubular photobioreactor. In addition, Tan(2015) cultured threemu- tant strains with 12% CO2, in which the mutant strain 3- A10 showed the highest growth rate (0.118gL?1d?1). Ac- cording to Da Silva Vaz. (2016),spLEB 18, cultivated in 10% carbon dioxide, acquired the maximal biomass concentration (0.59±0.04gL?1), maximal yield (0.05±0.00gL?1d?1) and maximal specific growth rate (0.11±0.01d?1), which was relatively lower than our re- sult. Different CO2concentration, different initial biomass concentration and photobioreactors may affect the growth parameters (Duarte., 2020).

Fig.3 The change in the pH (A), biomass (B), chlorophyll a content (C), and protein content (based on dry biomass (DW)) (D) for five Spirulina strains under 10% CO2 aeration. Data are expressed as the mean value±SD (n=3). Different superscripts indicate signi?cant difference of five strains (Tukey’s test; P<0.05).

Compared with the culture condition in the absence of CO2, the specific growth rate of five strains all decreased after 10% CO2was aerated, indicating that the 10% CO2generated an inhibition effect on the growth of the expe- rimental strains. The range of the specific growth rate in the strains was 0.351d?1to 0.367d?1 in the absence of CO2,while the range of the specific growth rate was from 0.323d?1to 0.347d?1under 10% CO2culture condition. The strain4-5 showed the highest growth rate, while the strain 7-8 gave the lowest growth rate when cultured in the absence of CO2. However, the growth rate of the strain 7-8 was thehighest among them after CO2aeration. Our result also de- monstrated that the growth rate of strain 7-8 decreased from0.351d?1to 0.347d?1, which descended only 1.14%, whilethe strain 4-5 decreased from 0.367d?1to 0.323d?1, de- creasing by 11.9%. It was found that the strain 7-8 show- ed a strong tolerance to 10% CO2condition. Biomass pro- duction of the strains also showed similar trends.It had been reported that the biomass production ofin three different pH cultures controlled by CO2-fed were low- er than that in the normal Zarrouk medium (Mehar, 2019).

As shown in Fig.3C, the chlorophyllcontent of the five strains increased with growth time. The range of ini- tial chlorophyllcontent was from 1.75 to 2.49mgL?1 in the strains. On the 7th day, the highest chlorophyll a con- tent (18.22mgL?1) in the five strains was obtained from strain 7-8, while strain 208 showed the lowest content at 12.09mgL?1. The statistical result indicated that all strains had no significant difference in the chlorophyllcontent. Tan(2015) reported the chlorophyllcontent of mu- tants and the wild strain in three different CO2concentra- tions, in which the mutant strain 4-B3 achieved the high- est content (3.82mgg?1) in 12% CO2. A previous report mentioned that the synthesis of photosynthetic pigments could be affected by some external factors, including CO2concentration (Wang, 2019). Compared with the re- sult shown in Fig.2D, the chlorophyllcontent in all the strains decreased after 10% CO2was aerated. Although the strain 7-8 showed the highest chlorophyllcontent in the strains, the content was 49.23% lower than that in the ab- sence of CO2. In addition, the chlorophyllcontent of strain208 showed the largest reduction, which was 61.83% low-er than the content in the absence of CO2supplementation.

Fig.3D exhibits the protein content of five strains. The highest soluble protein content was obtained from strain 208 (46.05%). The strain 4-5 showed the lowest soluble protein content at 32.65%, showing a significant difference when compared with the strain 208. Furthermore, the five strains showed a significant difference in the phycocyanin content (Fig.3D), of which the strain7-8 showed the high- est phycocyanin content (7.06%) while strain 4-5 showed the lowest phycocyanin content (3.90%). Gordillo(1998) found the soluble protein and phycocyanin signi?- cantly decreased in 1% CO2condition. Compared with theobservations of Fig.2E, the protein content and phyco- cyanin content of the strains drooped except for strain 7-8. Although the strain 208 showed the highest protein con- tent in 10% CO2, its protein content was still 10.74% low- er than that in the absence of CO2. Moreover, the protein content of strain 4-5 showed a significant reduction (from 42.10% to 32.65%) with the change in the CO2content. The strain 208 showed the greatest reduction in phyco- cyanin content from 11.33% to 4.89%. The previous re- search also found the protein content ofde- creased with an increase in the CO2concentration and aera- tion time (Braga, 2018). However, the soluble pro- tein content of strain 7-8 was 43.35% in 10% CO2, which increased to 8.16% than of the content in the absence of CO2, while it’s the phycocyanin content was the highest in this strain. The change in the protein content was consis- tent with the growth, suggesting that the strain 7-8 had bet- ter tolerance to 10% CO2from the five strains.

Although the 10% CO2condition had different inhibit- ing effects on the growth of the five strains, these strains showed a potential to grow in 10% CO2. The strain 7-8 ex- hibited the maximal biomass (1.603gL?1), the higher so- luble protein content (43.35%), and the highest phyco- cyanin content (7.06%), which showed excellent tolerance and adaptability to 10% CO2among the five strains.

3.4 The Growth of Spirulina Strains in 100% Unfiltered Power Plant Flue Gas

The fivestrains were exposed to the coal-fired flue gas from the power plant to test the growth. As indi- cated in Fig.4A, the pH of the five strains dropped to 8.37 within 9h and then fluctuated between 8.18 and 8.24 from the third day to the end. However, Chen(2016) and Zhu(2020) reported that the optimal pH forwas 9.5. An optimum and steady culture pH could help microalgae to utilize more CO2, while the lower pH could restrain the growth potential of algae. The change in the pH may be due to the dissolution or consumption of CO2and the consumption of nutrients in the medium and the formation of other products (Grima, 1999). Some researches added phosphate buffer to alleviate the pH re- duction caused by CO2, NOx, and SOxin the flue gas (As- lam, 2017). When culturing microalgae strains with CO2or flue gas in the future, the pH may be a concern.

