Growth, ammonium metabolism, and photosynthetic properties of Ulva australis (Chlorophyta) under decreasing pH and ammonium enrichment

The responses of macroalgae to ocean acidification could be altered by availability of macronutrients, such as ammonium (NH4+). This study determined how the opportunistic macroalga, Ulva australis responded to simultaneous changes in decreasing pH and NH4+ enrichment. This was investigated in a week-long growth experiment across a range of predicted future pHs with ambient and enriched NH4+ treatments followed by measurements of relative growth rates (RGR), NH4+ uptake rates and pools, total chlorophyll, and tissue carbon and nitrogen content. Rapid light curves (RLCs) were used to measure the maximum relative electron transport rate (rETRmax) and maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm). Photosynthetic capacity was derived from the RLCs and included the efficiency of light harvesting (α), slope of photoinhibition (β), and the light saturation point (Ek). The results showed that NH4+ enrichment did not modify the effects of pH on RGRs, NH4+ uptake rates and pools, total chlorophyll, rETRmax, α, β, Fv/Fm, tissue C and N, and the C:N ratio. However, Ek was differentially affected by pH under different NH4+ treatments. Ek increased with decreasing pH in the ambient NH4+ treatment, but not in the enriched NH4+ treatment. NH4+ enrichment increased RGRs, NH4+ pools, total chlorophyll, rETRmax, α, β, Fv/Fm, and tissue N, and decreased NH4+ uptake rates and the C:N ratio. Decreased pH increased total chlorophyll content, rETRmax, Fv/Fm, and tissue N content, and decreased the C:N ratio. Therefore, the results indicate that U. australis growth is increased with NH4+ enrichment and not with decreasing pH. While decreasing pH influenced the carbon and nitrogen metabolisms of U. australis, it did not result in changes in growth.


