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Journal of Applied Phycology
 
ISSN 0921-8971
 
J Appl Phycol
DOI 10.1007/s10811-016-0914-4
A laser-scanning confocal microscopy study
of carrageenan in red algae from seaweed
farms near the Caribbean entrance of the
Panama Canal
Gloria Batista de Vega, Jorge
A. Ceballos, Andrea Anzalone, Michelle
A. Digman & Enrico Gratton
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A laser-scanning confocal microscopy study of carrageenan
in red algae from seaweed farms near the Caribbean entrance
of the Panama Canal
Gloria Batista de Vega1,2 & Jorge A. Ceballos3 & Andrea Anzalone4 &
Michelle A. Digman4,5 & Enrico Gratton4,5
Received: 20 October 2015 /Revised and accepted: 10 July 2016
# Springer Science+Business Media Dordrecht 2016
Abstract Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, a
red macroalga, is a commercial source of carrageenan, a wide-
ly used polysaccharide compound important in the food and
pharmaceutical industries, in nanotechnology, and in pharma-
cological applications. Carrageenan is found mainly in the cell
wall and in the intercellular matrix. This is the first study to
propose the characterization of carrageenans in vitro, using the
auto-fluorescence properties of the alga treated with different
polyamines: putrescine, spermidine, and spermine. This study
suggests a four-phase cultivation sequence for seaweed
farmers to enhance and assess the potential carrageenan yield
of their crops. In phase 1, seedlings were treated with each of
the polyamines. Explants were subsequently transferred
through two additional culture phases before being planted
on the sea farms in phase 4 and then harvested after 60 days
for analysis. Images from transverse sections of 11 representa-
tive cultured K. alvarezii samples were obtained at 561 nm
excitation wavelength for both the cell center and the cell wall
of each sample. Spectral data were also analyzed using the
spectral phasor algorithm of SimFCS developed at the
Laboratory for Fluorescence Dynamics (www.lfd.uci.edu). We
report on the identification of several spectral fluorescence
emission fingerprints from different auto-fluorescence
compounds spatially mapped using this technique. These finger-
prints have the potential to improve strain selection of explants
for enhanced carrageenan yield in seaweed farming operations
as well as to enable wholesale pricing to correspond with
crop quality.
Keywords Carrageenan .Kappaphycus alvarezii . In vitro .
Seaweed farms . Polyamines . Confocal microscopy .
Fluorescence emission . Fingerprints
Introduction
Carrageenan is a general name for polysaccharides extracted
from certain species of red algae (Rhodophyta), mainly
Gigartina stellata (Stackhouse) Batters, Chondrus crispus
Stackhouse, Eucheuma spp., and Kappaphycus spp.
Carrageenans are high molecular weight (MW = 200,000 to
800,000 Da) polysaccharides made up of repeating galactose
units and 3,6-anhydrogalactose with varying numbers of sul-
fate groups connected byα-1,3 and β-1,4 glycosidic linkages.
They are biocompatible compounds with specific gelling
properties useful in a multitude of ways, including food and
biotechnology programs and controlled drug release applica-
tions. Their reported anticancer, antioxidant, and antiviral ac-
tivities have potential in the pharmaceutical industry, and the
high metal binding capacities of carrageenans make them ex-
cellent candidates for nanoparticle synthesis and encapsula-
tion processes (Raman et al. 2015). There are three primary
* Gloria Batista de Vega
gloriabv@cwpanama.net
1 Smithsonian Tropical Research Institute, Ciudad de Colón, Panama
2 U.S.A. Centro Regional Universitario de Colón, Avenida Randolph,
Sector Arco Iris, Corregimiento de Cristóbal, Ciudad de
Colón, Panama
3 Confocal and Electron Microscopy Laboratory, Smithsonian
Tropical Research Institute, Panama, PO Box 0843-03092,
Balboa, Ancon Republic of Panama
4 Laboratory for Fluorescence Dynamics, Department of Biomedical
Engineering, University of California, 3210 Natural Sciences II,
Irvine, CA 92697-2715, USA
5 Centre for Bioactive Discovery in Health and Ageing, School of
Science and Technology, University of New England,
Armidale, Australia
J Appl Phycol
DOI 10.1007/s10811-016-0914-4
Author's personal copy
classifications of carrageenan, and the genus Kappaphycus
was segregated from Eucheuma primarily on the basis of car-
rageenan type (Doty et al. 1986). DNA re-association kinetics
were used to determine nuclear genomic organization and com-
plexity in the carrageenophyte Kappaphycus alvarezii (Doty)
Doty ex P.C. Silva (Kapraun and Lopez-Bautista 1997).
