The Effect of Nutrient Enrichment on three Species of Macroalgae as Determined by Photoacoustics

The efficiency of photorophs in storing light energy in products of photosynthesis, or the quantum yield of the process, determines the flux of energy into any ecosystem, and is readily affected by any stress factor, physiological or environmental. Hence it can serve as a sensitive reporter of imminent ecosystem perturbation. Current methods of estimating that parameter are either indirect, or tedious and labor consuming. The photoacoustic method is based on the direct sensing o f the variable fraction of light energy degraded to heat, that while absorbed by the plant cells or tissue, failed to be stored as energy rich photosynthate. Algae are the main primary producers in all water bodies, marine, freshwater, natural and manmade, and as such they provide the energy basis for all aquatic ecosystems. The biomass and vitality of the algal population responds rapidly to seasonal changes in environmental factors such as temperature, light, vertical mixing, eutrophication, pollution and nutrient limitation. Typical processes resulting from human activities such as non-point sources of agrochemicals, sewage outflows, and effluents from food, animal and other chemical processing industries as well as from urban and rural runoff, contribute considerable amounts of nitrogen and phosphorous, in many cases in addition to heavy metals, to natural and artificial water bodies. In this study, we examined by the photoacoustic method the effects of enrichment by nitrogen and phosphorus on the photosynthetic efficiency of three common Mediterranean seaweed species which were collected from the field. The application developed by us, allows the rapid and direct determination of phytoplankton photosynthetic activity, by relating the energy stored photochemically to the total light energy absorbed by the plant material. The aim of this work was the examination of the applicability of the photoacoustic system to ecological work with macroalgae, since until now it has been successfully used only in homogenous phytoplankton cultures.


Introduction
The seasonal changes in algae populations are brought about by the annual rhythms of temperature, light intensity, and enrich ment or depletion of nutrients in surface waters [1].
Un d er su fficient lig ht int ens ity , pho tosy nt hes is o f phytoplankton is limited primarily by the availab ility of es s en t ial n u t r ien t s , mo s t co mmo n ly n it ro g en an d phosphorus [2][3][4]. Therefore, any increase or decrease in the supply of these nutrients results in dramat ic changes in the concent rat ion o f phytop lan kton [1], [5]. Such chang es in nutrient concentrations, may stem fro m natural events like mixing of water bodies and upwelling cu rrents, or fro m anthropogenic sources like marine and lacustrine pollution [5].Since even a barely detectable increase in nutrients is amp lified by the phytoplankton into an easily quantifiab le change in their total bio mass and taxonomic co mposition, phytoplankton are sensitive pollution detectors. The capability to harvest nutrients fro m the external environ ment at both high and low concentrations is an important property of the ecology of algae.
These changes in bio mass and productivity have been traditionally recorded by measurements of absorption, fluorescence, oxygen evolution, and 14 C labeling [6], [7]. The potential of photoacoustics in quantifying and characterizing algae photosynhtesis and chlorophyll concentration had been reported [6], [7]. The photoacoustic method is based on the conversion of the light energy into heat. The application developed by us, allows the rapid and direct determination of algae photosynthetic activity, by relat ing the energy stored photochemically to the total light energy absorbed by the plant material.
For detailed description see "Methods and Materials". Seaweeds are "bentical algae" that live in the sea . [8].
Seaweeds grow in the photic zone which is in the range between the surface water levels (in the intertidal zone) and down to ~200 meters deep in the open ocean [10].
Ulva rigida C. Agardh, Hypnea musciformis (Wulfen in Jacquin) J.V. Lamourou x and Padina pavonica (Linnaeus) Thivy Agardh are green, red and brown algae respectively. These three macroalgae are very co mmon along the Israeli shore and main ly grow on the Kurkar intertidal abrasion platforms.
The rationale for the species selection was to compare the effects of the unique laser used previously [11], [12] to estimate photosynthesis efficiency only on phytoplankton , on species of macroalgae fro m three different phyla that have different types of thallus morphologies and d iverse pig met assortments. Ulva rigida has green flat sheet-like thallus with toothed marg ins. This thallus is co mposed of two cell layers [13]. Hypnea musciformis has red-green narrow, cylindrical, branched thallus morphology. Its branch apices are slightly upcurved, flattened hooks [14]. Padina pavonica is characterized by flat calcified "ear-like" blades which has circinnately inrolled apical margins [15].
The aim of this work was the examination of the applicability of the photoacoustic system, prev iously used only in ho mogenous phytoplankton cultures, to ecological work with macroalgae.

