A systematic review of the predatory contaminant Poterioochromonas in microalgal culture

Abstract

Contamination by zooplankton has to a certain extent limited the large-scale cultivation and industrial exploitation of microalgae. However, systematic research on these predators in microalgal culture is still lacking. The identification of zooplanktonic contaminants derived from microalgal cultures is a basis for conducting related studies. Moreover, knowledge of the ecological distribution of such predators is crucial for avoiding or reducing the risk of biological contamination in the management of large-scale microalgal cultures. Understanding the feeding behaviors of zooplanktonic contaminants contributes to the establishment of targeted prevention strategies and control methods. Early detection is essential to allow prevention and control measures to be implemented in a timely and effective way. Reducing the susceptibility of the cultured microalgae to predators through breeding strains selection, the potential of modern molecular methods, or a synthesis of these approaches will be indispensable to the management of zooplankton contamination. Furthermore, exploring the resource utilization of predators helps to understand this issue comprehensively and to turn hazard into wealth. The genus Poterioochromonas is a typical mixotrophic flagellate and has attracted increasing attention because of the dramatic damage it can inflict on a wide range of microalgal cultures, regardless of the culture system, season, or environment. This review explores our current understanding of the predator Poterioochromonas and the areas where further research is needed, which should stimulate reflection on what we still need to know about these predators from a microalgal culture perspective and how we can utilize them.

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Journal of Applied Phycology (2023) 35:1103–1114
https://doi.org/10.1007/s10811-023-02941-0
A systematic review ofthepredatory contaminant Poterioochromonas
inmicroalgal culture
MingyangMa1· ChaojunWei2· WenjieHuang1· YueHe1· YingchunGong3· QiangHu1,4,5
Received: 2 February 2023 / Revised and accepted: 1 March 2023 / Published online: 28 March 2023
© The Author(s) 2023
Abstract
Contamination by zooplankton has to a certain extent limited the large-scale cultivation and industrial exploitation of
microalgae. However, systematic research on these predators in microalgal culture is still lacking. The identification of zoo-
planktonic contaminants derived from microalgal cultures is a basis for conducting related studies. Moreover, knowledge
of the ecological distribution of such predators is crucial for avoiding or reducing the risk of biological contamination in
the management of large-scale microalgal cultures. Understanding the feeding behaviors of zooplanktonic contaminants
contributes to the establishment of targeted prevention strategies and control methods. Early detection is essential to allow
prevention and control measures to be implemented in a timely and effective way. Reducing the susceptibility of the cul-
tured microalgae to predators through breeding strains selection, the potential of modern molecular methods, or a synthesis
of these approaches will be indispensable to the management of zooplankton contamination. Furthermore, exploring the
resource utilization of predators helps to understand this issue comprehensively and to turn hazard into wealth. The genus
Poterioochromonas is a typical mixotrophic flagellate and has attracted increasing attention because of the dramatic damage
it can inflict on a wide range of microalgal cultures, regardless of the culture system, season, or environment. This review
explores our current understanding of the predator Poterioochromonas and the areas where further research is needed, which
should stimulate reflection on what we still need to know about these predators from a microalgal culture perspective and
how we can utilize them.
Keywords Poterioochromonas· Biological contaminant· Detection· Prevent and control· Resource utilization
Introduction
Microalgal cells can be rich in lipids, proteins, polysac-
charides, functional pigments, and other active substances,
and hence have broad potential in the production of bio-
mass 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 cultiva-
tion 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, bacte-
ria, 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 produc-
tivity 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
* Yingchun Gong
springgong@ihb.ac.cn
* Qiang Hu
huqiang@szu.edu.cn
1 Institute forAdvanced Study, Shenzhen University,
Shenzhen518060, China
2 Hydrobiological Data Analysis Center, Institute
ofHydrobiology, Chinese Academy ofSciences,
Wuhan430072, China
3 State Key Laboratory ofFreshwater Ecology
andBiotechnology, Institute ofHydrobiology, Chinese
Academy ofSciences, Wuhan430072, China
4 Faculty ofSynthetic Biology, Shenzhen Institute
ofAdvanced Technology, Chinese Academy ofSciences,
Shenzhen518055, China
5 CAS Key Laboratory ofQuantitative Engineering Biology,
Shenzhen Institute ofSynthetic Biology, Shenzhen Institute
ofAdvanced Technology, Chinese Academy ofSciences,
Shenzhen518055, China
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1104 Journal of Applied Phycology (2023) 35:1103–1114
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industrial researchers have devoted much effort to the estab-
lishment of a diverse range of methods for killing or con-
trolling contaminants of microalgal cultures (Montemez-
zani 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 zoo-
planktonic predators in microalgal culture, especially their
biological characteristics, is lacking, thereby limiting our
understanding.
