A fit-free approach for the analysis of fluorescence spectral components in the microscopy images which does not require an a priori knowledge of the basis spectra was used. This technique is non-invasive and could be utilized to quantitatively identify different molecular species in live samples . In this work, we present a biometric tool developed for identifying the presence and the concentration of carrageenans that occur naturally in the wall cell and in the intercellular matrix of red algae. This tool also has the potential to be useful for the identification of other seaweed-derived hydrocolloids. The tool detects spectral emission from explants using confocal microscopy, and it has been found to be a powerful method for identifying specific emission fingerprints of autofluorescence of compounds present in seaweeds. This study reports on the identification of several spectral fluorescence emission fingerprints from different auto-fluorescence compounds spatially mapped in the commercially important red algal species K. alvarezii. The explants imaged were cultivated in vitro and treated with the polyamines spermidine, putrescine,grow table and spermine.Phase 1: in vitro culture, grown in seawater in the laboratory. Phase 2: explants from phase 1 transferred to indoor culture tanks in the laboratory. Phase 3: explants from phase 2 transferred to outdoor cultivation tanks on the mainland. Phase 4: explants from phase 3 transferred to ropes anchored in the sea. In situ sea culture was carried out within the Maritime Concessions of the company BGracilarias de Panamá, S.A.^ in the Cativa area of the Province of Colon near the Caribbean entrance of the Panama Canal. One of the fourteen farms designated by polygons within the Maritime Concessions was selected for in situsea culture. It was identified as polygon 11. The selected polygon was located in the northern section of Largo Remo within a lagoon open to the sea and surrounded by mangrove trees; substratum was predominantly sandy bottom with patches formed by small communities of sea grasses.
Seaweed cultivation activities were originally established in this area to involve local community members with limited livelihood options in the development of a type of aquaculture considered Beco-friendly. The physical infrastructures of the farms were thought to potentially buffer mangroves and coral reefs from the negative impacts of extreme weather events . In addition, it was hoped that the long-term allocation of the marine common spaces for sustainable seaweed farming in the Concessions might shield this sensitive area from mega-scale development projects in the rapidly industrializing region . The Polygon 11 used for this study in phase 4 is located within one of the most closely monitored tropical coastal zones in the world . Environmental information has been collected throughout the area since the early 1900s, with an archive of historical in situ data recorded by the Smithsonian Tropical Research Institute and 80 years of data collected by the Panama Canal Authority. In 2000, the Coastal Research Institute of GKSS, Germany, installed an in situ high tech monitoring system to integrate environmental data at the site. The seaweed farm sites were selected based upon environmental parameter averages identified as ideal measures for successful seaweed cultivation. These were determined by geographical area and degree of variation by season . Data indicated an annual temperature range of 28 to 30 °C, pH range, 7.0– 9.0, and salinity of 32.8–34.8 ‰. All these data ranges have remained relatively stable over the last several years . Growth of K. alvarezii is favored when salinity is about 32 to 35 ‰ and it is inhibited below 28 ‰ . Kappaphycus alvarezii growth rates seem to be optimal in a pH range from 7.0 to 9.0 with explants exposed to solar light at 30 cm depth and solar light intensity 845–1837 μmol photons m−2 s −1 . . In all phases of this study, explants were planted in the sea on ropes similar to those used in commercial seaweed farms.
To observe carrageenan in the cell walls and within the center cellular matrix of the cultured algae by confocal microscopy, it was necessary to develop a model of in vitro culture. This was done in phase 1 at the laboratory level using incubators containing cultivated K. alvarezii seed stock obtained from the seaweed farms. The incubator was designed at the Galeta Point Marine Laboratory of the Smithsonian Tropical Research Institute constructed with ATT double glazed window glass consisting of two panels with an intermediate air chamber and double sealed with butyl and polysulfide or silicone. This insulation maintained a controlled temperature between 30 and 25 °C and constant humidity between 60 and 75 %. The incubator maintains a irradiation with two fluorescent light tubes at 40 W and two 20 W tubes, with a photoperiod of 12 h light and 12 h dark. The spectral emissions and confocal microscopy were explored as alternative technologies for measurement of carrageenan quality of seaweed. This histological analysis was performed at the Laboratory for Fluorescence Dynamics, University of California, Irvine, USA.This study combines an experimental approach to enhance commercial carrageenan productivity with the novel application of confocal laser-scanning microscopy, a technique capable of measuring and characterizing carrageenan content in vitro. We tuned the wavelengths of excitation and emission to record the emission fluorescence of carrageenans, and each color visually represents a different type of carrageenan although in this paper we have not identified the specific types. The identification of several spectral fluorescence emission fingerprints from different auto-fluorescence compounds from explants of K. alvarezii are reported and spatially mapped. These fingerprints have the potential to improve strain selection of explants with the aim to increase the carrageenan yield of seaweed farming operations and to potentially enable wholesale pricing to correspond with crop quality. Carrageenans were characterized using auto-fluorescence properties of the species of K. alvarezii subjected to treatment with different polyamines: putrescine, spermidine, and spermine.
