OPEN

ORIGINAL ARTICLE

Autotrophic and heterotrophic acquisition of carbonand nitrogen by a mixotrophic chrysophyteestablished through stable isotope analysis

Ramon Terrado1, Alexis L Pasulka2, Alle A-Y Lie1, Victoria J Orphan2, Karla B Heidelberg1and David A Caron1

1Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA and 2Division ofGeological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA

Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modesbetween autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutritiondiffer among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritionalflexibility, although most species generally fall towards the heterotrophic end of the mixotrophyspectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growingmixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence ofeither heat-killed bacteria enriched with 15N and 13C, or unlabeled heat-killed bacteria and labeledinorganic substrates (13C-bicarbonate and 15N-ammonium). The alga exhibited high growth rates (upto 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotopeanalyses revealed that Ochromonas obtained 84–99% of its carbon and 88–95% of its nitrogen fromconsumed bacteria. The chrysophyte assimilated inorganic 13C-carbon and 15N-nitrogen whenbacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient tosupport net population growth of the alga. Our use of nanoSIMS represents its first applicationtowards the study of a mixotrophic alga, enabling a better understanding and quantitativeassessment of carbon and nutrient acquisition by this species.The ISME Journal advance online publication, 19 May 2017; doi:10.1038/ismej.2017.68

Introduction

Aquatic microbial food webs encompass interactionsbetween bacteria, cyanobacteria, phototrophic andheterotrophic protists, and viruses (Azam andMalfatti, 2007; Sarmento, 2012; Worden et al.,2015). Models of these food webs generally char-acterize eukaryotic microbes as either phototrophic(capable of producing energy and fixing carbon forgrowth using only inorganic compounds and light)or heterotrophic (dependent on the consumption oforganic material for nutrition). These characteriza-tions greatly oversimplify trophic interactionsbecause many microbial eukaryotic algae are mixo-trophic, capable of using both inorganic and organicforms of carbon and major nutrients (Flynn et al.,2013). Although the mixotrophic nature of manyalgae is now recognized, the impact of this behaviorin terms of carbon and nutrient cycling, as well as itsecological significance for the algae that conduct this

behavior, is not widely acknowledged or understood.Some attempts have been made to classify mixo-trophs in a few conceptual models (Våge et al., 2013;Andersen et al., 2015; Mitra et al., 2016; Ward andFollows, 2016), but the importance of mixotrophicorganisms to food web structure and function has notyet routinely been entrained into mathematicalmodels of food web dynamics.

Mixotrophic species exhibit a range of nutritionalbehaviors. Some species are predominantly photo-trophic but supplement photosynthesis with a smallamount of heterotrophy, while other species arepredominantly heterotrophic and use photosynthesisonly for extending survival when prey are scarce(Jones, 1994, 2000; Rottberger et al., 2013). Mixednutrition appears to provide an advantage particularlyin oligotrophic ecosystems (Hartmann et al., 2012;Unrein et al., 2014), presumably because nutrients areoften limiting to purely phototrophic protists, whilestrict heterotrophs are limited by prey availability. Incontrast, mixotrophic protists may meet their elemen-tal and energy demands through a mixture of photo-synthesis and prey ingestion. Yet, mixotrophic algaedo not dominate all ecosystems, and therefore mixednutrition appears to impose tradeoffs that are not wellunderstood (Raven, 1997; Stoecker, 1998).

Correspondence: DA Caron, Department of Biological Sciences,University of Southern California, 3616 Trousdale Ave., AHF 301,Los Angeles, CA, 90089-0371, USA.E-mail: dcaron@usc.eduReceived 21 October 2016; revised 20 March 2017; accepted3 April 2017

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More than 100 mixotrophic species of Chrysophy-ceae have been described, and sequence-basedsurveys of natural samples point to potentially largerdiversity (Andersen, 2007; del Campo and Massana,2011). The group presents interesting model speciesfor investigating mixotrophy due to the widediversity of nutritional strategies among these spe-cies. Some, such as Uroglena americana, appear tobe primarily autotrophic but require a phospholipidthat is obtained via phagocytosis of prey (Kimuraand Ishida, 1989). A similar case has been reportedfor Dinobryon cylindricum, where phagocytosismight also provide essential growth factors and/ornutrients for photosynthetic growth (Caron et al.,1993). At the other end of the spectrum, species existthat exhibit primarily heterotrophic metabolismwhere phototrophy appears to be of minor signifi-cance (Andersson et al., 1989; Caron et al., 1990;Boraas et al., 1992; Sanders et al., 2001; Foster andChrzanowski, 2012; Chrzanowski and Foster, 2014;Wilken et al., 2014).

Experiments with mixotrophic algae have yieldedresults that outline the overall metabolism of theseorganisms (Sanders and Porter, 1988; Sanders, 2011),but most studies have not clearly defined the sources(phototrophy or heterotrophy) of carbon and nutri-ents in these species. Similarly, recent molecularstudies offer insights into the metabolic potential ofthese algae (Liu et al., 2015; Terrado et al., 2015; Liuet al., 2016), but do not specifically quantify thecontributions of photosynthesis and heterotrophy tomixotrophic nutrition. Stable isotope probing (SIP)offers a method to address this issue. Combining SIPwith high-resolution nanometer-scale secondary ionmass spectrometry (nanoSIMS), researchers havebeen able to quantify and map elemental andisotopic abundances at 50–100 nm resolution inorder to assess the metabolic activities and elementalflow within individual cells (Boxer et al., 2009; Gaoet al., 2016). The approach has been used tocharacterize the metabolic potential and anabolicactivity in environmental microorganisms (Orphanand House, 2009; Orphan, 2009), including theutilization of different carbon substrates by deepsub-seafloor bacterial cells (Morono et al., 2011),carbon and nitrogen utilization by cyanobacteria(Popa et al., 2007; Finzi-Hart et al., 2009), the flux ofnitrogen within coral symbionts (Kopp et al., 2013),the transfer of nitrogen from lysed algae to bacteria(Sheik et al., 2014) and the transfer of carbon tobacterial surface symbionts of a large cellulolyticprotist symbiont of a lower termite (Carpenter et al.,2013).