In our group’s long-term research surveying the cultiva-
tion 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 regard-
less 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 con-
taminants 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 mal-
hamensis (Pringsheim) Péterfi, photosynthesis only con-
tributes 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 Syn-
echococcus (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., tempera-
tures 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 suc-
cessively 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 break-
through of high-cell-density cultivation, the resource uti-
lization of Poterioochromonas has attracted quite a lot of
attention.
This paper reviews the advancements made in our under-
standing of Poterioochromonas as a harmful predator in
microalgal culture, including its identification, ecological
distribution, feeding behavior, early detection, and preven-
tion/control methods. Furthermore, its applications in the
bio-manufacturing of bioactive molecules and in control-
ling 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 solv-
ing the problem of contamination of microalgal cultures by
zooplankton.
Identication
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 analy-
sis are the two main approaches to identifying protozoan
contaminants in microalgal culture. Poterioochromonas
cells are highly variable in shape and are mostly spheri-
cal 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 Poteri-
oochromonas remains intact and distinct under autotrophic
conditions, while it becomes rather amorphous when Pote-
rioochromonas cells encounter abundant dissolved organics
or particulate prey (Guo and Song 2010; Andersen etal.
2017; Man etal. 2020). The chloroplast of Poteriooch-
romonas 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 dif-
ferentiating it from its sister genus Ochromonas, the latter
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1105Journal of Applied Phycology (2023) 35:1103–1114
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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 Poteriooch-
romonas, consisting of chitin, is easily observed after stain-
ing with Calcofluor White (Herth 1980).
Another key morphological characteristic of Poteriooch-
romonas 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.
Considering its small cell size and complex morpho-
logical variation, identifying Poterioochromonas based on
molecular data provides an alternative and reliable strategy.
Currently, the most commonly used marker genes for identi-
fying 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 avail-
able (Kim etal. 2019; Gastineau etal. 2021). These marker
genes could also be good candidates for use in early moni-
toring 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 differ-
ences 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 Pote-
rioochromonas 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 com-
mon species of Poterioochromonas in microalgal cultures
and related research is P. malhamensis; however, the varia-
tion 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 distrib-
uted in freshwater ecosystems. For example, the authen-
tic strain P. stipitata was isolated from a mountain lake in
Fig. 1 Diagram of key cell
morphological characteristics
of Poterioochromonas. Scale
bar = 5μm
flagella
mastigoneme
chloroplast
cup
stalk
foot
lorica
Fig. 2 Diagram depicting the cyst morphology of Poteriooch-
romonas. 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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1106 Journal of Applied Phycology (2023) 35:1103–1114
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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 Mal-
ham Tarn, which is a high-altitude mountain lake in Eng-
land (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 tradition-
ally considered as a freshwater genus, but this opinion is
gradually being challenged by more and more new discov-
eries. 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. malhamen-
sis 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 approxi-
mately 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).
Furthermore, Poterioochromonas has been consistently
found in other specific habitats, aided by the rapid advance-
ments 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 com-
mon 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 (Haplocla-
dium 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).
Table 1 Poterioochromonas
strains available in microalgal
culture collections
These strains can be obtained from the Culture Collection of Algae at Göttingen University (SAG), Ger-
many; the Central Collection of Algal Cultures (CCAC), Germany; the National Center for Marine Algae
and Microbiota (NCMA), USA; the Culture Collection of Algae at the Laboratory of Algology (CCALA),
Czech Republic; the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB),
China; the American Type Culture Collection (ATCC); the Microbial Culture Collection at the National
Institute for Environmental Studies (NIES), Japan. An asterisk means that the strain is now renamed as
Poterioochromonas sp.
