DISCUSSION
Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
DISCUSSION
The rising demand for seaweeds during the last 50 years has outstripped the
ability to supply the required biomass from natural (wild) stocks thus triggering a
dramatic growth of its production through extensive mariculture. Thus commercial
cultivation has overshadowed production of algae collected from natural sources.
According to FAO, (2012) there is a dramatic increase in the production of Eucheumoid
over the other species due to wider application of carrageenan. In 2010 approximately
5.7 million tonnes (wet weight) of Kappaphycus and Eucheuma, 5.1 million tonnes of
Laminaria japonica, 1.8 million tonnes of Gracilaria sp., 1.6 million tonnes of
Porphyra sp. and 1.5 million tonnes of Undaria pinnatifida were produced through
seaweed mariculture.
Kappaphycus production is often dependent on culture conditions, grazing,
epiphytism, outbreak of ice-ice disease and other environmental factors. An epiphytic
outbreak in Kappaphycus (Eucheuma) farm was reported in 1975 (Doty & Alvarez,
1975). However, not much interest was focused on these problems, until the other major
epiphyte outbreak and their impact on biomass production in 2004 (Critchley et al.,
2004; Hurtado et al., 2006 and Vairappan, 2006). Thus understanding the effect of these
factors and various other obstacles on the growth of K. alvarezii and carrageenan yield
is important. Therefore the need for advanced management in Kappaphycus cultivation
is required in the areas where intensive farming is practiced or in the new cultivation
area.
Kappaphycus Cultivation in the Field and Various Factors
The world’s geographical area for the Kappaphycus farming lies between 10°N
and 10°S latitude mostly in the Southeast Asian countries then extending to tropical
regions of East Africa and Brazil. However amongst the Southeast Asian region
Indonesia, Philippines and Malaysia contribute to nearly 60% of the global production
(Hayashi et al., 2011a). It is well known that Kappaphycus grows in a temperature
ranges between 20-35°C (Areces, 1995; McHugh, 2003 and Ask, 2006) but growth and
high biomass production is mostly found in a temperature range of 25-33°C in
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Southeast Asian farm areas (Glenn & Doty, 1981). Ohno et al., (1994) found that
commercial growth of Kappaphycus could not be obtained if the water temperature is
below 20°C. However Sahoo & Ohno, (2003) and Sahoo et al., (2003) cultivated
Kappaphycus as well as Eucheuma serra in deep seawater with much lower water
temperature. The effect of nutrients and salinity on Kappaphycus is not well known
although it can be assumed that they are also important to plant growth. The salinity
ranges from 30-35 ppt in most of the successful farm sites (Glenn & Doty, 1981).
Mandapam (Tamil Nadu) being a tropical region as located in similar topographic
position (Latitude 09°06′-09°14′ North and Longitude 78°53′-79°24′ East) have various
environmental parameters which are similar to other cultivation sites in the world. In
the present study, the seawater temperature ranged from 24.6°C-32.5°C, salinity ranged
between 26-34.3 ppt and various other environmental parameters are not only found to
be ideal for Kappaphycus cultivation but also for further expansion on large-scale
cultivation in the region.
The first effort of Kappaphycus (Eucheuma) cultivation was initiated by Dr.
Maxwell Doty of the University of Hawaii in conjunction with Marine Colloids (taken
over by FMC in 1977) in the mid-1960s (Ronquillo & Gabral-Llana, 1989). Since then
first successful farming of Kappaphycus started in Philippines during 1970 and the
cultivation technique had undergone several modifications. The commercial cultivation
for Eucheuma started in Philippines through monoline method during 1977.
Subsequently numerous systems developed through laboratory and field experiments
(Doty & Alvarez, 1975). Presently three basic forms of cultivation such as fixed offbottom, floating long line and rafts were practiced in the world (Trono, 1989). Fixed
off-bottom method is practiced in Indonesia (Adnan & Porse, 1987), Philippines (Lim
& Porse, 1981 and Sahoo, 2000), Fiji (Luxton et al., 1987), Vietnam (Hung et al., 2009)
etc. This system predominated primarily because it is simple, farm materials were
readily available, inexpensive and plants grew well (Trono, 1989). In India culture of K.
alvarezii was initiated in mid 1990s at Coast Okha, Gujarat (Mairh et al., 1995) but
subsequently the cultivation was shifted to Mandapam during 1995–1997. Initially netbag and monoline method were practiced for cultivation but due to heavy grazing in
monoline method and low productivity in net bags, these cultivation practices were
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
abandoned. Later bamboo rafts were introduced as a viable method. Even in the open
bamboo rafts the grazing was very predominant thus causing huge loss of biomass.
Therefore to protect Kappaphycus from grazing, the rafts were covered from the bottom
by net. Sahoo, (2011) published a training manual describing different aspects of
Kappaphycus farming which include site selection, seedling selection, relevant tools
and equipment, methods of rope, harvesting, construction of drying frames and drying
techniques. The manual highlights the cause, effect, and solutions to commonly
encountered problems such as grazing by herbivores, epiphytic algae and ice-ice
disease. These problems swamp the Mandapam coasts cutting down seaweed
production drastically (Pers.Com). Therefore, in the present study K. alvarezii was
cultivated in modified rafts where the raft was covered with net not only from below but
also from all sides of the raft. Using modified raft for cultivation (present study) have
several advantages such as enhanced growth rate, maximum protection from grazing,
epiphytism and biofouling over the local rafts as used by seaweed farmers.
Analysis of Growth rate
The growth of seaweed depends on the several environmental factors such as
irradiance, temperature, nutrients, water movements, pH and salinity (Doty, 1973).
