Controlling of flagellates and ciliates contaminations in Chlorella mass culture


 Background: Flagellates and ciliates are two common bio-contaminants which frequently cause biomass losses in Chlorella mass culture. Efficient and targeted ways are required to control contaminations in Chlorella mass cultivation aiming for biofuel production especially. Results: Five surfactants were tested for its ability to control bio-contaminations in Chlorella culture. All five surfactants were able to eliminate the contaminants at a proper concentration. Particularly the minimal effective concentrations of sodium dodecyl benzene sulfonate (SDBS) to completely eliminate Poterioochromonas sp. and Hemiurosomoida sp. were 8 mg L−1 and 10 mg L−1, respectively, yet the photosynthesis and viability of Chlorella was not significantly affected. These results were further validated in Chlorella mass cultures in 5, 20, and 200 m2 raceway ponds. Conclusions: A chemical method using 10 mg L-1 SDBS as pesticide to control flagellates or ciliates contamination in Chlorella mass culture was proposed. The method helps for a sustained microalgae biomass production and utilization, especially for biofuel production.


Background
Chlorella is a genus of unicellular green microalgae that has long been used as a model organism to study photosynthesis [1]. Chlorella biomass is rich in protein, vitamins, and minerals. The success of Chlorella mass culture during the late 1940s created a stable Chlorella industry, primarily for human nutrition and animal feed [2,3]. Recently, Chlorella is considered a candidate for bioenergy and bioremediation owning to its ability to grow fast, uptake nutrients in wastewaters, and synthesize a large amount of TAGs or carbohydrates in cells [4,5].
However, the current autotrophic technologies that are used for the mass production of Chlorella biomass are facing challenges from biological contamination. Biological contamination occurs frequently in Chlorella mass culture in the widely used cultivation systems including circular and raceway ponds [3,[6][7][8]. Zooplanktonic predators, such as ciliates, rotifers, amoeba, and agellates, are the most common contaminants as reported in the literature [8,9]. According to Ma et al's [10,11] surveys, contamination by the predatory agellate, Poterioochromonas malhamensis, in Chlorella culture occurs at any time throughout the year and no matter where the cultures are conducted only when there is air contact.
Ciliates are also widely spread and can cause serious problems to microalgae cultivation under broad climate conditions [12]. Chlorella cultures are so vulnerable that every "invasion" by these predators might devastate the cultivation. The most direct effect of such contamination is the reduction of biomass yield.
For example, the cell density of Chlorella has been shown to decrease from 4.0 × 10 8 cells mL − 1 to 1.0 × 10 8 cells mL − 1 within three days, whereas that of the grazer P. malhamensis increased to 1.1 × 10 6 cells mL − 1 from 0.1 × 10 4 cells mL − 1 [11]. Moreno-Garrido and Canavate [12] reported that grazing ciliates can visually clarify dense outdoor mass cultures of Dunaliella salina within 2 days. Similarly, over 60% of Chlorella biomass can be digested in a short time due to the explosive growth of grazers such as agellates or ciliates, according to the authors' experiences. Such catastrophic losses are unacceptable.
Thus, the control of biological contamination is very important for the mass production of Chlorella in open systems.
Biological contaminations are different in their occurrence, development, and contamination mechanisms [6]. Many contaminations have occurred in an associative or sequential manner and interacted with the target microalgae [13]. These factors make the control of biological contamination very complicated.
Methods have been suggested to overcome the challenges of biological contamination, such as ltration, changes of the environmental conditions such as medium pH, and use of chemical additives including quinine, formaldehyde, ammonia, and hydrogen peroxide [6,8,10,12]. These methods are helpful in controlling the different types of zooplanktonic contaminants. However, methods such as ltration and changes of medium pH are ine cient to apply in large scale, and chemical additives, for example, ammonia and ammonium bicarbonate, are not applicable in microalgal cultivation where nitrogen limitation is necessary to induce TAG or astaxanthin accumulation since the addition of such chemicals will relieve nitrogen de ciency. Thus more e cient and targeted ways are still required. Wang et al [6] suggested that strain selection (non-susceptibility/resistance to biological pollutants) is the most practicable approach to cope with biological invasions, yet it is very time-consuming because a single algal species is unlikely to excel in all the required characteristics, such as resistance to biological pollutants, rapid growth, high product content, wide tolerance of environmental conditions, and other qualities that facilitate industrial production.
Here, we report on a simple and e cient chemical method, using surfactant as a single additive, to control the contamination of agellates and ciliates in Chlorella mass culture. Flagellates and ciliates, speci cally Poterioochromonas sp. and Hemiurosomoida sp. in the present study, have several similarities in the context of contamination in Chlorella mass culture. They are both unicellular, and can swim and graze on Chlorella cells and especially lack a resistant structure outside the plasma membrane [14][15][16] in comparison to Chlorella. These characteristics create possibilities for the targeted control of Poterioochromonas and Hemiurosomoida without inhibition on Chlorella growth. Several surfactants were used as pesticides and their effects on agellates, ciliates, and Chlorella were investigated and compared. The application of this method was also discussed and recommended based on eld testing.