Strain name Isolator Time Site
P. malhamensis SAG 933-1a/ ATCC 11,532/UTEX 1297/
CCAP 933-1a/ NIES 2144
Yuezeng Chen
(T. Y. Chen)
1948 England
P. malhamensis SAG 933-1c/ CCAP 933-1c L. Provasoli 1951 England
P. malhamensis SAG 933-1d/ CCAP 933-1d R. A. Lewin 1950 USA
P. malhamensis SAG 933–8 E. G. Pringsheim Unknown Germany
P. malhamensis SAG 933–9 E. G. Pringsheim 1954 Germany
P. malhamensis CCMP 2718 * Robert A. Andersen 1988 Australia
P. malhamensis CCMP 2740 * Robert A. Andersen Unknown USA
P. malhamensis CCMP 2060 * Robert A. Andersen 1987 Belize
P. stipitata CCMP 1862 * Robert A. Andersen 1985 USA
P. malhamensis CCMP 3181 Robert A. Andersen 2006 Australia
Poterioochromonas sp. ISE1 Giuseppe Torzillo Unknown Italy
Poterioochromonas sp. DS/ CCAC 3498 Doris Springmann 1990 Germany
Poterioochromonas sp. CCAC 7156-B & 7157-B & 7158-B Michael Melkonian 2017 Vietnam
P. malhamensis CMBB-1 Mingyang Ma 2014 China
P. malhamensis CMBB-008 Yingchun Gong 2012 USA
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1107Journal of Applied Phycology (2023) 35:1103–1114
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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. mal-
hamensis was found to be the most abundant protozoan in
the liquid-filled leaves of the purple pitcher plant, Sarrace-
nia 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 coex-
ists 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 quan-
titative real-time polymerase chain reaction (qPCR), that
Poterioochromonas is ubiquitous in the atmospheric envi-
ronment (Wang etal. 2021). A recent study showed that
Poterioochromonas is an important component of periphy-
ton, which is widely distributed across paddy fields and
may have relevance to environmental phosphate manage-
ment (Zhang etal. 2021b). The occurrence of Poteriooch-
romonas in the soil environment has also been verified (Li
etal. 2022). In addition, Poterioochromonas can occur
in certain artificial ecosystems. For instance, using dena-
turing 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 observa-
tions, 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, atmos-
pheric, and soil environments, and coexist across both
plants and animals regardless of longitude or latitude. All
of this confirms that the mixotrophic protist Poteriooch-
romonas is globally distributed, and contamination of Pote-
rioochromonas can happen in microalgal cultures all over
the world. From the perspective of preventing contamina-
tion, the surrounding environment and biology of microal-
gal 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 ubiqui-
tously 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 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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1108 Journal of Applied Phycology (2023) 35:1103–1114
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Feeding behavior
Protozoa possess a vast complex of diverse feeding strate-
gies, 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 bacte-
ria, microalgae, fungi, and organic particles. Furthermore,
it can also feed on inorganic particles, such as latex par-
ticles (Zhang and Watanabe 1996). However, the grazed
inorganic particles are gradually excreted from the Poteri-
oochromonas cell. Therefore, it appears that food selectiv-
ity 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 zooplank-
ton 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. Poteri-
oochromonas can survive well at temperatures from 10°C
to 36.7°C, whereas the optimal temperature for Poteri-
oochromonas 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 fur-
ther 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 Poteriooch-
romonas is still a matter of debate. Poterioochromonas can
graze on prey both under dark conditions and light condi-
tions. Holen (1999) isolated one strain of P. malhamensis
that exhibited a higher ingestion rate under dark condi-
tions (9.4 bacteria flagellate−1 h−1) than under light condi-
tions (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 Pote-
rioochromonas 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 con-
sidered as one of the most promising approaches to prevent-
ing 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 Poteriooch-
romonas. 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 mor-
phology 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 Poteri-
oochromonas was negatively correlated with the specific
growth rate and cell wall thickness of Chlorella (Wei etal.