Carrageenan occurs naturally in the cell walls and in the in-
tercellular matrix of several red algae, and it has been studied
using diverse methodologies and measures (Gordon-Mills et al.
1978; Kapraun and Lopez-Bautista 1997; Lechat et al., 2000).
Biochemical analysis and Fourier transformed infrared spectros-
copy (FTIR) are the most commonly used tools for evaluation of
carrageenan quality (Chiovitti et al. 1996; Chopin et al. 1990;
Pereira et al. 2009; Pérez-Gonzáleza et al. 2009).
Kappaphycus alvarezii (Solieriaceae Rhodophyta) has
been actively farmed in many countries as a biomass source
for carrageenan production since the early 1970s (Doty and
Alvarez 1974). However, the present-day carrageenan indus-
try faces numerous challenges related to the quality and quan-
tity of raw materials. As a result, in 2000, of the first-recorded
occurrence of Polysiphonia sp. epiphytic filamentous algae
(EFA), annual K. alvarezii production and the number of sea-
weed farmers declined rapidly (Critchley et al. 2004; Hurtado
et al. 2006; Vairappan et al. 2007). This raised serious con-
cerns about the future stability of the algal resource that per-
sists to the present time. The outbreaks of EFA caused by
Polysiphonia sp. in the Philippines (Hurtado et al. 2006) and
the epiphyte Neosiphonia savatieri (Hariot) M.S. Kim & I.K.
Lee in the Philippines and Malaysia (Hurtado et al. 2006;
Vairappan 2006) caused a significant decrease in global
K. alvarezii production. This decrease is reportedly correlated
with drastic environmental changes in areas with low water
motion in both of these locations. Morphological studies per-
formed in China revealed that the main epiphyte on
K. alvarezii was N. savatieri and reduced photosynthetic ac-
tivity caused by extensive shading could be one of the primary
reasons for the decreased productivity of K. alvarezii infected
by N. savatieri (Pang et al. 2011).
A sustainable seaweed farms project was established in the
Caribbean Sea near the northern entrance of the Panamá Canal
by Gracilarias de Panamá S. A. The objective was to cultivate
species of red algae used by the communities of the
Panamanian Caribbean areas and to provide an important live-
lihood activity for residents. The first seaweed farms were
developed with the species Gracilaria domingensis
(Kützing) Sonder ex Dickie and Eucheuma sp. (Vega 2004a,
b). These two genera were reported to already inhabit this area
(Abbott and Norris 1985; Doty et al. 1986). In 2006, in vitro
culture methods were initiated at the Galeta Point Marine
Laboratory of the Smithsonian Tropical Research Institute,
which is located close to the areas where the seaweeds are
cultivated (Perez 2008; Soler and Batista 2007). Panama is
among the few countries in the Americas with commercial
farming of Kappaphycus sp. that has exported production to
Europe and Asia (Hurtado et al. 2014).
Expertise obtained through the development and operation
of the seaweed farms in the Caribbean of Panama revealed a
need to improve and standardize various aspects of cultiva-
tion. In addition, it was hypothesized that improved selection
processes for quality seedstock would result in a crop with
higher carrageenan content. This study presents a procedure
to identify and broaden knowledge of carrageenan content
located in the cell wall and in the cell centers of the red alga
K. alvarezii. Processes for enhancing carrageenan content
were explored through in vitro techniques with treatment by
three polyamines administered at different concentrations.
Carrageenan was imaged in the treated explants at the
Laboratory for Fluorescence Dynamics at the Department of
Biomedical Engineering, University of California, Irvine,
USA. 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 sam-
ples (Wright et al. 2012).
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 confo-
cal microscopy, and it has been found to be a powerful method
for identifying specific emission fingerprints of auto-
fluorescence of compounds present in seaweeds. This study
reports on the identification of several spectral fluorescence
emission fingerprints from different auto-fluorescence com-
pounds spatially mapped in the commercially important red
algal species K. alvarezii. The explants imaged were cultivat-
ed in vitro and treated with the polyamines spermidine, pu-
trescine, and spermine.
Materials and methods
Study area The culture system was developed in four phases
using three cultivation areas. 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 (phase 4) 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 poly-
gons within the Maritime Concessions was selected for in situ
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sea culture. It was identified as polygon 11. The selected poly-
gon 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 (Vega 2009;
Vega et al. 2004). Seaweed cultivation activities were origi-
nally established in this area to involve local community
members with limited livelihood options in the development
of a type of aquaculture considered Beco-friendly^ (Vega
2009; Vega et al. 2004). The physical infrastructures of the
farms were thought to potentially buffer mangroves and coral
reefs from the negative impacts of extreme weather events
(Contreras and Rosenthal 2003). 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 (Brooks 2015).
