Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A620/A280 of 4.78), allowing its usage for health-enhancing properties while eliminating other aspecific algal components. Proteomic investigation and HPLC analysis of purified AFA phycobilisomes revealed that, contrary to the well-characterized Synechocystis and Spirulina spp., only one type of biliprotein is present in phycobilisomes: phycocyanins with no allo-phycocyanins. Two subunit polypeptides of PC were also separated: the beta subunit containing two bilins as chromophore and the alpha subunit containing only one.     

一步柱层析纯化螺旋藻藻蓝蛋白

采用硫酸铵盐析结合疏水层析技术分离纯化螺旋藻中的藻蓝蛋白.试验结果表明,在磷酸盐缓冲体系下藻蓝蛋白粗提液经1.25 mol/L硫酸铵盐析处理后离心脱气,只需采用一步Macro-Prep Methyl 疏水层析,藻蓝蛋白的纯度（A620/A280）可提高到4.017,回收率为19.38%.特征吸收峰和荧光光谱证实纯化后的产物符合藻蓝蛋白的性质,Native-PAGE电泳只出现单一染色带,表明纯化得到的藻蓝蛋白是均一的;SDS-PAGE电泳出现分子量为15.4 kDa、17.3 kDa的2条染色带,分别为藻蓝蛋白的α亚基与β亚基.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis

C-phycocyanin was purified from Spirulina (Arthrospira) fusiformis by a multi-step treatment of the crude extract with rivanol in a ratio 10:1 (v/v), followed by 40% saturation with ammonium sulfate. After removal of rivanol by gel-filtration on Sephadex G-25, the pigment solution was saturated to 70% with ammonium sulfate. After the last step of purification, C-phycocyanin had an emission and absorption maxima at 620 and 650 nm, respectively and absorbance ratio A(620)/A(280) of 4.3, which are specific for the pure biliprotein. Its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding two bands of molecular masses 19500 and 21500 kDa, corresponding to alpha and beta subunits of the pigment, respectively. The yield of C-phycocyanin was approximately 46% from its content in the crude extract.     

THE PHOTOSYNTHETIC EFFICIENCY OF PHYCOCYANIN IN CHROOCOCCUS,AND THE PROBLEM OF CAROTENOID PARTICIPATION IN PHOTOSYNTHESIS

The absorption spectra of the principal pigment components extracted from Chroococcus cells have been measured, and their sum compared with the absorption of a suspension of living cells. The agreement was sufficiently close so that it was concluded the absorption spectra of the extracted and separated pigment components could be used to obtain estimates of the relative absorption of the various components in the living cells. The quantum yield of Chroococcus photosynthesis was measured at a succession of wave lengths throughout the visible spectrum, and the dependence of yield on wave length was compared with the proportions of light absorbed by the pigment components. This comparison showed beyond reasonable doubt that the light absorbed by phycocyanin is utilized in photosynthesis with an efficiency approximately equal to that of the light absorbed by chlorophyll. The light absorbed by the carotenoid pigments of Chroococcus seems for the most part to be unavailable for photosynthesis. The results leave open the possibility that light absorbed by the carotenoids is active in photosynthesis, but with an efficiency considerably lower than that of chlorophyll and phycocyanin. It is also possible that the light absorbed by one or a few of the several carotenoid components is utilized with a high efficiency, while the light absorbed by most of the components is lost for photosynthesis.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Relation between pigment content and photosynthetic characteristics in a blue-green algae

1. The blue-green alga Anacystis nidulans was cultured under steady state conditions at 25 and 39°C. and under several different light intensities to give five different types of cells. 2. Cells were submitted to pigment analysis based upon acetone extracts and aqueous extracts obtained by sonic disintegration. The different cell types show a threefold range of chlorophyll content and a fourfold range of phycocyanin content with only minor changes in the chlorophyll/phycocyanin ratio. Cells of highest pigment content were estimated to contain 2.8 per cent chlorophyll a and 24 per cent phycocyanin, the latter on a total chromoproteid basis. 3. Light intensity curves of photosynthesis were obtained for each of the cell types at 25 and at 39°C. The slopes of the light-limited regions of the curves are approximately linear functions of chlorophyll and phycocyanin contents. Maximum light-saturated rates of photosynthesis at 25 and 39° show no simple relation to pigment content.     