Here we apply stable isotope probing and nano-SIMS to an axenic culture of the mixotrophicchrysophyte, Ochromonas sp. strain BG-1 (Sanderset al., 2001). Incubations were conducted with thealga in the presence of either labeled inorganicsubstrates or labeled heat-killed bacteria, in order toidentify and quantify the sources of carbon andnitrogen utilized by this alga. Given its affinity for

bacterial prey (Sanders et al., 2001), we hypothesizethat this Ochromonas strain, in the presence of highbacterial abundances, would acquire an elevatedpercentage of its carbon and nitrogen requirementsthrough heterotrophy via the ingestion and assimila-tion of bacteria. Our analysis showed that whiledefined as a mixotroph, this strain of Ochromonasrelies primarily on bacterial phagotrophy for bothcarbon and nitrogen acquisition and that photo-synthesis remains a minor aspect of its nutrition.

Materials and methods

Bacterial isolation and production of heat-killedbacteriaHeat-killed bacteria (HKB) were prepared as non-living prey for all experimental work in this study toavoid uptake or release of organic or inorganicsubstances by bacteria in the axenic culture of thechrysophyte. Bacteria from a bacterized culture ofOchromonas BG-1 were streaked on petri disheswith a solid agar medium containing a minimal M9medium (Marley et al., 2001) using ammonium asthe sole source of nitrogen and glucose as the solesource of carbon. Individual colonies were trans-ferred to 25ml tubes containing the M9 medium(without agar), which were used to inoculate 1.5 l ofmedium and incubated at 20 °C on a shaker table inorder to obtain large numbers of bacteria for theproduction of HKB. Cultures were grown to station-ary phase, and cells were transferred into 200mlbottles and heat-killed in a water bath at 70 °C for atleast 1 h. Cells were transferred into 50ml sterileFalcon tubes and concentrated by centrifugationusing an Allegra 25 R centrifuge (Beckman Coulter,Inc., Brea, CA, USA) equipped with a TS-5.1-500swinging bucket rotor (Beckman Coulter) at 5000 r.p.m for 15min. Supernatant was discarded and cellsre-suspended using sterile Milli-Q water by vortex-ing. Cells were centrifuged and washed at least threetimes. After the final wash, cells were re-suspendedin sterile Milli-Q water, and aliquots were storedfrozen in 2ml Eppendorf tubes until use. Theeffectiveness of the heat-killing process was verifiedby inoculating aliquots of the HKB into rich nutrientbroth and monitoring for growth.

In order to label bacteria with stable isotopes, theglucose and ammonium in the M9 medium weresubstituted with D-glucose-1-13C (glucose 4% finalconcentration, 99 atom % 13C, Sigma-Aldrich Corp.,St Louis, MO, USA) and ammonium-15N chloride(18mM final concentration, 98 atom % 15N, Sigma-Aaldrich Corp., St Louis, MO, USA). Growth, heat-killing and processing of the labeled bacteriafollowed the same protocol described above.

Ochromonas cultures and experimental incubationsCultures ofOchromonas sp. strain BG-1 weremaintainedaxenic and bacteria-free on a modified DY-V recipe:medium was prepared following the Provasoli-Guillard

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National Center for Marine Algae and Microbiotaprotocol (see https://ncma.bigelow.org/algal-recipes)but without either MES buffer or nitrate, and withthe addition of sodium bicarbonate at 95 μM; the soleinorganic source of nitrogen in this medium wasammonium chloride at 50 μM. Prior to the experi-mental incubations, an axenic Ochromonas culturewas supplemented with HKB in order to increase theabundance of Ochromonas. The resulting culturewas used as the inoculum for the experimentalincubations once HKB had been depleted for 2–3 days (4–5 days of growth following inoculation).Three experiments combining different sources oflabeled substrates/prey and light conditions wereperformed:

Experiment 1: Ochromonas was grown in tripli-cate cultures with labeled-inorganic substrates;ammonium-15N chloride (98 atom % 15N, Sigma-Aldrich Corp.) was added as 50% of the totalammonium (25 μM ammonium-15N and 25 μM ammo-nium-14N) and sodium bicarbonate-13C (98 atom %13C, Sigma-Aldrich Corp.) was added as 100% of thetotal bicarbonate (95 μM). A separate set of triplicatecontrol cultures with unlabeled ammonium andbicarbonate was run in parallel. Initial abundancesof Ochromonas in these cultures were ~ 5× 103 cellsml−1. Unlabeled HKB were added at abundances of5 × 107 HKB ml− 1 to both sets of triplicate cultures atthe time of inoculum with the alga to ensure that preywas initially present at high abundance. Cultures wereincubated at 20 °C with constant illumination andconstant mixing using a magnetic stirrer to avoidsettlement of non-motile HKB. We chose stirring basedon preliminary work with other Ochromonas strainsin the lab that compared constant gentle stirring vsconstant gentle shaking. In these preliminary experi-ments, no significant differences were observed ingrowth rates between these methods of mixing.

Experiment 2: Ochromonas was grown in tripli-cate cultures with 13C- and 15N-labeled HKB.A separate set of triplicate control cultures withunlabeled HKB was run in parallel. Unlabeledammonium and bicarbonate were added to themedia at the same concentrations as in Experiment1, and initial abundances of the alga (~ 5×103 cellsml−1) and HKB (~ 5×107 HKB ml−1) were also thesame. Cultures were incubated at 20 °C with constantillumination and constant mixing using a magneticstirrer.

Experiment 3: Ochromonas was grown in tripli-cate cultures with 13C- and 15N-labeled inorganicsubstrates at the same concentrations previouslyused, unlabeled HKB at the same starting abun-dances, and culture conditions exactly as inExperiment 1, except that the incubation was car-ried out in continuous darkness. A set of controlcultures with unlabeled ammonium and bicar-bonate was incubated in parallel in continuousdarkness.