These factors may interact and regulate the growth of Kappaphycus. Exposure to
optimal amount of wavelengths of photosynthetically active radiation (PAR) is
undoubtedly as essential for rapid growth of Kappaphycus. Seawater temperature is the
main factor which influences the growth of Kappaphycus. Temperature affects both the
biochemistry of seaweeds and the rate of diffusion. However it has been reported that
Kappaphycus attained highest growth rate when compared to other seaweeds. This high
growth rate of Kappaphycus is due to wide range of tolerance to ecological parameters
(Dawes et al., 1994 and Nishihara & Terada, 2010), greater response to water motion
(Glenn & Doty, 1992) and its morphological variability (Doty, 1987). Kappaphycus
thalli have thick branches, which have been shown to reduce diffusion of materials into
the centre of the thallus, thus requiring greater water motion for their growth (Glenn &
Doty, 1992). Water motion enhances the growth rate of seaweeds by reducing the
thickness of the diffusion boundary layer around the algal surface (Wheeler, 1988). The
decreased boundary layer then increases the supply of inorganic carbon, phosphate,
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
nitrate and other micronutrients to the thallus (Wheeler, 1988 and Hurd, 2000) and
facilitates the removal of algal metabolic products such as oxygen and hydroxide ions
(Gonen et al., 1995), excess hydrogen peroxide and halogenated organic compounds
away from the plant surface (Mtolera, 1996). Growth rates of Kappaphycus cultivated
by different methods vary substantially. Fixed off-bottom monoline method yielded 3-
4% day-1 in Indonesia (Adnan & Porse, 1987), 3.5-3.7% day-1 in Fiji (Luxton et al.,
1987), 4.2-4.3% day-1 in Kenya (Wakibia et al., 2006), 2-3.2% day-1 in Philippines
(Hurtado et al., 2001) and 1.6-4.6 % day-1 in Vietnam (Hung et al., 2009). On the other
hand in the Bamboo raft method, the growth rate are 1.24 % day-1 in Philippines
(Samonte et al., 1993), 0.5-5.6 % day-1 in Tanzania (Msuya & Kyewalyanga, 2006),
4.5% day-1 in Philippines (Gerung & Ohno, 1997) and 4-5 % day-1 in Brazil (Bulboa &
Paula, 2005). The growth rates varied widely depending on the cultivation system
adopted but in general, seaweed cultivated in the usual fixed off-bottom system or in
rafts showed high values ranging between 0.2%-5.3% day-1 and 0.5%-10.7% day-1
respectively (Hayashi et al., 2011a). In the present study, the daily growth rate (DGR)
of K. alvarezii in modified raft was 4.85 % day-1 which is higher than those of local raft
3.66 % day-1. Further, highest growth rate was achieved during post monsoon season
followed by pre-monsoon and summer seasons. The growth curve showed exponential
stage upto 30 days and remained stationary in the later days of cultivation in all the
seasons. Similar growth curves with an exponential phase during the first few weeks of
culture and then a plateau at longer culture duration was observed by Aguirre-vonWobeser et al., (2001); Villanueva et al., (2011) in Kappaphycus sp. and Bezerra &
Marinho, (2010) in other seaweeds like Gracilaria sp. The growth rate of K. alvarezii at
different monsoon period is 4.85 % day-1 in post monsoon followed by 3.94 % day-1 in
pre monsoon and 2.26 % day-1 in summer. The daily growth rate of the present study is
above 3.5 % day-1 which is considered desirable for commercial cultivation. The
maximum growth rate observed during post monsoon season may be due to change in
the seawater temperature which is coupled with influx of more nutrients brought in by
fresh water from the land along with rain water. Another reason may be due to bringing
of more nutrients which is commonly called as upwelling during the North-east
monsoon. A slight decrease in growth rate observed during the pre-monsoon period is
due to the high wind velocity and high water current accompanied by South-west
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
monsoon (Eswaran et al., 2002). Minimum growth rate found in summer month is due
to extreme environmental factors such as high salinity, high temperature, and lower pH
leading to infestation of the plants by diseases which in turn lowers the photosynthesis
rate and other physiological function of the plants (Present work). Similar pattern in
growth rate during different seasons were also reported in various cultivation sites
(Glenn & Doty, 1981; Mairh et al., 1986; Msuya & Salum, 2006; Thirumaran &
Anantharaman, 2009 and Pellizzari & Reis, 2011).
On an economic perspective, harvesting during the end of exponential phase and
onset of stationary phase is ideal as minimal increment in biomass is expected beyond
this point. In the present study the harvesting period of K. alvarezii during the transition
phase between the exponential and stationary stage of the culture is 45 days after
planting which coincides with the actual optimized harvesting period in various other
studies (Hurtado et al., 2008 and Villanueva et al., 2011). In general, it has been found
that optimum harvesting time for carrageenan manufacturers is between 6-8 weeks after
planting (Barraca, 1999). Beyond this period the plants are proned to fragmentation of
thalli, herbivory by different grazers, epiphytism and ice-ice disease.
Grazing
Herbivores have a major influence on macroalgae in both temperate and tropical
shallow water communities (Lubchenco & Gaines, 1981). Herbivores occur chiefly in
warmer waters (40°N-40°S) and are especially significant in the tropics (Hay, 1981).
