Introduction

Microalgal cells can be rich in lipids, proteins, polysaccharides, functional pigments, and other active substances, and hence have broad potential in the production of biomass energy, food and feed, in medicine, and in health care (Tavakoli et al. 2022). However, microbial contamination is one non-negligible factor limiting the large-scale cultivation and industrial exploitation of microalgae (Mooij et al. 2015). In mass microalgal culture, it is rarely possible to achieve axenic cultivation, and open microalgal cultures will inevitably become contaminated with zooplankton, bacteria, fungi, and other microalgae (Wang et al. 2013). Among these contaminants, predatory zooplankton are considered the most destructive. Once a microalgal culture has been invaded by zooplankton, the microalgal biomass productivity can be reduced to an extremely low level within a few days (Gong et al. 2015; He et al. 2022). To overcome the challenges of biological contamination, academic and industrial researchers have devoted much effort to the establishment of a diverse range of methods for killing or controlling contaminants of microalgal cultures (Montemezzani et al. 2015; Molina et al. 2019). In this respect, having an all-round understanding of the predator is important to the basic management of the contaminating predator in the microalgal culture. However, systematic research on zooplanktonic predators in microalgal culture, especially their biological characteristics, is lacking, thereby limiting our understanding.

In our group’s long-term research surveying the cultivation of Chlorella, more than 90% of failed cultures were caused by the grazing of Poterioochromonas (Ma et al. 2018). Indeed, the contamination of Chlorella cultures with Poterioochromonas was ubiquitously observed regardless of the culture system, season, medium, or environment (Ma et al. 2018). This illustrates why Poterioochromonas, with wide food spectrum in terms of microalgal prey, high environmental tolerance, and high capacity for damage, should be regarded as one of the most damaging contaminants in the microalgal industry. Poterioochromonas (Ochrophyta, Chrysophyceae) is a mixotrophic protist and so can live autotrophically or heterotrophically, the latter meaning that it can graze on particulate organic matter or utilize dissolved organic substrates (i.e., osmotrophy) (Zhang and Watanabe 1996). For Poterioochromonas malhamensis (Pringsheim) Péterfi, photosynthesis only contributes approximately 7% of the total carbon budget of the alga in mixotrophic conditions (Sanders et al. 1990). Therefore, Poterioochromonas is also considered to be a predominately heterotrophic mixotroph (Caron et al. 1990). It can graze on many commercial microalgae as well as Chlorella, such as Synechocystis, Nanochloropsis, and Synechococcus (Touloupakis et al. 2016; Ma et al. 2018). The grazing rate of P. malhamensis on these microalgal cells increases as the prey diameter decreases, ranging from 0.7 to 4.4 cells predator−1 h−1. Furthermore, the grazing ability of Poterioochromonas on microalgal cells under culture conditions suitable for microalgal growth (e.g., temperatures of 20 °C–30 °C and pH of 5.0–9.0) remains strong. The growth rates of P. malhamensis fed on Chlorella under the aforementioned culture conditions ranges from 0.027 to 0.055 h−1. Recently, an increasing number of studies have indicated that the genus Poterioochromonas is a globally distributed protist and can occur in both freshwater and marine ecosystems (Zhang et al. 2021a), or even coexist with plants and animals (Tarayre et al. 2014; Feng et al. 2016). To eliminate the harmfulness of Poterioochromonas on microalgal growth, a diverse range of methods for early detection and prevention as well as control have been successively established. On the other hand, the benefits of Poterioochromonas, such as its grazing on the harmful genus Microcystis (Ma et al. 2022a) and its application in the bio-manufacturing of bioactive molecules (Ma et al. 2021), have been well explored. Moreover, with the breakthrough of high-cell-density cultivation, the resource utilization of Poterioochromonas has attracted quite a lot of attention.

This paper reviews the advancements made in our understanding of Poterioochromonas as a harmful predator in microalgal culture, including its identification, ecological distribution, feeding behavior, early detection, and prevention/control methods. Furthermore, its applications in the bio-manufacturing of bioactive molecules and in controlling harmful Microcystis blooms are also discussed. The aim of this review is to demonstrate that achieving a high level of knowledge about a protozoan predator can help in solving the problem of contamination of microalgal cultures by zooplankton.

