Algal Research 26 (2017) 436–444

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Algal Research

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Eﬀective control of Poterioochromonas malhamensis in pilot-scale culture of MARK Chlorella sorokiniana GT-1 by maintaining CO2-mediated low culture pH ⁎ Mingyang Maa,b,c, Danni Yuana,b,c, Yue Hea,b, Minsung Parka, Yingchun Gonga,b, , ⁎⁎ Qiang Hua,b,d,e, a Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c University of Chinese Academy of Sciences, Beijing 100049, China d SDIC Microalgae Biotechnology Center, China Electronics Engineering Design Institute, Beijing 100142, China e Beijing Key Laboratory of Algae Biomass, Beijing 100142, China

ARTICLE INFO ABSTRACT

Keywords: Although predators in microalgal culture can often be protozoa reducing biomass productivity and culture Chlorella sorokiniana stability, there are few effective approaches to control them. This study investigated the effect of culture pH (i.e., Contamination control 6.0, 6.5, 7.0 and 7.5) maintained by supply of compressed air bubbles containing various concentrations of CO2 Poterioochromonas malhamensis on death of the flagellate Poterioochromonas malhamensis and several other protozoa in the culture of the green CO 2 microalgae Chlorella sorokiniana GT-1. C. sorokiniana GT-1 grew well at pH 6.0 and 6.5 and a sustainable biomass pH − concentration of 1.61 g L 1 was obtained from the cultures maintained at pH 6.5. The cultures maintained at Protozoan predator pH 7.0 and 7.5 collapsed on days 7 and 4 of culture, respectively, as a result of contamination by P. malhamensis and to less extent by other protozoa (e.g., ciliates and amoebae). Further experiments revealed that it was the

actual dissolved CO2, not the low pH itself, or reduced dissolved oxygen in the culture medium that prevented

the occurrence of P. malhamensis. It is speculated that increased CO2 partial pressure in the culture media may

enhance diﬀusion of CO2 into the cytoplasm of P. malhamensis that lowers the intercellular pH, and thus results in cell death. The method developed in this study can be eﬀective in protozoan control in pilot-scale Chlorella culture in an open raceway pond. It is suggested that a low pH maintained temporarily or constantly by supply of

CO2 may be a promising approach to control P. malhamensis and alike in microalgal culture.

1. Introduction approaches have been effective in controlling or killing protozoa without affecting the growth of the microalgae [10]. Becker [11] ob- Many advantages of the use of microalgae as biomass for fuels and served that lowering algal culture pH to an acidic level (pH 3.0) for other valuable products have been extensively reviewed [1–3]. How- 1–2 h reduced zooplankton contamination. However, this method also ever, there has been limited success in applying microalgal biomass to impaired the growth of the targeted microalga in practice. It is therefore biofuel production on a large industrial scale, mainly as a result of high important to find a crop protection method that can reduce a predator costs [4]. Among the various factors that contribute to the high pro- population while at the same time maintaining a healthy culture of duction costs of microalgal biomass, the frequent crash of microalgal microalgae. cultures has devastating effects on their productivity [5]. The con- Chlorella is considered as one of the best microalgae for producing tamination of the cultivation system by predators such as protozoa [6] proteins for food and fish feed, and lipids for biofuels and non-fuel oils is one of the leading causes of such crashes. Once certain protozoa have [12]. During five years of Chlorella cultivation in the laboratory and invaded the microalgal culture and proliferated, the biomass pro- outdoors from 2011 to 2016, however, we found that Chlorella cultures ductivity can be reduced to zero within a few weeks to a few days [7]. often suffered from crashes caused by the flagellate Poterioochromonas Researchers have, therefore, been exploring various control methods malhamensis. P. malhamensis is a mixotrophic flagellate [13] that lives for protozoan predators because their presence in large-scale cultiva- either by photosynthesis or by grazing on bacteria and microalgae. It tion systems is inevitable [8,9]. However, only a limited number of can rapidly phagocytose a large number of Chlorella cells and the cell

⁎ Correspondence to: Y. Gong, Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. ⁎⁎ Correspondence to: Q. Hu, Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. E-mail addresses: [email protected] (Y. Gong), [email protected] (Q. Hu). http://dx.doi.org/10.1016/j.algal.2017.06.023 Received 13 February 2017; Received in revised form 24 June 2017; Accepted 25 June 2017 Available online 11 July 2017 2211-9264/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). M. Ma et al. Algal Research 26 (2017) 436–444 volume of P. malhamensis increases by 10–30 times after feeding on shown to be effective in maintaining the pH with minor fluctuations. microalgae [14]. P. malhamensis rapidly grazed Chlorella cells and the The rate of delivery of CO2 was adjusted using an air flowmeter (Model color of the culture suspension turned from green to yellow brown and LZM-6T, Cheng Xin, China). CO2/air rates of 10–30%, 6–15%, 1–6% deep brown within a few days after P. malhamensis occurred in the and 0.5–2% were used to maintain pH levels of 6.0, 6.5, 7.0 and 7.5, culture. Although no research has been reported on the destruction of respectively. mass cultivation of Chlorella by P. malhamensis, the contamination of large-scale culture of Synechocystis by Poterioochromonas was reported 2.4. Monitoring the growth status of Chlorella sorokiniana [15]. No safe and effective method has been developed to control P. malhamensis. Touloupakis [15] reported that a Synechocystis culture The growth parameters, including cell number density, cell dry maintained at pH > 11.0 avoided contamination by Poter- weight, OD730 and chlorophyll a concentration, were applied to ioochromonas, but the productivity of the culture decreased by 32%. Chlorella cultures maintained at different pH values. Each parameter The main objective of this study was to develop a method to control was measured in triplicate for each sample. P. malhamensis in Chlorella culture. The plausible cause of inhibitory and lethal effect of CO2 on P. malhamensis was investigated and po- 2.4.1. Cell number density of Chlorella sorokiniana tential application of industrial flue gas CO2 to microalgal culture for Cell number of C. sorokiniana GT-1 in a culture was counted by contamination control was discussed. microscope with a hemacytometer (Improved Neubauer, USA).

