CORE Metadata, citation and similar papers at core.ac.uk Provided by UNL | Libraries University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Natural Resources Natural Resources, School of 2015 Phycocyanin-specific ba sorption coefficient: Eliminating the effect of chlorophylls absorption Y. Z. Yacobi Kinneret Limnological Laboratory J. Köhler Leibniz Institut für Gewässerökologie und Binnenfischerei F. Leunert Leibniz Institut für Gewässerökologie und Binnenfischerei Anatoly A. Gitelson University of Nebraska - Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/natrespapers Part of the Natural Resources and Conservation Commons, Natural Resources Management and Policy Commons, and the Other Environmental Sciences Commons Yacobi, Y. Z.; Köhler, J.; Leunert, F.; and Gitelson, Anatoly A., "Phycocyanin-specific bsa orption coefficient: Eliminating the effect of chlorophylls absorption" (2015). Papers in Natural Resources. 594. http://digitalcommons.unl.edu/natrespapers/594 This Article is brought to you for free and open access by the Natural Resources, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Natural Resources by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. LIMNOLOGY and Limnol. Oceanogr.: Methods 13, 2015, 157–168 OCEANOGRAPHY: METHODS VC 2015 Association for the Sciences of Limnology and Oceanography doi: 10.1002/lom3.10015 Phycocyanin-specific absorption coefficient: Eliminating the effect of chlorophylls absorption Y. Z. Yacobi,*1 J. Kohler,€ 2 F. Leunert,2 A. Gitelson†3 1Israel Oceanographic and Limnological Research, Kinneret Limnological Laboratory, Migdal, Israel 2Leibniz Institut fur€ Gewasser€ okologie€ und Binnenfischerei (IGB), Muggelseedamm€ 301, Berlin, Germany 3School of Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska Abstract The applicability of algorithms for estimation of phycocyanin (PC) concentration based on light spectral reflectance heavily depends on the specific absorption of the pigment. But the determination of PC-specific absorption coefficient is not a straightforward task, as PC optical activity is overlapped by absorption of à chlorophylls. The aim of our study was to determine aPC(625)—the specific absorption coefficient of PC at 625 nm, in samples with PC concentrations ranging from 0.5 mg m23 to 126.4 mg m23 and varying propor- tions of chlorophylls a, b, and c in the samples. The effect of chlorophylls was subtracted from total absorp- tion at 625 nm using Chl a absorption at 675 nm as a reference; the contribution of the chlorophylls was computed on the basis of their relative absorption at 625 nm and concentrations. The major effect on the à precision of aPC(625) determination was imposed by Chl a absorption, but the effect of the accessory chloro- à phylls ought to be accounted for in order to arrive at reliable PC-specific absorption values. aPC(625) varied widely, from 0.002 mg m22 to 0.027 mg m22, and decreased with increase of PC concentration. As PC con- 23 à 22 centration exceeded 10 mg m , aPC(625) was almost invariable, slightly oscillating around 0.007 mg m and similar to PC specific absorption coefficient estimated by alternative methods. It is clear that a universal à 22 value is unfeasible, but aPC(625) of 0.007–0.008 mg m seems an appropriate value for use in algorithms destined for estimation of phycocyanin in typical mesotrophic and eutrophic inland waters dominated by cyanobacteria. A useful way to determine the potential of vegetation to culated by division of the absorption value at a given wave- capture light is by measuring mass-specific light absorption. length by the concentration of the pigment in question, and Practically, the value of specific light absorption is vital for in the case of Chl a is a straight forward task, as the absorption the development of analytical models for determination of peak is located beyond the optical activity of other pigments pigment concentration (Gons 1999), and for algorithms des- and is only slightly influenced by detritus and colored dis- tined to estimate phytoplankton primary productivity (Marra solved organic matter. The variation of the specific absorption et al. 2007). In the case of phytoplankton, it usually defined coefficient of Chl a was extensively studied and the inverse as chlorophyll a (Chl a)-specific absorption (Kiefer and Soo- relationship between pigment concentration and its specific Hoo 1982). Synthetic datasets of reflectance spectra showed absorption is well documented (Bricaud et al. 1995; Allali that the relationship between optical information at the red- et al. 1997; Dall’Olmo and Gitelson 2005; Gurlin et al. 2011). NIR domain, essential for development of algorithms for Chl Determination of specific absorption coefficient of other a estimation in productive waters, heavily depends on Chl a- pigment than Chl a is complicated due to overlapping specific absorption value at 665 nm (Gilerson et al. 2010). absorption of the pigments themselves, as well as by the The measurement of phytoplankton absorption is accom- effects