Identification

The identification of protozoan contaminants is a critical first step in establishing effective early warning systems and control methods that prevent or treat contamination. Cell morphological observation and molecular sequence analysis are the two main approaches to identifying protozoan contaminants in microalgal culture. Poterioochromonas cells are highly variable in shape and are mostly spherical or ovoid, and rarely pyriform (Pringsheim 1952). The cell size of Poterioochromonas is generally 5–12 μm, and cells have no cell wall or eyespot. The key identification characteristics of the genus Poterioochromonas include its flagella, chloroplast, and lorica (as shown in Fig. 2). Each Poterioochromonas cell has two unequal flagella, which play important roles in their feeding process. The long flagellum is 1 to 1.5 times the length of the cell body, while the short flagellum is less than half the length of the cell body. The long flagellum is covered by mastigonemes, while the short one has no mastigonemes (Schnepf et al. 1977).

A typical Poterioochromonas cell has a brown plate-like bilobed chloroplast, but there is great variation in the shape and color of the chloroplast among different growth stages and culture conditions. The chloroplast of Poterioochromonas remains intact and distinct under autotrophic conditions, while it becomes rather amorphous when Poterioochromonas cells encounter abundant dissolved organics or particulate prey (Guo and Song 2010; Andersen et al. 2017; Man et al. 2020). The chloroplast of Poterioochromonas is surrounded by four membranes and is usually connected with the nucleus through a joint outer envelope (Gibbs 1962).

The lorica is the crucial characteristic for identification of the genus Poterioochromonas, especially in terms of differentiating it from its sister genus Ochromonas, the latter having almost the same cell morphology described above except for the existence of a lorica. The lorica contains a cup, a stalk, and a foot (Fig. 1). The lorica of Poterioochromonas, consisting of chitin, is easily observed after staining with Calcofluor White (Herth 1980).

Fig. 1
figure 1

Diagram of key cell morphological characteristics of Poterioochromonas. Scale bar = 5 μm

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Another key morphological characteristic of Poterioochromonas is that it can form endogenous silicious cysts (also referred to as stomatocysts or statospores) under extreme conditions, as can other species of chrysophytes (Findenig et al. 2010; Man et al. 2020). Poterioochromonas cysts are smooth, globular spheres with a diameter of 10–15 μm. At the top of the cyst, there is a hole (0.5–1.0 μm) surrounded by a collar with three layers (Fig. 2). Sometimes, a cap covering the hole can be observed. Perhaps because of its capacity to form siliceous cysts, Poterioochromonas can survive globally and invade microalgal cultures repeatedly, meaning it is hard to completely avoid contamination with Poterioochromonas.

Fig. 2
figure 2

Diagram depicting the cyst morphology of Poterioochromonas. The numbers 1–3 denote the layers in the collar of the cyst: 1, first layer; 2-a, 2-b, 2-c, second layer; 3-a, 3-b, 3-c, third layer. The number 4 denotes the cap. Scale bar = 2 μm

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Considering its small cell size and complex morphological variation, identifying Poterioochromonas based on molecular data provides an alternative and reliable strategy. Currently, the most commonly used marker genes for identifying Poterioochromonas contain the nuclear small subunit 18S rDNA (SSU rDNA) (Boenigk et al. 2005), the internal transcribed spacer, the mitochondrial cytochrome oxidase subunit I (COI) gene, and the ribulose-1, 5-bisphosphate-carboxylase gene (in the NCBI database). Furthermore, the chloroplast genome of Poterioochromonas is also now available (Kim et al. 2019; Gastineau et al. 2021). These marker genes could also be good candidates for use in early monitoring of contaminants in microalgal cultures.

The grazing abilities of zooplankton vary considerably within and between species (Post et al. 2008). Therefore, accurate identification to the level of species is necessary when identifying contaminants of microalgal cultures. To date, there have only been three species reported in the genus Poterioochromonas—namely, P. malhamensis, P. stipitata, and P. nutans (Andersen et al. 2017). However, the differences between these three species are still a matter of debate, and there are no obvious cell morphological characteristics that allow them to be easily distinguished from one another (Péterfi 1969). The lorica morphology is the main difference. Compared to P. stipitata, the ratio of width to height of the lorica cup in P. nutans cells has been found to be larger (Jane 1944). The lorica foot of P. malhamensis has been found to generally be singular and with no branch, while that of P. stipitata has three or more branches (Peck 2010). However, according to a recent observational study of different Poterioochromonas species, there is uncertainty regarding the intraspecific differences in the lorica cup morphology and the number of branches of the lorica foot (Man et al. 2020). Therefore, it is difficult to accurately distinguish between the different species of Poterioochromonas based only on the characteristics of the lorica. Presently, the most common species of Poterioochromonas in microalgal cultures and related research is P. malhamensis; however, the variation in predation ability among different species and strains of Poterioochromonas remains a subject for further study.