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 (Cubit et al. 1988). Environmental infor-
mation 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 (Cubit et al. 1988)
and 80 years of data collected by the Panama Canal
Authority. In 2000, the Coastal Research Institute of GKSS,
Germany, installed an in situ hightech monitoring system to
integrate environmental data at the site. The seaweed farm
sites were selected based upon environmental parameter aver-
ages identified as ideal measures for successful seaweed cul-
tivation. These were determined by geographical area and
degree of variation by season (Vega et al. 2004). Data indicat-
ed 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
(Contreras and Rosenthal 2003).
Growth of K. alvarezii is favored when salinity is about 32
to 35 ‰ and it is inhibited below 28 ‰ (Paula et al. 2001).
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 pho-
tons m−2 s−1. (Vega 2009; Vega et al. 2004). 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 (Fig. 1) 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 labo-
ratory 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 tempera-
ture 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 (TLT
W/54RS) tubes, with a photoperiod of 12 h light and 12 h
dark. The spectral emissions and confocal microscopy
were explored as alternative technologies for measure-
ment of carrageenan quality of seaweed. This histological
ana lys i s was per fo rmed a t the Labora to ry fo r
Fluorescence Dynamics, University of California, Irvine,
USA.
Experimental phases
Selection of explants Kappaphycus alvarezii seed stock was
collected from seaweed farm, polygon 11, over a 3-year peri-
od from 2012 through 2014. From the collected seed stock,
explants were selected to be planted for phase 1 and those that
survived were transferred to phases 2, 3, and 4 until the 60th
day in each phase. For each phase, the explants treated with
the different polyamines in phase 1 were transferred to differ-
ent sites according to experimental design. The weight in
grams and length in centimeters of the explants were mea-
sured every 15 days from the day of planting. Beginning in
phase 2, width in centimeters was also measured for each
explant. Explants were selected by size in phase 1 to maintain
consistency with previously published reports from
K. alvarezii (Soler and Batista 2007; Perez 2008; Robaina
et al. 2008). All explants selected for the study appeared clean,
green, and healthy. The existence of color strains of
K. alvarezii with varied pigmentation (green, red, yellow,
and brown) has been reported in the literature for decades,
with differences in their physiological characteristics
Fig. 1 Cell walls and intercellular matrix ofKappaphycus alvarezii SEM
images obtained with Zeiss Model Evo40 VP. Images of explant treated
with spermidine 10−5 M
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including growth performance and photosynthesis reported
from laboratory studies (Dawes and Koch 1991; Aguiree-
Lipperheide et al. 1995; Hurtado et al. 2014; Pang et al. 2011).
Phase 1 began with the selection of explants of
K. alvarezii that measured 1.0 cm long and weighed
0.02 g and which were seeded in Berlin packaging jars
Wm Pp of 500 mL. All explants selected had a single
shaft axis at the end without tangible protuberances to
ensure consistency and proper counting of shoots and calli
on the first day of measurement. The explants were placed
in incubators after having been seeded into one of three
jars. Each jar was seeded with four explants and the ex-
perimental culture medium added. The culture medium
had been prepared previously and contained the poly-
amines, putrescine (Put), spermidine (Spd), and spermine
(Spm) at the following rates: 0 M (control); Put 10−5 M,
Put 10−7 M, and Put 10−9 M; Spd 10−5 M, Spd 10−7 M,
and Spd 10−9 M; and Spm 10−5 M, Spm 10−7 M, and Spm
10−9 M. Standard culture media sodium metasilicate,
Na2SiO3 was added. The pH was measured to be between
7.5 and 7.8. The polyamines described above were added
to the medium after being autoclaved, and the solution
further treated by filtration. Representative explants were
measured and weighed every 15 days until completion on
the 60th day of planting.
In phase 2, the surviving treated explants from phase 1
were transferred into each of two aquaria inside the labo-
ratory designated ponds 2A and 2B in a semi-protected
area, with a wall and a ceiling. Three explants were
planted on each of three ropes of 50 cm long with a space
of 12 cm between each seedling. Each culture tank had 12
algae in a total area of 12.5 cm2. Representative explants
were collected and measured every 15 days. In addition,
new apices and calli that grew on each individual explant
were counted and recorded.
For phase 3, explants obtained from phase 2 that ap-
peared healthy and felt solid to the touch were transferred
to the tanks. Each tank has an area of 1.0 m2 at a depth of
0.50 m. We planted four ropes of 0.80 cm long. In each
rope, we planted four seedlings spaced apart by 20 cm.