Cultures were sampled daily for determinations ofHKB and Ochromonas abundance and chlorophyll a

(Chl a) concentration. Samples for counts of HKBwere preserved with formaldehyde (3.7% finalconcentration) and stored at 4 °C prior to stainingwith DAPI using standard methods (Porter and Feig,1980). HKB were enumerated using an OlympusBX-51 epifluorescence microscope (Olympus Scien-tific Solutions Americas, Inc., Waltham, MA, USA).Algal densities were determined from 1ml samplespreserved with 50 μl of Lugol’s solution and stored at4 °C until enumeration by light microscopy using aPalmer-Maloney chamber. Chl a was measured from50ml samples filtered onto 25mm diameter glassfiber filters (GF/F, Whatman), extracted with 4ml100% acetone at − 20 °C overnight in the dark, andmeasured using a non-acidification protocol with aTrilogy Lab fluorometer (Turner Designs, Sunnyvale,CA, USA; Welschmeyer, 1994).

Samples for nutrients (ammonium and solublereactive phosphate) were collected at the timeof inoculation and periodically throughout thecourse of the experiments. Approximately 15 mlof sample was filtered through a 0.2 μm filter into aclean glass vial that had been previously triple-rinsed with 0.2 μm filtered Milli-Q water thentriple-rinsed with 0.2 μm filtered sample water.Duplicate blanks of 0.2 μm filtered Milli-Q waterwere stored and analyzed for each sampling date.The vials were stored frozen until analysis at theMarine Science Institute Analytical Lab at theUniversity of California Santa Barbara (MSI-UCSB)by flow injection analysis using a QuikChem 8000(Lachat Instruments Div., Zellweger Analytics, Inc.,Hach Company, Loveland, CO, USA).

Bulk particulate carbon and nitrogen, and13C- and 15N-isotopes, were determined from 30–50 ml of the cultures filtered onto pre-combustedglass fiber filters (GF/F, Whatman). Samples werecollected at the time of inoculation and when HKBabundance had been reduced three orders ofmagnitude. Carbon and nitrogen were also mea-sured in 50–150 μl aliquots of the stock suspen-sions of HKB. Blank filters consisting of 20 ml of0.2 μm filtered Milli-Q water were also analyzed.Filters were dried at 60 °C. Analysis of CHN wasdone at the MSI-UCSB using a CEC 440HA (ExeterAnalytical; EA) following standard protocols, andan acidification step with HCl to remove inorganiccarbon. The C- and N-isotopic composition of theparticulate organic matter was determined as CO2

and N2 released by flash combustion using aFinnigan DELTAplus Advantage in ContinuousFlow Mode coupled to an isotope ratio massspectrometer (IRMS) Costech ECS 4010 ElementalCombustion System.

NanoSIMS analysisNanoSIMS analysis was conducted in order toexamine the source of carbon and nitrogen (that is,inorganic vs HKB) incorporated by Ochromonas.Sample preparation for nanoSIMS included 2ml of

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culture collected at the time of inoculation and at48 h (the end of rapid growth of Ochromonas whenbacterial prey had been exhausted). Samples of HKBstocks were also collected. Samples were preservedwith 2X EM grade glutaraldehyde (2.5% finalconcentration) and stored at 4 °C until analysis. Cellswere deposited onto silicon wafers (7 × 7mm, 725+/− 25 μm thick, P-type/Boron 0.028 Ω-cm resistiv-ity; Active Business Company GmbH, MunichGermany), washed with Milli-Q water and air dried.NanoSIMS was performed at the Caltech Microana-lysis Center using a Cameca NanoSIMS 50 L instru-ment. Individual cells were located using thenanoSIMS CCD camera and random areas on thewafer were chosen for analysis. Algal cells were pre-sputtered with a 30 pA primary Cs+ beam current for1 frame (~12min) and then analyzed using a 1 pAprimary Cs+ beam current with a nominal spot size of100 nm.

For all analyses, the beam was rastered over asquare region of 10 × 10 μm for 12 min per analy-tical frame. Pre-sputtering was carried out on alarger region of at least 13 × 13 μm to make sure thatthe analytical frame was fully within the pre-sputtered area. Four masses, 12C2

−, 12C13C−, 12C14N−

and 12C15N−, were collected in parallel usingelectron multipliers. Secondary electrons were alsocollected during the analysis, providing secondaryelectron images of the cells. Ion images wererecorded at 512 × 512 pixel resolution with a dwelltime of 2.5 ms per pixel and at least 4 frames peranalysis, which were merged into a single frameduring post-processing. A total of 33 Ochromonascells from the end of the rapid growth phase of thealga were analyzed, with a minimum of 4 cellsanalyzed per treatment. Unlabeled HKB with aknown 15N and 13C composition were measured atthe start and end of all nanoSIMS measurements inorder to determine the magnitude of instrumentalisotope fractionation and drift during the analysis.The average nanoSIMS acquired 15N/14N and13C/12C ratios were then compared and correctedwith the elemental analysis-IRMS acquired valuesfor the unlabeled HKB. The offset between thenanoSIMS and the elemental analysis-IRMSresulted in a correction factor of 1.1165 for 13Cratios and 1.0724 for 15N ratios. 13C and 15N cellularenrichments measured by bulk elemental analysis-IRMS and single-cell nanoSIMS analyses for theHKB were: 15N FHKB was 0.779 ± 0.011 (bulkelemental analysis-IRMS) or 0.901 ± 0.001 (nano-SIMS) and 13C FHKB was 0.041 ± 0.00003 (bulkelemental analysis-IRMS) or 0.050 ± 0.001 (nano-SIMS). Error values indicate standard errors.