Ecological distribution

To analyze the source of a predator in microalgal cultures and to develop effective prevention strategies, it is crucial to understand the ecological distribution of the predator. Poterioochromonas has been found to be widely distributed in freshwater ecosystems. For example, the authentic strain P. stipitata was isolated from a mountain lake in Hungary, where the headwaters derive from the melting of ice (Andersen et al. 2017). The typical strain P. malhamensis SAG 933-1a was also isolated in 1948 from a rock in Malham Tarn, which is a high-altitude mountain lake in England (Pringsheim 1952). Based on an investigation along the middle–lower reaches of the Yangtze River in China, it was found that P. malhamensis was present in most of the 20 lakes investigated and tended to be more prevalent in eutrophic lakes compared to mesotrophic lakes (Shi et al. 2018). Therefore, Poterioochromonas is traditionally considered as a freshwater genus, but this opinion is gradually being challenged by more and more new discoveries. For instance, by combining light microscopy with 18S rDNA phylogenetic analysis, Poterioochromonas was detected in the South China Sea, where its cell morphology was found to vary greatly from that of normal freshwater strains (Li et al. 2019). The newly isolated P. malhamensis SZCZR2049, the chloroplast genome of which has been completely mapped, originated from a saltwater lake (Van Lake, Turkey) and can grow well in F/2 medium with a salinity of 20‰ (Gastineau et al. 2021). In a recent study, a surprising finding reported that P. malhamensis was found to be the dominant species of small eukaryotes (< 20 µm) in a high Arctic marine ecosystem with a salinity of approximately 35‰ and temperature of approximately 4 °C (Zhang et al. 2021a). Furthermore, our group has also found, using quantitative real-time PCR based on a specific primer, that P. malhamensis can contaminate cultures of the marine microalga Nannochloropsis oceanica (Wang et al. 2021). More importantly, Poterioochromonas was also found on the Tara Oceans expedition, where more than 35,000 samples of seawater and plankton were collected from around the world from September 2009 to December 2013 (Karlusich et al. 2020). These results indicated that Poterioochromonas is also ubiquitous in ocean ecosystems. Currently, 15 strains of Poterioochromonas have been isolated from different sites and preserved in different culture collections (Table 1).

Table 1 Poterioochromonas strains available in microalgal culture collections
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Furthermore, Poterioochromonas has been consistently found in other specific habitats, aided by the rapid advancements in high-throughput sequencing. For example, a strain of Poterioochromonas sp. was inconceivably isolated from the digestive tract of the termite Reticulitermes santonensis (Tarayre et al. 2014). Using amplicon sequencing analysis, an abundance of Poterioochromonas cells were found in a new type of Pomacea canaliculata lung nodule (a common nodule caused by infection with Angiostrongylus cantonensis), for which these cells were identified as the main cause (Guo et al. 2018). As well as occurring in the abovementioned animals, Poterioochromonas can also live on the surface of some plants. For example, two strains of chrysophytes were isolated from two bryophytes (Haplocladium strictulum and Timmiella anomala) and identified as P. malhamensis based on cell morphology and phylogenetic analyses of SSU rRNA and COI genes (Feng et al. 2016). Through 18S rDNA clonal analysis of the metagenome, it was found that Poterioochromonas was also present in the lichen Usnea longissima (Yunzhe 2012). In addition, P. malhamensis was found to be the most abundant protozoan in the liquid-filled leaves of the purple pitcher plant, Sarracenia purpurea, which was found by analyzing samples from 39 sites collected from northern Florida to Newfoundland and westwards to eastern British Columbia (Kadowaki et al. 2012). These results indicate that Poterioochromonas coexists extensively across both plants and animals.

Besides the above habitats, Poterioochromonas has also been detected in a range of other environments. For example, in a 16-month study of tropical airborne algae in the Hawaiian Islands, Poterioochromonas was found using high-throughput sequencing technology (Sherwood et al. 2020). Our group has also observed, based on quantitative real-time polymerase chain reaction (qPCR), that Poterioochromonas is ubiquitous in the atmospheric environment (Wang et al. 2021). A recent study showed that Poterioochromonas is an important component of periphyton, which is widely distributed across paddy fields and may have relevance to environmental phosphate management (Zhang et al. 2021b). The occurrence of Poterioochromonas in the soil environment has also been verified (Li et al. 2022). In addition, Poterioochromonas can occur in certain artificial ecosystems. For instance, using denaturing gradient gel electrophoresis band purification and DNA sequencing, it was found that Poterioochromonas dominated the eukaryotic community in the late stage of the formation of aerobic granular sludge in an annular gap bioreactor (Williams and de los Reyes 2006). And based on high-throughput sequencing and microscopic observations, P. malhamensis was also found in the membrane-attached biofilms in membrane bioreactors (Inaba et al. 2018).

