Enhanced Biomass and CO2 Sequestration of Chlorella Vulgaris

Process Biochemistry 90 (2020) 168–176 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Enhanced biomass and CO2 sequestration of Chlorella vulgaris using a new T mixotrophic cultivation method Qian Yua, Haonan Wanga, Xiao Lia, Yonghao Yina, Song Qinb, Baosheng Gea,* a State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, 266580, PR China b Key Laboratory of Coastal Biology and Biological Resource Utilization, Yantai Institute of Coastal Zone, Chinese Academy of Sciences, Yantai, 264003, PR China ARTICLE INFO ABSTRACT Keywords: CO2 sequestration using microalgae has been deemed as a promising way, which can fix CO2 and simultaneously Biomass produce valuable bio-products. However, large scale open-pond production of Chlorella vulgaris is normally Chlorella vulgaris cultured using acetic acid as a carbon source, which is costly and shows a low efficiency in CO2 fixation. Here, a Mixotrophy new mixotrophic culture strategy using both CO2 and acetic acid was developed and evaluated for CO2 se- Photoheterotrophy questration by a C. vulgaris culture in an open pond. Our results show that the growth rate of C. vulgaris under CO2sequestration this new mixotrophic condition reaches 0.24 g/L/d, which is higher than the 0.15 and 0.21 g/L/d of photoheterotrophic culture with acetic acids and photoautotrophic culture with CO2, respectively. The averaged CO2 fixation rate was determined as 0.29 g/L/d, which is much higher than heterotrophic method but slightlylower than photoautotrophic method. This result was further confirmed in a 1252 m open raceway pond. Physiological and biochemical characterization showed that the cell quality of C. vulgaris under mixotrophic conditions is better than those of photoautotrophic method and photoheterotrophic cultures. The enzyme activity assay and transcriptome sequencing analysis revealed that the metabolism of carbohydrates and amino acids was significantly enhanced under mixotrophic condition compared with other groups, which may attribute tothein- creased biomass and CO2 sequestration of C. vulgaris. Our results suggest that this mixotrophic strategy can be applied in large-scale cultivation of C. vulgaris for biomass production and CO2 sequestration. 1. Introduction rich in proteins, lipids, polysaccharides, and other biologically active metabolites [12], which shows its excellent applicability in the fields of The wide use of fossil fuels has significantly contribution to the food, health care, feed, and medicines. Besides, it is widely studied as a increase in atmosphere CO2 concentration [1], which causes many potential bio-energy feedstock. Therefore, culture of C. vulgaris has been environmental problems, such as global warming, climate changes and deemed as an ideal way for CO2 fixation and production of valuable deterioration of ecological environment [2]. Therefore, sequestration of bioactive substances. Although CO2 fixation using C. vulgaris has been CO2 from atmosphere has attained great attention [3]. Recently, var- widely studied, the large scale commercial application is still not rea- ious carbon sequestration methods have been developed, including lized, which is mainly due to the high cost in closed photo-bioreactor physical, chemical and biological methods [4]. However, CO2 seques- and low efficiency in open raceway pond [13]. tration with physical and chemical methods are costly and incompatible Recently, C. vulgaris has become the second largest cultured algal in with sustainable development requirements [5]. Biological CO2 se- China [14], and it is normally cultured in open raceway ponds using questration using microalgae or plants has been considered to be a acetic acid [15,16], where acetic acid act as a carbon source and also promising method [6], which can fix CO2 through photosynthesis and keeps the pH within neutral range [17]. Compared with acetic acid, meanwhile produce valuable bioactive products [7]. CO2 can also be used by C. vulgaris as carbon source with the ad- Chlorella vulgaris is a kind of single-cell eukaryotic green algae [8], vantages of higher CO2 fixation efficiency, and lower-risk ofcon- which has been reported to possess a high CO2 sequestration rate [9] tamination [18]. However, C. vulgaris cells cultured only with CO2 are and biomass productivity, favourable adaptability under different inferior to those with acetic acids or glucose in terms of cell size and temperature, nutrient and climatic conditions [10,11]. It can grow well density, and are difficult to harvest [19]. Therefore, improvement of in oceans, lakes, ponds and other water environments. C. vulgaris is also biomass production and CO2 sequestration rate is an area of great ⁎ Corresponding author. E-mail address: [email protected] (B. Ge). https://doi.org/10.1016/j.procbio.2019.11.022 Received 19 August 2019; Received in revised form 3 November 2019; Accepted 20 November 2019 Available online 20 November 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved. Q. Yu, et