The raw data from ion images were processedusing the open-source MATLAB plugin Look@Na-noSIMS (Polerecky et al., 2012). Ion images frommultiple frames were corrected for dead time andquasi-simultaneous arrival (QSA) effect, accumu-lated and aligned. Each cell was defined as a regionof interest (ROI) using the 14N12C− image and

compared with the corresponding secondary elec-tron image as a guide.

CalculationsThe growth rates of Ochromonas were calculatedbetween sampling intervals using the formula:

m ¼ ln Ochromonasf½ �–ln Ochromonasi½ �ð Þ= Tf�T ið ÞðiÞ

where μ is growth rate, (Ochromonasf) is the finalabundance of Ochromonas, (Ochromonasi) is theinitial abundance of Ochromonas and Tf and Ti arethe final and initial sampling times in hours,respectively. Ingestion rates were calculated for thealga up to time when bacterial abundances weregreatly reduced as in (Sanders et al., 2001), using theformula:

I ¼ HKB½ �i � HKB½ �f� �

= D Ochromonas½ �ð Þ= ln Ochromonasð �f= Ochromonas½ �i� �� �

= Tf�T ið ÞðiiÞ

where I is ingestion rate, Δ (Ochromonas) the changein abundance of the alga, and Tf and Ti are the finaland initial sampling times in hours, respectively.

Molar concentrations of nitrogen in HKB werecalculated from CHN values of known abundances ofHKB. Molar concentrations of nitrogen in Ochromo-nas were calculated from CHN values of knownabundances of the alga in the inoculum at time 0,and at successive time points. Molar concentrationsof dissolved inorganic nitrogen were obtained fromammonia concentrations in the cultures. Nitrogenmolar concentrations associated with both HKB andOchromonas biomass at the time of inoculum andwhen HKB were grazed to very low abundances(after 48 h, at which time nitrogen associated withHKB was negligible), were used to calculate grazingassimilation efficiency.

For nanoSIMS, all ion ratios were converted tofractional abundance using the relationship Fsample =Rsample/1+Rsample, where F is the fractional abun-dance and R is the ratio of either 13C/12C or 15N/14N.The carbon isotope data were collected using themolecular ions 13C12C- and 12C2- and to accuratelycalculate the 13C fractional abundance from our rawion counts, the Rsample was multiplied by 0.5.

Finally, to determine the sources responsible forthe isotopic enrichment in Ochromonas cells (fHKB orfInorg.), the following isotope mass balance was usedfor both carbon and nitrogen calculations:

FOchromonas ¼ FHKB � fHKB þ F Inorg: � f Inorg: ðiiiÞassuming that:

fHKB þ f Inorg: ¼ 1 ðivÞwhere FOchromonas is the 13C or 15N fractional abun-dance for the alga, FHKB is the 13C or 15N fractionalabundance for the heat-killed bacteria, and FInorg isthe 13C or 15N fractional abundance of the inorganicsubstrate. Nitrogen FInorg was assumed to be either0.49 for treatments with 15N-ammonium chloride

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(based on molar additions), or 0.00367 for non-labeled treatments (based on natural abundance);Carbon FInorg was assumed to be 0.98 for treatmentswith 13C-bicarbonate or 0.0109 for non-labeledtreatments. Using equations (iii) and (iv) we solvedfor fHKB (the fraction of HKB) and fInorg (the fraction ofinorganic nitrogen or carbon) in the different experi-mental treatments.

Results

Growth of Ochromonas sp. strain BG-1Initial abundances of Ochromonas in all experimen-tal incubations ranged from 5×103 to 1.2 × 104

Ochromonas cells ml−1. Rapid growth of Ochromo-nas lasted ~2 days (Figure 1a). The growth ratethroughout this entire growth phase ranged from 1.7

to 2.0 day− 1 (Table 1), and cell abundances increased30- to 60-fold (Figure 1a). HKB abundancesdecreased 3 orders of magnitude during this periodof rapid algal growth, from the average inoculum of5× 107 HKB ml− 1 to an average value of 9 × 104

HKB ml− 1 after 48 h of algal grazing (Figure 1b). HKBabundances were too low to be counted confidentlyafter 100 h of incubation. Although algal abundanceincreased many-fold in the first 48 h, total Chl aincreased only 1.4- to 3-fold for algae incubated inthe light (Figure 1c), effectively resulting indecreases in the chlorophyll concentration alga− 1

in all treatments (Figure 1d). Ammonium andphosphate during this period showed no netremoval, but rather accumulated substantially inthe medium during the first 48 h of the experiments(Figure 2). Blank measurements of 0.2-μm filteredMilli-Q water were consistently two orders of

Figure 1 Cell abundances of Ochromonas sp. strain BG-1 (a), abundances of heat-killed bacteria, HKB (b), Chl a concentration in thecultures (c) and Chl a alga− 1 (d) in three experiments. Average and standard error values for three replicates are represented. Solid linesrepresent cultures incubated in 24 h light regimes, dotted lines indicate cultures in continuous darkness. Circles represent culturesincubated with stable isotope labeled HKB and non-labeled inorganics, triangles indicate cultures with stable isotope labeled inorganicsand non-labeled HKB. Squares are control cultures grown with unlabeled inorganics and unlabeled HKB.

Table 1 Growth rates and ingestion rates of Ochromonas in three different experiments

Experiment 1 Experiment 2 Experiment 3

Control Labelled Control Labelled Control Labelled

Light Yes Yes Yes Yes No NoLabel None Inorganics None HKB None InorganicsGrowth rate 0–48 h (μ, per day) 1.74±0.08 1.72±0.04 2.04±0.04 1.99±0.12 1.76± 0.38 1.93±0.10Growth rate 48–148 h (μ, per day) 0.01±0.04 −0.01±0.05 -0.01±0.03 −0.01±0.03 −0.04± 0.03 − 0.07±0.09Ingestion rate (HKB per alga per h) 13.90±0.48 13.06±1.99 19.99±3.10 16.87±3.63 16.02± 1.71 15.00±1.35

Each experiment had triplicate unlabeled controls and triplicate labeled cultures. Labels for the inorganic constituents were 13C-bicarbonate and15N-ammonium. Growth rates for the alga during the first 48 h of incubation (0–48 h) and after HKB were depleted (48–148 h) are presented;ingestion rates are presented for the initial 48 h period as no ingestion was observed after HKB were depleted. HKB stands for heat-killed bacteria.