of the noncellular water constituents. Of particulate plished using cell suspension or particles captured on a glass importance is to establish real values for the water soluble fiber filter. The specific absorption of a pigment in vivo is cal- phycobilins, signature pigments for cyanobacteria (Rowan 1989); some representatives of that phylum are toxin- producers and thus confer a potential environmental risk *Correspondence to: [email protected] (Carmichael 1997). Cyanophytes often dominate phyto- †Present address: Department of Civil and Environmental Engineering, plankton in marine and freshwater ecosystems, and current Technion, Haifa, Israel knowledge leads to the conclusion that they will increase in 157 Yacobi et al. Phycocyanin-specific absorption coefficient importance in the foreseen future (Paerl and Huisman 2008). difference is probably the result of package effect as well as With the increasing importance of these organisms attempts various physiological states of algae (nutrient/light effects). are made to improve methods for monitoring, among others (4) The presence of pigments other than PC and Chl a, and by means of remotely sensed information, mostly by acquisi- the variable influence of Chl a on retrieved absorption at tion of spectral information in the visible and near-infrared 625 nm, are potential causes of error in PC retrieval. (5) Due domains (Simis et al. 2005, 2007; Hunter et al. 2009; Mishra to significant and systematic overestimation of Chl a by the et al. 2013, 2014; Ogashawara et al. 2013). In cyanobacteria algorithm developed by Gons et al. (2000) for Chl a < 20 dominating phytoplankton in inland waters, the major pig- mg m23, there are significant errors in subtraction of Chl a ment is mostly phycocyanin (PC). PC absorption peaks contribution at 625 nm in the Simis et al. (2005) algorithm. around 610–620 nm and impairs a blue-green color in combi- It is likely that non-phytoplankton absorption as well as nation with Chl a. Phycobilins are located in protein-based absorption by Chl c and Chl b may explain this tendency to structures, phycobilisomes, and the procedures applied for overestimate Chl a (Gitelson et al. 2008). extraction of phycobilins are those used for most proteins, Absorption of all chlorophylls partly overlaps and also i.e., in aqueous solutions (Colyer et al. 2005). Those proce- overlaps the range of the optical activity of PC. It is therefore dures are laborious and require specialized equipment, needed necessary to take into account the effect of Chl a and the for cell disruption for the liberation of protein cellular compo- accessory Chl b and Chl c absorption in the spectral range of nents. The existing laboratory methods for phycobilins extrac- PC absorption in order to achieve a reliable estimation of tion do not suit large-scale work, as mostly required for field PC-specific absorption. The goals of this study were: (1) to samples, and therefore, may introduce large margin of experi- determine the net effect of PC absorption at the wavelength mental error (Millie et al. 1992). Moreover, the bond between around 625 nm (where a peak is observed in absorption the phycobilins and the protein is not broken following cell spectra and a small but discernible trough in reflectance disruption, and the spectrum showed by the released phycobi- spectra); (2) to establish the quantitative relationship lin is species specific, and dependent on the interaction between PC concentration and specific absorption coefficient between the protein and the chromophore. of PC; (3) to define the minimum concentration of PC or rel- So far, a major effort was directed to develop an optical ative proportion of it to Chl a where the optical activity of technique for remote estimation of PC, a water-soluble pig- PC is detectable in natural samples. ment common in freshwater cyanobacteria. The absorption signal of PC is detectable from reflectance spectra in eutro- Materials and Procedures phic waters near 625 nm (Dekker 1993; Jupp et al. 1994; Sampling sites Gitelson et al. 1999; Schalles and Yacobi 2000). Following Water samples were collected in Lake Kinneret (Israel) in the same logic as for Chl a retrieval (Gitelson et al. 1985), a eight different campaigns (June, July, August, September, semianalytical algorithm for PC estimation has been pro- and November 2012 and June and July 2013) and in three posed (Simis et al. 2005). The algorithm attributed the campaigns in Berlin lakes (Germany) in August 2013. absorption signal in the 625-nm band to both PC and Chl a and to the absorption of Chl a around 670 nm. The initial Experimental work tests have shown that the algorithm generally provides sig- Collected water samples were kept in a dark box, shipped nificant overestimations of the PC, except when extremely to the laboratory, and processed within four hours from the high PC concentrations associated with massive blue-green first collection. Qualitative microscopic examination was algae blooms were observed in the lakes (Simis et al.