As shown in Fig. 3, Poterioochromonas has been found in more than 20 countries. It can occur in aquatic, atmospheric, and soil environments, and coexist across both plants and animals regardless of longitude or latitude. All of this confirms that the mixotrophic protist Poterioochromonas is globally distributed, and contamination of Poterioochromonas can happen in microalgal cultures all over the world. From the perspective of preventing contamination, the surrounding environment and biology of microalgal cultures (e.g., the aeration, water resource, surrounding soil, and possibly small animals and plants that may find their way into the cultures) should be carefully monitored to avoid invasion of the cultures by Poterioochromonas. The wide distribution of Poterioochromonas also indicates that other predatory zooplankton may also exist ubiquitously around the environment of microalgal cultures, and therefore omni-directional prevention is essential to avoid, or at least reduce, the risk of zooplanktonic contamination in microalgal cultures.

Fig. 3
figure 3

Global distribution map of Poterioochromonas. Data were collected from different microalgal culture collections and published articles. Red arrows point to locations where Poterioochromonas was found or isolated from marine ecosystems

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Feeding behavior

Protozoa possess a vast complex of diverse feeding strategies, and therefore knowledge of the feeding behaviors of protozoan contaminants contributes to the establishment of targeted control methods. In general, the mechanistic steps in protistan prey capture comprise searching, contact, capture, processing, ingestion, and digestion (Montagnes et al. 2008). Poterioochromonas is a flagellate that actively pursues prey and its feeding process has been well studied. Before capturing food, the flagella in Poterioochromonas rotate the food particle rapidly at the anterior of the cell (Pringsheim 1952). In the meantime, an empty vacuole (like a blister) forms on the cell surface and the food is enclosed within the open food vacuole with the help of the flagella. Finally, the food vacuole is withdrawn into the cytoplasm, and after the food has been digested, the residue is discharged.

The prey spectrum of Poterioochromonas is wide. It has been observed that Poterioochromonas can graze on bacteria, microalgae, fungi, and organic particles. Furthermore, it can also feed on inorganic particles, such as latex particles (Zhang and Watanabe 1996). However, the grazed inorganic particles are gradually excreted from the Poterioochromonas cell. Therefore, it appears that food selectivity occurs in the digestion process of Poterioochromonas rather than the ingestion process (Dubowsky 1974).

During the feeding process, the cell morphology and ultrastructure of Poterioochromonas cells vary greatly. The chloroplast of autotrophic Poterioochromonas is obvious and many lipid droplets can be observed (Ma et al. 2018). After feeding on prey, the cell size of Poterioochromonas increases greatly but the chloroplast becomes amorphous and the number of mitochondria increases (Guo and Song 2010). Considering that different predatory zooplankton exhibit a variety of feeding mechanisms, more effort should be devoted to studying the feeding behavior of predatory contaminants from microalgal cultures.

Environmental conditions (e.g., temperature, light, and pH) are some of the main factors affecting the growth of microalgae, and the feeding ability of zooplankton is also greatly affected by these environmental factors. Poterioochromonas can survive well at temperatures from 10°C to 36.7°C, whereas the optimal temperature for Poterioochromonas grazing on prey is usually 25°C (Hutner et al. 1957; Ma et al. 2018). A recent study revealed that Poterioochromonas can survive at the extremely low temperature of 4°C (Zhang et al. 2021a), but its grazing ability at such a low temperature remains a subject for further investigation. Moreover, Poterioochromonas grown at 33°C has been found to tolerate a short-term, high-temperature shock (i.e., 42°C for 16 min) (Schmitt 1984).