Introduction
Since the industrial revolution, the atmospheric CO 2 concentration has increased from 280 μatm to over 390 μatm, and about 30% of the additional CO 2 has been absorbed by the ocean [1].This results in ocean acidification, a term which describes the contemporary reduction in seawater pH by ca.0.1 units with an expected further reduction of 0.3-0.5 units by 2100 [2][3][4][5].In addition to ocean acidification, coastal regions receive inputs of excess nitrogen from aquaculture, agriculture, wastewater treatment, and the burning of fossil fuels [6,7].Excess nitrogen is the commonly regarded cause for green algal blooms world-wide, and they are typically dominated by macroalgae from the genus Ulva [8][9][10].Green algal blooms can impose negative effects on their ecosystems and local human communities by decreasing biodiversity and ecosystem services [11][12][13][14].Elevated nutrients can modify the effects of elevated pCO 2 /decreased pH on algal physiology [15][16][17][18][19][20][21][22] because nitrogen and carbon metabolisms are linked via the process of protein synthesis [23].In order to understand how nutrientopportunistic macroalgae, such as Ulva spp.will respond to future oceanic conditions, it is important to consider the interaction of elevated nutrients with decreasing pH.
Non-calcareous macroalgae have been shown to express a range of responses to future pCO 2 /pH conditions.Hizikia fusiforme growth rates increased under future pCO 2 /pH conditions while maximum photosynthetic rates were unchanged [24].Growth rates of Gracilaria chilensis and another Gracilaria sp. were enhanced by future pCO 2 /pH conditions [25].Gracilaria lemaneiformis growth rates were also enhanced under future pCO 2 /pH conditions, but only at an intermediate photon flux density (PFD) (160 μM photons m -2 s -1 ) [26].The growth rates of thirteen species of algae, including green, red, and brown algae, had no response to future pCO 2 /pH conditions with the exception of Hypnea musciformis, which exhibited negative growth rates [27].Ulva spp.growth rates have been shown to increase or be unaffected by future pCO 2 /pH conditions [21,[28][29][30].Differences in responses to elevated pCO 2 /decreased pH may be caused in part by species specific differences or by unsuitable nutrient concentrations, temperature, and/or PFD for the seaweeds to support higher growth rates.
Carbon concentrating mechanisms (CCMs) allow macroalgae to increase CO 2 at the site of carbon fixation and may be downregulated with elevated pCO 2 /decreased pH in Ulva spp.[31,32].This has been linked to increased energy availability for nutrient uptake, protein synthesis, and growth when nutrients are not limiting [31,33,34].Therefore, elevated pCO 2 / decreased pH might change nutrient uptake, assimilation, and storage capacity of macroalgae which utilize CCMs.For example, when CCMs were reduced with elevated pCO 2 /decreased pH in Pyropia haitanensis, growth rates and NO 3 -uptake rates increased, and photosynthetic rates increased with the combination of elevated pCO 2 and elevated NO 3 - [35].Further, nutrients mediated the effect of elevated pCO 2 /decreased pH on P. haitanensis by increasing growth rates and nitrate reductase activity (NRA) when grown with elevated CO 2 and NO 3 -enrichment [20].Ulva lactuca photosynthetic rates and NRA were increased with elevated pCO 2 / decreased pH, but only when temperature was sufficient (25˚C compared to 15˚C), while NO 3 -uptake rates were enhanced at both temperatures with elevated pCO 2 /decreased pH [21].Another algal species which utilizes CCMs, Hizikia fusiforme, was also found to have enhanced growth rates, NRA, and nitrate uptake rates with elevated pCO 2 /decreased pH [24].The interaction of NH 4 + enrichment and elevated pCO 2 /decreased pH increased growth rates of Ulva pertusa [22].These studies provide evidence that local (i.e., nutrient enrichment) and global (i.e., ocean acidification) drivers of environmental change could interact to change macroalgal growth and physiology.Ulva spp.are opportunistic under eutrophic conditions [9] and have potentially increased growth rates under elevated pCO 2 alone [15,28].Prior studies suggest nitrogen in the form of NO 3 -could have interacting effects with elevated pCO 2 /decreased pH in Ulva spp., but less is known regarding the effects of NH 4 + as a potential interacting driver [15,16,22,32].Typically, NH 4 + is the preferred form of nitrogen for Ulva spp.because it requires less energy for assimilation than NO 3 -, as NO 3 -must first be reduced via nitrate reductase activity (NRA) [36].
Although NO 3 -is the most abundant and common form of dissolved inorganic nitrogen (DIN) in the ocean, increasing human population densities on coasts, land use change, and decreasing ocean pH all increase the availability of NH 4 + in coastal areas [37].
To test the hypothesis that there will be an interacting effect of decreasing pH and elevated NH 4 + concentrations on the growth, nutrient, and photosynthetic physiology of Ulva australis, a laboratory growth experiment was conducted across a range of future pCO

Collection and acclimation
Ulva australis was collected from Blackmans Bay, Tasmania, Australia (42˚59'56"S 147˚19'8"E) in July 2015 (Austral winter).Algae were stored in plastic zip-lock bags with seawater on ice and transported to the laboratory in a cooler within five hours of collection.U. australis was identified using morphological characteristics.All visible epiphytes were carefully removed from the surface of the blades which were then rinsed with filtered seawater.The cleaned algal samples were kept in aerated seawater at 16.6˚C under 200 μmol photons m -2 s -1 (measured using a 4π Li-Cor LI-193 Spherical Quantum Sensor connected to a LI-250A portable light meter) with a 12h:12h light dark cycle for 3 days to acclimate to experimental light and temperature conditions.