2. Materials and methods 2.4.2. Dry cell weight of Chlorella sorokiniana Dry weight of C. sorokiniana GT-1 cells was determined gravime- 2.1. Microalgae strain and culture medium trically, using a method adopted from [17]. An aliquot (5–10 mL) of culture suspension, depending on cell density, was filtered through a Chlorella sorokiniana GT-1 was originally isolated from the SDIC preheated (105 °C, 24 h), pre-weighed glass microfiber filter (Whatman Microalgae Biotechnology Center R & D facilities in Hebei, China. The GF/C, 47 mm, UK). The filter was washed twice each with 20 mL of alga is preserved in the China General Microbiological Culture 0.5 M ammonium bicarbonate. The filter was weighed after drying at − Collection Center (No. 11801). The culture medium BG-11 [16] was 105 °C for 24 h to reach a constant weight. Dry weight (DW, g L 1) was used to cultivate C. sorokiniana GT-1. calculated using Eq. (1):

WW10− 2.2. Experimental design DW = V (1) fi The pH of C. sorokiniana GT-1 cultures were maintained at four where W1 and W0 are the weight of the lters at the end and start of different levels (pH 6.0, 6.5, 7.0 and 7.5) by adjusting the rate of de- cultivation, respectively, and V is the volume of the microalgae sus- fi livery of CO2 during a 10-day cultivation period. The experiments were pension ltered. conducted in a 12-L vertical flat-plate photobioreactor (PBR) (JLCTVS- 12S, Shanghai Joylab, China), which was made from a rectangular body 2.4.3. Determination of chlorophyll a of polymethyl methacrylate. C. sorokiniana seeds from a log-phase Chlorophyll a was determined using the method of [18] with a culture was obtained from glass columns (φ = 5 cm) with a working slight modification. An aliquot (5 mL) of culture suspension was filtered fi fi fi volume of 750 mL. The optical density (OD730) at inoculation was 0.4. through a glass micro ber lter (Whatman GF/C, 47 mm). The lter The culture temperature was 23 ± 1 °C and light was provided con- containing algae pellets was transferred to a 15-mL centrifuge tube − − tinuously at a light intensity of 140 μmol photons m 2 s 1. All ex- covered by silver paper. Pure methanol (10 mL) was added to the periments were conducted in duplicate. Four parameters, i.e., cell centrifuge tube, which was then placed in a water-bath at 75 °C for count, dry weight, OD730 and chlorophyll a concentration, were used to 20 min. Afterward the sample was centrifuged (4000 ×g, 10 min, 4 °C) measure algal growth. Occurrence and population dynamics of proto- and the supernatant was transferred to a cuvette for measurement of zoan in algal cultures were monitored daily. optical density at 653 nm (OD653) and 666 nm (OD666). The amount of − 1 Based on our previous laboratory experience of the naturally oc- chlorophyll a (Chl-a, mg L ) was calculated using Eq. (2): curring contamination of C. sorokiniana GT-1 by P. malhamensis,we (15.65×−×× OD666 7.34 OD653 ) V MeOH experimentally created contamination of the culture by simply letting Chl−=a Valgae (2) the culture grow for 10 days. The PBRs with cover-lids were first pre- treated with sodium hypochlorite (75 ppm) and then washed at least where VMeOH is the volume of methanol and Valgae is the volume of four times with distilled water before inoculating the seed culture. microalgae suspension used for the extraction of pigments. However, the PBRs were exposed to the environment as the lids cannot be sealed completely, which resulted in spontaneous contamination in 2.4.4. OD730 of Chlorella sorokiniana the laboratory setting. Therefore, our results were obtained from C. Cell density of C. sorokiniana culture was measured as optical den- sorokiniana GT-1 cultures that were spontaneously contaminated by P. sity at 730 nm (OD730) using a fluorescence spectrophotometer malhamensis, not from cultures pre-inoculated with a given number of (DR6000, HACH, USA). Samples were diluted to a concentration that

P. malhamensis. Our experiments were designed with the intention of gave a ﬁnal OD730 reading between 0.2 and 0.8. BG-11 medium was reproducing the natural initiation and progression of contamination as used as the blank solution. closely as possible. We also rationalized that our experimental design would allow us to gain additional insights into how C. sorokiniana GT-1 2.4.5. Observation of morphology of Chlorella sorokiniana population responds to the progression of contamination by P. mal- The color and turbidity of the C. sorokiniana cultures were observed hamensis. daily and a camera (Canon EOS 60D, Japan) was used to record the macro-morphological changes. The samples, in 50 mL colorimetric

2.3. Control of culture pH by supply of CO2 at different flow rates tubes, were photographed everyday under the same conditions with fixed camera parameters. The integrity and agglomeration of the cells To maintain a desired pH in the culture media, the rate of delivery were monitored every day using a microscope (BX53, Olympus, Japan) of CO2 was adjusted and recorded four times each day after monitoring to record the micro-morphological changes in C. sorokiniana during with a pH meter (Model FE20, Mettler Toledo, USA). This approach was cultivation. Appearances and colors of C. sorokiniana cultures at