Photoacoustics
The photoacoustic method is based on the conversion of absorbed light to heat. When the energy of the photons absorbed in a sample is degraded to heat the thermal expansion of the material causes a volume increase: V= H÷C p  (1) where  is the thermal expansivity, = 1÷V (V÷T) p , , the density, C p , the heat capacity of the mediu m and H is the heat liberated or enthalpy change [16], [17].
The photoacoustic method, allo ws the direct determination of the energy storage efficiency of photosynthesis by relating the energy stored by photosynthesis to the total light energy absorbed by the plant material [6], [11], [18].
Depending on the efficiency of the photosynthetic system, a variab le fraction of the absorbed light energy is stored, thereby affecting the heat evolved and the resulting photoacoustic signal. The h igher the photosynthetic efficiency, the larger will be the d ifference between the stored energy with and without ongoing photosynthesis [19].
By exposing the cells to continuous, saturating, background light, no storage of any of the pulse energy can take place, whereas in the absence of such light, a maximal fraction of the pulse energy is stored by photosynthesis. Thus the photosynthetic storage efficiency,  is determined as the complement of the rat ios of the photoacoustis signal generated by a weak pulse of light in the dark (PA dark ), to that obtained under strong continuous illu mination (PA light ) [7], [11].  = (PA light -PA dark) ) ÷PA light = 1-PA dark ÷PA light (2) We detect and follow the pressure wave in an aqueous suspension of algae over 10-20 microseconds after the laser pulse.
The e xperimental setup: The experimental setup is shown schematically in Figure 1. The overall procedure was similar to that described in detail in Pinchasov et al. [11]. The second harmonic of a Continuum Min ilite Q-Switched Nd-Yag laser at 532 nm was used. The signal was processed with a Tektronix TDS 430A oscilloscope. The submersible, stainless steel enclosed homemade detector contained a 10 mm d iameter resonating ceramic d isc (BM 500, Sensor, Ontario, Canada). The sample was placed in a 16 mm quart z glass cell (PAC). The laser (L) pulse, after passing through a pair of 1 mm wide slits (S) is incident upon the suspension of algae whose pigments absorb part of the laser light. Depending on the experimental conditions a variab le fraction of the absorbed light pulse is stored in the products of photosynthesis. The remainder of the absorbed light is converted to heat producing an acoustic wave. This is intercepted by a detector (D), containing the above ceramic d isc. A small portion of the laser pulse is deflected by a beam splitter (BS) and used to trigger the Tektronix TDS 430A oscilloscope, where the amp lified (A mptek A-250 Preamp and Stanford Research A 560 A mp) photoacoustic signal is recorded. The signal contains a noisy background and later reflections from the walls of the vessel as well as fro m impedance mismatch within the detector. Signal to noise was imp roved by averaging over 128 pulses, and by taking RMS values over the time of the recorded signal (~10 µs). Weak (~20 µJ cm -2 ), 5ns pulses at 532 n m wavelength, were used as a probe for ongoing photosynthesis. The source of the background light (B), was a quartz-halogen illu minator (Cole -Parmer 4971). The intensity of the background light was adjusted to the 2000 E m -2 s -1 by neutral density filters and measured with a LiCor light meter equipped with a cosine quantum sensor (for technical details see Pinchasov et al. [11]).