Herbivorous fishes accounted for only 5% (1,660/29,500) of the total number of fish
species known globally. Of this quantity, 89% are from tropical waters (1,441/1,660)
while 3% are temperate species (49/1,660) (Froese & Pauly, 2007 and Pablico et al.,
2008). On coral reefs it is estimated that 60-97% of the primary production is consumed
by herbivores (Hay & Fenical, 1988). Herbivory influences the growth rate and
reproductive output of algae (Berner, 1990) and also play a role in determining
diversity, abundance, and species composition of seaweeds in shallow water benthic
communities (Hay et al., 1988 and Alstyne, 1989). Grazing is one of the major
problems observed during the present study. The major effect of grazing is loss of
biomass due to fragmentation of the thalli from the rope. Wounds made by grazers
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weaken the thalli and are easily detached from the main thallus by water motion. The
absence of epidermal and cortical layer in grazed thalli found in anatomical studies can
be easily infested by epiphytes. The heavy infection of various epiphytes on grazed
thalli can also be correlated to low defence mechanism in grazed thalli.
Lubchenco & Gaines, (1981) define expected herbivore damage to individual
seaweed, in a given space, at a given time as the probability of being encountered, the
probability of tissue consumption once encountered and reduction in fitness after
herbivore attack relative to degree of undamaged plant left. Seaweed grazers are
commonly classified based on their grazer taxon, feeding mode, size group, diet
specificity and typical habitat (Potin, 2012). Herbivores diet specificity are mainly of
two types namely generalist herbivores such as fish, sea urchins, and many gastropods
which consume a wide variety of food plants (Lubchenco & Gaines, 1981) and
specialist herbivores which by contrast feed on a few specific plant species or certain
part of the plant. Specialist herbivores of marine macroalgae are rare (Hay & Fenical,
1988 and Hay et al., 1989) than the generalist herbivores.
Kappaphycus cultivation site in the present study is dominated by generalist
herbivores such as Scarus sp., Gobiid sp. Siganus sp. and mesograzers like Gammarus
sp. and Idotea sp. Pablico et al., (2008) highlighted the incidence of most common
marine herbivores such as Blenniidae (combtooth blennies), Scaridae (parrot fishes),
Acanthuridae (surgeon fishes), Pomacentridae (damsel fishes), Kyphosidae (sea chubs),
Siganidae (rabbit fishes) and Aplodactylidae (marble fishes) in the seaweed farming site
of Philippines. Trono & Valdestamon, (1994) and Hurtado et al., (2001) reported
excessive grazing by Siganids, sea urchins (Echinothrix sp., Diadema setosum etc) and
various macrobenthic grazer such as starfish (Protoreaster nodosus) as the most
common grazers. The presence of wide variety of grazers in Kappaphycus farming site
is due to the occurrence of favourable habitat i.e. seagrass bed which are dominated by
Thalasiya hembruchi, Cymodacia, Syrincodium isotifolium and Halophilo ovalis or the
herbivores which become endemic to that particular site (Present work).
Athiperumalsami et al., (2008) reported rich diversity of seagrasses in various sites in
Gulf of Mannar, Southeast coast of India. Many grazers favour the area where
seagrasses are abundant for their habitat only. Most seagrass beds which have been
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studied have a higher faunal biomass and biodiversity than unvegetated neighbouring
habitats (Homziak et al., 1982; Wells et al., 1985; Carpenter & Lodge, 1986 and
Nakamura & Sano, 2005). Migrations of fishes in seagrass beds and other coastal
habitats may be another reason for the occurrence of grazers in the area where
seagrasses are dominant (Hindell et al., 2000; Scott et al., 2000; Guest et al., 2003;
McArthur et al., 2003; Chittaro et al., 2005; Dorenbosch et al., 2005 and Unsworth et
al., 2007). Kikuchi, (1966) classified common grazer based on their occurrence such as
seasonal grazer (which occurs during certain seasons) and territorial grazer (which
occurs in certain places). The present work reported the occurrence of seasonal grazers
in Kappaphycus cultivation sites and thalli were found to be colonized by different life
stages of the grazers. This finding correlates with the work of Kochzius, (1999) where
seasonal grazers inhabit seagrass and seaweeds for shelter during certain seasons of the
year or during certain life stages in tropical region.
Herbivores usually prefer seaweeds rather than the seagrasses. Among the
seaweed, class Rhodophyceae (33 families, 145 species) are the most often reported
component of herbivore diets, followed by the class Phaeophyceae (20 families, 81
species), dominated by
families Dictyotaceae and Sargassaceae and class
Chlorophyceae (14 families, 33 species), mainly from the family Ulvaceae (Pablico et
al., 2008). The grazers prefer the filamentous red algae rather than the brown,
encrusting, calcareous macroalgae and other seagrasses found in cultivating raft for
food (present study). Similar feeding habits with most preferring red and green foliose
macroalgae were also reported by Horn et al., (1982, 1990); Barry & Ehret, (1993);
Caceres et al., (1994) and Horn & Ojeda, (1999). Seagrasses, on the other hand are
more evolved group of plants compared to seaweeds and they have well-developed
defence mechanisms. Therefore, seagrasses are not consumed by fishes as their food.
This pattern of choosing the seaweeds by grazers for their food and shelter is well
supported by a wide array of studies (Tsuda & Bryan, 1973; Von Westernhagen, 1974;
Montgomery, 1977 and Ganesan et al., 2006). The occurrence of fishes and
mesograzers are also reported by Montgomery & Gerking, (1980) in Sargassum sp.,
Ulva sp. and Eucheuma sp., Smit et al., (2003) in Gracilaria sp. and Marroig & Reis,
(2011) in Kappaphycus cultivation farm. The reason behind preferring the red algae
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
over brown algae by many herbivores is due to their types of food reserves present in
macroalgae. The main food reserves in macroalgae are different types of
polysaccharides varying in its linkage between the monosaccharide’s subunits.