Fig.4 The change in the pH (A) and protein content (based on dry biomass (DW) (B) for five Spirulina strains during unfiltered coal-fired power plant flue gas aeration. Data are expressed as the mean value±SD (n=3). Different superscripts indicate signi?cant differences of five Spirulina strains (Tukey’s test; P<0.05).

Table 2 displays the biomass, specific growth rate, and productivity of the strains in unfiltered power plant flue gas.Compared with that under 10% CO2culture, the growth of the five strains in the unfiltered power plant flue gas re- duced significantly. The specific growth rate of strain 208 decreased from 0.337 to 0.170d?1with a decrease of 49.55%, indicating that the flue gas showed a significant inhibitory effect on the growth of all experimental strains. The ma- ximum specific growth rate ofspLEB 18 cul- tivated in combustion gas for 12d was 0.06d?1(Da Silva Vaz., 2016), which was lower than our result.In ad- dition, our experiment result wasconsistent with the re- sult of Duarte(2017) who reported the specific growthrate (0.14±0.04d?1) ofin CO2flue gas. It was also reported that the maximum productivity of thesp. LEB 18 cultivatedwith simulated coal ?ue gas in intermittent mode was 0.08±0.01gL?1d?1(Radmann, 2011). These results also showed that the flue gas had significant inhibitory effects on the growth of. The low growth and biomass of strains in unfiltered power plant flue gas may be due to the complex ?ue gas composition, the longtime aeration, and the poor pH va- lue. The high concentration of SOx, NOx, and particulate material could inhibit the growth of some algae species (Lee, 2002; Kumar., 2014). The previous re- search reported that the reduction of pH could affect the activities of enzymes involving carbon sequestration, such as extracellular carbonic anhydrase and low pH restrained cell growth (Tang, 2011). According to our result, there were significant differences in the extent of the de- cline among the five strains (Table 2). For example, the specific growth rate of strain 4-5 decreased by 43.65% from 0.323 to 0.182d?1, and the specific growth rate of strain 7-8 decreased by 56.48% from 0.347 to 0.151d?1. These results indicated that the tolerance of microalgae strains to flue gas was different. Although these five strains had low growth, it was possible to obtain suitablestrains for CO2fixation in coal-fired power plants flue gas through directional screening.

As for the proteins, the previous study reported that pro- tein content could reach 46%–63% in(Aika- wa, 2012). In our study, the strain 6-10 displayed the highest soluble protein content at 66.02% and the protein content of strain 208 was 60.37% (Fig.4B).strain7-8 attained the highest phycocyanin content (9.28%), which was significantly higher than the others. The phy- cocyanin content of strain 208 was 7.5%, followed by that of strain 7-8. The strain 4-5 who showed the highest bio- mass had the lowest protein content (38.31%) and phyco- cyanin accumulation (4.72%). Inversely, the strain 7-8 show- ed a high phycocyanin content but a low biomass. The fast growth of algae may need more substance and energy to support, which may consume protein. Chen(2010) reported that a fast growth could consume lots of nitrogen, which may cause a reduction of in the phycocyanin con- tent offering nitrogen source. Moreover, when compared with that in Fig.3D, the content of soluble protein and phy- cocyanin in the five strains was significantly higher than that in 10% CO2. The soluble protein content of strain 6-10increased from 42.61% to 66.0%, while the protein of strain 4-5 changed from 32.65% to 38.31%, thus increas- ing by 17.34%. In addition, the phycocyanin content of strain 7-8 was 31.44% higher than that in 10% CO2. Pre- vious research reported that the production of protein in- creased when the microalgae were cultured with CO2in flue gas containing fly ashes (Braga, 2019).

When five strains were cultured in unfiltered flue gas, the strain 4-5 showed the best growth with a specific growth rate at 0.182d?1. Although the biomass in the five strains was non-ideal, the protein content in the strains was high. These results indicated that the strains might have the po- tential to fix CO2in unfiltered flue gas through directional screening. Furthermore, the method of elevating the growthof strains in flue gas needs to be explored in the future,which is important to directly use coal power plant flue gas under large-scale cultivation.

4 Conclusions

The changes in the growth status and physiological in- dexes ofstrains under 10% CO2and unfiltered coal-fired power plant flue gas culture conditions were test- ed in order to determine the ideal strain with strong tole- rance to CO2and coal-fired power plant flue gas. The re- sults revealed that the experimental strains could tolerate 10% CO2. When the strains were cultured in 10% CO2, the highest biomass was 1.603gL?1, obtained from strain 7-8.The unfiltered coal power plant flue gas couldsignificant- ly inhibit the growth of all these strains, while it showed no negative impact on their protein accumulation ability.strain 4-5 was the superior strain in unfiltered coal power plant flue gas based on the growth. All experi- mental strains cultured in CO2or coal-fired power plant flue gas showed different tolerance abilities. Our results in-dicated that it is possible to obtain suitablestrains to fix coal-fired power plants flue gas through directional screening. Excellent strains could be cultured in large-scale open pond to fix CO2, relieving environmental pressure, and generating economic benefits. In the future, it would be important to improve the productivity and carbon se- questration benefits of microalgae in flue gas.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2016YFB0601 001). We also thank the reviewers for their valuable and constructive comments and all the staff at the Laboratory of Applied Microalgae Biology for their help during the experiment.

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