Phytoplankton Nutrition and Related MixotrophyJ A Raven, University of Dundee at SCRI, Dundee, UKS C Maberly, Centre for Ecology & Hydrology, Lancaster, UK

ã 2009 Elsevier Inc. All rights reserved.

Introduction

Phytoplankton are algae and cyanobacteria whichgrow free-floating in a water body and consequentlyrely on the water column for their resources. Mostphytoplankton are photolithotrophs, which meansthat light provides the energy and inorganic mol-ecules and ions provide the materials, including car-bon, required for growth. Some phytoplankton areunable to use external organic carbon as a supplemen-tary or sole source of energy and carbon, a conditiontermed obligate photolithotrophy. However, someobligate photolithotrophs can take up and metabolizelow molecular mass nitrogen-containing organic car-bon compounds (e.g., urea, amino acids), thus contri-buting to the nitrogen budget of the organisms.Otherscan exploit external reserves of organic nitrogen orphosphorus by producing extracellular enzymes thatconvert the organic molecule into an inorganic mole-cule that is subsequently taken up.Although phytoplankton are the main primary pro-

ducers in at least larger bodies of inland water, asignificant number of these organisms are able touse external organic compounds. These organismscan use external organic compounds as a supplemen-tal source of energy and carbon, or of nitrogen orsulfur, and are termed mixotrophs. Some mixotrophstake up organic compounds into the cytosol on amolecule-by-molecule basis across the plasmalemma;these are sapromixotrophs (e.g., the green alga Chla-mydobotrys, and some species of Chlamydomonas).Other mixotrophs (e.g., some chrysophytes, crypto-phytes and dinoflagellates, and some species of theprasinophyte flagellate Pyramimonas) take up organicparticles, with digestion of the complex organic mole-cules in food vacuoles followed by uptake of individualmolecules across the food vacuole membrane in thecytosol. These organisms are phagomixotrophs, andcan obtain nitrogen, phosphorus, sulfur, iron, andother elements from their particulate food.Some mixotrophic algae are able to grow in the

absence of light. This characteristic is known aschemoorganotrophy, and the algae and cyanobacteriawhich exhibit it are termed facultative chemoorgano-trophs. Some organisms that are very closely relatedto photosynthetically competent algae have, duringevolution, lost the capacity to photosynthesize andare obligate chemoorganotrophs and live as saproor-ganotrophs, e.g., Astasia, or phagoorganotrophs, e.g.,

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Peranema. Both of these chemoorganotrophs are closerelatives of the photosynthetic Euglena.

The precise definitions of these different terms,which are used to categorize the nutritional charac-teristic s of phy topla nkton, are give n in Box 1. It isnow possible to suggest how these characteristics areinvolved in determining the ecological occurrence ofthe algae. However, almost all the work describingnutritional characteristics of phytoplankton has beenobtained from laboratory cultures with inherent lim-itations of using a small subset of phytoplanktonfound in inland waters that can be readily cultured,using vessels with a large solid surface area in relationto the volume of water and non-natural spectral com-position of light and levels of ultraviolet radiation.

Photolithotrophy by Phytoplanktonin Inland Waters

Background

To describe the process of photolithotrophy in phyto-plankton in inland waters, the mechanisms by whichelectromagnetic radiation as photosynthetically activeradiation (PAR; 400–700nm) is converted to chemi-cal energy is first described. The sources, mechanismsof uptake and uses of the essential elements are thenconsidered in turn in (approximate) order of abun-dance (by atoms) in the phytoplankton of inlandwaters, i.e., H, C, O, N, P, K, S, Mg, Ca, Cl, Fe,Mn, Cu, Zn, Mo, Ni, Co, and, for some heterokontssuch as diatoms, Si.