Retrieval of phycocyanin concentration from remote-sensing reflectance using a semi-analytic model in eutrophic lakes

With the rapid development of the economy in recent years, massive algal (blue-green algae in particular) blooms have often observed in Chinese eutrophic lakes. The concentration of the cyanobacterial pigment phycocyanin (PC), an accessory pigment unique to freshwater blue-green algae, is often used as a quantitative indicator of blue-green algae in eutrophic inland waters. The purpose of this study was to evaluate the semi-analytic PC retrieval algorithm proposed by Simis et al. and to explore the potential to improve this PC algorithm so that it is more suitable for eutrophic lakes, such as Taihu Lake. In this paper, we recalculated the correction coefficients γ and δ to calculate the absorptions of chlorophyll-a at 665 nm and the absorptions of phycocyanin at 620 nm in terms of in situ measurements and observed that the values of these coefficients differed from the values used by Simis et al. and Randolph et al. The two coefficients are site dependent due to the different bio-optical properties of lakes. We also observed that the specific PC absorption at 620 nm a_pc*(620) decreases exponentially with an increase in PC concentrations. Therefore, a non-linear power–function of a_pc*(620), instead of a constant value of a_pc*(620) as used by Simis et al., was proposed for our improved PC retrieval algorithm in Taihu Lake, yielding a squared correlation coefficient (R²) of 0.55 and a root mean square error (RMSE) of 58.89 μg/L. Compared with the original PC retrieval algorithm by Simis et al., the improved retrieval algorithm has generally superior performance. In evaluating the limitation of the PC retrieval algorithms, we observed that the ratio of the total suspended solids to phycocyanin can be used as a primary measure for retrieval performance. Validation in Dianchi Lake and an error analysis proved that the improved PC algorithm has a better universality and is more suitable for eutrophic lakes with higher PC concentrations.     

The protein-chromophore bond in B phycoerythrin from Porphyridium cruentum. Radiosulfur labeling experiments

Red algae of the species Porphyridium cruentum were grown in a minimum sulfate medium containing 35SO42-. 35S-labeled phycoerythrin was extracted. B Phycoerythrin, b phycoerythrin and R phycocyanin could be separated from other proteins by using a carrier-free electrophoresis on columns. The final ratio A545/A280 of B phycoerythrin thus obtained was greater than or equal to 5. 35S-labeled B phycoerythrin was digested proteolytically with trypsin and pepsin. The resulting 35S-containing bilipeptides were separated by isoelectric focusing. Zones of enhanced chromophore concentration always showed an enhanced radioactivity. Peptide fractions with a low molar ratio sulfur/chromophore (1.1-1.8) were purified to remove sucrose and the carrier ampholyte. A modified, optimized Edman degradation followed. A butylacetate-soluble, red Edman product was obtained that contained most of the chromophore and the bulk of the radioactivity. This product was purified by two-dimensional thin-layer chromatography. The main spot of the chromatogram was subjected to acidic hydrolysis. The major part of the radioactivity in the hydrolysate cochromatographed with cysteine. That proves cysteine to be the binding amino acid in all cases investigated.     

CHARACTERIZATION OF PIGMENT MUTANTS IN A BLUEGREEN ALGA,ANACYSTIS NIDULANS1

Characteristics of 2 types of pigment mutants of the bluegreen alga, Anacystis nidulans, are described. “Yellow-green” mutants (YG) which have normal chlorophyll but only half the phycocyanin of the parent are similar to the parent in number of reaction centers/cell, number of chlorophylls/reaction center, maximum turnover rate of the reaction centers, quantum yields at 620 and 686 nm and specific growth rate; they have a reduced action at 620 nm. “Blue” mutants (BL) with somewhat higher phycocyanin but only one-third the chlorophyll of the parent are dissimilar to the parent. BL's have fewer reaction centers/cell, a smaller number of chlorophylls/reaction center, a higher maximum turnover rate of reaction centers, and a lower specific growth rate. BL's show ca. half the quantum yield of the parent at 620 nm and at 686 nm show a “red rise” rather than a “red drop.” The consequences attending low chlorphyll in the BL's are more drastic than those attending the low phycocyanin of the YG's.     

Untersuchung der Beziehung zwischen Pigmentzusammensetzung und CO2-Gaswechsel bei der Blaualge Anacystis nidulans