Each tank contained 16 seedlings m−2.
In phase 4, all surviving explants from phase 3 were
transferred to the seaweed farm in three lines with 16
explants attached to each line, which were attached to
structures anchored to the sea floor. Two sets of structures
were anchored one beside the other, spanning an area of
about 40 m2. We randomly selected four explants to mea-
sure and observe every 15 days until the 60th day.
Daily growth rate Thirty-six explants were randomly select-
ed from a population of approximately 96. Each explant was
weighed in grams and measured in centimeters at 15-day in-
tervals in order to determine the daily growth rate
(DGR = % day−1. The Sokal and Rohlf (1995) model was
applied.
W f ¼ W oeμt
μ ¼ lnW f−lnW oð Þt−1

  100
where,Wf = final weight,Wo = initial weight, μ = growth rate,
and t = planted day, until reaching the last date of the research.
This variable was then used as the dependent variable. If there
were more than two classifications, a Fisher’s analysis of var-
iance test was performed for completely randomized designs
with Tukey’s HDS test multiple comparisonmethods. All tests
were with a 5 % significance level.
Statistical analysis SYSTAT statistical software package was
used to run repeated measure analysis of variance on each of
the variables, length, weight, and number of apices and calli
were tested for normality and homogeneity of variance.
Analysis of variance (ANOVA) and multifactorial analysis
of variance were also conducted to determine the differences
in growth rate (alpha = 0.05). Out of the nine replicates that
were treated, 1 and 3 were selected randomly to be used in the
analyses for a balanced design.
Confocal microscopy Two samples were used to detect fluo-
rescence of carrageenan powder from two manufacturing
companies known to produce good-quality carrageenan pow-
der. The first sample was from Malaysia and the second from
Japan Marine Science. A small portion of each powder was
mounted on a slide with a drop of water. Observations and
image acquisition were made with a confocal microscope
(Olympus FV 1000) at the laboratory of electronic microsco-
py of the Smithsonian Tropical Research Institute in Panama
to determine the emission spectra of carrageenan. Auto-
fluorescence was detected using the Lambda acquisition mode
of the Olympus microscope with laser excitation of 546 nm
and photo-counting method. The emission was scanned every
3 nm from 560 to 660 nm.
Approximately 117 samples of explants from the cultiva-
tion phases were selected at random and recorded in a data-
base by seed number, seed type, days of growth, and treat-
ment. All samples were stored between 10 and 20 °C. They
were later transported to the Laboratory for Fluorescence
Dynamics at the University of California in Irvine for imaging
and corresponding analysis. Upon detecting emission, we
proceeded to check the fluorochrome in the library of the
microscope to identify the carrageenan within the cells of
the cultivated algae with auto-fluorescence. We then
proceeded to make cross-sections of several samples of algae
to observe auto-fluorescence from within the walls and
throughout the centers of the cells.
Transverse sections of calli and apexes from explants of
K. alvarezii were prepared from 11 explants in total,
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representing samples cultured in the treatments. Observations
of the spectra of carrageenan in the cell walls and within the
intercellular matrix were made at the Laboratory for
Fluorescence Dynamics, UCI, using the confocal microscope
Zeiss LSM710, ×40 NA 1.2 water immersion objective.
Samples of K. alvarezii were as a follows: Callus samples
15, 17, and 86 were treated with 10−5 M spermidine. Apex
samples 16 and 85 were treated with 10−5 M spermidine.
Callus sample 91 and apex samples 88 and 107 treated with
10−7 M spermidine. Callus samples 83 and apex 87 were
given control treatment. Sample 114 was a dried powder from
the untreated parent alga.
For each of the samples described, cross-sections were made
in order for the cell walls and intercellular matrices to be ob-
served using confocal microscopy Zeiss LSM710, ×40 NA1.2
water immersion objective. Images of the cell wall and the cel-
lular matrix of each sample were obtained at 561 nm excitation
wavelength. Emission data was collected with the spectral mode
of the Zeiss 710LSM in the range 562–722 nm about every 3 nm
for a total of 51 spectra. The spectral image of the Zeiss Zen
software was used to display the images color coded according
to the spectral color palette. Spectral data were also analyzed
using the spectral phasor algorithm of SimFCS (Laboratory for
Fluorescence Dynamics, www.lfd.uci.edu).
The spectral phasor approach The spectral phasor consists
of a mathematical transformation of the emission spectrum
into the Fourier domain as described by the following equa-
tions (Cutrale et al. 2013).
xcoordinate ¼ g ¼
X
λ
I λð Þcos 2πnλ
.