Experimental design
Three Ulva australis thalli with a total fresh weight of 1.07 ± 0.02 g (mean ± SEM) were placed in 650 mL chambers filled with 600 mL of seawater that was UV-sterilized and filtered through a 1 μm-filter (Polyester Felt Filter Bags, NETCO, Hobart, Australia).Peristaltic pumps (FPU500, Omega Engineering, USA) were used to provide fresh seawater to each of the 24 growth chambers at a rate of 6-8 mL/min.The pH T of seawater pumped to each tank was maintained using an automated pH control system [38].Seawater was equilibrated using a membrane contactor (Micromodule, model 0.5X1, Membrana, USA) where the appropriate mix of N 2 and CO 2 gas was achieved using three pairs of mass flow controllers (MFCs) set to pH T s of 8.05, 7.85, and 7.65 (FMA5418A and FMA545C, Omega Engineering, USA).The flow rate of each MFC was proportional to the input voltage, which was supplied by an analog output module housed in a USB chassis (NI9264 and cDAQ-9174, National Instruments, USA) using a control system similar to that described in Bockman [39].Each of the three MFCs were randomly assigned to four ambient NH 4 + and four enriched NH 4 + growth chambers for a total of 24 chambers.The pH T within each culture chamber was measured every 1.5-3 hours throughout the week-long experiment, monitoring the effect of U. australis photosynthesis and respiration on seawater pH T .The seaweed biomass: seawater volume ratio affected the pH T of the culture chambers so the average pH T of each chamber was denoted by measurements of pH T during the dark cycle throughout the entire experiment which resulted in a continuous range of pH T s (

pH T and total alkalinity measurements
A syringe pump (V6 pump with valve 24090, Norgren, UK) and two 12-port rotary valves (23425 valve driver with valve 24493, Norgren, UK) were used to sample seawater directly from each growth chamber.For each spectrophotometric pH measurement, a reference spectrum was acquired after flushing 25 mL of seawater through a 1 cm flow-through quartz cuvette.A spectrum (400-800 nm) was acquired using an LED light source and a UV-Vis spectrometer (BluLoop and USB2000+, Ocean Optics, USA).A dye + seawater spectrum was then obtained after mixing 200 μL of 2 mM metacresol purple sodium salt dye (211761-10G, Sigma Aldrich, Australia) with an additional 25 mL of seawater within the syringe pump.The two spectra were used to calculate an absorbance spectrum.pH T was calculated using the quadratic fits of the absorbance spectra between 429-439 nm, 573-583 nm and a background signal averaged between 750-760 nm.When compared to calculations based on a single wavelength, the quadratic fit approach leads to a three-fold improvement in measurement precision [38].
Each recorded pH T was the average of four replicate measurements, which took approximately three minutes to obtain.The temperature of each sample was recorded with a PT100 temperature sensor and a high-precision data logger (PT-104, PICO Technology, UK).All instrument control, spectra manipulations, and pH T calculations were done using LabVIEW 2014 (National Instruments, USA).Total alkalinity (AT) samples were calculated from water samples collected in October 2015 using seawater from the same region (Taroona, Tasmania, Australia) as was collected in July 2015 for the experiment.AT samples were poisoned with mercuric chloride (0.02% vol/vol [40]) and were analyzed at the Australian National University, using an automatic built in-house titrator (consisting of a 5 mL Tecan syringe pump (Cavro X Calibur Pump), a Pico USB controlled pH sensor, and a TPS pH electrode).AT values were then calculated using the Gran technique [40].

Growth rates
Ulva australis thalli were blotted with tissue to remove excess water and weighed before the start of the experiment and after seven days.The total weight of the three thalli from each chamber was used for the analysis.The RGR, expressed as % day -1 , was calculated as RGR = ln (FW f /FW i ) x t -1 x 100 where FW i is the initial fresh weight, and FW f is the final fresh weight after t days.