437 M. Ma et al. Algal Research 26 (2017) 436–444 different pH values were still observed after the experimental period for measured. Detailed treatments were similar with that described above, longer time (from days 11 to 15). except that purified C. sorokiniana and P. malhamensis cultivated in log phase were used to measure the intracellular pH values. The in- 2.5. Identification and quantification of protozoa in contaminated Chlorella tracellular pH values of C. sorokiniana and P. malhamensis were mea- cultures sured after cells being treated with low pH level for 2 h. Intracellular pH values of C. sorokiniana and P. malhamensis were determined by dye The main contaminant P. malhamensis was identified based on mo- BCECF-AM [2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, acet- lecular information (GeneBank Accession No. KY432752, submitted by oxymethyl ester] (Sigma, USA) using flow cytometer with the modified the authors) and live morphological observation under the microscope. method of Franck [21]. After being incubated in medium at low pH, C. − Other protozoa were determined by morphological observation [19]. sorokiniana (final concentration for dye, 2 × 107 cells mL 1) and P. − The cell number of P. malhamensis stained with 1% Lugol's iodine was malhamensis (final concentration for dye, 5 × 106 cells mL 1) were determined using a hemacytometer with a phase-contrast microscope at centrifuged (2000 ×g, 5 min) and resuspended in BG-11 medium 400× magnification. The numbers of other protozoa were counted containing 10 μM BCECF-AM. The suspension was incubated at 35 °C with the same microscope using a zooplankton-counting chamber (1- for 30 min, and then was washed with BG-11 medium twice. Fluores- mL) at 200× magnification; the cells were pre-stained with 1% Lugol's cence intensity (50,000 individual cells) was analyzed using a flow iodine [20] for P. malhamensis. cytometer (FC500, Beckman Coulter, USA) with an excitation wavelength of 488 nm and emission wavelength pairs of 535 nm/660 nm. 2.6. Preliminary test on control mechanisms The analysis parameters for the flow cytometer for one species during whole experiment were stereotyped. Before analysis with the flow To determine the cellular mechanism by which lowering the culture cytometer, a fluorescence microscope (BX53, Olympus, Japan) was ff pH to pH 6.0 or 6.5 by supply of CO2 inhibits or kills P. malhamensis,we used to test di usivity of dye BCECF-AM in C. sorokiniana cell and P. tested the inhibitory effect on P. malhamensis with three different malhamensis cell, using an excitation wavelength of 488 nm and an treatments. The first treatment was to maintain the culture at pH 6.0 by emission wavelength of 535 nm. + bubbling of compressed air containing a dissolved oxygen (DO) con- For in vivo calibration, stained cells were transferred into high [K ] −1 ff ff – centration of 6.0 mg L using 25% CO2. The second treatment bu ers at di erent gradient pH values (pH 6.2 6.8) created at 0.2 pH (Fig. 1B) maintained the culture at the same pH of 6.0 and the same DO unit intervals. To equilibrate the intracellular pH of stained cells to the − concentration of 6.0 mg L 1, but using 1 M HCl or 1 M NaOH and 25% pH of external buffers, 13.8 μM nigericin (Sigma, USA) and 5 μM vali- ff N2, respectively, instead of 25% CO2. The third treatment (Fig. 1C) nomycin were added into calibration bu ers. After incubation in dark maintained the culture at a pH of 6.0 with 1 M HCl or 1 M NaOH and for 30 min, fluorescence intensities of samples were collected by flow − + adjusted the DO concentration to 8.0 mg L 1 with 99% air. All the cytometry. High [K ]buffers were prepared by mixing appropriate experiments were conducted in a 250-mL conical flask with a working proportions of 135 mM KH2PO4/20 mM NaCl and 135 mM K2HPO4/ volume of 200 mL and each treatment was performed in triplicate. 20 mM NaCl. BCECF-AM and valinomycin were dissolved in di- Purified P. malhamensis was isolated from the contaminated Chlorella methylsulfoxide, and nigericin was dissolved in absolute ethanol. All culture by gradual dilution and the seed was preserved in sterile BG-11 chemicals were stored at 4 °C. medium. In this experiment, P. malhamensis was also cultivated in the

Chlorella culture. The initial cell number of P. malhamensis was 2.7. Use of increased levels of CO2 to control protozoa in pilot-scale cultures − 2.00 × 106 cells mL 1. The cell numbers of P. malhamensis, the pH and the DO concentration were measured once every hour for 6 h. The DO Chlorella sorokiniana GT-1 was cultivated in a 100-L raceway pond concentration was measured using an SG6 DO analyzer (Mettler To- with a working-depth of 150 mm in a greenhouse, and the average ledo). The cell numbers of P. malhamensis were determined as described temperatures of culture medium during our cultivation were in Section 2.5. The culture conditions were as described in Section 2.2, 17.2 ± 2.1 °C. The pond was circulated at a velocity of − − − except that the light intensity was 50 μmol photons m 2 s 1. 0.25 ± 0.05 m s 1 using a high-density polyethylene plastic paddle-

To further explore the mechanism by which high concentration CO2 wheel with 6 blades. Pure industrial CO2 was added continuously into inhibits or kills P. malhamensis, while showing little eﬀect on C. sor- cultures to maintain the culture at pH 6.8 ± 0.2. Contamination of okiniana, intracellular pH values of C. sorokiniana and P. malhamensis protozoa in the culture was monitored daily, and when harmful pro- under low pH level (pH 6.0) created by 25% CO2 or HCl/NaOH were tozoa occurred in cultures, a higher level of CO2 was added to decrease

Fig. 1. Schematic diagram of the three diﬀerent treatment approaches to the control of Poterioochromonas malhamensis.

438 M. Ma et al. Algal Research 26 (2017) 436–444

A 8.0 3. Results and discussion

7.5 3.1. Growth of Chlorella sorokiniana GT-1 cultivated at four diﬀerent pH levels maintained by supplying various amounts of CO2 per volume of 7.0 compressed air