L
 
X
λ
I λð Þ
ð1Þ
ycoordinate ¼ s ¼
X
λ
λð Þsin 2πnλ
.
L
 
X
λ
I λð Þ
ð2Þ
Where I(λ) is the emission spectrum measured over a
wavelength range L at each pixel of the image and n is 1
in our case. A fundamental property of the phasor trans-
formation achieved by Eqs. (1) and (2) is that if the spec-
trum at each pixel of the image is due to the sum of two
spectral components, then the phasors must align along a
straight line joining the phasors of the pure components.
Instead, if the spectrum at each pixel contains molecular
species form more than two components, the phasor
values cannot be aligned. Therefore, the spectral phasor
analysis is a very simple way to obtain a specific finger-
print of a sample of unknown composition. This property
is used in this manuscript 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.
Results
Results of phase 1 showed statistical significance in length
and weight with p value = 0.001 among treatments cultivated
in vitro for 60 days. The explants treated with Spd 10−5 M had
the longest length at 1.2–2.0 cm and the greatest weight in-
crease at 0.05–0.90 g. They differed from the other treatments
which obtained lengths ranging between 0.5 and 1.1 cm) and
weights ranging between 0.013 and 0.040 g (Fig. 2a, b).
The growth rate in centimeters per day showed significant
differences among treatments p value = 0.005 while growth
rate in grams showed no significant differences p value = 0.11
(Fig. 2c, d). The explants treated with Spd 10−5 M obtained
the greatest rate in centimeters (1.05–1.4 % cm day−1) and
also in grams (2.72–4.5 % g day−1), although not significant
(Fig. 2c, d).
Statistical significance was also found in new formations of
callus among treatments p value = 0.025 and apexes p val-
ue = 0.046, Fig. 2e, f. The greatest number of new callus
formation was six, in explants treated with Put 10−5 and the
most apexes, four in Spd 10−7.
In phase 2, selected explants from phase1 treated with Put
10−7 M and Put 10−9 M; Spd 10−5 M, Spd 10−7 M, and Spd
10−9 M; and control were cultivated in outdoor pond sites for
60 days. Differences in length and weight showed statistical
significance with p value = 0.0001 and 0.001 respectively
among treatments. The explants treated with spermidine
10−7 M presented greater length (3.0 cm) and weight
(0.11 g) while explants treated with putrescine 10−9M showed
the shortest length (0.94 cm) and lowest weight (0.02 g)
Fig. 3a, b.
The growth rate (% day−1) in grams also showed sta-
tistical significance between treatments p value = 0.0001.
Even though the explants treated with Put 10−7 M in-
creased 1.86 (% day−1) and Spd 10−7 M 2.77 (% day−1)
the most significant difference occurred in explants treat-
ed with Spd 10−5, which presented the highest growth rate
in grams 4.26 (% day−1) (Fig. 3c). Statistical significance
was not found in new callus formation or apex develop-
ment among treatments p value = 0.902 and apexes p
value = 0.646 (Fig. 2d, e).
In phase 3, selected explants from phase 2 treated with Put
10−5 M, Put 10−9 M, Spd 10−5 M, and control were cultivated
in outdoor tanks for 60 days. The weight and length of the
explants were not statistically significant between treatment
means. p values = 0.515 and 0.257, respectively (Fig. 4a, b).
Growth rates of explants in cm and in g (% day−1), showed
statistical significance between treatments p value = 0.001 and
0.033, respectively. The explants treated with Spd 10−5 M
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obtained the greatest range increase in centimeters, 4.37–5.17
(% day−1) and in grams, 11.74–15.23 (% day−1) (Fig. 4c, d).
Statistical significance was also found with respect to
new formations of callus among treatments p val-
ue = 0.025, while the number of apexes presented no
statistical significance p value = 0.98 (Fig. 4e, f). The
explants treated with Spd 10−5 M had the greatest number
of callus (24), and the number of apices (7) was statisti-
cally similar to the control.
In phase 4, explants selected from phase 3 treated with Put
10−5, Put 10−9, Spd 10−5, Spm 10−7, Spm 10−9, and the control
were transferred to seaweed farms for a duration of 60 days. In
this phase, all explants planted on the farms increased in
weight, length, and number of apices and calluses. Statistical
significance was determined among all treatments for length
(cm) with a p value = 0.0001, weight (g) p value = 0.0001. A
wide centimeter variable was added which also showed statis-
tical significance among all treatments with p value = 0.0001.
