Acta Protozool. (2014) 53: 3–12 http://www.eko.uj.edu.pl/ap ActA doi:10.4467/16890027AP.13.0020.1117 Protozoologica Special issue: Marine Heterotrophic Protists Guest editors: John R. Dolan and David J. S. Montagnes Review paper Protozoa and Oxygen Tom FENCHEL Marine Biological Laboratory, University of Copenhagen, Denmark Abstract. Aerobic protozoa can maintain fully aerobic metabolic rates even at very low O2-tensions; this is related to their small sizes. Many – or perhaps all – protozoa show particular preferences for a given range of O2-tensions. The reasons for this and the role for their distribution in nature are discussed and examples of protozoan biota in O2-gradients in aquatic systems are presented. Facultative anaerobes capable of both aerobic and anaerobic growth are probably common within several protozoan taxa. It is concluded that further progress in this area is contingent on physiological studies of phenotypes. Key words: Protozoa, chemosensory behavior, oxygen, oxygen toxicity, microaerobic protozoa, facultative anaerobes, microaerobic and anaerobic habitats. INTRODUCTION low molecular weight organics and in some cases H2 as metabolic end products. Some ciliates and foraminifera use nitrate as a terminal electron acceptor in a respira- Increasing evidence suggests that the last common tory process (for a review on anaerobic protozoa, see ancestor of extant eukaryotes was mitochondriate and Fenchel 2011). had an aerobic energy metabolism. While representa- The great majority of protozoan species, however, tives of different protist taxa have secondarily adapted depend on aerobic energy metabolism. Among pro- to an anaerobic life style, all known protists possess ei- tists with an aerobic metabolism many – or perhaps ther mitochondria capable of oxidative phosphorylation all – show preferences for particular levels of oxygen or – in anaerobic species – have modified mitochon- tension below atmospheric saturation. This represents dria that play a different role in energy metabolism or an important niche component and an important deter- at least maintain some other functions of mitochondria; minant for the spatial distribution of microorganisms these modified mitochondria are termed hydrogeno- in nature. There are several reasons for this including somes and mitosomes, respectively (Van der Giezen oxygen toxicity, correlation between oxygen tension 2011). Most anaerobic protozoa depend on different and the distribution of preferred prey items, and oxygen types of fermentative metabolism producing various requirements of symbionts (Fenchel and Finlay 2008). Address for correspondence: Tom Fenchel, Marine Biological In the tradition of bacteriologists we can distinguish Laboratory, Strandpromenaden 5, DK-3000 Helsingør, Denmark; between obligate anaerobes that only possess an an- E-mail: [email protected] aerobic energy metabolism and that are to a variable 4 T. Fenchel 3/4 9/4 degree sensitive to exposure to oxygen (Fenchel and ume) or (r’) (Fenchel and Finlay 1983), Km will be 1.25 Finlay 1990), microaerophiles, and aerobes. Here I ar- proportional to (r’) . Thus Km decreases with decreas- bitrarily define microaerophiles as species that grow ing cell size and small aerobic organisms can thus cope best and show chemosensory preference for O2 tensions with very low oxygen concentrations. Especially larger somewhere within the range 0–10% atmospheric oxy- cells typically diverge from a spherical shape, but then gen saturation. In at least some cases these show dimin- some linear dimension instead of r’ will still approxi- ished growth rates or other signs of decreased fitness mately apply. at higher oxygen tensions. Finally I refer to aerobes as Figure 1 shows an example of data on the respira- forms that normally occur at higher O2-tensions. Facul- tory rate of a ciliated protozoan, Euplotes sp. (Fig. 2b) tative anaerobes are species that can grow aerobically, as function of ambient O2 tension and Fig. 3 shows but are also capable of sustained balanced growth under empirically determined values of Km for different sized strict anaerobic conditions, albeit with correspondingly unicellular organisms and for isolated mitochondria as lower growth rates and cell yields (Bernard and Fenchel function of their linear dimensions. The conclusion of 1996). In all studied cases, protozoa show chemosen- this is that small aerobic protozoa can approach their sory motile responses to O2-tension, something that maximum O2 uptake rate even at very low ambient O2 may be a universal trait of motile microbes (Fenchel concentrations. and Finlay 2008). Why are so many protozoa microaerophiles? The present paper discusses the physiological and That oxygen toxicity for microaerophilic prokary- behavioral responses to pO2 and habitats character- otes is caused by the formation of oxygen radicals com- ized by O2-gradients in nature. It