Growth and biochemical composition of microalgae are aﬀected by 6.5 culture pH [22]. However, the culture medium pH changed con- pH tinuously with the growth and metabolism of the microalgae during 6.0 cultivation. In this study, the pH of Chlorella cultures was adjusted to and maintained at pH 6.0, 6.5, 7.0, or 7.5 during a 10-day cultivation 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5 period by continuous supply of compressed air with 10–30, 6–15, 1–6

or 0.5–2% CO2 (v/v), respectively. Fig. 2A shows that the measured pH 5.0 values ﬂuctuated slightly around the desired pH values ( ± 0.2) during 012345678910 the cultivation period. The growth kinetics of the C. sorokiniana cultures B under four diﬀerent levels of pH was divided into two types (Fig. 2B and C) based on the changes in the color of the cultures and cell morphology of C. sorokiniana (Fig. S1). The color of the cultures at pH 6.0 and 6.5 turned from light green to dark green and remained dark green until the end of the cultivation period (even after the experimental period from days 11 to 15), whereas that of the cultures at pH 7.0 and 7.5 turned from light green to yellow green to brown or light green from days 7 and 4, respectively (Fig. 2B). In accordance with the changes in the color of the cultures, the concentrations of chlorophyll a in the cultures at pH 6.0 and 6.5 increased considerably in the log-phase (from days 1 to 4) and remained stable or increased further slightly in the stationary phase (from days 5 to 10), whereas the chlorophyll a concentrations of the cultures at pH 7.0 and 7.5 decreased gradually from days 5 and 3, respectively (Fig. 2C). The same trends were obtained for the changes in

cell dry weight and OD730 nm of the cultures (Supplementary Fig. S2). These results indicated that Chlorella cultures grew more rapidly at pH 6.0 and 6.5 than that at pH 7.0 and 7.5. Moreover, the concentration C 70 pH 6.0 of chlorophyll a,OD730 nm, and dry weight of the growth parameters pH 6.5 measured in the cultures at pH 6.5 were greater than those of the cul- 60 pH 7.0 pH 7.5 tures at pH 6.0, although the cultures at both pH 6.0 and 6.5 showed ) the same growth pattern (Fig. 2C; Supplementary Fig. S2). The highest -1 50 biomass productivity was obtained from the cultures at pH 6.5 main- – 40 tained with 6 15% CO2. It was concluded that the optimal pH of C. sorokiniana culture for maximum sustainable biomass productivity is 30 pH 6.5. Three reasons could explain these results. Firstly, our Chlorella 20 strain is an acidophilus species. Under heterotrophic growth conditions, a C. sorokiniana GT-1 culture at pH 6.5 resulted in the greatest biomass

Chlorophyll-a (mg L 10 productivity compared with those maintained at either pH 6.0 or 7.0 (data not shown). Fig. 2C shows that two days before the protozoa 0 appeared, the biomass concentration of the C. sorokiniana cultures at 012345678910 pH 6.5 was significantly higher than that at lower (e.g., pH 6.0) or Time (d) higher pH (e.g., at pH 7.0 and 7.5) (Fig. 2C), indicating that the optimal pH of C. sorokiniana GT-1 is pH 6.5 for both heterotrophic and photo- Fig. 2. Changes in (A) pH, (B) color and (C) concentration of chlorophyll a in cultures of Chlorella sorokiniana over a 10-day period at four different levels of pH adjusted or autotrophic growth. The acid-tolerance of Chlorella may be attributable maintained by a continuous supply of compressed air with different concentrations of to its ability to maintain an optimum intercellular pH at 7.3 over the

CO2. The pH was monitored and adjusted four times each day during cultivation. The external pH range from 5.0 to 7.0 [23]. horizontal lines in (A) are the expected pH values. (For interpretation of the references to Secondly, C. sorokiniana can tolerate high concentrations of CO2. color in this ﬁgure legend, the reader is referred to the web version of this article.) The inhibitory eﬀect of CO2 on Chlorella generally occurs when the concentration is > 10% [24,25]. However, our results showed that C.

sorokiniana GT-1 grew well at high concentrations of CO2 (6–15% CO2). the pH and maintain the culture at pH 6.0 ± 0.2 until all the harmful Yun et al. [26] observed that the CO2 tolerance of Chlorella increased protozoa had been killed. The input of CO2 was then stopped until the with gradually increasing CO2 concentrations from 5 to 30%, whereas pH of the culture recovered to 6.8 ± 0.2. In order to evaluate the eﬀect the Chlorella biomass decreased when a high concentration of CO2 of control, number of protozoa was counted every one or 2 h using the (> 10%) was supplied continuously. method as described in Section 2.5 while cultures were treated with Thirdly, lowering culture pH to an acidic level (e.g., 6.0–6.5) by

CO2. supply of high concentrations of CO2 eliminated or reduced the occurrence of harmful protozoa in the culture. Large error bars of the growth curves obtained from those cultures at pH 7.0 and 7.5 indicated that individual cultures at the suboptimal pH were problematic with regard to protozoan contamination, i.e., timing of occurrence and

439 M. Ma et al. Algal Research 26 (2017) 436–444

3.2. Eﬀect of low pH maintained by CO on mortality of P. malhamensis A 3.5 2 C. sorokiniana ) and other protozoa -1 ) 2.0 pH 6.0

-1 P. malhamensis 2.8 It is challenging to obtain sustainable cultivation of Chlorella free of 1.6 contamination by harmful protozoa, especially P. malhamensis, under cells mL cells 6 cells mL cells

8 2.1 outdoor conditions, particularly in summers when warm temperatures 1.2 increase diversity and population density of protozoa in microalgal

(×10 cultures. Fig. 3C and D showed that the cell density of C. sorokiniana 1.4 0.8 cultures at pH 7.0 and 7.5 decreased dramatically from days 5 and 3, respectively, with concomitant of P. malhamensis increased in popula- 0.4 0.7 tion density. As the Chlorella concentration declined to a low level of − 3×107 cells mL 1, P. malhamensis population decreased coincidently C. sorokiniana 0.0 0.0 P. malhamensis (×10 (Fig. 3D). It is likely that the collapse of the Chlorella culture was at- 012345678910 tributable to the booming of P. malhamensis population. As expected, no 3.5 P. malhamensis was found in the Chlorella cultures at pH 6.0 and 6.5 and B C sorokiniana )