al. Process Biochemistry 90 (2020) 168–176 concern for application of C. vulgaris. Recently, mixtrophic cultivation 2.3. Determination of chlorophyll and protein content of microalgae has been proved to be a preferable cultivation mode for biomass, bioenergy production and bioremediation [20]. Tamarys et al. The chlorophyll content of C. vulgaris was determined as previously [16] showed that the mixotrophic culture of Chlorella protothecoides reported [24]. Briefly, 2 mL algae culture samples were centrifuged at using glucose and acetate grew better than under autotrophic condi- 6000 rpm for 10 min to harvest the cells. Then 96% methanol was tions. Monika et al. showed that biomass and lipid productivity of added, and the sample was ultrasonicated for 40 min on ice. The sample Chlorella pyrenoidosa increased significantly under mixotrophic culture was centrifuged again, the supernatant was collected and absorbance conditions of sodium acetate and glycerol. was measured at 653 (OD653) and 666 nm (OD666), respectively, with Here we report a new mixotrophic cultivation of C. vulgaris, which 96% methanol as blank. The chlorophyll content of chlorophyll a (Ca) contains two stages, one photoautotrophic cultivation stage with CO2 as and b (Cb)was calculated according to the following formula [24]: carbon source during daytime, and another heterotrophic cultivation Ca (mg/L) = (15.65 × OD666 - 7.34×OD653) × dilution factor (1) stage using acetic acid during nighttime. The biomass production, CO2 Cb (mg/L) = (27.05 × OD653 -11.21×OD666) × dilution factor (2) bio-fixation rate, and transcriptome analysis were used to evaluate the The protein content of C. vulgaris cells was determined using the effectiveness of the new strategy and its potential application forthe Bradford method [25]. large-scale carbon sequestration using C. vulgaris. 2.4. Lipid extraction and FAME (Fatty acid methyl ester) analysis 2. Materials and methods Total lipid extraction from microalgal cells was determined according to the following procedure. The lyophilized cells (W1) were 2.1. Strains and culture conditions suspended in 5 mL of chloroform/methanol (2/1, v/v) and sonicated for 30 min in an ultrasonic cleaner (KQ-100KDE, Kunshan, China) at 25 °C. The C. vulgaris strain was purchased from the Freshwater Algae After agitation, the mixture was centrifuged at 8000 g for 20 min at 4 °C Culture Collection at the Institute of Hydrobiology, Chinese Academy of and the supernatant was transferred to another pre-weighed tube (W2). Sciences (http://algae.ihb.ac.cn/), and cultured in conical flasks con- The residues were further extracted twice and the chloroform phase taining BG11 medium [21]. The cultures were divided into four groups: was collected together. After evaporating and drying to constant weight 1) photoautotrophic culture where 5% CO2 (95% air) was bubbled under nitrogen atmosphere, the lipids were gravimetrically quantified continuously into the BG11 medium with gas flow rate of 10 L/h; 2) (W3). The lipid content was expressed as % dcw (dry cell weight) as photoheterotrophic culture where 0.05 M of acetic acid was added to according to the following formula: act as carbon source and regulate the pH of BG11 medium within Lipid content(% dcw)=(W3-W2)×100/W1 (3) 6.5–7.5; 3) mixotrophic culture, where 5% CO2 was continuously The fatty acid content of C. vulgaris was characterized according as bubbled during daytime, and acetic acid was added at night to regulate previously reported [26,27]. Briefly, 20−35 mg algae pellets were the pH value within neutral range; 4) control group, where C. vulgaris thoroughly mixed with 5 mL of sulfuric acid-methanol solution in a 10 was cultured in BG11 medium without supplement of CO2 or acetic mL round bottom flask. After the solution was methylated using con- acid. All the algal cultures were incubated under LED fluorescent lamp densation reflux at 70 ℃ in a water bath for 1 h. The sample was cooled with illumination intensity of 120 μmol photons m−2 s−1, and day: to room temperature, 0.75 mL of deionized water and 2 mL of n-hexane night cycle at 12 h:12 h. The culture temperature was maintained at 25 was added into the flask for extraction. Then the upper liquid wasex- ± 1 °C by the temperature-controlled incubator. tracted and analyzed on an Agilent 1100 gas chromatography. The pilot scale cultivation was conducted in a 125 m2 open raceway pond by mixing with a six-blade paddlewheel. The C. vulgaris cells were 2.5. Carbonic anhydrase and Rubisco activity assay cultured under natural light and temperature conditions with the depth of 20 cm. The 5% CO2 was continuously bubbled during daytime, and The activity of carbonic anhydrase (CA) was determined using the acetic acid was added at night to regulate the pH value within 6.5-7.5. following procedure: moderate amounts of algal cells were harvested The pond cultured with acetic acid was chosen as control. and suspended in 10 mM Tris-HCl buffer (pH 8.3) [28].

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