Algae Samples
The samples of three co mmon macroalgae species: Ulva rigida, Hypnea musciformis and Padina pavonica were collected fro m the intertidal abrasion platforms at Bat Yam (located in the middle of the Israeli Mediterranean) during spring 2010.
All samples were kept at 22 ± 0.1℃ in 100 mL Erlyn mayers during 192 hours under continuous irradiance at ~200 ± 5.0 µI m -2 s -1 . Initial volu me of mediu m in Erlyn mayers was 75 mL. For every species were processed 3 duplicate samples.
Chlorophyll was determined by photoacoustic method, based on the proportionality of the photoacoustic signal to the amount of pigment. Fo r all experiments (include control) were used slices of common.
The samples were exposed to 3 treat ments: nitrogen (was added as NaNO 3 , 10 mL at concentration of 50 ml/ L fro m the stock of 75.0 g/ L), phosphorus (was added as NaH 2 PO 4 ,10 mL at concentration of 5 ml fro m the stock of 5.0 g/L), and nitrogen and phosphorus together. Controls were kept in seawater alone.

Results and Discussion
We have used the effects of nutrient limitation and of eutrophication on the photosynthetic efficiency of seaweeds as experimental variables in a demonstration of the applicability of photoacoustics in aquatic ecology. Nutrient limitat ion on the one hand and anthropogenic eutrophication on the other, are among the most important factors determining the overall ecological status of water bodies.
Since nutrient status is known to rapidly affect algal growth and photosynthesis we chose that environmental factor for our study In general, in all samples (Figs. 2, 3 and 4), photosynthetic efficiency and chlorophyll concentration (photoacoustic signal) decreased with time, during the 192 hours of incubation..
As is evident from Figures 2, 3 and 4, the photoacoustic signal rapidly decreased , and within 190 hours, the controls declined to some 70 % in P. pavonica (Fig. 2) and H. musciformis (Fig. 4), and ~ 50 % in U. rigida (Fig. 3), of the initial values. The addition of nutrients slowed down, but did not prevent, such decline (~ 20 % in U. rigida (Fig. 3) and ~ 50 % and 30 % in P. pavonica and H. musciformis, respectively (Figs. 2 and 4). The decline in all treat ments, including in the P and N enriched ones is likely due to the requirement fo r addit ional nutrients besides nitrogen and phosphorus.
Similar effects of nutrient limitation on photosynthetic apparatus and chlorophyll concentration have been reported by Villares et al. [20] and Litch man et al. [21]. Our results suggest that the added nitrogen prevented this nutrient fro m becoming limiting, and, therefo re, the observed declines in photosynthesis and chlorophyll were due to the incipient phosphorus limitation.
Samples with added nitrogen and phosphorus together were usually the slowest to show a decrease in photosynthetic efficiency.
A decrease in chlorophyll concentration under nutrient limitat ion has been observed for both marine and freshwater algae [21][22][23].
Reduction trend in chlorophyll concentration in nutrient-enriched samples may be explained by the availability of nutrients required for the synthesis of pigments (mostly nitrogen), and membranes (both nitrogen and phosphorus).
The general conclusion fro m these data, which was obtained by photoacoustics, is that in the sampled algae, nitrogen was the first nutrient that became limiting followed by phosphorus. In all cases the addition of phosphorus and nitrogen alone, or together slowed down the decline in photosynthetic efficiency, that was the fastest in the unsupplemented controls.
Our results, which were obtained by the photoacoustic method, agree with others, previously described in literatu re [24][25][26] showing that the nitrogen and the phosphorus additions mit igated the effects of nutrient limitation.
Our results show that photoacoustics can be a useful tool in ecophysiological studies of macroalgae, providing sensitive and immed iate evidence for any deleterious processes affecting seaweeds. Based on our results we are working on expanding the method's applicability to various additional phototrophs such as seagrasses, turf algae, algal mats and zoo xanthellate corals..
The directness of our method holds the potential for development of novel tools for mon itoring changes in photosynthetic efficiency related to water quality in marine and freshwater benthos communities, The photocoustic method can provide early warning alerts to incipient negative ecosystem developments, as well as report recovery processes in response to the implementation of remed iation measures