Fig. 2 a Length (cm), b weight (g), c growth rate (% day−1) length (cm),
d growth rate (% day−1) weight (g), e number of callus, and f number of
apexes of Kappaphycus alvarezii explants cultivated in vitro, phase 1
under different molar concentrations of polyamines; 0 M (control); Put
10−5 M, Put 10−7 M, and Put 10−9 M; Spd 10−5 M, Spd 10−7 M, and Spd
10−9 M; and Spm 10−5 M, Spm 10−7 M, and Spm 10−9 M until 60 days.
Values presented are averages (n = 4). Bars show polyamines with
different concentrations. The vertical bars show the standard error.
Uppercase letters represent significant differences among the treatments
according to the repeated measures analysis of variance using Tukey’s
HSD test under the hypothesis that all population means are equal
considering p < 0.05
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Number of calluses and apexes were also significant among
all treatments p value = 0.017 and 0.019, respectively.
The mean length at 11.23 cm, weight at 34.0 g, width
at 14.8 cm, number of calluses (20), and number of apex-
es of explants treated with spermidine 10−5 M were sta-
tistically different with higher values from the other ex-
plants (Fig. 5a–f). In that treatment, growth rate in grams
also had a higher range of 2–11.0 % day−1 (Fig. 5d). The
growth rate presented no statistical significance in grams
(% day−1) with p value = 0.19. The explants treated with
spermina 10−9 presented the lowest values in number of
both callus formation (2) and apices (3), as well as in
length (0.43 cm) and weight (0.067 g). The lowest width
(2.88 cm) was observed in Spm 10−7 M (Fig. 5a–c, e, f).
Confocal microscopy
For the confocal microscopy study, the following samples of
K. alvarezii were analyzed: 15, 16, 17, 83, 85, 86, 87, 88, 91,
107, and 114 from the same mother alga no. 28. Images were
obtained at 561 nm excitation wavelength of both the cell
Fig. 3 a Length (cm), bweight (g), c growth rate (% day−1) weight (g), d
number of calli, and e number of apexes of Kappaphycus alvarezii in
phase 2. The explants from phase 1 were transferred into ponds outside
the laboratory in a semi-protected area under different molar
concentrations of polyamines: 0 M (control); Put 10−7 M and Put
10−9 M; and Spd 10−5 M, Spd 10−7 M, and Spd 10−9 M until 60 days.
Values presented are averages (n = 4). Bars show polyamines with dif-
ferent concentrations. The vertical bars show the standard error.
Uppercase letters represent significant differences among the treatments
according to the repeated measures analysis of variance using Tukey’s
HSD test under the hypothesis that all population means are equal
considering p < 0.05
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center and the cell wall of each sample, except for sample no
114, the algae powder, for which only a single image was
obtained. The samples with numbers in bold are reported in
the figures of the manuscript.
Kappaphycus alvarezii samples are shown in Figs. 6, 7, 8,
and 9, with the image colored according to the spectral scale
and the intensity as provided by the ZEN software. These are
shown together with the spectral phasor and the image colored
according to the three-cursor selections as shown in the spec-
tral phasor plot. Both cell center and cell wall are shown for
each sample.
Each of the panel of figures shows the image colored
according to the spectral palette, the spectral phasor im-
age, and the image according to the colors blue-green-
red as shown by the cursor selection in the respective
spectral phasor. These cursors correspond to pixels in
the spectral region 580 nm (blue), 586 nm (green),
and 591 nm (red). The pie graphs in Figs. 6, 7, 8,
and 9 show the percentage of pixels in the three-
cursor selections for each image. In the phasor plot, if
the pixels are aligned along a line, this implies that a
pixel contains the linear combination of the spectra at
the extreme of that line. We note that the pixels in the
phasor plot are not aligned, indicating that we need
more than two basic spectra to describe the emission
of these samples. Since the emission spectrum is mainly
from carrageenan, this indicates that there are more than
two species of carrageenan present in these samples.
The color maps in Figs. 6, 7, 8, and 9 reveal the region
of the sample where these species are most abundant.
Fig. 4 a Length (cm), b weight (g), c growth rate (% day−1) length (cm),
d growth rate (% day−1) weight (g), e number of calli, and f number of
apexes of Kappaphycus alvarezii in phase 3. The explants from phase 2
0 M, Put 10−5 M, Put 10−9 M, and Spd 10−5 M, were transferred into
outside tanks until 60 days of being planted. Values presented are
averages (n = 4). Bars show polyamines with different concentrations.