NH 4 + uptake rates
At the end of the seven-day incubation period, one of the three Ulva australis thalli (0.43 ± 0.03 g of FW) was removed from each chamber to an Erlenmeyer flask containing 200 mL of filtered seawater with overhead light of 200 μmol photons m -2 s -1 .The seawater in each flask was obtained from the automated pH control system shortly before the start of the experiment so the seawater pH T in the flasks was representative of the seawater in the chambers the algae came from.The initial NH 4 + concentration of 20 μM was obtained with the addition of NH 4 Cl to ambient seawater.Flasks were placed on an orbital shaker (RATEK OM7, Victoria, Australia) set to 80 rpm and continuously stirred to induce water motion and reduce boundary layer effects [41].A 10 mL sample of the water was taken at 0 and 30 minutes, and frozen at -20˚C, until defrosted and analyzed for NH 4 + concentration using a QuickChem 8500 series 2 Automated Ion Analyzer (Lachat Instrument, Loveland, USA).The uptake rate (V) was determined according to Pedersen [42]] using the formula where S i and S f are the initial and final NH 4 + concentrations (μM) over a period of time (t), vol is the seawater volume in the flask and FW is the fresh weight (g) of the algae.

Internal soluble NH 4 + pools
The boiling water extraction method was used to determine the internal soluble NH 4 + pool [43].Ulva australis tissue (0.18 ± 0.01 g FW) was put in a boiling tube with 20 mL of deionized water then placed in a boiling water bath for 40 minutes.The liquid was cooled, decanted, and then filtered through a 0.45 μm Whatman filter (GF/C).This process was repeated on the same algal piece three times and the concentration of internal soluble NH 4 + pools was calculated using the sum of the NH 4 + concentrations of the three water samples of each algal piece.
NH 4 + concentrations were measured as stated above.

Photosynthetic pigments
Following the experiment, a 0.04 ± 0.001 g FW piece of Ulva australis from each experimental chamber was kept at -20˚C pending analysis.Each sample was then ground in 5 mL of 100% ethanol with a ceramic mortar and pestle in dim light and with the samples shaded.The extract was poured into 10 mL centrifuge tubes and placed in the dark at 4˚C for six hours.Samples were then centrifuged for 10 min at 4000 rpm at 4˚C.Total Chl a and b concentrations in the supernatant were determined according to the quadrichroic formula from Ritchie [44] using a spectrophotometer (S-22 UV/Vis, Boeco, Germany).

Rapid light curves
Chlorophyll fluorescence of photosystem II was measured using a Pulse Amplitude Modulation fluorometer (diving-PAM, Walz, Germany) to generate rapid light curves and obtain measurements of the maximum quantum yield of PSII photochemistry (F v /F m ), which is used as an indicator of stress [45].On day seven of the experiment, one thallus from each chamber was dark adapted for 20 minutes before exposure to a flash of saturating light to obtain maximum fluorescence (F m ).Then a rapid light curve was generated by increasing exposure to photosynthetic active radiation (PAR) ranging from 0-422 μM photons m -2 s -1 .F v /F m was calculated by the equation F m -F 0 /F m , were F 0 is the fluorescence under measuring light conditions (ca.0.15 μmol photons m -2 s -1 ) and F m is the maximum fluorescence under saturating light conditions.Relative ETR (rETR) was calculated by the equation rETR = Y Ã PAR Ã 0.5.A hyperbolic curve was fit to the rETRs generated by each rapid light curve using a modified equation of Walsby [46]: where rETR c is the calculated rETR, rETR max is the maximum ETR at light saturating PFDs, α is the initial slope of the curve during light-limiting PFDs, and β is the slope of photoinhibition at high PFDs.The coefficients used in the equation were calculated using a least squares method [46].

Total carbon and nitrogen content
A 0.35 ± 0.03 g FW section was dried at 60˚C overnight, ground to a fine powder, and then analyzed for total tissue carbon and nitrogen content.Samples were weighed into pressed tin capsules (5x8 mm, 0.2 mg; Sercon, U.K.).Carbon and nitrogen content were determined using a Fisons NA1500 elemental analyzer coupled to a Thermo Scientific Delta V Plus via a Conflo IV.Combustion and reduction were achieved at 1020˚C and 650˚C respectively.Percent C and N composition was calculated by comparison of mass spectrometer peak areas to those of standards with known concentrations.