Electromagnetic Energy

Photolithotrophy by oxygen-producing organisms,including algae and cyanobacteria, proceeds accord-ing to the general equation [1]:

2H2�Oþ CdO2þ � 8 photons ð400� 700 nmÞ ! ðCH2

dOÞþH2

dOþ �O2 ½1�This apparently simple equation carries two signifi-cant messages compared to other representations.One important point is the occurrence of the photonson the left-hand (substrate) side of the equation; insome representations light is written above the reac-tion arrow, which is where catalysts should be placed.The photons are substrates for photosynthesis just asare water and carbon dioxide.

Box 1 Definitions of Terms Related to TrophicModes of Organisms

Chemoorganotroph: An organism which obtains the energyfor growth and maintenance from organic carbon, which alsoserves as the source of carbon skeletons for growth.

Mixotroph: An organism which combines photolithotrophy(qv) and chemoorganotrophy (qv).

Phagomixotroph: A mixotroph (qv) which obtains itsorganic carbon as do phagotrophs (qv).

Phagotroph: A chemoorganotroph (qv) which obtains itsorganic carbon and other resources by ingesting particulatematter.

Photolithotroph: An organism which obtains its energy forgrowth and maintenance from electromagnetic radiation, andits carbon from inorganic carbon, by photosynthesis. Otherelements are taken up in the available chemical form by uptakeof individual molecules across the plasmalemma. Photolithotro-phy is combined in many phytoplankton cells with a requirementfor external supply of one or more of the vitamins Vitamin B12,Thiamine, and Biotin, in that decreasing order of the number ofspecies which require them.

Sapromixotroph: A mixotroph (qv) which obtains its organicmatter as do saprotrophs (qv).

Saprotroph: A chemoorganotroph (qv) which obtains itsorganic carbon, and other nutrients, by uptake of individualmolecules across the plasmalemma.

Algae (Incl. Cyanobacteria) _ Phytoplankton Nutrition and Related Mixotrophy 193

A second important point is the presence of twowater molecules on the left-hand side and one watermolecule on the right-hand side. The reason for this isshown by the superscripts which indicate how theoxygen from water (*) ends up in the evolved oxygen,while the oxygen from carbon dioxide (d) ends up incarbohydrate and water. Although this equation indi-cates carbohydrate as the product of photosynthesis,and much of the organic products of photosynthesisare stored momentarily, or for a longer time, in car-bohydrate, a significant fraction is taken from thephotosynthetic carbon reduction cycle (PCRC) as amore oxidized compound, 3-phosphoglycerate, in thesynthesis of lipids and of many amino acids and pyr-imidines. Notwithstanding the diversity of ultimateend products of photosynthesis, i.e., all organic cellconstituents and organic matter lost to the medium,eqn [1], descr ibes the ap proximate stoichi ometry ofcell growth in acid-base terms, although differentnitrogen sources alter the stoichiometry. Maintainingintracellular acid-base balance even when bicarbon-ate (see below) is the inorganic species take up by thecells requires excretion of about one hydroxyl ion foreach bicarbonate ion taken up.A third important point is that a minimum of eight

photons are needed to reduce one carbon dioxide tocarbohydrate, using four electrons from water. Therequirement for the energy of two photons to transfereach electron from water to carbon dioxide is that

two different photochemical reactions are involved inseries, each using one photon to transfer one electron.

Light energy is absorbed by pigments associatedwith proteins in or on thylakoid membranes which,in eukaryotic algae, are located within organellesknown as plastids. Chlorophyll a, a ring-shaped mol-ecule with a central magnesium atom, is the mainphotosynthetic pigment in all oxygen-evolving organ-isms such as algae and cyanobacteria, and absorbsblue and red light most strongly. The taxonomicdiversity of algae and cyanobacteria is reflected inthe diversity of the other thylakoid-associated pig-ments (secondary pigments) which absorb awide vari-ety of wavelengths and pass the energy on tochlorophyll a in reaction centers. In each of the twotypes of reaction center photochemistry takes place,with the energy of photons converted into oxidation-reduction (chemical) energy. Nonphotochemical reac-tions, involving among other things a cycling ofprotons across the thylakoid membranes, result in theenergy from the photochemical reactions being con-verted into the energy used to phosphorylate ADP toproduceATP, and to reduceNADPþ toNADPH.Muchof the energy available from reactions involving ATPand NADPH is used to power the PCRC mentionedabove in which CO2 is reduced to carbohydrate. ThePCRC enzymes occur in the stroma of the plastids ofeukaryotes, and in the cytosol of cyanobacteria.