The vertical bars show the standard error. Uppercase letters represent
significant differences among the treatments according to the repeated
measures analysis of variance using Tukey’s HSD test under the
hypothesis that all population means are equal considering p < 0.05
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Taken together, the analysis of all samples shows that
different samples can be characterized by at least three
emission components that seem to be representative of
the carrageenan content and treatment. The exact source of
this emission is unknown at present time, but we show that we
can use an unsupervised classification and obtain a quantification
of the abundance of different species based on the spectral phasor
plot.
Control sample no. 83, callus from an explant of
K. alvarezii, shown in the pie graph (Fig. 6), has a
wider distribution of the green cluster presented both
at the cell center and at the cell wall. This suggests a
Fig. 5 a Length (cm), b weight (g), c width (cm), d growth rate
(% day−1) weight (g), e number of calli, and f number of apexes of
Kappaphycus alvarezii in phase 4 from phase 3. The explants 0 M
(control); Put 10−5 M and Put 10−9 M; Spd 10−5 M; and Spm 10−7 M
and Spm 10−9 M cultivated in seaweed farms until 60 days of being
planted. Values presented are averages (n = 4). Bars show polyamines
with different concentrations. The vertical bars show the standard error.
Uppercase letters represent significant differences among the treatments
according to the repeated measures analysis of variance using Tukey’s
HSD test under the hypothesis that all population means are equal con-
sidering p < 0.05
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distinct characteristic, compared with the callus treated
with spermidine 10−5 M. Control sample no. 87, apex
from explant (Fig. 7), shows a wider distribution of the
green cluster in the center of the cell, and the red clus-
ter at the cell wall also suggests a distinct characteristic
compared with the apex treated with spermidine
10−5 M.
Callus and apex from sample no. 15 (Fig. 8) and sam-
ple no. 85 (Fig. 9), respectively, treated with spermidine
10−5 M showed that the callus had a much larger contri-
bution of the red cluster in the cell wall. A wider distri-
bution of green cluster at the center of the cell showed
distinct color characteristics compared with the apex that
was treated similarly to sample no. 15. This sample dem-
onstrated a different morphology than the sample in
Fig. 6. 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.
Discussion
This study combines an experimental approach to enhance
commercial carrageenan productivity with the novel applica-
tion of confocal laser-scanning microscopy, a technique capa-
ble 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 al-
though in this paper we have not identified the specific types.
The identification of several spectral fluorescence emission
fingerprints from different auto-fluorescence compounds from
Fig. 6 Images obtained from a callus of an explant of Kappaphycus
alvarezii cultivated in vitro treated as a control by confocal microscopy
Zeiss LSM710, ×40 NA1.2 at both center and cell wall. a, b Images
shown here are colored according to the spectra palette. Scale bar is
5 μm. c, d Images shown here are painted according to the spectral
scale and intensity as provided by the ZEN software of (a) and (b)
images. e, f Phasor plot histogram that corresponds to pixels in the
spectral region: 580 nm (blue), 586 nm (green), and 591 nm (red) of (c)
and (d) images, respectively. g, h Pie graphs show the percentage of
pixels in the three cursors for each image
Fig. 7 Images obtained from an apex from explant treated as a control of
Kappaphycus alvarezii cultivated in vitro by confocal microscopy Zeiss
LSM710, ×40NA1.2 at both center and cell wall. a, b Images shown here
are colored according to the spectra palette. Scale bar is 5μm. c, d Images
painted according to the spectral scale and intensity as provided by the
ZEN software of (a) and (b) images. e, f Phasor plot histogram that
corresponds to pixels in the spectral region: 580 nm (blue) 586 nm
(green) and 591 nm (red) of (c) and (d) images, respectively. g, h Pie
graphs show the percentage of pixels in the three cursors for each image
J Appl Phycol
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explants of K. alvarezii are reported and spatially mapped.
These fingerprints have the potential to improve strain selec-
tion 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 (C. Agardh) J. Agardh, and the calculation of meta-
chromatic indices gave a higher value for the medullary cell
walls in the tetrasporophyte than in the female gametophyte
(Gordon-Mills et al. 1978).
During culture in the laboratory, 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 (PGR) treatments
that were expected to favor the development of cystocarps.
Particularly, Spm promoted sporulation in several
Rhodophytes such as Hydropuntia cornea (formerly
Gracilaria cornea) and Grateloupia imbricate (García-
Jiménez and Robaina 2015).