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magnitude lower than culture samples, or below thedetection limit.

Ochromonas abundances in cultures incubated inthe light did not increase significantly beyond thefirst 48 h of incubation (Figure 1a). Nevertheless,total Chl a concentrations continued to increase inthose cultures after 48 h, resulting in increases inchlorophyll content cell−1 (Figures 1c and d). Con-currently, ammonium concentrations decreased incultures receiving light following 48 h of rapid algalgrowth, although phosphate concentrations contin-ued to increase (Figure 2). Cultures incubated incontinuous darkness exhibited low total Chl aconcentrations throughout the entire experiment, aswell as very low cellular Chl content (dotted lines inFigures 1c and d). Both ammonium and phosphateconcentrations continued to increase throughout theexperiment in Ochromonas cultures incubated incontinuous darkness (Figure 2).

Consumption of HKB by Ochromonas averaged 15bacteria cell−1 h− 1 through the 2 days of rapid growthof the alga (Table 1). Ingestion rates were higher forthe first 24 h, with values up to 50 bacteria cell−1 h−1.Ochromonas reduced HKB abundances three ordersof magnitude after 2 days, to less than 105 HKB ml− 1.A grazing efficiency for nitrogen by the alga wasestimated from algal and bacterial abundance mea-surements and the molar concentrations of nitrogencontained in HKB and Ochromonas. On the basis ofthose estimates, ~ 50% of nitrogen contained in HKB

was assimilated by Ochromonas when grown in thelight; cultures that were grown in the dark presenteda slightly lower nitrogen transfer efficiency, with upto 30% of nitrogen in HKB assimilated by Ochromo-nas (Figure 3).

Stable isotope analysisIncubations were carried out with either labeledinorganic nitrogen and carbon (as 15N-ammoniumchloride and 13C-bicarbonate) or labeled heat-killedbacteria (HKB; previously grown on labeled sub-strates; 15N-ammonium and 13C-glucose) in order todetermine the sources of nitrogen and carbonincorporated by Ochromonas. Isotopic ratios mea-sured by nanoSIMS were consistent both withinsingle cells and between cells within the sametreatment. The error associated with repeated mea-sures of the same cell (measured as a Poisson error)was always at least 2 orders of magnitude lower thanthe measured isotopic fractionation. For cells withinthe same treatment, the standard error was at least 1order of magnitude lower than their average isotopicratio. At the end of the period of rapid growth, allthree experiments presented a significantly higherfractional abundance of both 15N and 13C in repli-cates incubated with an enriched isotopic substratecompared to the control replicates (ANOVA, for eachtreatment and isotope Po0.01). Ochromonas wasmost enriched in the incubations with labeled HKB(fractional abundance for 15N=0.793± 0.007,13C= 0.044 ± 0.001; Figures 4a and b) followed bythe incubations amended with 15N-ammonium and13C-bicarbonate in the light (fractional abundance for15N=0.028± 0.003, 13C= 0.017 ± 0.001; Figures 4aand b). The experiment performed with labeledinorganic substrates in the dark yielded low values(fractional abundance for 15N=0.012 ± 0.0004,13C= 0.012 ± 0.0001; Figures 4a and b).

There was good agreement between nanoSIMSand bulk elemental analysis-IRMS measurementsshowing the same trends in enrichment (Figures 4cand d), although there was a somewhat greater offsetbetween these measurements for samples that werehighly enriched, particularly for 13C carbon. Addi-tional bulk elemental analysis-IRMS measurementswere collected after 48 h, at time points beyond theperiod of rapid growth of the alga when HKB hadbeen reduced to insignificant abundances. Thesemeasurements revealed a decrease in F for 13C in thetreatments with labeled HKB (Experiment 2 inFigure 5), while the treatments with labeled inor-ganic nitrogen and carbon in the light exhibited aslight increase in fractional 15N (Experiment 1 inFigure 5). Fractional abundances for the cultureincubated in continuous darkness did not showsignificant changes after the depletion of HKB(Experiment 3 in Figure 5).

We used the stable isotope mass balance calcula-tions (iii) and (iv) described in the Material andMethods in order to determine the relative

Figure 2 Ammonium and phosphate concentrations (μM) in threeexperiments with Ochromonas grown with heat-killed bacteria(HKB) and inorganic nutrients. Averages and standard errors forthree replicates are represented. Solid lines represent culturesincubated in 24 h light regimes, dotted lines cultures in contin-uous darkness. Circles represent cultures incubated with stableisotope labeled HKB and non-labeled inorganics, triangles indicatecultures with stable isotope labeled inorganics and non-labeledHKB, and squares are control cultures grown with both unlabeledinorganics and unlabeled HKB.

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contributions of either organic or inorganic nitrogenand carbon sources during mixotrophic growth ofOchromonas. The nanoSIMS measurements of 15Nand 13C enrichment of algal cells recovered from thedifferent incubation conditions revealed that most ofthe nitrogen and carbon assimilated by Ochromonaswas derived through bacterivory of the HKB, ratherthan CO2 fixation and ammonium assimilation. Theresults of Experiment 1 (incubated in light) andExperiment 3 (incubated in continuous darkness)using heavy isotope-labeled inorganics and unla-beled HKB indicated that 95 or 98% of algalnitrogen, and 99.4 or 99.9% of algal carbon,respectively, was derived from HKB. Experiment 2,using heavy isotope-enriched bacteria and incubatedin the light, yielded slightly lower values ofincorporation of nitrogen (88%) and carbon (84%)from HKB, but bacteria were still the major source ofnitrogen and carbon in all experiments.