2020). Besides these various factors, the prey microorgan-
isms 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 graz-
ing 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 Poteriooch-
romonas (Blom etal. 2010; Callieri etal. 2016). However,
the grazing-resistance mechanisms of microalgae against
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1109Journal of Applied Phycology (2023) 35:1103–1114
1 3
zooplankton have been less well studied, and yet knowl-
edge 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 productiv-
ity of microalgal cultures. Monitoring methods for con-
taminants 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 zoo-
plankton grazers and culture health, but such an approach
is time consuming and tedious. Poterioochromonas malha-
mensis, with its small cell size (< 10μm) and variable mor-
phology, 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 eas-
ily 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 auto-
matically distinguish and quantify the Poterioochromonas
in Chlorella cultures. Results showed that FlowCAM was
able to rapidly detect Poterioochromonas even at a concen-
tration 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 cul-
tures 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. mal-
hamensis 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 overly-
ing 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 Chlo-
rella vulgaris cultures, the variation in spectral features at
708nm was found to be associated with chlorophyll catabo-
lism 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 infor-
mation 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 detec-
tion 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 catego-
rized as physical, chemical, and biological control methods
(Carney and Lane 2014; Montemezzani etal. 2015; Lam
etal. 2018). There are different advantages and disadvan-
tages for each type of control method. For instance, physi-
cal 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 inex-
pensive, 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 Chlo-
rella (its prey) possesses a thick cell wall. Therefore, the
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1110 Journal of Applied Phycology (2023) 35:1103–1114
1 3
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. How-
ever, 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 pro-
ductivity 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 strat-
egy 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 reduc-
ing the probability of P. malhamensis occurring in C. soro-
kiniana cultures (Ma etal. 2017). However, the mechanism
of the latter method was not the low concentration of dis-
solved 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 micro-
algal 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 inhibit-
ing 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 dem-
onstrated well that the minimal effective concentration
of sodium dodecyl benzene sulfonate (SDBS) required
to completely eliminate Poterioochromonas sp. in Chlo-
rella 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 micro-
algal 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 exam-
ined under light and electronic microscopy. For this rea-
son 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 impor-
tant (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. malhamen-
sis culture was verified to be capable of defending itself
against P. malhamensis both under laboratory and out-
door 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 con-
tamination in microalgal cultures.
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1111Journal of Applied Phycology (2023) 35:1103–1114
1 3
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 bio-
logical 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 cya-
nocobalamin (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, malhamen-
silipin A, and certain antimicrobial compounds. Chrysolami-
narin, 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). Com-
pared 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 rain-
bow trout (Oncorhynchus mykiss), dietary supplementation
with P. malhamensis containing abundant β-1,3-glucan may
change the bacterial diversity and the composition of intes-
tinal 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 deter-
mined as 89% of total carotenoids (Withers etal. 1981).
By optimizing cultivation conditions, the fucoxanthin con-
tent 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. malhamen-
sis 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 dis-
play a diverse range of bioactive functions, including mod-
erating protein tyrosine kinase inhibition, acting as an antivi-
ral, 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 prac-
tical applications of malhamensilipin A derived from either
biosynthesis or chemosynthesis.
Other antimicrobial compounds may also be produced by
Poterioochromonas. Biomass extracts and culture superna-
tants 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 com-
pounds, 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 Pote-
rioochromonas 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 Pote-
rioochromonas 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 zoo-
planktonic 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 dev-
astating threat to microalgal cultures and therefore requires
systematic research. Poterioochromonas should be regarded as
one of the most damaging contaminants in the global micro-
algal industry owing to its wide food spectrum in terms of
microalgal prey and high environmental tolerance. Poteriooch-
romonas can be identified precisely based on the characteris-
tics of its cell morphology, including its flagella, chloroplast,
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1112 Journal of Applied Phycology (2023) 35:1103–1114
1 3
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 Poteriooch-
romonas 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 bio-
logical control agent for Microcystis blooms, owing to its abil-
ity 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 cul-
ture. Also, more broadly, it underlines in an applied biology
context how knowing your enemy can help protect against it.
Authors’ contributions Mingyang Ma: Literature search, Funding
acquisition, Conceptualization, Writing – original draft. Chaojun Wei:
Data analysis, Visualization. Wenjie Huang and Yue He: Visualization.
Yingchun Gong and Qiang Hu: Funding acquisition, Writing – review
& editing, Supervision.
Funding This work was funded by the National Key Research and Develop-
ment Program of China (No. 2018YFD0901504), National Natural Science
Foundation of China (No. 32002413, No. 31872201, and No. 31772419)
and China Postdoctoral Science Foundation (No. 2019M662749).
Data availability Data sharing is not applicable to this article as no new
data were created or analyzed in this study.
Declarations
Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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