Total pH and seawater carbonate parameters
The pH T given for each treatment is the average value from the dark cycle pH T measurements in each culture chamber.The measurements oscillated around the gas mixers' set points due to algal metabolism: during the light period pCO 2 decreased, increasing pH T ; during the dark period cellular respiration produced CO 2 , decreasing pH T , with pH T being relatively stable throughout the dark cycle (Fig 1

Interactive effects
The slopes for all dependent variables, with the exception of E k , were indistinguishable between ambient and enriched NH 4 + treatments as indicated by the non-significant interaction terms (pH T and NH 4

+
) in the ANCOVAs (Table 2).The following results of those dependent variables with a non-significant interaction are reported as ANCOVAs with the interacting term dropped from the model.

Photosynthetic pigments
The total chlorophyll concentration (Chl a + b) content was higher in Ulva australis from enriched       + treatment (dashed lines) were tested for an interaction using an ANCOVA. https://doi.org/10.1371/journal.pone.0188389.g006

Discussion
The growth, nutrient, and photosynthetic physiology of Ulva australis with increased pCO 2 / decreased pH did not depend on the NH 4 + treatment, with the exception of E k .This was counter to the hypothesis that NH 4 + enrichment and increased pCO 2 /decreased pH would interact to change U. australis growth and physiology.This study demonstrates that U. australis growth rates are more likely to be influenced by nutrient enrichment, rather than ocean acidification, as NH 4 + enrichment increased activities of PSII and NH 4 + pools and ultimately increased growth rates.N-deficiency has been shown to lower the ability of Ulva rotundata to photoacclimate to changing light regimes and can lead to declines in rETR max and α in U. lactuca [47,48].NH 4 + enrichment increased total chlorophyll concentrations, rETR max , F v /F m , and α increased with NH 4 + enrichment indicating N-deficiency inhibited photosynthesis.
Photoinhibition (β) and differences in β between NH 4 + enriched and ambient treatments were small at the highest PFDs measured which suggests an increased range of PFD would be better suited for demonstrating effects on β.Nutrient enrichment increased growth and photosynthetic characteristics of U. australis which has been shown with many macroalgae [8].
In this study, decreased pH influenced photosynthetic physiology as demonstrated by total chlorophyll, rETR max , E k , and F v /F m .With pH being reduced by the addition of pCO 2 , the increase in the total dissolved inorganic carbon (DIC) concentration in seawater likely contributed in the increased activity and efficiency of PSII.However, this did not result in increased growth rates.A decoupling of the photosynthetic characteristics and growth rates is not uncommon because growth is linked to multiple components of algal metabolism, not just a single process (i.e., photosynthesis).In this experiment, this decoupling may represent a tradeoff between nitrogen resources for improved photosynthetic efficiency (higher concentration of chlorophyll) or growth (resulting in dilution of chlorophyll with cellular division).Here, it was demonstrated that Ulva australis grown with NH 4 + enrichment was better acclimated to various pH conditions with regards to E k , as there was no relationship between E k and pH.When grown in the ambient NH 4 + treatment, E k increased with increasing CO 2 /decreased pH.In future pH conditions, U. australis growing in low NH 4 + seawater may be able to increase their potential habitat range to include those with higher light levels.However, given enough nutrients, light limitations would be reduced and pH would have no effect on E k .The supposition that macroalgal growth rates may increase with future pCO 2 /pH conditions due to energy savings from downregulation of CCMs [33,49,50] is likely not a pervasive feature of CCM utilizing macroalgae.Enhanced growth with pCO 2 enrichment is probably the result of the influence of light levels on CCMs [51].Energetic constraints on carbon acquisition at low PFDs increases dependence on passive CO 2 diffusion, while CCMS are more efficient at high PFDs [33].When PFD is low, the carbon demands of photosynthesis can be saturated by diffusion alone and CCMs are not needed.For example, pCO 2 enrichment only enhanced Gracilaria lemaneiformis growth rates at an intermediate PFD [26].Young and Gobler [32] found that Ulva spp.growth rates increased with pCO 2 enrichment but varied by season, primarily increasing only in summer months.Assuming their findings are representative of Ulva spp.seasonal growth dynamics in a temperate location, then the results of the current study likely represent a less productive time of year for U. australis.Considering other environmental variables such as season, temperature, and light intensity are important for building a comprehensive framework from which we can elucidate patterns of ecological relevance from laboratory studies.NH 4 + enrichment increased RGRs to approximately twice that of Ulva australis grown in non-enriched seawater.Increased RGR with increasing nutrient concentrations is common for Ulva spp.[47,52], but it is also dependent on seasonal changes in light supply and ambient nitrogen levels [53].For example, Lapointe and Tenore [54] showed that when Ulva fasciata was not grown with sufficient light, the enhancement of growth with NO 3 -was eliminated.