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 cultiva-
tion techniques by establishing and adopting viable methods
Fig. 9 Images obtained from an apex of explant treated with spermidine
10−5 M from of Kappaphycus alvarezii cultivated in vitro by confocal
microscopy Zeiss LSM710, ×40 NA1.2 at both center and cell wall. a, b
Images shown here are colored according to the spectra palette. Scale bar is
5 μm. c, d Images shown here are painted according to the spectral scale
and intensity as provided by the ZEN software of (a) and (b) images. e, f
Phasor plot histogram that corresponds to pixels in the spectral region:
580 nm (blue) 586 nm (green), and 591 nm (red) of (c) and (d) images,
respectively. g, h Pie graphs show the percentage of pixels in the three
cursor for each image
Fig. 8 Images obtained from a callus of explant treated with spermidine
10−5 M from Kappaphycus alvarezii cultivated in vitro by confocal
microscopy Zeiss LSM710, ×40 NA1.2 at both center and cell wall. a,
b Images shown here are colored according to the spectra palette. Scale
bar is 5 μm. c, d Images shown here are painted according to the spectral
scale and intensity as provided by the ZEN software of (a) and (b)
images. e, f Phasor plot histogram that corresponds to pixels in the
spectral region: 580 nm (blue) 586 nm (green), and 591 nm (red) of (c)
and (d) images, respectively. g, h Pie graphs show the percentage of
pixels in the three cursors for each image
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of enhancing strains of commercially important species
(Yokoya et al. 2004). Zitta et al. (2013) found that the thick
wall of the cells, which are composed in part of callus fila-
ments, showed the presence of acidic polysaccharides, sug-
gesting 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 respi-
ration and thus energy metabolism (Schmidt 2009),
supporting subsequent cell divisions and the formation of lat-
eral branching. According to Doty (1988), K. cottonii thalli
are compressed to flatten above the basal segment; prostate,
irregular in form, or with linear segments with irregular oc-
currences of protuberances or branches.
The images obtained in this study by the confocal mi-
croscopy 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 (Figs. 8 and 9). These samples dem-
onstrated 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 fluores-
cence 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 seedstocks in the natural marine habi-
tat. 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 ben-
efits to the industry.
This study suggests a novel technique for seaweed cultiva-
tors 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. Chemical products extracted from
macroalgae account for most of the remaining value. The var-
ious species possess high levels of secondary metabolites and
structural polysaccharides of commercial value (Roesijadi
et al. 2010). These include alginates from brown algae and
agar and carrageenans from red algae. Today’s growing de-
mand for carrageenan and the need for more sustainable farm-
ing practices is a large challenge. There is an urgent require-
ment to understand and detect differences among types and
qualities of carrageenan as a means of managing higher yield-
ing, economically viable, long-term farming practices
(Vairappan et al. 2007).
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 farmgate value increasing from US$72 million
to US$1.4 billion. Major carrageenan seaweed farming coun-
tries include Indonesia, the Philippines, the United Republic
of Tanzania, Malaysia, and China (Valderrama et al. 2013).
Despite the high demand for carrageenans, seaweed scien-
tists, farmers, buyers, and processors still lack accessible tools
to accurately and inexpensively assess the carrageenan con-
tent 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 com-
mercial 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 (Valderrama et al.
2013). 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 corre-
lation between the seasonal weather patterns and the quality of
the product (Vega 2009). We note that this area of the
Caribbean seems to be protected from the adverse impacts of
hurricanes (Contreras and Rosenthal 2003; Cubit 1994). This
J Appl Phycol
Author's personal copy
unique condition is an important consideration regarding the
importance of commercially viable seaweed farms in this
region.
Acknowledgments Early stages of this work were sponsored by the
NGO Friendship Foundation and the United Nations Development
Program (UNDP) Small Grants Program for the Association of
Seaweed Producers and the Seaweed Farming Project with the Guna
Community of Cativa, Panama. The Colon Monitoring Coastal Center
Association for the Caribbean (ACEMOC) provided environmental real-
time data of the area. Samuel and Guillermo Liberman through Sociedad
Latinoamericana de Inversiones and Gracilarias de Panama provided gen-
erous funding and continuous support for this work. The sponsors played
no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript, but without their support, this research
would not have been possible. Thanks are given to Dr. James Norris,
Emeritus Scientist of the Department of Botany of the Smithsonian
Institute, for facilitating the use of The Algal Collection of the U.S.
National Herbarium, which is among the largest in the world for tropical
marine algae and currently houses a significant collection from Caribbean
Panama, including type specimens relevant to the research. Thanks to
Suzzane Lao MSc, Computer Program Analyst from the Smithsonian
Tropical Research Institute, for analyzing the in vitro data; Raúl Alegre,
Data Analyst from Market Research Collective, for designing tasks; and
Lynn Cornish MSc of Dalhousie University in Nova Scotia, Canada, for
edits to the paper and recommendations. EG, AA, and MAD acknowl-
edge funding from NIH P41-GM103540 and NIH P50-GM076516.
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