The effect of light on the feeding behavior of Poterioochromonas is still a matter of debate. Poterioochromonas can graze on prey both under dark conditions and light conditions. Holen (1999) isolated one strain of P. malhamensis that exhibited a higher ingestion rate under dark conditions (9.4 bacteria flagellate−1 h−1) than under light conditions (5.2 bacteria flagellate−1 h−1), which was also further verified in a more recent study (Weisse and Moser 2020). However, another study produced the opposite result (Ma et al. 2018). In contrast to these results, another study found that ingestion rates of P. malhamensis under dark or light conditions were similar (Zhang and Watanabe 2001). This indicates that the grazing abilities of different species of Poterioochromonas might respond differently to changes in illumination. Regarding pH, the feeding ability of Poterioochromonas has been found to be negatively correlated with the pH of the growth medium within pH 6.0–9.0 (Ma et al. 2018), and Poterioochromonas has been shown to retain its grazing ability even at the low pH of 3.5 (Moser and Weisse 2011). Above pH 11.0, the feeding behavior or even cell viability of Poterioochromonas is completely inhibited (Touloupakis et al. 2016). Therefore, a high pH (above 11.0) is also considered as one of the potential methods for controlling Poterioochromonas contamination in microalgal culture (Touloupakis et al. 2016). In general, ecologically based selective environments are currently considered as one of the most promising approaches to preventing and controlling contamination in microalgal cultivation (Mooij et al. 2015).

In addition to environmental conditions, many biotic factors can also affect the grazing ability of Poterioochromonas. For instance, the larger the prey, the lower its grazing ability. Indeed, generally, Poterioochromonas can only graze on prey that is smaller than itself (< 10 μm). The abundance and richness of prey can also affect the feeding behavior of Poterioochromonas (Saleem et al. 2013). Moreover, the biochemical composition and morphology of the prey are considered to be other factors affecting the grazing ability of Poterioochromonas. For example, it was found that the grazing ability of Poterioochromonas was negatively correlated with the specific growth rate and cell wall thickness of Chlorella (Wei et al. 2020). Besides these various factors, the prey microorganisms may evolve different strategies to defend themselves against the grazing of Poterioochromonas. For example, the presence of an S-layer in the cell wall of prey bacteria was found to be an important factor affecting the grazing ability of Poterioochromonas (Tarao et al. 2009), and the prey bacteria in an aquatic environment might form flocs or microcolonies to resist the grazing of Poterioochromonas (Blom et al. 2010; Callieri et al. 2016). However, the grazing-resistance mechanisms of microalgae against zooplankton have been less well studied, and yet knowledge of such mechanisms is crucial if specific control methods targeting predatory zooplankton in microalgal cultures are to be established.

Early detection

Early detection is essential for reducing or even preventing the negative effects of predators on the biomass productivity of microalgal cultures. Monitoring methods for contaminants in microalgal cultures can mainly be divided into direct methods (e.g., microscopy, continuous flow cytometry and in situ microscopy, and oligonucleotide markers) and indirect methods (e.g., spectral markers, metabolic markers, and photosynthesis-based markers) (Deore et al. 2020). An effective detection technique should be able to accurately recognize the potential predators at low concentrations when they are not yet having any great effect on the growth of the microalgal culture. Microscopic observation is a common daily monitoring method for assessing the presence of zooplankton grazers and culture health, but such an approach is time consuming and tedious. Poterioochromonas malhamensis, with its small cell size (< 10 μm) and variable morphology, is difficult to observe. For example, a study of P. malhamensis contamination in Chlorella cultures concluded that by the time the cell concentration of P. malhamensis reached a density that was sufficient to allow it to be easily observed under the light microscope (> 105 cells mL−1), the Chlorella biomass was already beginning to be affected and would soon decrease to a negligible level if no effective control method was undertaken (Ma et al. 2018). Therefore, microscopy is unsuitable for the early detection of these small protozoan contaminants.

In terms of other methods, to achieve early detection, Wang et al. (2017) used a FlowCAM flow-cytometer to automatically distinguish and quantify the Poterioochromonas in Chlorella cultures. Results showed that FlowCAM was able to rapidly detect Poterioochromonas even at a concentration as low as 10 cells mL−1. Compared to traditional counting using a hemocytometer under light microscopy, FlowCAM can provide a 4-day early warning system for microalgae farmers, enabling them to take effective action towards controlling Poterioochromonas and preventing cultures from crashing. However, the accuracy of FlowCAM, which achieves cell classification and enumeration based on pixel intensity, is poor, especially at high concentrations of microalgal cells. Furthermore, the flow-through channel has been found to be easily blocked when the microalgal concentration exceeds 108 cells mL−1 (Day et al. 2012). Recently, a qPCR method based on the mitochondrial COI gene has been established for detecting the occurrence of P. malhamensis in Chlorella cultures (Wang et al. 2021). This method was found to be effective even when the P. malhamensis concentration was lower than 0.07 cells mL−1, thereby demonstrating a high level of sensitivity. Moreover, the specificity of the primer designed for detecting P. malhamensis was also high. The method was also used to track the origin of P. malhamensis in a Chlorella culture, and the results showed that there were three possible ways for P. malhamensis to invade the culture—namely, via the overlying air, the culture medium, and the algal seed.