. -1 ) 2.0 pH 6.5 therefore the growth of C. sorokiniana at pH 6.0 and 6.5 followed a -1 P. malhamensis typical growth curve of microalgal culture, reaching the maximum cell 2.8 8 −1 1.6 concentration of 1.8 × 10 cells mL under the given culture condi-

cells·mL tions (Fig. 2C; Fig. 3A and B; Supplementary Fig. S2). 6 cells mL

8 2.1 1.2 Based on previous investigations [5,27,28], certain amoeba and ciliates are also common, dangerous predators that can deteriorate (×10 1.4 microalgal cultures. In this study, when the culture pH was raised from 0.8 pH 6.5 to 7.0, a ciliate (i.e., Tetrahymena thermophila) was observed in the contaminated Chlorella cultures, in addition to the ﬂagellate P. 0.7 0.4 malhamensis (Fig. S3, Fig. 4C). And its maximum population density of − 1.63 × 103 cells mL 1 was measured when a signiﬁcant decline in the C. sorokiniana 0.0 0.0 P. malhamensis (×10 Chlorella population occurred (Fig. 4C). As the culture pH increased 012345678910 further to pH 7.5, the maximum population density of T. thermophila C 3.5 4 − 1 C. sorokiniana ) was not only greater (i.e., 8.23 × 10 cells mL ) but also occurred -1 ) 2.0 pH 7.0 P. malhamensis -1 sooner than that at pH 7.0. Furthermore, two more ciliates (i.e., Colpoda 2.8 sp. and Vennalla sp.) and an amoeba (i.e., Sterkiella sp.) were detected in 1.6 contaminated Chlorella cultures (Fig. S3, Fig. 4D). cells mL cells 6 cells mL cells

8 2.1 1.2 3.3. Mechanism of lethal eﬀect of high concentrations of CO2 on P. (×10

(×10 malhamensis 1.4 0.8 The above results showed that occurrence and proliferation of P. 0.4 0.7 malhamensis and several other protozoa can be prevented or reduced in – sorokiniana Chlorella culture at pH 6.0 6.5 maintained by CO2. To investigate . ﬀ C 0.0 0.0 P. malhamensis possible mechanism of inhibitory or lethal e ect of CO2-maintained low 012345678910 culture pH (e.g., pH 6.0–6.5) on P. malhamensis,weﬁrst speculated that

D 3.5 ) death of P. malhamensis might be due to the low culture pH (pH 6.0).

C. sorokiniana -1 ) 2.0 pH 7.5 However, maintaining the same pH level by HCl in C. sorokiniana cul- -1 P. malhamensis tures exerted little lethal eﬀect on P. malhamensis, suggesting that the 2.8 1.6 low pH by itself in the cultures was not responsible for death of P. cells mL

6 malhamensis (Fig. 5). Previously, one species of Poterioochromonas was cells mL 8 2.1 isolated from the digestive tract of termites [29], which was an acid 1.2

(×10 condition. Sanders [13] reported that P. malhamensis grew well in (×10 1.4 cultures at pH 4.0, showing its tolerance to extremely low pH condi- 0.8 tions. As aerating Chlorella cultures with compressed air containing high 0.7 0.4 concentrations (15–25%) of CO2 may partially stream oﬀ dissolved

sorokiniana oxygen (DO) from the cultures, we then thought that reduced DO might . P. malhamensis C 0.0 0.0 inhibit respiratory activities of P. malhamensis, and eventually resulted 012345678910 in death of the zooplankton. To verify our speculation, we set up an Time (d) experiment in which the culture pH 6.0 was maintained by HCl and mixing the cultures with 1% CO2 blended with N2 (instead of air). As Fig. 3. Population dynamics of Chlorella sorokiniana and the main contaminant shown in Fig. 5, the P. malhamensis population in the Chlorella cultures Poterioochromonas malhamensis over 10 days of cultivation at four different pH levels: (A) pH 6.0; (B) pH 6.5; (C) pH 7.0; and (D) pH 7.5. No P. malhamensis was observed in the continued steady, indicating that drastically reduced DO in the cultures cultures maintained at pH 6.0 and 6.5. did not cause death of the zooplankton. Fenchel [30] reported that some protozoa, especially small flagellates with chloroplasts (e.g., P. degree to which negative effects of harmful protozoa on microalgae malhamensis), can tolerate very low DO concentrations. The authors varied somewhat among individual batch cultures under more or less suggested that a high rate of O2 uptake can be achieved even at ex- similar culture conditions. tremely low DO concentrations because large surface to volume ratios of small zooplanktons can greatly increase diffusion of O2 into the cells, and also O2 generated as by-product of photosynthesis in those

440 M. Ma et al. Algal Research 26 (2017) 436–444

A 105 A pH 6.0 Tetrahymena thermophila 8 Colpoda sp. 4 Vannella sp. ) 10 Sterkiell a

-1 sp. 7 103

6 pH 102

5 25% CO + 75% Air (pH = 6.0) 101 2

Cell density (cell mL density (cell Cell 25% N + 74% air + 1% CO + HCl 2 2 99% Air + 1% CO + HCl 2 100 4 012345678910 0123456

B 5 10 B pH 6.5 Tetrahymena thermophila Colpoda sp . 8 10 4 Vannella sp. ) Sterkiella sp. -1

) 7 -1 10 3

6 10 2 DO (mg L

1 10 5 25% CO 2+ 75% Air (pH = 6.0) Cell density (cell mL (cell density Cell 25% N2+ 74% air + 1% CO2 + HCl 99% Air + 1% CO + HCl 0 2 10 4 012345678910 0123456 C 105 Tetrahymena thermophila C pH 7.0 Colpoda sp . Vannella )

4 sp. -1 ) 10 2.0

-1 Sterkiella sp.