Rhodophyta group being rich with floridean starch which is characterized by alphalinked polysaccharides and their extracellular compounds are dominated by diverse
polysaccharides composed of galactose. The hydrolysis of this starch is more
susceptible to amylases enzymes which are rich in the guts of different types of grazers
including fishes. In contrast, phaeophyta consists of beta-linked laminarin and mannitol
as the main food reserve and the enzymes for this digestion are very rare in fishes
(Montgomery & Gerking, 1980). Therefore, herbivores choose the food which is easily
digestible to them. These finding is coupled with the observation in the present study
where the presence of different forms of polysaccharides in Kappaphycus is confirmed
by the histochemical studies.
In the present study it was observed that biomass loss in the rafts used by local
farmers started from the initial day of cultivation. This may be due to differential
pattern of grazing where grazers initially target on the growing tips containing the
apical meristems later moved to lateral branches and main axis of the thalli (present
study). This preferential pattern of grazing correlates with the theory proposed by
different groups (Nicotri, 1977; Smit et al., 2003; Ganesan et al., 2006 and Pablico et
al., 2008) in various other seaweeds. The problems of grazing and biomass loss can be
controlled upto certain level using modified raft by fitting net not only from below but
also raising the nylon net upto 6" from all four sides of the raft. This modified raft with
net has several advantages and can overcome grazing and entanglement by other
seaweeds (present work). Earlier attempts were made to avoid grazing by using bags
made up of chemical such as parahydrobenzene which was tied at different intervals in
the rope on the farm (Doty, 1978). Ask & Azanza, (2002) proposed a few method to
eliminate the impact of herbivores by Avoidance (Plant outside herbivores’ natural
habitat, e.g. float to avoid urchins and bottom dwelling fish, away from coral heads to
avoid reef bound herbivorous fish), Barriers (using Gill nets, cages, barrier nets),
Auditory (Underwater noise makers to frighten fish), Visual (Scare lines or
plastic/wood models of predators), Electrical (Electrical signals emulating predators)
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
and Chemical Compounds (that deter herbivores such as those produced by algae that
are not susceptible to herbivores) Recently, a new farming technique using tubular nets
has been practiced in Brazil (Paula & Pereira, 1998 and Góes & Reis, 2011) to
overcome various problems related to seaweed cultivation.
Epiphytism and Biofouling
Among the common problems associated in Kappaphycus cultivation,
epiphytism is widely known. Epiphytism is a phenomenon where one plant lives on the
surface of another plant. These epiphytes may be either macro- (> 1 mm long) or mesoepiphytes (< 1mm long) depending on their size. Most but not all seaweeds studied in
natural ecosystems are plagued by epiphytes, endophytes and other eukaryotic parasites
that cause no or a few apparent disease symptoms. The surface of healthy macroalgae is
consistently colonized by microbial communities as the macroalgal surface is an
important habitat for microorganisms (Bolinches et al., 1988). Various types of
interactions can be established between the host and the epiphytes and these interactions
can have both positive effects such as providing ecological niches, food and protective
habitat for animals (Pavia et al., 1999; Karez et al., 2000; Viejo & Åberg, 2003 and
Bittick et al., 2010) and the negative effects such as competition with the host for light,
nutrient absorption, reducing the photosynthesis, consequently the growth and
reproduction of the host plants (D’Antonio, 1985; Cebrian et al., 1999 and Honkanen &
Jormalainen, 2005) and favouring the attraction of grazers to the host alga (Bernstein &
Jung, 1979). The damage caused by the epiphyte can be highly variable and is mainly
influenced by the type of anatomical association and the incidence of the epiphyte
(Fletcher, 1995). These epiphytic interactions are more concern when they impact the
mariculture of economically important seaweeds (Friedlander, 1992; Critchley et al.,
2004; Hurtado et al., 2006; Vairappan, 2006 and Gachon et al., 2010). Different types
of host-epiphyte interfaces were studied and their relative abundance and temporal
variability were monitored to unravel the mechanisms of host recognition and host
damage that could explain the loss of crops and the negative effects of epiphytism in
Gracilaria chilensis farming (Leonardi et al., 2006). The host epiphytic associations are
classified into five types based on their anatomical relationships (Hurtado & Critchley,
2006). Infection type 1 include the epiphytes weakly attached to surface of the host and
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not associated with damage of host tissues (Present work Hincksia sp., Enteromorpha
sp., Caulerpa sp., Sargassum sp., Padina sp., Dictyota sp. Lyngbya sp., Hypnea
musciformis, Acanthophora sp.). Infection type 2 includes those epiphytes strongly
attached to surface of the host but not associated with any host tissue damage (Present
work Clavelina sp. Officinalis sp.). Infection type 3 includes all the epiphytes that
penetrate the outer layer of host wall without damaging its cortical cells. Infection type
4 included epiphytes penetrating deep into the host cell wall, disorganizing the cortical
tissue. Infection type 5include epiphytes that penetrated deeply into the cortex, reached
the medullary tissue, and cause destruction of the host’s cells in the area around
infection (Present work Ceramium sp. and Neosiphonia sp.). The last group of epiphyte
invades the tissue of host by growing intercellularly and they are associated with
destructions of both cortical and medullary cells (Ceramium sp., Neosiphonia sp. and
Acrochaetium sp. etc). The first three types are not as harmful as others. They only
entangle on the Kappaphycus thallus and compete for space, nutrients and sunlight. The
last two types of epiphyte are very destructive to Kappaphycus cultivation as they
invade the cortical and medullary layers of the host plant. Different forms of penetration
by epiphytes are also observed in the transverse section of the thalli. Acrochaetium sp.
show endophytic type of association and they penetrate via intracellular gap reaching
the medullary layers of the thalli, which are most destructive to the host plant while
some other epiphytes anchor on their host by penetrating rhizoids. Similar cell wall
digestion by Acrochaete operculata was also described in Chondrus crispus by Correa
& McLachlan, (1994). Epiphytes such as Neosiphonia sp. form haustoria like insertion
in the cortical cells. Their presence weakens the host and increases its susceptibility to
other microbial pathogens (present study). Occurrence of different red filamentous
epiphytes like Neosiphonia sp. and Polysiphonia sp. has also been reported by Largo et
al., (1995a); Hurtado et al., (2006); Vairappan, (2006); Vairappan et al., (2008) and
Borlongon et al., (2011). Seasonality in occurrence of epiphytes on K. alvarezii was
found to be highest during summer season. The prevalence of high temperature, low pH
and salinity during this season coincide with favourable conditions for the epiphytic
growth (Present work). Similar correlative findings were also reported by Vairappan,
(2006); Vairappan et al., (2010) and Hutardo & Critchley, (2006).
