Semi-continuous cultivation of indigenous Chlorella sorokiniana for biomass and pigment production

. The characteristics of microalgae, the composition of the growth medium, cultivation parameters, and the design of photobioreactors should be considered when obtaining biomass and biologically active substances from microalgae. Continuous and semi-continuous cultivation of microalgae at optimal hydraulic retention time (HRT) is one of the most promising approaches to optimizing the accumulation of biomass and desired metabolites. The continuous nutrient supply to photobioreactors avoids nutrient limitation and maintains algal biomass productivity at its maximum level. This study reports the effect of HRT on the growth of Chlorella sorokiniana and nutrient uptake by algal cells. The maximum cell density in the photobioreactor was observed during cultivation at HRT of 5 days, while the concentration of pigments and ammonium uptake remained at a high level at HRTs of 5 – 2.5 days. The obtained results demonstrate that C. sorokiniana can grow efficiently under semi-continuous cultivation conditions and can be considered to produce valuable metabolites.


Introduction
Microalgae are a group of photosynthetic microorganisms of great scientific and industrial interest.These organisms have high metabolic activity and great potential for various biotechnologies.The incredible diversity of microalgae makes it possible to select biotechnologically promising strains with the characteristics necessary to obtain the desired products (biomass, proteins, polyunsaturated fatty acids, pigments, antioxidants, and vitamins).In addition, different species of microalgae are effectively used in bioremediation and carbon dioxide sequestration processes [1,2].
Currently, interest in the cultivation of microalgae as well as cyanobacteria has increased, one of the reasons for which is their ability to exhibit high radical scavenging activity [1,3].The main antioxidants found in algae are vitamins, phenolic compounds, carotenoids (e.g., β-carotene, zeaxanthin, and neoxanthin), chlorophylls, and fatty acids [1,[3][4][5][6].Microalgal pigments, such as carotenoids and chlorophylls, are often used in industries such as food, nutraceutical, pharmaceutical, and cosmetic [7].One of the advantages of obtaining antioxidants from microalgae is the versatility of their cultivation as well as the possibility of applying many technological improvements to enhance the quality of the target products.
Green algae (Chlorophyta), as well-known representatives of photoautotrophic microalgae, efficiently convert the main inorganic nutrients (carbon dioxide, ammonium, nitrate, phosphate, and sulfate) into biomass enriched with lipids, proteins, carbohydrates, pigments, and other valuable compounds of photosynthesis.Chlorella is one of the most popular and best studied genera of green microalgae (Chlorophyta; Chlorellales; Chlorellaceae).Chlorella cells show a high growth rate, ease of cultivation, and high nutrient assimilation, including when cultured in various wastewater systems.Chlorella cells contain high levels of proteins (including essential amino acids), polyunsaturated fatty acids, carbohydrates, pigments, and vitamins.Chlorella extracts can be used as supplementary foods for humans and animals [8][9][10].Species of greatest scientific interest include Chlorella vulgaris and Chlorella sorokiniana.Successful use of C. sorokiniana culture in various biotechnologies is possible due to its cultural features.The competitive advantage lies in the fact that this algal species is characterized by relatively high growth rates [11][12][13].
Obtaining algal biomass directly depends on the choice of cultivation regimens and the composition of the culture medium.Many studies have been carried out on the selection of methods of cultivation to improve microalgal growth and the content of useful metabolites such as proteins, lipids, and antioxidants [14][15][16].In batch cultures, it is impossible to maintain a constant level of nutrients.Another disadvantage of implementing batch culture is the time required to complete one batch [17,18].These shortcomings can be overcome with semi-continuous or continuous cultivation regimens since biomass productivity will be achieved within hours or days of cultivation and can be maintained for a longer period in an optimized state.Under continuous conditions, cell density can be controlled and kept constant by varying the dilution rate in the reactor.Continuous cultivation maintains a constant level of nutrients in the environment, ensuring stable production of biomass and metabolites.To optimize cultivation in this mode, it is necessary to establish the relationship between the availability of nutrients and cell growth rate, as well as the hydraulic retention time (HRT).Hydraulic retention time is the period of retention of the growth medium in the system, determined by the daily volume of the supplied medium.HRT values can vary from 20 to 2 days.Many researchers demonstrated that the biomass productivity during continuous cultivation of individual microalgae was higher compared to the values obtained in batch regimens [19,20].
The maximum productivity of algae, as well as the efficiency of nutrient uptake, depend on the HRT values of semi-continuous and continuous operations.The effect of HRT on the growth and productivity of microalgae will also be related to cultivation parameters such as temperature, light level, CO2 supply, nutrient content in the growth medium, and the characteristics of the microalgae themselves.Therefore, the aim of the present work was to determine the optimal values of the HRT for obtaining high levels of biomass and pigments by the green microalga Chlorella sorokiniana under semi-continuous operations.