3 10 1.5 cells mL cells 6

102 (×10 1.0

Cell density (cell mL (cell density Cell 10 25% CO + 75% Air (pH = 6.0) 0.5 2

25% N2+ 74% air + 1% CO2 + HCl 100 99% Air + 1% CO2 + HCl 012345678910 0.0 P. malhamensis 0123456 D 105 pH 7.5 Tetrahymena thermophila Colpoda sp. Time (h) Vannella sp. ) 104 -1 Sterkiella sp. Fig. 5. Changes in (A) pH, (B) dissolved oxygen (DO) and (C) cell density in the cultures of P. malhamensis under diﬀerent culture conditions over a six-hour period. Error bars represent the standard error of the mean (n = 3). 103

chloroplast-containing ﬂagellates can provide internal supply of O2 for 102 respiratory activities of the ﬂagellates. Finally, we hypothesized that it may be dissolved/gaseous mole-

cular CO2 rather than the low culture pH per se that causes inhibitory or 101 Cell density (cell mL (cell density Cell lethal effect on P. malhamensis. We speculated that molecular CO2 can penetrate into naked or cell wall-less P. malhamensis cells via passive 0 10 diffusion. Once in the cells, CO2 molecules are partially converted to 012345678910 2– − CO3 and HCO3 , which are weak acids lowering the cytoplasm pH, Time (d) resulting in denaturing macromolecules like proteins, RNA/DNA and eventually cell death. The green microalga C. sorokiniana cells, on the Fig. 4. Diversity and population dynamics of protozoa (except for Poterioochromonas malhamensis) during 10 days of cultivation of C. sorokiniana at four different levels of pH. other hand, possess rigid, thick cell walls, which may reduce or prevent (A) pH 6.0; (B) pH 6.5; (C) pH 7.0; and (D) pH 7.5. The protozoa included Tetrahymena penetration of molecular CO2 into the cytoplasm. The mechanism of the thermophila, Colpoda sp., Vannella sp. and Sterkiella sp. There was no contamination in inactivation of bacteria in foods by high-pressure CO2 has been well culture (B) at pH 6.5. studied [31–33]. Linsey [34] reviewed that high-pressure CO2 in- activated bacteria mainly due to diffused CO2 decreasing the intracellular pH, followed by inhibiting key enzymes and disordering

441 M. Ma et al. Algal Research 26 (2017) 436–444

Fig. 6. Determination of intracellular pH values of P. malhamensis A B (A, C, E) and C. sorokiniana (B, D, F) with stain BCECF-AM. A–B, diffusivity of dye in cells, fluorescence was excitated at 488 nm and emission wavelength was 535 nm, scale bars represent 2 μm; C–D, In vivo calibration of intracellular pH in cultured cells, fluorescence ratio was calculated based on double emission wa- velengths (535 nm/660 nm) with fixed excitation at 488 nm. E–F, intracellular pH values of P. malhamensis and C. sorokiniana in-

cubated at low pH (pH 6.0) created by high concentration CO2 C 6 Y = 0.932 X - 1.56 D 13 Y = 2.7334 X - 9.2821 (25%) and HCl for 2 h. R2 = 0.9405 R2 = 0.9774 11 5 9 4 7 3 5 Fluorescence Ratio Fluorescence Ratio 2 3 6.26.46.66.87.07.27.47.67.8 6.26.46.66.87.07.27.47.67.8 Intracellular pH Intracellular pH E 8.0 F 8.0 7.5 7.5

7.0 7.0

6.5 ** 6.5

6.0 6.0 Intracellular pH Intracellular pH 5.5 5.5

5.0 5.0 CO HCl CO 2 2 HCl

10 established successfully. Corresponding to our expectation, high CO2- ) ) -1 10 P. malhamensis -1 maintained low culture pH can reduce the cytoplasmic pH of P. mal- Ciliates 8 hamensis to 6.55 (Fig. 6E), which was outside of normal cytosolic pH 8 Amoeba between 7.0 and 7.4 for most eukaryotic cells [37]. On the other hand,

cells mL the cytoplasmic pH of P. malhamensis incubated in low pH medium cells mL 4 6 3 created by HCl was maintained at pH 7.36. However, the intracellular 6

(10 pH values of C. sorokiniana in low culture pH created by CO2 or HCl 4 were both maintained surround pH 7.10 (Fig. 6F). It demonstrated that 4 high CO2-maintained low culture pH can reduce the cytoplasmic pH of P. malhamensis but not that of C. sorokiniana. 2 2

3.4. Application of increased levels of CO to the control of protozoa in P. malhamensis 0 0 2 02468 Ciliates/Amoeba(10 pilot-scale microalgal culture Time (h) To test the effectiveness of this method in a pilot-scale culture Fig. 7. Dynamics of protozoa naturally occurring in contaminated Chlorella sorokiniana system, we cultivated C. sorokiniana in 100-liter open raceway pond culture cultivated in an outside raceway pond (100 L) during treatment at a low pH outdoors at pH 6.8 ± 0.2. When P. malhamensis occurred, and parti- – (5.8 6.0) maintained by pure CO2. cularly when its population developed rapidly with concomitant decrease in the Chlorella population, we lowered the culture pH to their metabolism and intracellular electrolyte balance. Hypothetically, pH 6.0 ± 0.2 by increasing CO2 supply into the cultures. As shown in the mechanism of CO2 controlling P. malhamensis could be the same as Fig. 7, the P. malhamensis population decreased considerably and that of that of high-pressure inactivation of bacteria by CO2. By introducing a several ciliates and amoeba species were eliminated from the cultures chemical agent BCECF-AM, which was a common pH probe [35,36], the within 8 h of treatment. The Chlorella culture grew normally again after cytoplasmic pH values of P. malhamensis and C. sorokiniana incubated in this treatment. According to our observation, this lethal effect lasted medium at low pH level (pH 6.0) created by 25% CO2 and HCl were more than one week, and once or twice treatments were enough to determined. The stain BCECF-AM was shown to diffuse well into P. control the contamination during the cultivation period of three weeks. malhamensis cell (Fig. 6A) and C. sorokiniana cell (Fig. 6B). Auto- Although the elevated CO2 was proven to be an effective method to fluorescence of the chloroplast was shown to have little influence on the control the contaminant in Chlorella culture, attention should be paid to calculated ratio, since standard curves (Fig. 6C and D) on the re- the following points in terms of practical application in large scale fl lationship between intracellular pH and uorescence ratio were cultivation: 1) cost of CO2 and 2) effective methods to deliver CO2 to