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Biofouling is another factor which affects the growth of Kappaphycus by
competing with the host for space, light and nutrients. Biofoulers such as Enteromorpha
intestinalis, Caulerpa scalpelliformis, Hincksia sp., Hypnea musciformis, Acanthophora
specifera, Sargassum myriocystum, Dictyota dichotoma and Padina tetrastromatica
were found to be entangled with Kappaphycus thalli and on bamboo rafts (Present
study). Hurtado et al., (2001) also reported biofoulers such as Enteromorpha, Ulva,
Hypnea, Dictyota, and Hydroclathrus and they compete directly with K. alvarezii for
space on cultivation rope and removal of nutrients and inorganic carbon from the water
column. As a consequence, the shading effect of these fouling seaweeds is detrimental
to K. alvarezii resulting in yield reduction (Kuschel & Buschmann, 1991). Besides all
the above type of biofoulers observed in Kappaphycus cultivation farm, some common
seagrasses such as Thalasiya hembruchi, Cymodacia, Syrincodium isotifolium and
Halophilo ovalis also entangled with cultivated plants only for space, nutrient and light.
Similar seasonal weeds were noted as a serious problem from the beginnings of
Eucheuma sealant cultivation (Parker, 1974) but the problem is still not well studied.
Fletcher, (1995) provided a review on the impact of pest weeds on Gracilaria
cultivation that is relevant to the situation with Eucheuma sealants. Patterns of epiphytic
assemblage are also strongly dependent on the intensity of grazing by various
invertebrates (Lubchenco & Gaines, 1981). Epiphytic bryozoan (Membranipora
membranacea) that infests kelps causing fragmentation and loss of fronds has been
reported in Gulf of Maine and Atlantic coast of Nova Scotia. (Dixon et al., 1981; and
Krumhansl et al., 2011). Neish, (2003) highlighted that if epizoa is prominent in
Kappaphycus cultivation farm it should be removed immediately and be replaced with
new propagules. Different types of fauna from class Ascidia, hydroid and some
common epizoa such as Tunicates and Clavalina sp. showed heavy infestation mostly
on branching point between main axis, primary axis and secondary axis. Sponges,
oyster pearl (Pincatada fucata) and other Mollucs group also get attached to the basal
part of primary and secondary branches of Kappaphycus. Correa & Sanchez, (1996)
reported preferential affection of Endophyton sp. on basal segment of Mazzaella
laminarioides frond. This association indicates the biochemical interactions between the
organisms which are also coupled with chemical substances released by seaweeds when
under attack by some herbivores for defence mechanism. These substances act as
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communicator between wide varieties of species in a common food chain (Toth &
Pavia, 2000 and Toth, 2007). These findings are well supported by recent studies of
Marroig & Reis, 2011 on the occurrence several biofoulers in K. alvarezii cultivation
site in Brazil. Buschmann et al., (1994) reported large profuse settlements of barnacle’s
spp., bivalves and mussel on the thalli and branches of K. alvarezii. Similar
specialization pattern of infection was also reported by Ballantine, (1979) in which most
epiphytes was heaviest on the basal portion of host both in number and abundance of
epiphytic species decreases with proximity to the meristematic region.
Ice-ice disease
The term “ice-ice” was coined by Filipino farmers to describe the dying tissue in
the thallus of Eucheuma which are devoid of pigments that ultimately causes break up off
baranches. Doty, (1978) identified stress of light, temperature, salinity and pH as the
major factors promoting ice-ice disease and drew a correlation between its occurrence and
epiphytism. Uyenco et al., (1981) noted high population of bacteria found on thallus
infected with ice-ice but concluded that they were secondary to the problem. Doty, (1987)
noted that the occurrence of ice-ice was seasonal and was correlated with change in the
monsoon. Largo et al., (1995a, b) showed that certain types of bacteria appeared to be
capable of inducing ice-ice in stressed propagules and noted that several abiotic factors
could generate such symptoms. Mtolera et al., (1996) observed that E. denticulatum
produced volatile halocarbons (VHC) in high-light and low-CO2 environments. Pedersen
et al., (1996) demonstrated that high VHC levels produced diseases in Gracilaria cornea
and a similar phenomenon may occur with Eucheuma seaplants, especially in low water
flow and unmixed boundary layer conditions. Ice-ice can be triggered by stressful
conditions of abiotic factors that may be acting synergistically along with biological
agents such as epiphytes, biofoulers and bacterial pathogens. Andrews, (1979) described
ice-ice as an algal disease which states that ice-ice is caused by both abiotic and biotic
agents causing abnormal, injurious and continuous interference with physiological
activities of the host, resulting in loss of economic value.