At low PAR fluxes, the rate of photosynthesisand growth increases proportionately with PAR. AsPAR increases further there is a relatively abrupt tran-sition to rates of photosynthesis being independent ofthe incident PAR (light saturation). Further increasesin PAR can cause a decrease in the photosynthetic rateas a result of photoinhibition. This process can occureven at low, rate-limiting, fluxes of PAR but at highlight additional photodamage can occur when the rateof absorption of light energy exceeds the capacity forphotochemistry to dissipate this excess energy. Algaeand cyanobacteria have a number of mechanisms,such as state transitions, which limit the fraction ofabsorbed energy which reaches the most sensitive site,and nonphotochemical quenching processes such asxanthophyll cycles, which limit photodamage. Theyalso possess repair mechanisms which can cause a netreduction in photodamage; this repair is most obviousafter incident PAR flux has decreased.

The relevance of these relationships to the ecology ofthe phytoplankton of inland waters is that PAR canmarkedly change with time and depth. The temporalchanges result from annual and diel changes in PAR atthe water surface made less predictable by variablecloud cover and atmospheric attenuation. Light is lostby reflection at the water surface (particularly at lowsolar angles) and declines with depth as a result of

194 Algae (Incl. Cyanobacteria) _ Phytoplankton Nutrition and Related Mixotrophy

attenuation by dissolved and particulate material,including the algae themselves, within the water col-umn. Thus, an individual algal cell may, over a day,experience rapid changes in incident PAR from limitingto photoinhibiting as it is moved through differentdepths by water currents. In inland waters, ultravioletradiation tends to be attenuated more rapidly withdepth than does PAR, particularly where dissolvedor ga nic c ar bo n i s p re se nt . C on se qu ent ly, e xpos ur e t oultraviolet radiation relative to PAR is greater at thesurface of the water column than at the depth.Ne t p rimary productivity is the d if ference betwe en

gr oss p roduction and loss es ov er the full d ay– ni g htperiod fr om respiration (as carbon dioxide ) and as sol-uble or ganic carbon. Dark re spir ation is es sential forgr owth a nd ma intenance of photolithotrophs. Lo ss ofd is solv ed org an ic matte r gen era lly ha s n o o bv iou s fu nc-tion as a p os sible c on tributor to evolutiona ry fitness ; a nex cep tion is th e se cretio n of sid ero ph or es by cy an ob ac-ter ia which are inv olv ed in iron a cq uisition. The differ-ence betwee n g ros s and n et productivity mea ns that themaximum de pth at which prima ry productiv ity ca noccur (in the absence of vertical water movements) isless than the d epth at which sig nifica nt g ros s prima ryproductivity ca n occ ur, ta king into a ccount una voida bleback-reactions in the photosynthetic mechanism. Furtherpr oc es se s s uc h a s g ra zi ng , s inki ng , a nd hy dr au li c f lu sh-ing can lead to losses of phytoplankton from a system.