A four-phase cultivation pipeline is presented for enhancing and assessing the carrageenan content of seaweed crops encompassing in vitro culture techniques. Measurements of carrageenan fluorescence using bright field microscopy detected carrageenan in both the tetrasporophyte and in the female tissues of Eucheuma isiforme J. Agardh, and the calculation of metachromatic indices gave a higher value for the medullary cell walls in the tetrasporophyte than in the female gametophyte . During culture in the laboratory,vertical rack we followed the explants growth by measuring size, weight, and number of new apices and calluses every 15 days after seeding. We used polyamines Put, Spd, and Spm as plant growth regulator treatments that were expected to favor the development of cystocarps. Particularly, Spm promoted sporulation in several Rhodophytes such as Hydropuntia cornea and Grateloupia imbricate . Investigations into the effects of PGR on algal growth and development are necessary to understand the physiological basis of algal growth, callus formation, and regeneration. This information can then lead to improved seaweed cultivation techniques by establishing and adopting viable methodsof enhancing strains of commercially important species . Zitta et al. found that the thick wall of the cells, which are composed in part of callus filaments, showed the presence of acidic polysaccharides, suggesting a large content of carrageenan and neutral polysaccharides. In this study, calli and new apices were counted from day 15. Calli observed gave rise to irregularly branched uniseriate filaments, and we presume callus formation was initiated in the first week of seeding in phase 1. The new apical structures were elongated and we assumed that cell elongation occurs in the presence of large amounts of disorganized mitochondria and chloroplasts. The increased number of these organelles could be related to an increase in the process of cellular respiration and thus energy metabolism , supporting subsequent cell divisions and the formation of lateral branching. According to Doty , K. cottonii thalli are compressed to flatten above the basal segment; prostate, irregular in form, or with linear segments with irregular occurrences of protuberances or branches. The images obtained in this study by the confocal microscopy of the control callus and apices of an explant of K. alvarezii indicated a broader distribution of the green cluster at the cell center and at the cell wall of the callus. The apices showed a higher percentage of red color at the cell wall while color at the center of the cell was very similar to the callus. This suggests a distinct characteristic compared with the callus and apex treated with spermidine 10−5 M . These samples demonstrated a different morphology than the sample shown in Figs. 6 and 7. This type of morphology seems to result in a large contribution of the green component present both at the center and at the cell wall of the apex and more red color in the cell wall of the callus. It is possible that the content and type of carrageenan may also correspond to specific parts of the algal structure, and these could possibly be impacted by various environmental conditions. Therefore, the spectral phasor analysis is a very simple, time-sensitive way to obtain a specific fingerprint of a sample of uncertain carrageenan composition. The fluorescence properties of carrageenan are used in this study to show that there are more than two spectral components in each sample and that each sample can be classified in terms of percent of pixels corresponding to at least three spectral components. While the primary focus of this research was carried out utilizing different laboratory culture techniques, the success of the approach also depends on the efficacy of polyamine treated seed stocks in the natural marine habitat.
We found in our study that improvements in seedlings produced in the laboratory also held in the natural marine habitat. If this success is shown to be repeatable, then our approach could be utilized to improve the quality and quantity of the product from the farms with obvious benefits to the industry. This study suggests a novel technique for seaweed cultivators to assess the potential carrageenan yield of their crops in order to better select in vitro cultured explants prior to planting out on farms at sea. Consideration must also be given to these commercial possibilities beyond the usage of carrageenan in the food processing industries, such as in pharmacology, where high-quality parameters are required. Food products for human consumption, mainly associated with the Asian market, account for 83 to 90 % of the total value of macroalgae.The various species possess high levels of secondary metabolites and structural polysaccharides of commercial value.Today’s growing demand for carrageenan and the need for more sustainable farming practices is a large challenge. There is an urgent requirement to understand and detect differences among types and qualities of carrageenan as a means of managing higher yielding, economically viable, long-term farming practices . According to FAO statistics, world carrageenan seaweed farming production increased from less than 1 million wet tonnes in 2000 to 5.6 million wet tonnes in 2010, with the corresponding farm gate value increasing from US$72 million to US$1.4 billion. Major carrageenan seaweed farming countries include Indonesia, the Philippines, the United Republic of Tanzania, Malaysia, and China . Despite the high demand for carrageenans, seaweed scientists, farmers, buyers, and processors still lack accessible tools to accurately and inexpensively assess the carrageenan content of seaweed crops and associated products throughout all stages of growth and refinement. We continue to hone our studies related to the application of in vitro culture techniques of red seaweeds of commercial importance. Our efforts will focus on developing new plant biotechnology applications for the prevention of diseases of genera such as Kappaphycus, Gracilaria, and others of commercial importance. Finally, carrageenan seaweed farming in particular, has evolved into a successful commercial endeavor in a number of tropical countries endowed with clear, unpolluted intertidal environments and protected beach locations . The area of farming at the entrance of the Caribbean side of the Panama Canal has a very specific annual weather pattern with a rainy season that lasts 9 months with 3 months of dry season. During our studies, we have not found a correlation between the seasonal weather patterns and the quality of the product . We note that this area of the Caribbean seems to be protected from the adverse impacts of hurricanes . This unique condition is an important consideration regarding the importance of commercially viable seaweed farms in this region.