Results
Toxic effects of surfactants on grazer growth and reproduction The successive transfer cultures of the two grazers (Poterioochromonas sp. and Hemiurosomoida sp.) were established rst as described in Methods. Using these successive transfer cultures, the e cacies of the ve selected surfactants for controlling Poterioochromonas sp. and Hemiurosomoida sp. were evaluated. Toxic effects on both Poterioochromonas sp. and Hemiurosomoida sp. were observed for all ve surfactants, namely SDBS, CDEA, SDS, AEO-7, and AES.
As shown in Fig. 1 Toxic effects of the ve surfactants on Hemiurosomoida sp. were also observed (Fig. 2). The viability of Hemiurosomoida sp. was shown by an increased in cell densities, which were more than 40% higher in comparison to the initial density in the culture without surfactant supplementation. Hemiurosomoida sp. densities decreased signi cantly after surfactants addition. Taking the SDBS treatment as an example, almost 60% decrease in the Hemiurosomoida sp. density, from 1.6 × 10 3 cells mL − 1 to 680 cells mL − 1 , was obtained when 4 mg L − 1 SDBS was supplemented into the culture. A further increase in the SDBS concentration (10 mg L − 1 ) led to the complete elimination of Hemiurosomoida sp. and no living cells were observed under the microscope. The general trends of decreasing cell densities with increasing surfactant concentrations were also detected for the ve surfactants. However, the e cacies against Hemiurosomoida sp. were not the same as that for Poterioochromonas sp. The most powerful one was AEO7, which eliminated Hemiurosomoida sp. at a concentration of 8 mg L − 1 . The next ones were SDBS and CDEA, the minimal effective concentrations of which were 10 mg L − 1 and 15 mg L − 1 , respectively.  Fig. 3 as an example; other data concerning CDEA, SDS, AES, and AEO7 are provided in Additional le 1.
The time courses of the Chlorella biomass DW showed no signi cant difference when the SDBS concentration was less than 20 mg L − 1 (Fig. 3a). The biomass DW of the culture having no SDBS supplementation reached 0.72 g L − 1 on day 3, with an average growth rate of 0.84 d − 1 . Smaller but insigni cant biomass DW (0.67 g L − 1 ) and growth rate (0.82 d − 1 ) were obtained in the culture with 20 mg L − 1 SDBS supplementation. However, the biomass DW was only 0.41 g L − 1 with a signi cantly decreased growth rate of 0.66 d − 1 when the SDBS concentration was further increased to 40 mg L − 1 .
The photosynthetic activity of Chlorella ( Fig. 3b) showed that in comparison to the SDBS-free culture, the changes in the photochemical yield of Chlorella cells were very small after 3 days of exposure to 20 mg L − 1 SDBS. The ratio between variable uorescence and maximum uorescence (F V /F M ) of Chlorella was 0.72 in the SDBS-treated (20 mg L − 1 ) culture in the present study. This value fell into the general F V /F M range of dark-adapted green microalgae [17], suggesting that the photosynthetic activity of C.
pyrenoidosa XQ-20044 was not in uenced by SDBS at concentrations lower than 20 mg L − 1 .
FDA staining (Fig. 3b, 3c) clearly showed membrane integrity and viability of the Chlorella cells, with similar uorescein uorescence intensities in both the SDBS-treated (20 mg L − 1 ) and the contrast culture. All of the above results suggested that Chlorella biomass yield may be reduced due to over exposure to SDBS, but the in uences of SDBS was negligible at a concentration not higher than 20 mg L − 1 .