Furthermore, growth rates of Ulva lactuca more than doubled with the addition of NH 4 + or NO 3 -when collected from an oligotrophic site, but an increased growth rate with nutrient enrichment was not evident when algae were collected from a nutrient enriched site [55].
In the present experiment, internal NH 4 + pools and tissue N content were nearly twice as large in the NH 4 + enriched treatments as in the ambient treatments, indicating light and nutrients were sufficient for nutrient assimilation and growth, while the ambient NH 4 + treatments were N-limiting.In the NH 4 + enriched treatment, Ulva australis NH 4 + uptake rates were slower than in the ambient NH 4 + treatments, which supports the theory that nutrient histories influence nutrient uptake capabilities by feedback inhibition as internal N pools increase [56][57][58][59][60][61].U. australis from the NH 4 + enriched treatments, were still capable of NH 4 + uptake despite growth under high nutrient availability and relatively concentrated NH 4 + pools.This has also been demonstrated with Ulva expansa and Ulva intestinalis with varying nutrient histories [61] and shows their ability to take up surplus nutrients under growth with low and high nutrient concentrations.
The increase in tissue N, decrease in the C:N ratio and increase in E k in the ambient NH 4 + treatment with decreasing pH in this experiment indicate that decreased pH may provide relief from nutrient limitation.An increase in chlorophyll content and tissue N with decreasing pH support that NH 4 + was assimilated to produce nitrogenous compounds such as chlorophyll, protein, and amino acids and not stored in internal NH 4 + pools during this experiment.We did not detect changes in NH 4 + uptake rates with decreasing pH, which corresponds to the absence of changes in NH 4 + pools and growth rates.This contrasts that findings of increased NO 3 -uptake rates under future pCO 2 /pH conditions in Ulva rigida, Hizikia fusifome, and Gracilaria spp.[15,24,25], and increased NH 4 + uptake rate future pCO 2 /pH in Hypnea spinella [62].The effect of pCO 2 /pH on N uptake rate may also be sensitive to temperature, as NO 3 uptake rates in Ulva lactuca increased with CO 2 enrichment at 25˚C, but not 15˚C [21].Based on our results, it is unlikely NH 4 + enrichment (a local-scale environmental change) will interact with ocean acidification (a global-scale environmental change), to affect Ulva australis growth, nutrient, and photosynthetic physiology.We were able to demonstrate that increased growth rate with NH 4 + enrichment could be explained by cellular changes in NH 4 + and photosynthetic physiology.However, physiological responses to pH were more complex, where Ulva australis growth rates did not change under future pCO 2 /pH conditions, despite the fact that rETR max , F v /F m , and tissue N increased.These changes in photosynthetic and nutrient physiology could potentially lead to increased growth rates in macroalgae [63].It was also demonstrated that decreased pH may reduce nutrient limitation and increase E k under low NH 4 + conditions.Therefore, growth rates have the potential to increase with future pCO 2 /pH conditions under a more favorable set of environmental conditions where PFD and/or season may interact to influence U. australis growth rates in future pCO 2 /pH conditions.In summary, the concern that ocean acidification may contribute to the increasing the biomass of green-tide blooms along anthropogenically influenced coastlines world-wide is not supported, despite changes in photosynthetic and nutrient physiology that could favor increased growth.However, NH 4 + enrichment significantly increased growth rates of the opportunistic macroalga U. australis.This is likely to contribute to increases in the severity of green-tide blooms in areas where land-use change and development are leading to increases in NH 4 + concentrations in seawater.
(mean ± SEM) (n = 7).Salinity is assumed to be 35%.Temperature is assumed to be 16.5˚C (the average temperature throughout the experiment) MFC = mass flow controller.DIC = dissolved inorganic carbon.(PDF)