is emphasized that bined with a limited capacity for detoxifying them is responses to O2-tension is a significant aspect of pro- tozoan ecology. well established in the case of prokaryotes (e.g. Krieg and Hoffman 1986), but evidence is more limited in the case of eukaryotic microbes. It was found that while PHYSIOLOGICAL ASPECTS the O2 uptake of the Euplotes sp. (Fig. 1) increased with increasing ambient O2 concentration up to 100% atm. sat., the growth rate and cell yield were maximized Respiration rate as function of ambient O concen- 2 at an ambient O tension of 4–5% atm. sat., and cell tration 2 yield as well as the growth rate constant decreased by The uptake rate of a solute by a spherical cell R is about 30% when grown under atmospheric saturation given by R = 4Dπr´[C(∞) – C’] where D is the diffusion (Fenchel et al. 1989). This perhaps represents the en- coefficient of the solute (here for O2 in water), r’ is the ergetic costs of detoxification of oxygen radicals. The radius of the cell, and C(∞) and C’ are the O2 concen- response to O2 tension of the freshwater ciliate Loxo des tration far away from the cell (bulk O2 concentration) is light dependent. In darkness the ciliate has a prefer- and the O2 concentration at the cell surface, respec- ence for an O2 tension of 5–10%, but at a sufficiently tively (Berg 1983). Obviously, the maximum uptake high light intensity it prefers anoxia (Fenchel and Fin- is achieved by minimizing C’. There will, however, al- lay 1984). This ciliate is a facultative anaerobe that can ways be a maximum potential rate of O2 uptake, Rm (di- use nitrate reduction under anoxia (Finlay et al. 1983). mension T–1) that is realized under otherwise optimal The reason for the increased oxygen sensitivity when conditions and when an ambient O2 concentration is not exposed to light may relate to the fact that the pig- limiting. If the maximum uptake rate, Rm, is sufficiently ments of Loxodes causes photochemical generation of high, the cells could in principle be able to reduce C’ to superoxide when illuminated in the presence of oxy- zero. However, Rm is finite and we can then assume that gen (Finlay et al. 1986). Whether this explanation also the oxygen uptake as a function of ambient O2 is given applies to photophobic responses of other pigmented by R = 4Dπr’RmC(∞)[1 – R/Rm]. Solving for R we find ciliates such as species of Blepharisma and Stentor that R = RmC(∞)/[Km + C(∞)] where Km = Rm/(4Dπr’) (e.g. Matsuoka 1983) remains to be studied. It has been and [C(∞) – C’] = C(∞)Km/[Km + C(∞)]. This is Monod shown that some anaerobic protozoa in the presence of kinetics and Km is the half saturation constant; that is, oxygen have an O2 uptake that is not coupled to energy the bulk O2 concentration that allows for an uptake that conservation and this has been interpreted as a protec- is half that of Rm. Assuming that Rm scales as (cell vol- tion mechanism (Fenchel and Finlay 1990; Lloyd et al. Protozoa and Oxygen 5 20 –6 15 x10 –1 s 2 nl O 10 5 Fig. 1. Respiration rate of a Euplotes sp. as function 0 of bulk O tension fitted to a Monod function (R = 2 m 0 5 10 15 20 25 2.2 × 10–5 nl s.–1; K = 0.8% atm. sat.). Data from m O (% atm. sat.) Fenchel et al. (1989). 2 Fig. 2. Some microaerobic and anaerobic marine ciliates. a – Uronema filificum that prefers an O2-tension of 1–2% atm. sat.; b – Euplotes sp. (Fenchel et al. 1989, Bernard and Fenchel 1996) that prefers an O2-tension of 4–5% atm sat.; c – Plagiopogon loricatus that occurs at low O2-tensions in both the stratified water column and in sediments; d – Metopus contortus is an obligate anaerobe with hydogenosomes; it occurs in nearly all marine anaerobic habitats; e – Cyclidium cf. flagellatum is a microaerophile that prefers an O2-tension around 2%, but is also capable of sustained growth under anaerobic conditions (Bernard and Fenchel 1996). All scale bars: 10 µm. 1982). The effect of oxygen toxicity in protozoa merits ductivity due to chemolithotrophic bacteria that oxidize further investigations. reduced compounds diffusing upwards from the anoxic In many cases O2 preferences of protozoa correlate zone (Fenchel 1969, Fenchel et al. 1995). The ciliate with those of their prey. Thus many ciliates feed pref- Kentrophoros carries extracellular symbiotic sulfur bac- erentially on colorless sulfur bacteria that are typically teria on parts of its cell surface; the bacteria serve as food found in the chemocline in the narrow zone where the for the host and in accordance with the requirements of presence of sulfide overlap with the presence of oxygen sulfur bacteria for the simultaneous presence of low con- in stratified water columns and in sediments. In general, centrations of sulfide and oxygen the ciliate prefers mi- the chemocline represents a region of high bacterial pro- croaerobic conditions (Fenchel and Finlay 1989). 6 T. Fenchel 10 1 ) 2 0.1 (% atm.