Application of sodium dodecyl benzene sulfonate (SDBS) as a pesticide to control agellates and ciliates grazing on Chlorella in raceway pond The SDBS surfactant was further tested outdoors to validate the laboratory data. According to its performance in the raceway ponds, more technical details with respect to its outdoor application are discussed.
According to our observation, naturally occurring contaminations of Poterioochromonas sp. or Hemiurosomoida sp. can be observed generally on days 2-4 of a newly inoculated Chlorella culture in an outdoor raceway pond (unpublished results). This trend was successfully mimicked by the addition of Poterioochromonas sp. or Hemiurosomoida sp. "seeds" into the Chlorella culture ponds (Fig. 4). 18S rDNA based metagenomic data for identi cation of the contaminating species can be seen in Additional le 2. As soon as continued increases in grazer densities were observed for 3-4 days, for example, the grazer Hemiurosomoida sp. increased continually from 1.0 × 10 5 cells L − 1 on the 4th day to 2.7 × 10 5 cells L − 1 on the 5th day and 6.4 × 10 5 cells L − 1 on the 6th day, and further increased to 1.4 × 10 6 cells L − 1 the next day, the cultures were treated with 10 mg L − 1 SDBS to control Hemiurosomoida sp. or Poterioochromonas sp., and the other parallel cultures allowed contaminations to develop.
As shown in Fig. 4, cell densities of the grazers Poterioochromonas sp. and Hemiurosomoida sp., increased regularly for 3 or 4 days. The target microalgae C. pyrenoidosa XQ-20044 also showed a quick increase in cell density (indicated by Chl a content) during this period because the grazer populations were not large enough to have a signi cant grazing effect on Chlorella. The increase in grazer densities continued thereafter in the cultivations without SDBS addition, with the majority of grazers swallowing plenty of Chlorella cells and enclosing in their bodies. When the densities of Poterioochromonas sp. and Hemiurosomoida sp. reached approximately 3.6 × 10 7 cells L − 1 and 6.4 × 10 5 cells L − 1 , respectively, the Chlorella density decreased due to grazing. By comparison, almost all the Poterioochromonas sp. and Hemiurosomoida sp. cells disintegrated and disappeared in one day in the cultivations with SDBS addition (10 mg L − 1 ) on the 6th day and 7th day, respectively, with the Chlorella growth kept as normal.
Overall, the nal Chlorella biomass concentration reached 0.6 g L − 1 after a 12-day cultivation applying SDBS pesticide. It was only 0.26 g L − 1 if the Poterioochromonas-contamination was not controlled and 0.17 g L − 1 if the Hemiurosomoida-contamination was not controlled (Fig. 5). These data suggest that by applying 10 mg L − 1 SDBS as a pesticide to control Poterioochromonas sp. or Hemiurosomoida sp. contamination, the reduction in Chlorella biomass yield, which was estimated to be greater than 60% owning to grazer contamination, can be avoided. Actually, economic loss caused by biological contamination was much bigger than expected because the residual Chlorella biomass could only be used as low-quality raw materials when no effective steps were taken to manage the contaminations.
The working concentration of SDBS (10 mg L − 1 ) was slightly higher than the minimal effective concentration to eliminate Poterioochromonas sp. in the laboratory. This was to simplify the application that using one uniform concentration to control both Poterioochromonas and Hemiurosomoida contaminations.