DiscussionGrowth and nutrient dynamics of Ochromonas sp.,strain BG-1Phagotrophy by phototrophic flagellates is believedto confer significant ecological advantages on algaethat exhibit the behavior (Flynn and Mitra, 2009), butthere also appear to be limitations (although largelyuncharacterized) for conducting mixed nutritionsimultaneously in a single cell (Raven, 1997). Theseconstraints may involve costs or tradeoffs for main-taining dual cellular machineries (compared tospecialized competitors), or perhaps cross-talkbetween anabolic and catabolic biochemical

pathways that confound the performance of bothactivities simultaneously. Unfortunately, there isvirtually no quantitative information on the costsand benefits of mixotrophic behaviour, nor whetherboth processes are carried out sequentially orsimultaneously by these species. Temporal partition-ing might be a mechanism for maintaining bothabilities in minute cells (albeit putting one process‘on hold’ at any given time). Assessing whether bothbehaviors are occurring simultaneously, the specificphysiological benefit(s) for heterotrophic nutrition inmixotrophic algae, and the responsiveness of thisbehavior to abiotic and biotic variables are aspects ofmixotrophic nutrition that have been difficult toestablish using traditional approaches and methods.

We studied the mixotrophic chrysophyte Ochro-monas sp. strain BG-1 because it can be grown inaxenic culture (bacteria-free) and it has been pre-viously reported to be a predominantly heterotrophicorganism that attains high growth rates only in thepresence of bacteria (Sanders et al., 2001). Growth inaxenic culture prevented potentially complicatingbiological interactions and elemental flows resultingfrom the activities of other living microorganisms inthe cultures, thereby enabling a comparison betweennanoSIMS and bulk IRMS measurements and abetter understanding of the specific sources ofcarbon and nitrogen used for growth by the alga.

Additionally, the use of a single inorganic sourceof nitrogen simplified our experimental design sothat the only sources of nitrogen in the medium wereammonium or HKB. Algae generally possessmechanisms for the uptake and assimilation of bothammonium and nitrate, but transcriptomic dataindicate that some chrysophytes, including

Figure 3 Nitrogen mass balance for the three different experiments with Ochromonas. All nitrogen at the beginning of the experimentswas either present as organic nitrogen (associated with Ochromonas or HKB) or inorganic nitrogen in the medium (ammonium). Nitrogenassociated with Ochromonas or the inoculum of HKB was calculated from CHN analysis of the two populations and cell abundances in thecultures, with values transformed to μM. Dissolved ammonium was measured and transformed to μM concentrations. After ~ 48 h ofincubation, virtually all HKB were consumed by Ochromonas, and the nitrogen contained within HKB was assimilated into cells (area ofcolumn with dots at time 48 h) or released into the medium. Arrows represent an estimate of the percentage of nitrogen associated withHKB at time 0 and assimilated by Ochromonas at time 48 h, assuming that the excess nitrogen was released as ammonium.

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Figure 4 (a) Examples of cells analyzed by nanoSIMS, showing both unlabeled and labeled Ochromonas. The first row shows images ofthe alga obtained through secondary ion emission, the second row shows the fractional abundance for 13C, and the third row shows imagesof the fractional abundance for 15N. Note the different panel scales for 13C and 15N fractional abundances: the 13C fractional abundancecolor scale ranges from 0.02 to 0.1; the 15N color scale range varies among the four panels as differences were too wide to represent with asingle scale for all panels. Columns in (a), from left to right, show cells in the control treatment (no stable isotope labeling), cells grown inthe presence of stable isotope labeled inorganic carbon and nitrogen with unlabeled HKB (Experiment 1), cells grown in the presence ofstable isotope labeled HKB and unlabeled inorganic carbon and nitrogen (Experiment 2), and cells grown in the presence of stable isotopelabeled inorganic carbon and nitrogen with unlabeled HKB but incubated in continuous darkness (Experiment 3). (b) Plot of average (± SE)fractional abundances for 15N and 13C measured by nanoSIMS in cells from inoculum HKB, inoculumOchromonas and Ochromonas at theend of rapid growth (448 h). Values for the three experiments are presented, including the stable isotope labeled and non-labeledreplicates; note that error bars are often smaller than the data points. (c) and (d) show the fractional abundances for 15N and 13C,respectively, measured by nanoSIMS vs the fractional abundances measured by bulk IRMS. Samples for nanoSIMS and IRMS werecollected at the same time. Diagonal lines in the plots correspond to a 1:1 equivalence.

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Ochromonas sp. strain BG-1, might be missing thegenetic capability for nitrate assimilation (Terradoet al., 2015; Lie et al., 2017). Furthermore, MESbuffer was not added to the medium in ourexperiments because this might have provided analternative source of nitrogen. MES might also haveconstituted a source of organic carbon for the alga(Sanders et al., 2001), and its elimination ensuredthat the only sources of carbon in the medium wereeither bicarbonate or HKB. Transcriptomic analysisfrom experiments carried out in the same way as theones presented in this manuscript show that thephotosynthetic machinery of Ochromonas sp. strainBG-1 is expressed and upregulated in the presence oflight (Lie et al., 2017). This simplified approach,combined with stable isotope labeling, allowed us todetermine which source(s) of nitrogen and carbonwere used for growth by the alga.

Significant growth rates of Ochromonas in thepresent study were achieved only while HKB wereabundant as prey (Figure 1a). Chl a dynamics in theculture also reflected the high growth rates due tograzing on HKB, as the concentration of Chl a cell−1

decreased by one order of magnitude during the first48 h of incubation for cultures grown in the light aswell as in continuous darkness (Figure 1d). Thesechanges in cellular chlorophyll might be related to adilution of Chl a inside the cells due to the highgrowth rates of the alga (Hansen et al., 2000), so thatthe reduction of Chl a cell−1 was a consequence offast growing rates of the alga and not a directreduction of chlorophyll biosynthesis rate.