During the present study, it was observed that the occurrence of ice-ice disease in
Kappaphycus is highest during April to June i.e. the summer season when the seawater
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temperature was high between 32.3°C-32.5°C as compared to other months. Besides this,
the incidence of epiphytism was also quite rampant. Thus the present study confirms the
observation made by Doty, (1987). Amongst the epiphytes, Ceramium sp., Neosiphonia
sp. and Acrochaetium sp. were found to be dominant in ice-ice infected thalli. The
development of ice-ice disease in Kappaphycus during summer months coupled with low
tides, high light intensity and low water movement led to lower growth rate (present
study). These findings coincide with those of previous studies conducted in Kappaphycus
farms in the Philippines, Indonesia, Malaysia and Tanzania (Uyenco et al., 1981; Trono
& Ohno, 1989; Critchley et al., 2004; Hurtado et al., 2006; Largo, 2006; Vairappan, 2006
and Tisera & Naguit, 2009). Hurtado et al., (2012) attempted to combat ice-ice disease
using Acadian Marine Plant Extract Powder (AMPEP) on the thallus surface.
Improper Management Practice during Cultivation and its remedy
Besides grazing, epiphytism, biofouling and ice-ice disease in Kappaphycus,
other management practices play an important role in success of seaweed cultivation.
Santelices, (1999) highlighted the productivity of an established farm depends on the
management efficiency. Ask & Azanza, (2002) explained role of various factors for
successful mariculture among which farm-ecology, farm-system and maintenance at
cultivation site are primary concern. Msuya et al., (2007) reported several management
problems at Kappaphycus cultivation farm at Tanzania. In the present study loss of
biomass was observed due to negligence in the detection of ice-ice disease and grazing
at the early stage. Damage of rafts due to underlying rock or death of coral bed, damage
of nets from lower side enhanced grazing and exposed the whole plants to direct
sunlight which resulted in bleaching of tips of the thallus during low tide. Drying of the
harvested thalli on the sand led to contamination thus reducing the quality of
Kappaphycus. Hence, in the present study it is being suggested that the following steps
should be implemented for the better management of the farm by the seaweed farmers.
1. Proper crop management knowledge
The information on the biology and production ecology of Kappaphycus,
updated farming techniques and health management (disease) should be aware to
farmers by conducting frequent workshops, training programme etc.
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2. Importance of the site for Raft Culture
Proper site selection is one of the most important steps for seaweed culture. The
selection of suitable site for seaweed culture should be well aware to the seaweed
farmers. The site should be protected from strong tidal and wind generated waves. The
water should be clear to allow light penetration and effective photosynthesis for better
production.
3. Maintenance of seaweed culture Raft
Maintenance of seaweed culture raft in the right spot assumes great significance
for better growth and yield. The seaweed farmers should be aware of management
practices to be followed during the culture period. The growing seaweeds should be
monitored regularly during the culture period of 45 days. The other maintenance
activity includes checking of culture ropes tied in the raft, removal of unwanted algae
and other fouling organisms. Prevention of grazing by fishes should be maintained
regularly. The seaweeds were gently washed when found with silt deposits on their
surface to ensure proper photosynthesis.
4. Harvesting of Seaweed
Good quality seed materials i.e. well branched propagules with numerous tips
should be selected for replanting from the harvested seaweed. Harvested seaweed
should be rinsed several times in the sea to remove sediments or any attached foreign
particles. Seaweeds should be dried 2 – 3 days in the sun on a raised platform. Turning
the seaweeds up and down frequently will accelerate drying.
Anatomical, Histochemical and Ultrastructural Studies
The thallus of Kappaphycus alvarezii is pseudoparenchymatous and is
differentiated into a single layered epidermis covered by a thick extracellular mucilage
layer, 10-12 layered cortex and central medulla of small, filamentous cells. Cortex is
again divided into 3-4 layered cortical cells (outer cortex) of smaller size and 7-8 layered
sub-cortical cells (inner cortex) which are of larger size. Rao & Rao, (1999) differentiated
the thallus of K. cottonii collected from Andaman and Nicobar Island only into outer
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cortex of pigmented cells, inner cortex of large pseudoparenchymatous cells and a central
medulla of small cells. In the present study it was found that the epidermal cells are
elongated and the cortical cells are small and gorged with floridean starch grains and
proteins whereas the sub-cortical cells are large and showed peripheral distribution of
floridean starch grains and proteins. Tissue differentiation observed in the present study
represents a highly specialized division of labour and adaptation of plant to various stress
conditions. Nashier, 1996 also reported in Soliera robusta, that the differentiation of
thallus into epidermis, cortex and medulla along with their associated floridean starch
grains and cytoplasmic proteins is a remarkable adaptation which confers on the plant
both ecological and physiological survival strategies to withstand tidal fluctuations.