Changes in the spectral reflectance of microalgal cultures when contaminated with Poterioochromonas or diatoms may also be used for detection. For example, in a study on Chlorella vulgaris cultures, the variation in spectral features at 708 nm was found to be associated with chlorophyll catabolism as a result of contamination with Poterioochromonas (Reichardt et al. 2020). This study concluded, therefore, that contamination of Poterioochromonas in Chlorella cultures could be detected early through use of a multi-channel and fiber-coupled spectroradiometer. Unlike sampling-based approaches, this method does not require either an on-site laboratory or skilled support staff. Furthermore, it may avoid secondary contamination because it is an inherently non-contact method (Podevin et al. 2018). However, mass Chlorella cultures can be contaminated by other protists with similar pigments and pigment fluorescence features, in which case this method would not provide precise information on the contaminants in the Chlorella culture. In the future, combining a spectral method and a FlowCAM method could offer a promising approach for the early detection of contaminants in microalgal cultures in a quick and precise manner.

Prevention and control methods

To control zooplankton in microalgal cultures, a diverse range of methods have been successively established. These methods, which have been well reviewed, can be categorized as physical, chemical, and biological control methods (Carney and Lane 2014; Montemezzani et al. 2015; Lam et al. 2018). There are different advantages and disadvantages for each type of control method. For instance, physical methods are universally effective against a wide range of contaminants, but their capital and operational costs are high and most are difficult to use on an industrial scale. Chemical methods are usually easy and relatively inexpensive, but drug residue is a challenging problem (Day et al. 2017). Biological methods are safe and inexpensive; however, the range of their application is narrow and few successful cases have been reported (Kim Hue et al. 2019).

In terms of controlling Poterioochromonas, Wang et al. (2018) observed that it lacks a cell wall whereas Chlorella (its prey) possesses a thick cell wall. Therefore, the susceptibilities of Poterioochromonas cells and Chlorella cells to mechanical pressure are different. Indeed, they found that an ultrasonication treatment with a power of 495 W at 100% amplitude and lasting 1 h at a frequency of once every day was effective in preventing Poterioochromonas outbreaks in Chlorella culture with a volume of 60 L. However, the facility cost was high and it was difficult to apply in a large-scale culture system.

Adjustment of the pH or the concentration of dissolved oxygen or carbon dioxide (CO2) in the culture medium has also been studied as a potential control method. Although Poterioochromonas can live and retain its grazing ability even at the low pH of 3.5, it is more sensitive to alkaline environments (Ma et al. 2022a). Touloupakis et al. (2016) found that contamination with Poterioochromonas was eliminated in Synechocystis cultures grown in a medium with a high pH (above 11.0); however, the microalgal productivity at pH 11.0 decreased by 32% compared to that at pH 7.5. Furthermore, the carbohydrate and lipid content of Synechocystis cells grown at pH 11.0 decreased by a third. Low oxygen has also been considered as an effective strategy to inhibit the grazing ability of protozoan predators (Montemezzani et al. 2017), and a high concentration of CO2 (15%–30%) was demonstrated to be effective in reducing the probability of P. malhamensis occurring in C. sorokiniana cultures (Ma et al. 2017). However, the mechanism of the latter method was not the low concentration of dissolved oxygen created by the elevated CO2. In-depth study revealed that, as they were mixotrophic flagellates with chloroplasts, the P. malhamensis could produce oxygen by photosynthesis and tolerate a very low concentration of dissolved oxygen, but the elevated CO2 could reduce the cytoplasmic pH of P. malhamensis and result in cell death (Ma et al. 2017). From the prospective of practical application, however, the cost of supplying pure CO2 on an industrial scale would be high. To reduce the cost of elevated CO2 and achieve the large-scale application of this method, flue gas containing a high concentration of CO2 could be considered as an alternative.