442 M. Ma et al. Algal Research 26 (2017) 436–444 cultures, which are interconnected. Therefore, we need to devise eco- References nomical, yet, effective methods to deliver high concentration of CO2 to cultures, the open raceway pond in particular. The issue of effective [1] H.M. Amaro, A.C. Guedes, F.X. Malcata, Advances and perspectives in using mi- delivery can be partially, if not completely, addressed by using venturi croalgae to produce biodiesel, Appl. Energy 88 (2011) 3402–3410. [2] G.C. Dismukes, D. Carrieri, N. Bennette, G.M. Ananyev, M.C. Posewitz, Aquatic system, and microbubble generators or including a deep end at one end phototrophs: efficient alternatives to land-based crops for biofuels, Curr. Opin. of the algae pond. Although CO2 cost is something that we must con- Biotechnol. 19 (2008) 235–240. sider for large scale production of algal biomass, the direct use of flu gas [3] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, A. Darzins, ff Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and to prevent algal cultivation from zooplankton contamination o ers advances, Plant J. 54 (2008) 621–639. various advantages that can compensate the cost of CO2. The dual use [4] P.M. Schenk, S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H. Mussgnug, C. Posten, ffi of flue gas CO2 for photosynthesis and contamination control can be O. Kruse, B. Hankamer, Second generation biofuels: high-e ciency microalgae for – considered as an effective mechanism for biological carbon capture and biodiesel production, Bioenergy Res. 1 (2008) 20 43. [5] H. Wang, W. Zhang, L. Chen, J. Wang, T. Liu, The contamination and control of utilization to address the challenges of climate change. Thus, the op- biological pollutants in mass cultivation of microalgae, Bioresour. Technol. 128 erators might be eligible to apply for carbon credit to partially com- (2013) 745–750. pensate the CO cost. Compared to other measures such as chemicals [6] L. Peng, C.Q. Lan, Z. Zhang, C. Sarch, M. Laporte, Control of protozoa contamina- 2 tion and lipid accumulation in Neochloris oleoabundans culture: effects of pH and that are used to control contamination, CO2-based method is more dissolved inorganic carbon, Bioresour. Technol. 197 (2015) 143–151. environmentally friendly and green. Most importantly, the long term [7] I. Moreno-Garrido, J. Canavate, Assessing chemical compounds for controlling and overall productivity of the contamination-free pond can be sub- predator ciliates in outdoor mass cultures of the green algae Dunaliella salina, Aquac. Eng. 24 (2001) 107–114. stantially higher than that of untreated pond. Therefore, CO2-based [8] V. Pradeep, S.W. Van Ginkel, S. Park, T. Igou, C. Yi, H. Fu, R. Johnston, T. Snell, zooplankton control can improve the final economics of a given algal Y. Chen, Use of copper to selectively inhibit Brachionus calyciflorus (predator) farm. Finally, more studies need be taken to explore how widely ap- growth in Chlorella kessleri (prey) mass cultures for algae biodiesel production, Int. J. Mol. Sci. 16 (2015) 20674–20684. plicable this approach may be for the safety of mass cultivation of other [9] D. Rego, L. Redondo, V. Geraldes, L. Costa, J. Navalho, M. Pereira, Control of algal taxa. Moreover, this method is anticipated to still be effective to predators in industrial scale microalgae cultures with pulsed electric fields, control contamination in other microalgae cultures since protozoa are Bioelectrochemistry 103 (2015) 60–64. [10] V. Montemezzani, I.C. Duggan, I.D. Hogg, R.J. Craggs, A review of potential common harmful contaminants for mass cultivation of many micro- methods for zooplankton control in wastewater treatment high rate algal ponds and algaes [38]. Excitingly, elevated CO2 has been shown to be effective to algal production raceways, Algal Res. 11 (2015) 211–226. control unwanted microorganisms and maintain a desirable abundance [11] E.W. Becker, Microalgae: Biotechnology and Microbiology, Cambridge University of target microalgae Scenedesmus dimorphus [39]. Press, New York, 1994. [12] A. Richmond, Q. Hu, Handbook of Microalgal Culture: Biotechnology and Applied Phycology, second ed., John Wiley & Sons, Oxford, 2013. [13] R.W. Sanders, K.G. Porter, D.A. Caron, Relationship between phototrophy and 4. Conclusions phagotrophy in the mixotrophic chrysophyte Poterioochromonas malhamensis, Microb. Ecol. 19 (1990) 97–109. Chlorella sorokiniana GT-1 cultures maintained at pH 6.5 ± 0.2 by [14] X. Zhang, M.M. Watanabe, I. Inouye, Light and electron microscopy of grazing by Poterioochromonas malhamensis (Chrysophyceae) on a range of phytoplankton taxa1, bubbling of compressed air mixed with high concentrations of CO2 can J. Phycol. 32 (1996) 37–46. result in both optimum biomass productivity and low probability of [15] E. Touloupakis, B. Cicchi, A.M.S. Benavides, G. Torzillo, Effect of high pH on contamination and devastating impact by P. malhamensis and other growth of Synechocystis sp. PCC 6803 cultures and their contamination by golden – ff algae (Poterioochromonas sp.), Appl. Microbiol. Biotechnol. 100 (2016) 1333 1341. protozoa. It was the actual CO2, which can di use into the cytoplasm [16] R. Rippka, J. Deruelles, J.B. Waterbury, M. Herdman, R.Y. Stanier, Generic as- and reduce the intracellular pH of P. malhamensis, rather than the low signments, strain histories and properties of pure cultures of cyanobacteria, J. Gen. pH itself or low DO concentration in the culture medium that has in- Microbiol. 111 (1979) 1–61. ff [17] C. Zhu, Y. Lee, Determination of biomass dry weight of marine microalgae, J. Appl. hibitive or lethal e ect on P. malhamensis in C. sorokiniana GT-1 cul- Phycol. 9 (1997) 189–194. tures. [18] A.R. Wellburn, The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution, J. Plant Physiol. 144 (1994) 307–313. Declaration of authors' contributions [19] D.J. Patterson, Free-living Freshswater Protozoa: A Color Guide, Manson Publishing Ltd, London, 1996. [20] R. Leakey, P. Burkill, M. Sleigh, A comparison of fixatives for the estimation of Mingyang Ma designed and performed most of the experiments, abundance and biovolume of marine planktonic ciliate populations, J. Plankton analyzed the data, and wrote the paper. Danni Yuan identified the Res. 16 (1994) 375–389. protozoa and monitored their population dynamics. Yue He counted the [21] P. Franck, N. Petitipain, M. Cherlet, M. Dardennes, F. Maachi, B. Schutz, L. Poisson, P. Nabet, Measurement of intracellular pH in cultured cells by flow cytometry with cell number of Chlorella. Minsung Park gave critical revisions of the BCECF-AM, J. Biotechnol. 46 (1996) 187–195. article. Yingchun Gong and Qiang Hu obtained the funding, designed [22] N.R. Moheimani, Inorganic carbon and pH effect on growth and lipid productivity of Tetraselmis suecica and Chlorella sp. (Chlorophyta) grown outdoors in bag pho- the experiments, drafted the article and revised it critically, and gave – fi tobioreactors, J. Appl. Phycol. 25 (2013) 387 398. nal approval for submitting the article. [23] K.A. Gehl, B. Colman, Effect of external pH on the internal pH of Chlorella sac- charophila, Plant Physiol. 77 (1985) 917–921. [24] Y. Watanabe, N. Ohmura, H. Saiki, Isolation and determination of cultural char-