Hydrogen

Essentially all of the hydrogen in photolithotrophscomes from water. While water is the most abundantmolecular species in inland waters, regulating the netinflux of water poses problems both in low-osmolarityinland waters and in those of very high salinity. For thelow-osmolarity (fresh) waters the cell contents neces-sarily have a higher osmolarity than the medium, andcellular volume regulation involves energy input eitherto pump water out of the cells if there is no effectivecell wall (as in flagellates such as chrysophytes anddinoflagellates), or to build the cell wall which resistspressure in turgid cells. For the hypersaline inlandwaters there is a very low diversity of phytoplankton(mainly Dunaliella s pp. ). Th e v ol um e r eg ul at ion p r ob-lem here is maintaining the internal osmolarity equalto the external osmolarity using organic compounds(compatible solutes) which permit cell function. This isenergy-expensive, even using glycerol, the compatiblesolute with the lowest molecular mass.

Carbon

There is great vari ability in the concent ration, an dspeciati on, of inorga nic carbon in inland wat ers.

Inorgani c carbon compr ises carbon dioxide, bicar-bona te and carbo nate interlinked by equilibriawhich are related to pH. When a lake is in equ ilib-rium with the atmo sphere , the concent ration of inor-ganic carbon is co ntrolled largely by the geolog y ofthe catch ment, produci ng a sp atial variation amonglakes. Many lakes, however, can be far from equ ilib-rium. When averag ed over time, most lakes haveexces s CO2 and lose it to the atm osphere becau se ofinput from the catchment of CO2 , or organi c carbonwhich is bro ken down phot ochem ically and biol ogi-cally, to produce CO2. In productive lakes there areperiods, typically during summer stratification, whenrates of photosynthetic inorganic carbon uptakeexceed rates of resupply. This removal of whatamounts to CO2 increases the pH, and the near zeroCO2 concentrations can potentially limit rates of pho-tosynthesis. However, net photosynthesis can continueif algae make use of bicarbonate and/or have an effec-tive carbon concentrating mechanism (see below). Inpr odu ct iv e la ke s, pH c an v ar y b y 1 or 2 uni ts du ri ng24 h. Spatially, pH can vary from between about 1 inwaters influenced by volcanic activity to 10 or 11in waters of high acid neutralizing capacity and/orthat are experiencing severe inorganic carbon deple-tion. Stratified productive lakes also show strongdepth gradients of inorganic carbon and pH withlower concentrations and higher pH in the productivee p i l im ni on .

The core carboxyl ase of photo synthesis in alloxygen -producin g organisms is ribul ose-1,5-bi sphos-phate carboxylase-oxygenase (Rubisco). This enzymeuses carbon dioxide as the inorganic carbon sub-strate, and reacts not only with carbon dioxide butalso, competitively, with oxygen. The phylogeneti-cally variable, but always relatively low, affinityof Rubisco for carbon dioxide means that the carbondioxide concentration in inland waters can be rate-limiting for gross photosynthesis if the supply of theinorganic carbon substrate to Rubisco is by diffusionof carbon dioxide. The majority of the investigatedphytoplankton from inland waters have inorganiccarbon concentrating mechanisms (CCMs). TheseCCMs can be based, depending on the energizedtransport across a membrane or membranes, on car-bon dioxide, bicarbonate or protons. The main inlandwater taxa known to lack CCMs are the chrysophytesand synurophytes.

Oxygen

Oxygen in the organic matter of photolithotrophscome s mai nly from carbon dioxi de (eqn [1]) ; aminor component comes from molecular oxygen viaoxygenase reactions.