Fig 2 .
Fig 2. Relative growth rates (% day -1 ) for Ulva australis under ambient and enriched NH 4 + treatments across a range of pH T .Grey points represent ambient NH 4 + treatments and black points represent enriched NH 4 + treatments.The slope of RGR with decreasing pH T for each NH 4 + treatment (dashed lines) were tested for an interaction using an ANCOVA.https://doi.org/10.1371/journal.pone.0188389.g002

Fig 3 . 4 +
Fig 3. (A) NH 4 + uptake rates (μmol g -1 FW hour -1 ) in 20 μM NH 4 + seawater for 30 minutes and (B) internal NH 4 + pools (μmol g -1 FW) for Ulva australis grown under ambient and enriched NH 4 + treatments across a range of pH T s.Grey points represent ambient NH 4 + treatments and black points represent enriched NH 4 + treatments.A plus symbol (+) indicates an outlier which was removed for statistical analysis.The slopes of NH 4+ uptake rates and internal NH 4 + pools with pH T for each NH 4 + treatment (dashed lines) were tested for an interaction using an ANCOVA.https://doi.org/10.1371/journal.pone.0188389.g003

Fig 4 .
Fig 4. (A) Total chlorophyll (mg Chl a + b g -1 FW), (B) rETR max from rapid light curves, and (C) F v /F m from rapid light curves for Ulva australis grown under ambient and enriched NH 4 + treatments across a range of pH T .Grey points represent ambient NH 4 + treatments and black points represent enriched NH 4 + treatments.The slopes of total chlorophyll, rETR max , and F v /F m with decreasing pH T for each NH 4 + treatment (dashed lines) were tested for an interaction using an ANCOVA.https://doi.org/10.1371/journal.pone.0188389.g004

Fig 5 .
Fig 5. (A) Light saturation point (E k ), (B) initial slope of the curve (α), and (C) slope of photoinhibition at high photon flux densities (β) from rapid light curves for Ulva australis grown under ambient and enriched NH 4 + treatments across a range of pH T .Grey points represent ambient NH 4 + treatments and black points represent enriched NH 4 + treatments.The slopes of E k , α, and β with decreasing pH T for each NH 4+ treatment (dashed lines) were tested for an interaction using an ANCOVA.https://doi.org/10.1371/journal.pone.0188389.g005

Fig 6 .
Fig 6. (A) Tissue C (%DW), (B) tissue N (%DW), and (C) C:N ratio of samples of Ulva australis under ambient and enriched NH 4 + treatments across a range of pH T .Grey points represent ambient NH 4 + treatments and black points represent enriched NH 4 + treatments.The slopes of tissue C, tissue N, and the C: N ratio with decreasing pH T for each NH 4+ treatment (dashed lines) were tested for an interaction using an ANCOVA.
Multiple components of carbon and nitrogen metabolisms were measured with the aim of describing how changes in these processes integrate at the organismal level (i.e., growth).With elevated pCO 2 /decreased pH and NH 4 + enrichment Ulva spp.should have adequate internal supply of nitrogenous and carbon skeleton precursors and may have increased growth rates, potentially leading to increases in the severity of frequency of green tide blooms.
). Dark cycle pH T values were correlated to light and whole cycle pH T values (Pearson correlation: r = 0.70, p = 0.0002 and r = 0.92, p <0.0001, respectively).Mean values for each chamber during light, dark, and whole day cycles throughout the experiment are reported in Table1.Seawater carbonate parameters are described in the S1 Table.