Microalgal strain
The microalgal culture used in this study was the indigenous freshwater Chlorella sorokiniana strain AM-02.Microalga was isolated from a waterbody located in the city of Kazan (Republic of Tatarstan, Russia).The taxonomic affiliation of a pure culture of microalga was determined based on the analysis of the nucleotide sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene.The culture was maintained and stored on a standard medium BBM (Bold's basal medium [21]) with the addition of antibiotics (ampicillin and kanamycin) to reduce the risk of bacterial contamination.

Pre-cultivation conditions
Prior to the start of the experiments, single colonies of microalga were placed in 250 mL Erlenmeyer flasks containing standard BBM under sterile conditions.The inoculum was grown with shaking at 120 rpm at 26°C under continuous illumination (400 μmol photons m −2 s −1 ) for 5 days.Then biomass was concentrated by centrifugation at 5000 × g for 3 min and used for experiments in a photobioreactor.

Experimental setup for semi-continuous
C. sorokiniana cells were semi-continuously cultivated in a Labfors 4 Lux photobioreactor (total volume 3.6 L, working volume 2.6 L; Infors HT, Bottmingen, Switzerland) with regular monitoring of the level of carbon dioxide, photosynthetic photon flux density, temperature, and stirring of the culture medium.The photoautotrophic algae cultivation was carried out in a sterile modified BBM containing NH4Cl as the sole source of nitrogen (NH4 + : ~220 mg L -1 ).This modified medium was chosen because of the enhanced growth of algae observed in our early experiments [12].The use of ammonium ions as a nitrogen source is more appropriate since it is this form of nitrogen that, participating in nitrogen metabolism, entails less energy loss by cells [22,23].
Aeration (0.8 L min -1 ) was performed using a compressor through a 0.20 µm Midisart 2000 filter with a polytetrafluoroethylene membrane (Sartorius Stedim Biotech, Göttingen, Germany).The addition of carbon dioxide was provided by a thermal mass flow meter and controller (Vögtlin Instruments, Aesch, Switzerland).Air and CO2 were mixed and injected into the reactor (98/2, v/v).In all experiments, the photosynthetic photon flux density was 1200 µmol photon m -2 s -1 (provided by Gro-Lux tubes), and the stirring speed was 120 rpm.The light intensity on the surface of the vessel was measured using a photosynthetically active radiation meter (Apogee Instruments, USA).The light intensity and carbon dioxide level were selected based on previous experiments performed to observe C. sorokiniana cell growth and nutrient removal rates at various light intensities and carbon dioxide levels [24].pH was measured using an EasyFerm Plus PHI K8 200 electrode (Hamilton, OH, USA) and kept constant at 7.0 ± 0.05.When foam was detected in the photobioreactor, a sterile 2% defoamer (Antifoam B, Sigma-Aldrich, St. Louis, MO, USA) was used.Light intensity, temperature, pH, pressure, carbon dioxide flux, and percentage of the released molecular oxygen and carbon dioxide were measured by Infors devices (Infors HT, Bottmingen, Switzerland).
To study cultivation in a semi-continuous mode, before the culture reached the stationary phase of growth, a certain volume of growth medium was taken from the culture vessel and a new portion of the nutrient medium was added, maintaining a total working volume of 2.6 L in the Labfors 4 Lux photobioreactor.Thus, 5.0, 4.0, 3.5, 3.0, and 2.5-day hydraulic retention times were tested.The present work consisted of studying the role of hydraulic retention time on the efficiency of the cultivation of planktonic algal cells in a semi-continuous mode.To test reproducibility, three independent experiments were conducted.

Analytical Methods
Algal growth was monitored daily.Algal growth was estimated by daily measurement of the optical density at 750 nm (OD750nm) with a spectrophotometer (Lambda 35, Perkin Elmer, Singapore).For estimation of total weight, the biomass of microalgae was precipitated by centrifugation (at 5000 × g for 5 min), washed twice with sterile distilled water to remove salts from the nutrient medium, and centrifuged again.The resulting biomass was dried at +80°C until a constant weight was reached.The final biomass yield (g L -1 ) was calculated as the difference between the weight of a centrifuge tube with dried microalgal sediment and the weight of a tube without biomass.Total solids and volatile solids were analyzed using a drying oven and a muffle oven, respectively [11,12].
The concentration of chlorophylls a and b, as well as total carotenoids (mg L -1 ), was determined using spectrophotometric techniques.Pigments from an algal sample were extracted with dimethyl sulfoxide, and calculations were conducted according to the equations reported by Wellburn [25].The cell numbers were counted under a microscope with a hemocytometer.Total ammonium nitrogen was measured with Nessler reagent (Sigma-Aldrich, St. Louis, MO, USA) using a Lambda 35 spectrophotometer.The absence of a bacterial contribution to the removal of ammonium ions was confirmed by microscopic and culture methods.All analyses were measured in triplicate, and the means are presented along with standard deviations.The analysis of variance (ANOVA, Tukey method, α = 0.05) was used to compare differences.