Algae (Incl. Cyanobacteria) _ Phytoplankton Nutrition and Related Mixotrophy 195

Nitrogen

The reduced nitrogen in organic matter in photolitho-trophic phytoplankton from inland water comesmainly from ‘combined’ nitrogen, e.g., ammonium,nitrite, nitrate and dissolved low molecular massorganic nitrogen. All phytoplankton can apparentlyuse ammonium as their nitrogen source, most can usenitrate and nitrite, andmany can use organic nitrogen.The exceptions are filamentous cyanobacteria (e.g.,Anabaena) with heterocysts which are specialized inusing molecular nitrogen by nitrogen fixation, i.e.,diazotrophy. The heterocysts restrict access of molec-ular oxygen, an irreversible inhibitor of the enzymenitrogenase, to this key enzyme in the first step ofnitrogen fixation. The extent to which the availabilityof combined nitrogen limits phytoplankton produc-tivity in inland waters is still a matter of debate. It iswidely held that phosphorus is the most frequent lim-iting element for growth of inland water photolitho-trophic phytoplankton. This situation would agreewith the geochemical prediction that a shortage ofcombined nitrogen relative to other nutrients requiredfor photolithotrophic growth would be corrected byincreased growth of diazotrophic organisms. How-ever, in some lakes nitrogen is limiting or colimitingwith phosphorus, possibly because the environment isunfavorable for nitrogen-fixing organisms.The present situation is distorted by anthropogenic

inputs of combined nitrogen to a greater extent, rela-tive to the quantities needed for phytoplankton growth,than that of phosphates. This combined nitrogen inputincludes nitrate inputs from agricultural run-off, andoxidized and reduced nitrogen from atmospheric pol-lution. How this reflects the ‘natural,’ preindustrialsituation is not clear, since at least the atmosphericcomponent of anthropogenic inputs is quantitativelywidespread over many remote inland waters.

Phosphorus

The oxidized phosphorus in organic matter in photo-lithotrophic phytoplankton in inland waters comesfrom inorganic and organic phosphates, and to a verylimited extent from phosphonates, dissolved in the epi-limnion. As mentioned in the section Nitrogen, phos-phorus is predicted fromgeochemical considerations tolimit phytoplankton productivity in inland waters.However, there are many cases in which combinednitrogen limits phytoplankton growth in inland watersand diazotrophy does not correct this limitation.

Potassium

Potassium has occasionally been suggested as agrowth-limiting nutrient in the phytoplankton of

inland waters of low salinity, although there are fewdata which support this contention.

Sulfur

Atmospheric inputs of anthropogenic sulfur dioxide,largely derived from burning of high-sulfur coal in theabsence of scrubbing this gas from the flue gases, haveincreased the sulfate concentration in many inlandwaters. However, there are still a number of inlandwater habitats, all of low salinity, in which sulfuravailability can be close to limiting the primary prod-uctivity of phytoplankton. Sulfate is very abundant insome saline inland waters.

Calcium and Magnesium

These elements do not seem to be a limiting nutrientfor phytoplankton primary productivity in inlandwaters.

Iron

There are some examples of iron limitation of phyto-plankton primary production in inland waters withcombined nitrogen as the nitrogen source. While dia-zotrophy has a higher iron requirement than does theuse of nitrate, nitrite or, especially, reduced nitrogensources, there is little evidence of specific iron limita-tion of nitrogen fixation in inland waters.

Molybdenum

The requirement for molybdenum by inland waterphytoplankton mainly relates to nitrogenase in diazo-trophs, and to a quantitatively smaller extent togrowth with nitrate as the nitrogen source. Highsulfate concentrations in some saline inland waterscould competitively inhibit the uptake of molybdate,the predominant natural source of molybdenum. Fordiazotrophs, there are also organisms which canexpress nitrogenases which use vanadium, or (addi-tional) iron, instead of molybdenum.

Chlorine, Manganese, Copper, Zinc, Nickel,

and Cobalt

There seem to be no cases in which these elementslimit phytoplankton primary productivity in inlandwaters.

Silicon

Silicon is only essential for the growth of diatomsand, probably, many chrysophytes, synurophytes,

196 Algae (Incl. Cyanobacteria) _ Phytoplankton Nutrition and Related Mixotrophy

and perhaps prasinophytes with deposits of silica.Silicon is taken up as silicic acid, and the supply ofthis nutrient can limit the extent to which silicon-requiring organisms contribute to phytoplankton pri-mary productivity in productive habitats at somehabitats at some times, often in spring.