Nevertheless, it is also possible that there was someregulation of chlorophyll biosynthesis when HKBwere present as transcriptomic analysis on this algasuggests upregulation of genes related to chlorophyllsynthesis in the presence of light when HKB weredepleted (Lie et al., 2017). In any case, theseobservations are in agreement with previous studiesof Ochromonas (Pringsheim, 1952; Sanders et al.,2001) that observed a well-developed heterotrophiccapability, suggesting that carbon fixation andperhaps other cellular structures and processesinvolved in photosynthesis are reduced when grow-ing mixotrophically, as observed for some otheralgae (Wan et al., 2011).

Dissolved ammonium, as well as phosphate,accumulated in the Ochromonas cultures duringthe first 48 h of the experiments, when HKB wereactively grazed (Figure 2). This result indicates thatexcess nitrogen and phosphorous from grazed HKBwere excreted by the alga. Mass balance calculationsbased on changes in prey/algal abundances and theircellular nitrogen contents indicated that up to 50%of the nitrogen contained in consumed HKB wasassimilated by the alga, while a considerable amountof the excess nitrogen was released primarily asammonium during the period of active bacterialgrazing (Figure 2). These values for nitrogen assim-ilation (and excretion) are consistent with assimila-tion efficiencies of heterotrophic protists of similarsize (Taylor, 1982; Caron and Goldman, 1990),consistent with the conclusion that Ochromonaswas growing predominantly as a heterotroph when

Figure 5 Plots of average ( ± s.e.) fractional abundances for 15N and 13C measured by bulk IRMS for inoculum HKB, inoculumOchromonas, and Ochromonas at the end of rapid growth ( ~ 48 h) plus two additional values for Ochromonas after the period of rapidgrowth (448 h). Numbers above symbols indicate the sampling time (h); non-labeled time stamps have been removed for ease ofvisualization because they presented similar values around the natural isotopic abundance of 15N and 13C (red symbols). Values for thethree experiments are presented, including the labeled and non-labeled replicates. Note that axis scales among the three panels differ.

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prey were abundant. Further, transcriptomic ana-lyses have demonstrated that different ammoniumtransporters are expressed by Ochromonas sp. strainBG-1 growing on HKB compared to growth afterbacteria have been grazed to very low abundances(Lie et al., 2017). It therefore appears that transpor-ters for the export of ammonium out of the cell maybe different than those used for ammonium uptake,as has been observed for other organisms(Shnaiderman et al., 2013).

Interestingly, concentrations of ammonium, butnot phosphate, decreased in the medium once theHKB had been removed by grazing (that is, after 48 h;Figure 2) when Ochromonas was grown in the light.This result appears to indicate that the alga activelytook up ammonium (but not phosphate) from themedium when bacteria were no longer available andphotosynthesis was induced (Figure 1d). In contrast,both ammonium and phosphate in the culturesgrown in the dark continued to rise throughout theexperiment (dotted lines in Figure 2). No significantnet algal population growth occurred followingdepletion of prey even in the light, and theexplanation for the dichotomy in the uptake of thesetwo elements is unclear. We speculate that ammo-nium was taken up because it was specificallyrequired for rebuilding the photosynthetic machin-ery of the cell.

The continued appearance of phosphate in theculture medium during the later part of the experi-ments contrasts with studies of some other Ochro-monas species that have reported an uptake ofphosphate when the alga is growing autotrophically(Rothhaupt, 1996). The lack of phosphate uptake bystrain BG-1 might indicate that this Ochromonas wasincapable of effective phosphate uptake (whichmight also explain, in part, the poor phototrophicgrowth capacity of this strain), or that phosphoruswas not needed in significant amounts for cellularreorganization associated with the change to photo-trophic growth, and therefore uptake was notstimulated by the change to phototrophy. It isunlikely that the continued increases in the concen-tration of phosphate were due to the decompositionof dissolved organic phosphorus compounds in themedium because the cultures had no live bacteria.

Inferences from stable isotope probing experimentsStable isotope analysis (nanoSIMS and bulk ele-mental analysis-IRMS) revealed that both inorganic(13C-bicarbonate and 15N-ammonium) substrates and13C/15N-labeled HKB were assimilated by Ochromo-nas, contributing to 15N and 13C cellular enrichmentafter 48 h of incubation (Figure 4). However, themagnitude of enrichment from inorganic substratesor HKB indicated that, during mixotrophic growth,the primary sources of carbon and nitrogen werederived from phagotrophy. Isotopic mass balanceindicated that 88–95% of the nitrogen and 84–99%of the carbon were derived from HKB when

Ochromonas was growing mixotrophically in thelight. An enrichment of 13C was observed in algaegrown in the light compared to the dark (Experiment1 vs Experiment 3) when 13C-bicarbonate wasavailable (Figure 4). However, the calculated con-tribution of photosynthetic carbon fixation was only1–10% of the carbon assimilated into biomass. Thegreater efficiency of nitrogen incorporation fromprey observed in the light relative to continuousdarkness (Figure 3; Experiments 1, 2 vs Experiment3) suggests that light played a role, albeit a minorone, in phagotrophic efficiency of the alga. There-fore, despite putative reductions in the photosyn-thetic machinery of Ochromonas growingphagotrophically on HKB (as evidenced by low cellquotas of Chl a; Figure 1d), light did have a minorand positive impact on algal nutrition. Because theamount of carbon fixed by photosynthesis repre-sented a small fraction of carbon assimilated bythe alga when growing on HKB, we speculate thatthe photosynthetic apparatus might be providingenergy rather than carbon for cellular material, as ithas been hypothesized for Ochromonas danica(Wilken et al., 2014).