Acknowledgments acteristics of microalgae which functions under CO2 enriched atmosphere, Energy Convers. Manag. 33 (1992) 545–552. [25] L. Yue, W. Chen, Isolation and determination of cultural characteristics of a new Min S. Park was funded by Chinese Academy of Sciences Presidents' highly CO2 tolerant fresh water microalgae, Energy Convers. Manag. 46 (2005) International Fellowship Initiative (No. 2016VBA023). This work was 1868–1876. partially funded by China Electronics Engineering Design Institute, the [26] Y.-S. Yun, J.M. Park, J.-W. Yang, Enhancement of CO2 tolerance of Chlorella vulgaris – State Development & Investment Corporation, China (No. Y34115-1- by gradual increase of CO2 concentration, Biotechnol. Tech. 10 (1996) 713 716. [27] A.T. Ma, E.F. Daniels, N. Gulizia, B. Brahamsha, Isolation of diverse amoebal gra- Z01-0007). We thank Key State Laboratory of Freshwater Ecology and zers of freshwater cyanobacteria for the development of model systems to study Biotechnology at Institute of Hydrobiology, Chinese Academy of predator–prey interactions, Algal Res. 13 (2016) 85–93. Sciences for equipment support. [28] Y. Gong, D.J. Patterson, Y. Li, Z. Hu, M. Sommerfeld, Y. Chen, Q. Hu, Vernalophrys algivore gen. nov., sp. nov.(Rhizaria: Cercozoa: Vampyrellida), a new algal predator isolated from outdoor mass culture of Scenedesmus dimorphus, Appl. Environ. Microbiol. 81 (2015) 3900–3913. Appendix A. Supplementary data [29] J. Destain, É. Haubruge, P. Thonart, D. Portetelle, F. Francis, J. Bauwens, C. Tarayre, C. Brasseur, C. Mattéotti, M. Vandenbol, Isolation of an amylolytic Supplementary data to this article can be found online at http://dx. chrysophyte, Poterioochromonas sp., from the digestive tract of the termite Reticulitermes santonensis, Biotechnol. Agron. Soc. Environ. 18 (2014) 19–31. doi.org/10.1016/j.algal.2017.06.023.

443 M. Ma et al. Algal Research 26 (2017) 436–444

[30] T. Fenchel, Protozoa and oxygen, Acta Protozool. 53 (2014) 3–12. [35] E. Gibbin, S. Davy, Intracellular pH of symbiotic dinoﬂagellates, Coral Reefs 32 [31] G. Ferrentino, S. Spilimbergo, High pressure carbon dioxide pasteurization of solid (2013) 859–863. foods: current knowledge and future outlooks, Trends Food Sci. Technol. 22 (2011) [36] N.M. Mangan, A. Flamholz, R.D. Hood, R. Milo, D.F. Savage, pH determines the

427–441. energetic eﬃciency of the cyanobacterial CO2 concentrating mechanism, Proc. Natl. [32] O. Erkmen, Inactivation of Salmonella typhimurium by high pressure carbon dioxide, Acad. Sci. 113 (2016) 5354–5362. Food Microbiol. 17 (2000) 225–232. [37] I.H. Madshus, Regulation of intracellular pH in eukaryotic cells, Biochem. J. 250 [33] M. Cappelletti, G. Ferrentino, S. Spilimbergo, High pressure carbon dioxide on pork (1988) 1–8. raw meat: inactivation of mesophilic bacteria and eﬀects on colour properties, J. [38] A. Richmond, Outdoor mass cultures of microalgae, Handbook of Microalgal Mass Food Eng. 156 (2015) 55–58. Culture, 1986, pp. 285–330. [34] L. Garcia-Gonzalez, A. Geeraerd, S. Spilimbergo, K. Elst, L. Van Ginneken, [39] M.J. Giannetto, A. Retotar, H. Rismani-Yazdi, J. Peccia, Using carbon dioxide to J. Debevere, J. Van Impe, F. Devlieghere, High pressure carbon dioxide inactivation maintain an elevated oleaginous microalga concentration in mixed-culture photo- of microorganisms in foods: the past, the present and the future, Int. J. Food bioreactors, Bioresour. Technol. 185 (2015) 178–184. Microbiol. 117 (2007) 1–28.

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