In addition to the above methods, researchers have paid attention to screening different chemical compounds that are effective at controlling microbial contaminants. Recently, several chemical compounds have been screened to control Poterioochromonas contamination in microalgal cultures. A recent study showed that addition of phosphite (20 mM) in Synechococcus elongatus culture resulted in a significantly reduced grazing impact of P. malhamensis (Toda et al. 2021). It is notable, however, that the beneficial effect of chemical phosphite on inhibiting Poterioochromonas was only studied under laboratory conditions, with the effect of the chemical on microalgal growth remaining unknown. Moreover, this method is only applicable to cultures of microalgae having a high tolerance to an excessive concentration of phosphite. Our team reported that ammonium bicarbonate (NH4HCO3) at concentrations of 400–800 mg L−1 can be effective in controlling P. malhamensis in Chlorella cultures, with the 24-h mortality of P. malhamensis in indoor experiments and outdoor ponds being 94% and 90%, respectively (He et al. 2021). Compared to the untreated group, the biomass of Chlorella in the NH4HCO3 treatment group increased by 95% in outdoor ponds. Furthermore, it has been demonstrated well that the minimal effective concentration of sodium dodecyl benzene sulfonate (SDBS) required to completely eliminate Poterioochromonas sp. in Chlorella culture is 8 mg L−1, and that the photosynthesis and viability of Chlorella is not significantly affected (Wen et al. 2021). These results were validated in an outdoor raceway pond system with a maximum volume of 40,000 L. However, SDBS readily forms excessive bubbles in microalgal cultures and this leads to a decrease in microalgal biomass.

Other chemicals may also have potential as control agents. The effects of exogenous chemical compounds on the cell reproduction, cell viability, and cell morphology and ultrastructure of P. malhamensis can be easily examined under light and electronic microscopy. For this reason P. malhamensis was once considered as an effective and rapid test system allowing a variety of chemicals to be screened and preliminary predictions made about their potential ecological or health effects, which might be useful in toxicology, ecotoxicology, and pharmacology (Roderer 1986). Many compounds having toxicological effects on Poterioochromonas were therefore tested in the 1960s to 1990s (Isenb et al. 1962; Isenberg et al. 1963; Robinson and Quader 1980) and these compounds should be considered as potential candidates for controlling Poterioochromonas contamination in microalgal cultures.

Besides these control methods whose aim is to kill or inhibit Poterioochromonas, screening for microalgal species with low predation susceptibility is also important (Day et al. 2017). To date, few species of microalgae have been found to be successfully resistant against the grazing of zooplankton, especially in large-scale culture. Recently, however, one strain of C. sorokiniana (strain CMBB-146) isolated from a contaminated P. malhamensis culture was verified to be capable of defending itself against P. malhamensis both under laboratory and outdoor conditions (Ma et al. 2019). It was found that P. malhamensis could ingest Chlorella sorokiniana CMBB-146 cells but could not digest them, and this predation resistance of C. sorokiniana CMBB-146 was attributed to the particular composition of its cell wall. This finding indicates that breeding selected strains is one possible practicable approach towards controlling protozoan contamination in microalgal cultures.

Resource utilization

Although predatory zooplankton species are considered as contaminants in microalgal cultures, they also have many beneficial roles, such as the use of rotifers as important live prey in aquaculture (Kim et al. 2018) and the use of zooplankton grazing on harmful algae as a potential biological control of water blooms (Pal et al. 2020). In the middle of the last century, P. malhamensis was used as a standard bioassay system to measure the contents of cyanocobalamin (vitamin B12) in a variety of bio-samples, because of the linear relationship between its growth rate and the vitamin B12 concentration in the medium (Ford and Hutner 1955). Up to now, however, the utilization of Poterioochromonas as a resource has been mainly based on its biochemical composition as well as its feeding behavior.

Poterioochromonas cells can biosynthesize many active substances, such as chrysolaminarin, fucoxanthin, malhamensilipin A, and certain antimicrobial compounds. Chrysolaminarin, a type of storage polysaccharide, is widely distributed in golden algae and diatoms. The chrysolaminarin extracted from P. malhamensis was long ago found to be composed of β-1,3-glucan (Archibald et al. 1963), which commonly serves as an immunostimulant (De Marco Castro et al. 2021). Compared to the commercial β-1,3-glucan products derived from cereal and yeast, the chrysolaminarin from P. malhamensis has several advantages, including a higher content (more than 50% of dry weight), easier extraction, higher water-solubility, and higher bioactivity (Ma et al. 2021). For juvenile rainbow trout (Oncorhynchus mykiss), dietary supplementation with P. malhamensis containing abundant β-1,3-glucan may change the bacterial diversity and the composition of intestinal microbes in such a way that disease resistance against Aeromonas salmonicida is increased (Liu et al. 2022).