In the present study the cell wall and the intercellular region of K. alvarezii
reacted positively with Toluidine Blue (TBO) because of the presence of sulphated
polysaccharides. Schmidt et al., (2009) and Paula & Pereira, (1998) also observed
positive reaction with TBO in the cell walls of the K. alvarezii. The presence of
sulphated polysaccharides in the cell wall was also seen in many other macroalgae such
as Hypnea musciformis (Ramarao, 1970; Saito & Oliviera, 1990 and Bouzon, 2006),
Soliera robusta (Nashier, 1996), Gracilaria verrucosa and Gracilariopsis megaspore
(Sahu, 2004), Grateloupia filicina (Baweja, 2001). In the present study the occurrence
of metachromatic reaction in the cell walls of cortical and sub cortical cells are also
supported by the study in other macroalgae, Chondrus crispus (Gordon & McCandless,
1973), Chondria tenuissima (Tsekos et al., 1985) and Hypnea Musciformis (Bouzon,
2006). This metachromatic reaction in the cell walls is the indicator of the presence of
acidic polysaccharides of carboxyl and sulphate groups (McCully, 1968). Many starch
grains are also observed in the cortical and subcortical cells as an indication of
occurrence of photosynthesis in these cells (Schmidt et al., 2009). Talarico et al., (1990)
concluded that the fibrillar component together with matrix polysaccharides in the
intercellular space are involved in regulating mechanical and osmotic factors related to
environmental stress. The sulphated polysaccharides are mostly hot water soluble
polymers that form amorphous materials. This soluble polymer consists of galactose or
modified galactose units (Percivel, 1978). The presence of polysaccharides in the cell
walls of a number of brown algae has been demonstrated by differential staining
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techniques (McCully, 1966) and by X-Ray diffraction (Frei & Preston, 1962). This
sulphated polysaccharides are found only in marine algae and are absent in higher
plants. Thus, the present study concludes that the intercellular matrix is responsible for
the strength and flexibility of the thallus which protects against damage, water loss and
environmental stress conditions. In K. alvarezii the cell walls stained intensely with
Periodic Acid-Schiff (PAS) as compared to TBO and CBB indicating the presence of
high amount of insoluble polysaccharides (present study). Schmidt et al., 2009 also
reported similar type of PAS positive reaction in the cell wall of K. alvarezii. These
insoluble polysaccharides form structural polymers that are fibrillar in nature.
In the present study proteins are mainly concentrated in the cytoplasm of both
cortical and sub-cortical cells only. The cortical cells are metabolically more active than
the other cells in K. alvarezii which was observed by the strong positive reaction with
Coomassie Brilliant Blue (CBB). The same observation is also found by Schmidt et al.,
(2009) in K. alvarezii histochemical study. Pit-connections stained with CBB
suggesting the proteinaceous nature of pit connections. It is assumed that during the
final stage of cell division, a pore remains between the two daughter cells and this pore
is closed by the deposition of large amount of proteinaceous substances which is later
called as pit plug (Pueschel, 1980). Pit connection study using CBB was also done in
Griffithsia pacifica (Ramus, 1971), Gigartina teedii (Tsekos, 1983) and in Hypnea
musciformis (Bouzon, 2006).
Scanning Electron Microscopy
The thalli when observed under scanning electron microscope showed the
cluster of tightly packed epidermal cells without any breakage. The cortical cells were
elongated and compactly arranged one after the other, while the subcortical cells next to
cortical cell are spherical in shape and filled with granules. The cell layers of infected
Kappaphycus thalli were found to be disintegrated and many epiphytes were attached
on the surface of the thalli. Different ways of penetration from the surface towards the
cells was observed in the present study. Similar way of insertion was also reported by
Heesch & Peters, (1999) in Laminaria saccharina. The presence of goose bump like
feature was reported by Critchley et al., (2004) and Hurtado et al., (2006) in
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Polysiphonia and Neosiphonia savatieri infected thalli. SEM observation revealed the
emergence of 2-4 branches of epiphytes from one location in the present work which is
also reported by Vairappan, (2006). Presence of slight depression or cracks for the entry
of epiphytes weakens the host plants, making them vulnerable to thallus breakage and
bacterial attack (present work).
Transmission Electron Microscopy
At the ultrastructural level, cell wall showed arrangement of microfibrils in
concentric layers with different degree of compression (present study). The production
of fibrillar components in the cell wall is similar in many red algae and seems to be
formed by major fibrous vacuoles, endoplasmic reticulum and later by the addition of
vesicles derived from Golgi bodies (Bouzon, 2006). The nature and arrangement of cell
wall was also reported in Kappaphycus by Schmidt et al., (2009, 2010) and in other
seaweeds by Bouzon et al., (2011) in Hypnea musciformis, Schmidt et al., (2010) in
Chondracanthus teedii and Santos et al., (2012) in Gracilaria domingensis. But in case
of infected thalli the compressed nature of microfibrillar layer is decreased and layers
are fragmented in some region (present study). The chloroplasts in healthy K. alvarezii
thalli are larger, elongated and the thylakoids are flat, unstacked, evenly placed in the
cells (present study) which are typical structure of red algae. Floridean starch grains are
scattered in the cytoplasm. In case of infected thalli the chloroplasts showed structural
changes including modification in the size and organization of the thylakoids. The
thylakoids are disrupted and the number of plastoglobuli increased in the chloroplasts
(present study). According to Holzinger et al., (2009) when algae are subjected to any
form of stress, the synthesis of protein-containing cell are suppressed and the synthesis
of lipids starts which led to increase in plastoglobuli in infected or stressed thalli. This
increase in amount of lipid can be considered as a change in metabolism which in turn
results in the reduction of cell proliferation and decrease in growth rate. Similar findings
were also reported in red macroalgae by Schmidt et al., (2009, 2010) in K. alvarezii,
Holzinger et al., (2004) in Palmaria palmata and Odonthalia dentate and Steinhoff et
al., (2008) in brown macroalgae Laminaria hyperborean. When the cells are heavily
infected the protective mechanism of the cells failed to perform its functions leading to
imbalance in the cellular mechanisms. These imbalance lead to conformational changes
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in the biochemical functions of the cell finally leading to decrease growth and death of
the plant (Bowler et al., 1992 and Schmidt et al., 2009).
The pit-connection is an important feature of red algae except a few members of
Bangiales. The cells are interconnected through distinct pit-connections. In the present
work medullary cells are connected by pit-connections. The size of the pit-connections
varies depending on the cells, with the wider cell having larger pit-connections. The pit-
connection is electron dense at the periphery and electron translucent at the centre
(present work). This finding correlates with the work of Pueschel, (1977) and Bouck,
(1962) which described pit-connection as a membrane-bound plug-like structure across
the wall and has a dense peripheral zone and a lighter central region. Li et al., (2006)
described the role of pit connection as nuclear acid transfer and signal transduction
between two adjacent cells. Duckett et al., (1974) stated that the pit connections are
mechanical structures which anchor the cytoplasm in place and are a means of
increasing the strength of the thallus by providing a network of cross bridges between
cells which are otherwise only loosely interwoven.