Results and discussion
In the present work, the growth of the indigenous freshwater green microalga C. sorokiniana in the photobioreactor under a semi-continuous cultivation regimen was evaluated.HRT was decreased from 5.0 to 2.5 days by replacing one portion of the growth medium with cells with a new portion of fresh medium.The growth efficiency of the culture and pigment accumulation by the culture were evaluated upon reaching the maximum optical density (OD750nm) and dry weight in three independent experiments (experiments lasted for 30 days).With a decrease in HRT to 2.5 days, the biomass level began to decline (Table 1).In our experiments, cultivation at HRT below 5.0 reduced the growth parameters of the culture of Chlorella sorokiniana (Table 1).Thus, the average values of the optical density of the suspension decreased from 11.73 ± 1.23 (at HRT of 5.0) to 9.20 ± 0.51 (at HRT of 2.5).When using the method of direct cell counting, it was also noted that in experiments with shorter HRT, the number of cells in the photobioreactor was lower (data not shown).Dry weight data obtained from semi-continuous culture indicate that the decrease in HRT values was accompanied by a decrease in algal biomass weight as well.Interestingly, regardless of the hydraulic retention time applied, the pigment concentrations (chlorophyll a, chlorophyll b, and total carotenoids) were maintained at high levels and did not vary significantly between treatments.When the HRT was shortened from 5.0 to 2.5 days, the removal of ammonium ions (NH4 + ) was also high and was in the range of 97-100% (Table 1).Our findings demonstrate that high levels of chlorophylls and carotenoids production and ammonium removal by Chlorella sorokiniana cells are possible under semicontinuous operations.
Hydraulic retention time describes the theoretical average time that the medium resides in a reactor.The selection of the optimal HRT is the subject of many studies.Thus, Gao et al. [26] noted that HRT has a direct impact on biomass production and nutrient removal by Chlorella vulgaris and Scenedesmus obliquus in membrane photobioreactors fed with secondary effluent from municipal wastewater treatment plants.The authors demonstrated that the HRT of 2 days was optimal for biomass productivity.Shorter HRT values (1 day) were suitable for microalgae growth but not sufficient for the removal of nutrients.Liu et al. [20] evaluated the growth efficiency and productivity of C. sorokiniana during continuous cultivation in 10 L bioreactors at various pH and HRT values under natural light conditions.The researchers used wastewater with a high content of ammonium and phosphate ions as a growth medium.The authors noted that after the start of continuous cultivation, the yield of algal biomass increased, but subsequently decreased, which was due to the low value of the selected HRT, which was not enough to obtain optimal algal growth under natural light conditions.After the increase in HRT, the productivity of microalgae increased.In another study, 4 days of HRT were required to completely reduce Cr (VI) from water bodies in a continuous operation using C. vulgaris cells [27].Another study explored the potential of using attached Chlorella sp. for biomass production and investigated the effects of HRT levels on microalgal growth, biomass composition, and nutrient removal using synthetic municipal secondary effluent.The authors showed that the highest microalgal growth rate was found at 2-day HRT [28].The change in HRT was also investigated in a study of the co-cultivation of green algae C. vulgaris and cyanobacteria Arthrospira platensis for wastewater treatment [29].Longer HRT (4.6 days) was proven to show better cell growth and nutrient removal than shorter HRT (HRT of 2.0 and 1.4 days).Our results are consistent with the results of previous studies on the importance of adjusting the dosage of the medium for the efficient growth and productivity of biotechnologically promising microalgal species [20,[26][27][28][29][30][31].
The present study demonstrated the feasibility of semi-continuous cultivation of C. sorokiniana to obtain biomass, chlorophylls, and carotenoids, as well as to utilize nutrients from the growth medium.The dilution rate of the system affected biomass yield, while the level of pigments did not change significantly.Finally, the maximum dry weight of algae reached the highest concentration of 3.08 ± 0.31 g L -1 with the longest HRT of 5.0 days, whereas the average concentration of total pigments was in the range of 71-79 mg L - 1 .Algae immobilization should also be tested in future experiments under semi-continuous and continuous cultivation conditions.