Fucoxanthin has many bioactive functions, as it can act, for example, as an antiobesity agent, an antioxidant, an antitumor agent, and an anti-inflammatory. Commercial fucoxanthin is mainly extracted from brown seaweeds, but the content of fucoxanthin in seaweed is lower than 0.1% of dry weight (Yang et al. 2020). However, an early study showed that P. malhamensis cells can also biosynthesize fucoxanthin, and the fucoxanthin percentage was determined as 89% of total carotenoids (Withers et al. 1981). By optimizing cultivation conditions, the fucoxanthin content in P. malhamensis cells can reach up to 0.34% of dry weight (Ma et al. 2022b). Recently, our group established high-cell-density heterotrophic cultivation of P. malhamensis by optimizing a series of cultivation parameters, and the maximal dry weight biomass was 32.8 g L−1 under optimal cultivation conditions (Ma et al. 2021). Therefore, P. malhamensis could also be considered as an alternative bioresource for biomanufacturing fucoxanthin in the future.

Malhamensilipin A, a type of chlorosulfolipid, was first isolated from P. malhamensis in 1994 and was found to display a diverse range of bioactive functions, including moderating protein tyrosine kinase inhibition, acting as an antiviral, and displaying antimicrobial activity (Chen et al. 1994). After elucidating its structure and identifying its absolute configuration, the chemical synthesis of malhamensilipin A has been achieved (Bedke et al. 2010; Pereira et al. 2010). However, to date, there have been no further reports of practical applications of malhamensilipin A derived from either biosynthesis or chemosynthesis.

Other antimicrobial compounds may also be produced by Poterioochromonas. Biomass extracts and culture supernatants of Poterioochromonas have both been shown to exhibit strong antibiotic effects on freshwater bacterial isolates (e.g., the genus Flectobacillus) (Blom and Pernthaler 2010), and this effect has also been verified using other bacteria in subsequent studies (Semary et al. 2013; Schuelter et al. 2019). However, the type and structure of these antibiotic compounds derived from Poterioochromonas remain a subject for further study.

Finally, as well as its role in synthesizing useful compounds, Poterioochromonas may also play a role in the control of Microcystis, which is one of the main genera of harmful cyanobacteria with a worldwide distribution and the ability to produce the toxin microcystin in aquatic environments. Laboratory data have suggested that Poterioochromonas can graze rapidly on toxic Microcystis and efficiently degrade microcystins (Ou et al. 2005; Kim and Han 2007; Zhang et al. 2008). However, the biomass of Poterioochromonas in these studies was insufficient to evaluate the viability and controlling effect on Microcystis blooms of Poterioochromonas in the field. Taking advantage of the high biomass derived from heterotrophic fermentation, our group found that chemoheterotrophic P. malhamensis can live in the aquatic environment of a Microcystis bloom and promote the sedimentation of colonial Microcystis cells (Ma et al. 2022a).

Given the scope for utilizing Poterioochromonas as a resource, as described above, it is possible that other zooplanktonic contaminants derived from microalgal cultures could also be considered in the biomanufacturing of specific active substances, or in solving ecological problems.

Conclusion

Biological contamination by zooplankton is a potentially devastating threat to microalgal cultures and therefore requires systematic research. Poterioochromonas should be regarded as one of the most damaging contaminants in the global microalgal industry owing to its wide food spectrum in terms of microalgal prey and high environmental tolerance. Poterioochromonas can be identified precisely based on the characteristics of its cell morphology, including its flagella, chloroplast, lorica, and silicious cysts. Numerous reports have indicated that Poterioochromonas is a globally distributed mixotrophic protist that can survive in freshwater, marine, atmospheric, and soil environments, as well as coexist across both plants and animals, regardless of longitude or latitude. The feeding mechanism and factors affecting the growth and grazing of Poterioochromonas have been well studied. The development of robust methodologies for the early detection of Poterioochromonas in microalgal cultures, especially qPCR, allows for the timely implementation of best management practices to prevent/reduce the damage caused by its predation. In addition to a diverse range of physical and chemical methods, selective breeding of strains has also been demonstrated as a practicable approach in the control of Poterioochromonas contamination in microalgal cultures. On the other hand, Poterioochromonas has been offer potential as a “cell factory” for the biomanufacturing of bioactive compounds, such as the immunostimulant β-1,3-glucan. Moreover, Poterioochromonas can also serve as a biological control agent for Microcystis blooms, owing to its ability to rapidly graze on toxic Microcystis cells and efficiently degrade microcystins. This review of Poterioochromonas helps to highlight the importance of systematic research to the management of zooplankton contamination in microalgal culture. Also, more broadly, it underlines in an applied biology context how knowing your enemy can help protect against it.