Summary Changes in culture conditions caused strong changes in the pigment composition in the blue-green alga Anacystis nidulans. Under normal illumination (white light; 0.6·10³ erg/cm²·sec) the relation between the amounts of chlorophyll a and phycocyanin was 1:6.6. In a high light intensity (20.8·10³ erg/cm²·sec) the phycocyanin content was reduced and the relations thus changed to 1:1.9. Growing the algae in red light of high intensity (20·10³ erg/cm²·sec) increased the phycocyanin content; the chlorophyll a: phycocyanin relation was then 1:12.1.The action spectrum of apparent photosynthesis showed a minimum at 473 nm in all three cultures. The maximum of photosynthesis in low light cultures fell in the absorption region of phycocyanin at 621 nm. The action spectrum of the red light culture showed a reduced rate of photosynthesis in the same region. The strong light culture had an action spectrum similar to that of the red light culture with a maximum at 651 nm. The differing action spectrum of the low light culture may be a result of interruption in the energy transfer from phycocyanin to chlorophyll a within pigment system II.The transients of CO₂ exchange are independent of the pigment composition. Two different types of transients were found depending on the wavelength of the incident light. In red light of 550–650 nm a higher stationary rate was reached after a maximum of photosynthesis at the beginning of the illumination period. In blue and far red light a lower rate was found after the first maximum. Following a illumination period in blue or far red light a CO₂ evolution in the dark was observed. On the other hand, this CO₂ evolution was not found after illumination with red light. These effects are possiblt caused by a decarboxylation reaction (photorespiration) which occurs only in blue and far red light.     

Effect of light quality on the C-phycoerythrin production in marine cyanobacteria Pseudanabaena sp. isolated from Gujarat coast, India

The isolated cyanobacterium containing biopigments like chlorophyll-a, phycoerythrin, phycocyanin, and carotenoid was cultured under different quality of light modes to ascertain biomass and pigment productivity. On the basis of 16S rRNA gene sequence, the isolate was identified as Pseudanabaena sp. Maximum biomass concentration obtained in white-, blue-, and green-light was 0.82, 0.94, and 0.89 g/L, respectively. It was observed that maximum phycoerythrin production was in green light (39.2 mg/L), ensued by blue light (32.2 mg/L), while phycocyanin production was maximum in red light (10.9 mg/L). In yellow light, pigment production as well as the growth rate gradually declined after 12 days. Carotenoid production decreased in blue-, white-, and red-light after 15 days, while in green light it had increased gradually. The present communication suggests that Pseudanabaena sp. can be used for commercial production of phycoerythrin when grown under green light.     

Optimization of phycocyanin extraction from Spirulina platensis using factorial design

Phycocyanin extraction from cyanobacteria Spirulina platensis was optimized using factorial design and response surface techniques. The effects of temperature and biomass-solvent ratio on phycocyanin concentration and extract purity were evaluated to determine the optimum conditions for phycocyanin extraction. The optimum conditions for the extraction of phycocyanin from S. platensis were the highest biomass-solvent ratio, 0.08 gmL(-1), and 25 degrees C. Under these conditions it's possible to obtain an extract of phycocyanin with a concentration of 3.68 mgmL(-1) and purity ratio (A(615)/A(280)) of 0.46.     

Photochemical apparatus organization in Synechococcus 6301 (Anacystis nidulans). Effect of phycobilisome mutation

The photochemical apparatus organization in Synechococcus 6301 (Cyanophyceae) was investigated under various experimental conditions. Wild type (WT) Synechococcus produced phycobilisomes (PBSs) containing normal levels of phycocyanin (Phc) and allophycocyanin (Aphc). The ratio of reaction centers(RC) RCII/RCI of 0.4 was the same in WT and the mutant strain AN112, whereas RCH/PBS was 1.9:1 in WT and 1:1 in AN112. Excitation of WT cells with broad-band 620 nm light, which is absorbed primarily by Phc and Aphc and to a much lesser extent by chlorophyll (Chl), sensitized the RC of photosystem (PS) II at about 15 times the rate it sensitized RCI. This implies that PBSs are associated exclusively with PSII complexes and that PBS excitation is not transferred to PSI. The AN112 mutant of Synechococcus produced smaller PBSs consisting of the Aphc-containing core and of only six Phc-containing hexamers, respectively. It lacked about 65% of the Phccontaining rod substractures. Under our experimental conditions, the effective absorption cross section of the mutant PBS was only about half that of the WT. In agreement, the rate of RCII excitation by 620 nm light was also about half of that measured in the WT. Thus, the rate of light absorption by PSII depends directly on PBS size and composition. The low rate of RCI excitation with 620 nm light was the same in WT and AN112 cells, apparently independent of the PBS effective absorption cross section. We propose a strict structural-functional association between PBS and PSII complex. PSI is a structurally distinct entity and it receives excitation independently from its own Chl light-harvesting antenna.Abbreviations PBS phycobilisome - Phc phycocyanin - Aphc allophycocyanin - PS photosystem - RC reaction center - P700 reaction center of PSI - Q primary electron acceptor of PSII - Chl chlorophyll - MV methyl viologen - Tris Tris(hydroxymethyl)-aminomethane - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421

Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as α and (β subunits by SDS-PAGE and MALDI-TOF. HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that α and β-subunits contained one and two phycocyanobilin groups as chromophores, respectively.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

Photosynthetische carboxylierungsreaktionen verschieden pigmentierter Anacystis-zellen

Summary CO₂ exchange, ¹⁴CO₂ fixation and ¹⁴C-labelled photosynthetic products of differently pigmented Anacystis nidulans (strain L 1402-1) were studied during the induction period at +30°C. The algae were grown at +35° C in an atmosphere of 0.04 vol.-% CO₂ and measured under the same low CO₂ concentrations. Changing the culture conditions caused alterations in the pigment composition. Under normal illumination (white light; 0.6×10³ erg/ cm² s) the relation between amounts of chlorophyll a and phycocyanin was 1:7 to 1:10. In a high light intensity (30.8×10³ erg/cm² s) the phycocyanin content was reduced (1:5 to 1:2). When the cells were grown in red light of high intensity (20×10³ erg/ cm² s) phycocyanin synthesis increased; the pigment ratio varied between 1:20 and 1:33. Anacystis cells grown under strong white light were filamentous.Photosynthetic CO₂ uptake, measured with an infrared gas analyzer, was very low in algae grown in high light intensity. The pattern of ¹⁴C-labelled photosynthetic products of these algae was very similar to those of the Calvin cycle. In Anacystis cells grown under low intensities of white light or in red light ¹⁴CO₂ was, at the beginning of the light period, incorporated mainly into aspatate and glycerine/serine. The enzyme activities of NAD⁺-specific malate dehydrogenase, ribulose-1,5-diphosphate carboxylase, aspartate and alanine aminotransferase decreased with increasing phycocyanin content. NADP⁺-specific malic enzyme activities showed practically no change. In contrast, phosphoenolpyruvate carboxylase activity increased with a higher rate of phycocyanin synthesis. In another series of experiments the behaviour of the PEP carboxylase activity after breakdown of the Anacystis cells was tested in differently pigmented cultures. In all cases the enzyme activities very rapidly decreased within two hours. The results obtained are discussed with reference to the correlation of pigment composition and CO₂ fixation of the phosphoenolpyruvate system.

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Abkürzungen Asp Aspartat - Gly/Ser Glycin/Serin - PGS 3-Phosphoglycerat - ZmP Zuckermonophosphat Herrn Professor Dr. Andre Pirson in Verehrung gewidmet     

Light dependent oxygen uptake by the blue green alga Anacystis nidulans.

Cells of Anacystis nidulans consume oxygen when illuminated with 750 nm light. The same process occurs with 675 nm light when the photosynthetic production of oxygen has been halted by gentle heating of the cells. These reactions do not require the addition of artificial redox compounds. There seem to be two separate systems, one activated by 750 nm light, the other by 675 nm light. Polarographic action action spectra reveal that the 675 nm system utilises pigments of the photosynthetic apparatus, excluding phycocyanin. Fluorescence excitation spectra suggest that only the pigment P750 is involved in the 750 nm system. Purified P750 recombines spontaneously with extracted pigment-free cell fragments. After recombination the P750 has the same spectroscopic properties as the pigment in vivo.     

Transfer of the Excitation Energy in Anacystis nidulans Grown to Obtain Different Pigment Ratios

The blue-green alga, Anacystis nidulans, was grown in lights of different colors and intensities, and its absorption and fluorescence properties were studied. Strong orange light, absorbed mainly by phycocyanin, causes reduction in the ratio of phycocyanin to chlorophyll a; strong red light, absorbed mainly by chlorophyll, causes an increase in this ratio. This confirms the earlier findings of Brody and Emerson (12) on Porphyridum, and of Jones and Myers (8) on Anacystis. Anacystis cultures grown in light of low intensity show, upon excitation of phycocyanin, emission peaks at 600 mmu and 680 mmu, due to the fluorescence of phycocyanin and chlorophyll a, respectively. Changes in the efficiency of energy transfer from phycocyanin to chlorophyll a are revealed by changes in the ratios of these two bands. A decrease in efficiency of energy transfer from phycocyanin to chlorophyll a seems to occur whenever the ratio of chlorophyll a to phycocyanin deviates from the normal. Algae grown in light of high intensity show, upon excitation of phycocyanin, only a fluorescence band at 660 mmu and no band at 680 mmu. This suggests reduced efficiency of energy transfer from phycocyanin to the strongly fluorescent form of chlorophyll a (chlorophyll a(2)) and perhaps increased transfer to the weakly fluorescent form of chlorophyll a (chlorophyll a(1)).