Our isotopic mass balance calculations have twocaveats. First, we did not control the pH evolutionwithin the cultures, which would have given betterinsights on the carbonate equilibrium that mighthave been affected by respiration and carbon fixa-tion, and exchange with the atmosphere. As such,our estimate based on the experiments using labeledinorganic carbon might have underestimated theamount of inorganic carbon fixed by Ochromonas sp.BG-1 (1% according to the isotopic mass balance). Inany case, the experiment using labeled HKB shouldnot have been affected by this caveat, and theestimate of 10% of carbon derived from inorganicsubstrate is likely realistic. Second, Ochromonas isconsidered an inefficient carbon fixer due to missingcarbon concentration mechanisms (Maberly et al.2009) that increase CO2 concentration via thetransport of CO2 and/or bicarbonate to the RubisCOenzyme (Raven et al., 2008). On the other hand,transcriptomic analysis have shown that the photo-synthetic machinery of strain BG-1 is functional (Lieet al., 2017), and our experiments using labelledbicarbonate showed a significant enrichment of the13C fractional abundance in Ochromonas (Figures 4and 5); therefore, Ochromonas sp. strain BG-1 hassome capability to use inorganic carbon, albeitinefficiently.

Nevertheless, the strong heterotrophic activity ofOchromonaswhile growing mixotrophically is likelyto increase the intracellular CO2 pool as well as itsflux. As a rule of thumb, it is considered thatheterotrophic protists assimilate 40% of ingestedorganic matter, while they release 30% and respireanother 30% (Sleigh, 1989). Based on this, the totalamount of carbon released by Ochromonas as CO2

during exponential growth could be as high as thetotal bicarbonate added at the beginning of the

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incubation, which has consequences for the isotopicmass balance we have presented. If we assume thatthe isotopically enriched CO2 derived from the HKBrespiration is available at the same levels as thedissolved inorganic carbon, the incubation per-formed with Ochromonas and labeled HKB indi-cated that ~ 84% of carbon was derived from theHKB. Up to 20% of the assimilated carbon derivedfrom HKB could actually correspond to carbon thatwas initially respired and then fixed by Ochromonas.If correct, respiration derived from the phagotrophicactivity would act as a carbon concentration mechan-ism of sorts for this Ochromonas. While the primarysource of carbon for Ochromonas growing mixotro-phically was derived from HKB, a non-negligibleamount might have been derived from the respirationand then CO2 fixation of the bacterial biomass.

Ochromonas shifted its metabolism towards auto-trophy when incubated in the light, but only after ithad depleted the HKB in the cultures (≈ 48 h ofgrowth). This shift was reflected in the bulk IRMS13C fractional abundance for the treatment usinglabeled HKB where a reduction was observedbetween 48 h and 145 h (Experiment 2 in Figure 5),indicative of the incorporation of unlabeled carboninto algal biomass via light processes. Chrysophytesare generally considered poor autotrophic carbonfixers due to poor carbon concentration mechanisms(Maberly et al., 2009). Nonetheless, these resultsindicate that there was a significant level ofinorganic carbon assimilation. A comparison ofcultures grown in the light (Experiment 1 inFigure 5) and continuous darkness (Experiment 3in Figure 5) revealed that the 15N fractional abun-dance of the dark cultures did not change after 95 hwhile cultures in the light continued to enrich in 15N,indicating that Ochromonas continued to assimilatenitrogen to maintain its metabolism once HKB weredepleted. These results were consistent with observeddecreases in the concentration of ammonium in themedium during this time period (Figure 3a), althoughthe continued appearance of phosphate in the med-ium during this period is unexplained.

We obtained good overall agreement between thebulk isotope measurements and nanoSIMS measure-ments for nitrogen, consistent with observations inprevious studies (Popa et al., 2007; Orphan andHouse, 2009; Kopf et al., 2015; Figure 4c). However,the bulk measurements were somewhat lower interms of fractional abundance values for carbon,particularly for highly enriched samples (Figure 4d).We speculate that the differences between nanoSIMSand the bulk isotope measurements for carbon maybe related to the fact that nanoSIMS samples werepreserved with glutaraldehyde, whereas the samplesfor bulk analysis were not. Fixation has been shownto influence cellular carbon (Musat et al., 2014),although we might have expected this to dilute the13C in the nanoSIMS measurements relative to thebulk values. A more likely explanation is that thesingle-cell measurements are not influenced by

cellular debris in the culture that may be lessenriched. The bulk 13C value may be diluted bythese components relative to the nanoSIMS mea-surements, implying that the nanoSIMS data maymore accurately reflect carbon and nitrogen uptakeby the algae. However, cell to cell variability mayalso have contributed to the minor differencesbetween the bulk and nanoSIMS measurements.

The use of nanoSIMS in this study represents itsfirst application towards the study of carbon andnutrient fluxes in a mixotrophic alga, and hasenabled a better understanding of carbon and energyacquisition by this species, and cellular metabolism.Our findings expand information available fromtraditional analyses of Ochromonas sp. strain BG-1grown under various conditions of light and preyavailability (Sanders et al., 2001), confirming thatmost of the nitrogen and carbon used for growth areobtained through its bacterial prey. Although theresults cannot be directly extrapolated to all speciesalong the continuum of algae with different mixo-trophic strategies, our work validates the use ofstable isotope probing experiments and nanoSIMS tobetter understand the metabolic underpinnings ofmixotrophy in one species. Moreover, it provides anapproach for assessing mixotrophic nutrition inenvironmental samples. Ochromonas sp. strainBG-1 provided an ideal model system for comparingbulk isotope analysis to nanoSIMS because bacteriawere quickly removed by grazing within the first48 h of the experiments. The agreement betweenthese two measurements demonstrates that nano-SIMS accurately captured the dynamics of carbonand nutrient acquisition in this mixotroph, andcould therefore be more broadly applied to exploremixotrophy in complex mixed communities wherebulk measures would be insufficient to capture thesedynamics. This and future detailed studies willcontinue to yield improvements in our understand-ing of the nutrition of mixotrophic algae.

Conflict of Interest

The authors declare no conflict of interest.

AcknowledgementsThis collaborative research was supported by the Gordonand Betty Moore Foundation through Grant GBMF3299 toDAC and KBH at USC, and Grant (GBMF3780) to VJO atCalifornia Institute of Technology.

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