Discussion
Surfactants as novel pesticide for controlling biological contamination in Chlorella culture In the present study surfactants were used as pesticide to control ciliates and agellates contaminations in Chlorella culture. Among the selected ve surfactants, SDBS, SDS, and AEO7 met the basic requirements of a pesticide for the control of Poterioochromonas sp. and Hemiurosomoida sp. in Chlorella culture. First, the complete control (elimination) of the two grazers could be achieved by the addition of any one of the tested surfactants at a proper concentration (Figs. 1 & 2). Second, the surfactants SDBS, SDS, and AEO-7, which eliminated the two grazers at the minimal effective concentrations, had little effects on Chlorella growth. Particularly, the minimal effective concentrations of SDBS for the complete elimination of Poterioochromonas sp. and Hemiurosomoida sp. were as low as 8 mg L − 1 and 10 mg L − 1 , respectively. However, SDBS concentrations as high as 20 mg L − 1 had no effect on photosynthetic activity, cell membrane integrity, and biomass accumulation of C. pyrenoidosa XQ-20044 (Fig. 3).

Possible mechanisms of SDBS pesticide for controlling bio-contaminations
Previous studies regarding the aquatic toxicity of anionic surfactants [20][21][22][23] showed that green algae were more tolerant to anionic surfactant (such as SDBS) exposure compared to invertebrates including daphnia, ciliates, agellates, and bacteria. These results were consistent with those of the present study. Such differential tolerance between Chlorella and the two grazers provide evidence that these surfactants can be used as pesticides to control contamination in Chlorella mass cultures.
One remaining question is why did the surfactants only eliminate grazers such as Poterioochromonas sp. and Hemiurosomoida sp. rather than Chlorella? Why is there a different tolerance? Microscopical observation at 24 h after the addition of surfactants showed that the grazers decreased in numbers or even disappeared from the Chlorella culture. In fact, these changes occurred in less than 10 min after the addition of surfactants. Continuous microscopic monitoring (Additional le 3) revealed that the grazer cells, whether it was Poterioochromonas sp. or Hemiurosomoida sp., disintegrated shortly once the SDBS concentration got close to the minimal effective concentration. However, the free-living Chlorella cells that were not swallowed retained their morphological and physiological integrity (Fig. 3).
Surfactants (or 'surface active agents') are organic compounds that can modify the solution properties both within the bulk of the solution and at the solid/water interface [24], and they have been recognized as having certain cytotoxicity [21,22,25]. Cell membranes are the primary target for the toxicological effects of surfactants on cells, which are known to be loss of cell viability and cell lysis [26,27]. So, surfactants such as SDBS, caused the disintegration of Poterioochromonas sp. and Hemiurosomoida sp. cells in the present study. Poterioochromonas and Hemiurosomoida are unicellular organisms that lack a rigid or resistant structure (they are composed of insoluble non-hydrolysable biopolymers) outside the plasma membrane [14][15][16]. These cells were so sensitive that the lipid bilayers were disrupted immediately when enough surfactants were available in the medium.
One of the cell structures that differs Chlorella from the two grazers (Poterioochromonas sp. and Hemiurosomoida sp.) is its cell wall. Numerous species of green microalgae including C. pyrenoidosa have a two-layer cell wall with a classical polysaccharidic layer that is proximal to the cytoplasmic membrane and a thin outer layer [28,29]. The outer layers are often trilaminar organized (termed as the trilaminar sheath, TLS) and are composed of insoluble non-hydrolysable biopolymers exhibiting an unusually high resistance to non-oxidative chemical degradation [30,31]. In a study concerning biotoxicity of environmental chemicals, Gwenael Corre et al [28] found that the presence of a TLS in C. emersonii was associated with a very high resistance to anionic (DBS) and nonionic (TX-100) detergents at all growth stages and the net photosynthesis was not signi cantly affected in that species. This is also the reason why the photosynthetic capacity and viability of C. pyrenoidosa were not signi cantly affected by 20 mg L − 1 SDBS in the present study. The TLS of C. pyrenoidosa may have worked as a protective structure against SDBS.
Applications of SDBS pesticide in Chlorella mass culture SDBS is one of the most commonly used anionic surfactants for cleaning application, degreasing preparations, and emulsion polymerization [32]. This surfactant is easy to manufacture, store, transport, and handle [32], making the production industry and consumption market well-developed. It is forecasted that the world surfactant market will grow from the 2018 level to $66.4 billion by 2025 [33]. As one of the most commonly used surfactants, the current price of SDBS is only $1.4 per kilogram. Owning to its nearuniversal application, SDBS and its variants are also the most researched and documented, especially in terms of their fate in the environment. SDBS is generally regarded as a biodegradable surfactant and its degradation rate may be as high as 97-99% under aerobic conditions [34]. We harvested the SDBStreated Chlorella in other study to nd if there was residual SDBS in the harvested Chlorella biomass, and no SDBS was detected. Our previous studies have shown that the surfactants such as SDBS are unable to induce changes in algal lipid synthesis [35,36]. Therefore, it will be very cheap, convenient, and safe to use SDBS as a new pesticide in microalgal mass cultivation, especially for biofuel production. The surfactant SDBS directly acts on the unprotected plasma membrane of the grazers,therefore, the e cacy of SDBS as a pesticide may be general, and it might be possible to apply SDBS in control of contaminations caused by other grazers in Chlorella mass culture.
Avoiding target biomass reduction is a necessary principle for biological contamination control in microalgal cultivation. Grazer reproduction and Chlorella biomass loss are both becoming faster and bigger with the extension of time for a contaminated Chlorella cultivation. So, early detection and treatment are crucial for minimizing algal biomass reduction. From this point of view, 10 mg L − 1 SDBS should be added as soon as grazers are observed microscopically to prevent further reproduction of grazers. Since cell densities of the grazer Poterioochromonas and Hemiurosomoida are relatively low at this time, 10 mg L − 1 SDBS would be adequate for completely eliminating the contaminants.
The tolerable SDBS concentration for C. pyrenoidosa is 20 mg L − 1 , which is at least two times that of the minimal effective concentration for eliminating the grazers Poterioochromonas sp. and Hemiurosomoida sp. Such a difference is very helpful for outdoor application. Even the SDBS pesticide is required once again in a short time, the Chlorella will not be affected negatively. Besides, the surfactant is inevitably degraded by bacteria and fungi in the open culture, resulting in a decreased effective term. Fortunately the disintegration effect of SDBS on grazers will not be signi cantly weakened because the grazers can be eliminated in less than one day.