The Role of Mixotrophy in thePhytoplankton of Inland Waters

Sapromixotrophy

Dissolved organic matter in inland waters originatesin terrestrial primary productivity (see section Hydro-gen), from photolithotrophs (see section Electromag-netic Energy) and from downstream processes basedon phytoplankton primary productivity. The extentto which sapromixotrophy can contribute to phyto-plankton primary productivity in inland waters isunclear. Globally, the conclusion seems inevitablethat there is a net efflux of dissolved organic matterfrom phytoplankton to the epilimnion of inlandwaters. This conclusion does not preclude the occur-rence of locations in which the reverse is true, at leastat some times. An example is acidic lakes in disusedopen-cast lignite mines.

Phagomixotrophy

In a manner in some ways resembling that for sapro-mixotrophs, the global net flux of particulate organicmatter to consumers is fromphytoplankton to nonalgalphagotrophs in inland waters, with a relatively smalluptake by phagomixotrophs.Of course, the planktonicphagomixotrophs only ingest the smaller particles, andso are best considered as part of the ‘microbial loop.’Here, as well as picophytoplankton, the prey for smallphagotrophs (including phagomixotrophs) includesbacteria growing on dissolved organic matter, thusgiving a link with the dissolved organic carbon pool(see sections Hydrogen and Sapromixotrophy).Locally, e.g., in some lakes in Antarctica, phagomixo-trophic algae are predominant (in biomass and in spe-cies number) members of the plankton.While sapromixotrophy deals solely with organic

carbon and nitrogen, phagomixotrophy involves theuptake into food vacuoles of elements in the ratio inwhich they occur in the prey. This means that phago-trophy could do much more than supply organic

carbon as a carbon and energy source: it is also anitrogen, phosphorus and iron source. While someevidence is consistent for a role of phagomixotrophyin the supply of nitrogen, phosphorus and iron forthe organisms, the evidence overall is somewhatequivocal. It is also known that, at least in thelaboratory, phagomixotrophs can acquire iron fromparticulate/colloidal iron not readily available to non-phagotrophic phytoplankton.

Conclusions

Phytoplanktonic algae and cyanobacteria face a widevariation in availability of energy andmaterials amongdifferent lakes, and within a given lake at differenttimes and depths. This variation is matched by thedifferent strategies employed in obtaining resourcesby different taxa, at a phylogenetic or functionalgroup level, and by the flexibility to exploit differenttypes of resources depending on their relative avail-ability. Seen as a ‘compound organism,’ the phyto-plankton are important as ‘ecosystem engineers’ thatalter the availability of the materials within an inlandwater body.

See also: Algae; Phytoplankton Productivity; Protists.

Further Reading

Falkowski PG and Raven JA (2007) Aquatic Photosynthesis.

2nd edn. Princeton, NJ: Princeton University Press.

Jaworski GHM, Talling JF, and Heaney SI (2003) Potassium depen-dence and phytoplankton ecology: An experimental study. Fresh-water Biology 48: 833–840.

Laybourn-Parry J, Marshall WA, and Marchant HJ (2005) Flagel-

late nutritional versatility as a key to survival in two contrastingAntarctic saline lakes. Freshwater Biology 50: 830–838.

Maberly SC (1996) Diel, episodic and seasonal changes in pH and

concentrations of inorganic carbon in a productive lake. Fresh-water Biology 35: 579–598.

Maberly SC, King L, Dent MM, Jones RI, and Gibson CE (2002)

Nutrient limitation of phytoplankton and periphyton growth in

upland lakes. Freshwater Biology 47: 2136–2152.Raven JA (1997) Phagotrophy in phototrophs. Limnology and

Oceanography 24: 198–205.

Reynolds CS (2006) The Ecology of Phytoplankton. Cambridge:

Cambridge University Press.Titell J, Bissinger V, Gaedke U, and Kanjuke N (2005) Inorganic

carbon limitation and mixotrophic growth in Chlamydomonasfrom an acidic mining lake. Protist 156: 63–75.