Conclusions
All ve selected surfactants were effective for eliminating Poterioochromonas sp. and Hemiurosomoida sp. contamination in the laboratory. Further studies indicated SDBS (10 mg L − 1 ) is an e cient pesticide to control the contaminations without damaging Chlorella. One of the principles for SDBS pesticide application is early detection and treatment of contaminations. The surfactant SDBS directly acts on the unprotected plasma membrane of the grazers; therefore, the e cacy of SDBS as a pesticide may be general. The authors expect a broad spectrum of anti-bio contaminations to be developed using the method outlined in the present study.

Methods
Chemicals used for the control of biological contaminants  . Detailed information about the raceway ponds and general cultivation parameters can be seen in our previous study [37]. Chlorella pyrenoidosa XQ-20044 was rstly cultivated in greenhouse-covered raceway ponds (5 m 2 , 1000 L) using BG-11 medium and solar irradiation. Then, approximately 2 L of the Poterioochromonas sp./Hemiurosomoida sp. culture suspension was added empirically into each pond on the 2nd or 3rd day. The grazer cultures acted as seeds to bring about Poterioochromonas sp./Hemiurosomoida sp. contamination, which was validated later by microbial community analysis using metagenomics data. 1 − 2 days after the addition, the grazers increased in density and could be easily observed under the microscope and counted using a counting chamber. Such development was very similar to the natural occurrence of Poterioochromonas sp. or Hemiurosomoida sp. contamination in Chlorella mass cultivation. After several days of cultivation and development when marked increases in the grazer density were observed, 10 mg L − 1 SDBS was added to the ponds. For the control experiments, the development of the two grazers was not interfered with by any extra operation. The experiments were conducted in parallel. Chl a content and grazer density were monitored every day to indicate Chlorella growth and grazer development, respectively.
The SDBS pesticide was also applied in a 20 to 200 m 2 cascade culture of Chlorella in October 2019. The cultivations were performed according to our previous study [37] and continued for 20 days. For the rst 10 days, the cultivation was conducted in a greenhouse-covered 20 m 2 raceway pond (4000 L cultural volume) and then the culture suspension was transferred to an 200 m 2 open raceway pond (40000 L cultural volume) to inoculate a new cultivation. Solar irradiations during the culture period is given in Additional le 4. The cascade culture was microscopically monitored twice a day and two rounds of naturally occurring bio-contamination were observed. The SDBS pesticide (10 mg L − 1 ) was used to control these contaminations.

Measurements
Biomass dry weight (DW) and Chlorophyll a (Chl a) content were measured to evaluate Chlorella growth. About 10 mL of algal suspension was ltered through a pre-dried lter paper (0.45 µm). The lter paper holding the cells was washed with 10 mL ddH 2 O, dried at 105 °C for 4 h and weighed to calculate the microalgal DW (g L − 1 ). Chl a was extracted from live cells with hot DMSO (70 °C) and was quanti ed spectrophotometrically [38]. An equation (DW g L − 1 = 38.14 × Chl a mg mL − 1 , R 2 = 0.9979) was estimated from an uncontaminated Chlorella mass culture and used to calculate Chlorella DW for those samples that had contamination during the eld test. with Lugol's solution. Only 1 µL of Lugol's solution (10%) was used for each 10 mL of sample to inhibit grazer swimming but avoid cell disruption. At least three independent countings were conducted for each sample. To study the morphological changes of grazer cells exposure to surfactant, the cells were continuously monitored under microscope, and a small device was used to assist video recording.
Description of the device and the recorded videos can be seen in Additional le 3. The outdoor samples were also subjected to metagenomic sequencing to evaluate whether the microbial community was consistent with that expected.
All the above analytical experiments were performed in triplicate and the results were analyzed for variance using SAS 9.13 at a signi cance level of α = 0.05. Tukey's multiple comparison tests were done where applicable.

Declarations
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Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les.    Comparison of biomass yields between the contaminated cultivations with and without SDBS treatment.