Nutrient Analysis
Marine algae exhibit high content of ash mainly due to the presence of sodium,
potassium, calcium and magnesium cation (De Boer, 1981). The ash content in most land
vegetables is usually much lower than in seaweeds with an average value of 5-10
gm/100gm dry weight (USDA, 2001). In the present study ash content ranges between 2735% in all the thalli (healthy, fish grazed and ice-ice infected). The present finding also
correlates with the earlier report of Kappaphycus in Indian coast by Bindu, (2011) and Sahu
et al., (2011) where the ash content was 27% and 21.2% respectively. While lower ash
content of 19.7% in the same plant form different site was reported by Fayaz et al., (2005).
In contrast higher ash content were found in Eucheuma sp. by Matanjun et al., (2009) in
Malaysia (46.19%), McDermid & Stuercke, (2003) in Hawaii (43.6%) and Renaud & Van,
(2006) in Australia (43.8%). Freile-Pelegrin & Robledo, (2008) also reported 52.6% of ash
content in E. isoforme in Mexican coast. McDermid & Stuercke, (2003) reported the ash
value in other red seaweeds such as 25% in Porphyra vietnamensis, 53% in Gracilaria
coronopifolia, 48% in G. parvispora and 52% in G. salicormia.
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Carbohydrate constitutes the most important component of the seaweeds. In
most seaweed species, the major component was soluble carbohydrate (Chlorophyta
range 2.5-25.8%, Phaeophyta range 8.4-22.2% and Rhodophyta range 18.7-39.2%). In
the present study the total soluble carbohydrate content showed a lot of variation in both
healthy and infected plants of K. alvarezii. The maximum carbohydrate content in 45
days old healthy thalli is 42.3% but in infected thalli the value decreased to 30.3%
(present study). Fayaz et al., (2005) and reported lower carbohydrate content of 27.4%
in K. alvarezii. In contrast lower carbohydrate content were also found in Eucheuma sp.
by Matanjun et al., (2009) in Malaysia (26.49%), McDermid & Stuercke, (2003) in
Hawaii (28.0 %) and Renaud & Van, (2006) in Australia (30.6%). Freile-Pelegrin &
Robledo, (2008) also reported 34.5-53.8% of carbohydrate in E. isoforme in Mexican
coast. Similar range of carbohydrate contents (47.42%) and (44.5%) were also reported
by Kumar et al., (2011) and Sahu et al., (2011) in K. alvarezii respectively.
Protein content ranges between 10.13-11.38% in all the healthy thalli while in
case of fish grazed and ice-ice infected thalli the protein content varied between 5.13-
7.58% (present study). These findings were consistent with other studies that reported
similar ranges of protein by Matanjun et al., (2009) (9.76%) in Malaysia and Freile-
Pelegrin & Robledo, (2008) (6.3-13.3%) in Mexico. The present protein values are
comparatively higher than the values reported by McDermid & Stuercke (2003) (4.9%)
and Renaud &Van, (2006) (5.0%). Simultaneously Kumar et al., (2011) and Fayaz et
al., (2005) reported the protein content of 14.84% and 16.24% respectively in K.
alvarezii. The biochemical compositions (ash, carbohydrate, protein) in all the infected
thalli were found to be lower because the biochemical and physiological activities of the
thalli were ceased due to heavy infestation. Similarly variations in ash, carbohydrate
and protein content can be explained by the seasonal or geographical changes,
occurrence of several varieties in a given species. Another reason may be due to the
effects of various environmental factors.
Seaweeds are usually rich source of various types of minerals such as sodium,
potassium, calcium, magnesium, iron, copper etc. Mineral contents in seaweeds are
generally very high ranging between 8% - 40%. They are very important for various
biochemical activities in the body as a co-factor of enzyme (Ensminger et al., 1995).
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Some edible seaweed contain significant amount of carbohydrate, protein, lipid, vitamin
and minerals (Fayaz et al., 2005). This wide range of mineral contents in seaweeds is
generally not found in edible terrestrial plants. This is related to factors such as seaweed
habitat, maturity, geographical origin, seasonal, environmental and physiological
variations (Lobban et al., 1985; Jensen, 1993; Mabeau & Fleurence, 1993; Dawes, 1998
and Rupérez, 2002).
Amongst the macronutrients present in healthy thalli, sodium content was found
to be maximum followed by potassium and calcium, whereas magnesium was in least
(present work). The present value of both macronutrient and micronutrient fall within
the range of earlier report in Kappaphycus by Kumar et al., (2011) and in other red
seaweeds by Rupérez, (2002). The Na/K ratio is below 2 in the present work which is
good from the nutritional point of view since the diet with high Na/K ratio are related
to the incidence of hypertension. This observation is supported by previous studies
where the Na/K ratio was found to be below 1.5-2 (Rupérez, 2002; Matanjun et al.,
2009 and Rajasulochana et al., 2010). But the present observation is contradictory from
the study of Santoso et al., (2006) in Indonesia where the maximum macronutrient in K.
alvarezii is potassium followed by sodium, calcium whereas Rajasulochana et al.,
(2010) reported calcium as the highest macronutrient in Kappaphycus sp. This variation
in the mineral composition among the seaweeds and from one place to another may be
due to different geographical origin, variation in seasonal, environmental, physiological
conditions and type of processing. Arasaki & Arasaki, (1983) reported values ranging
from 0.9-7.0 percent for sodium, 0.73-6.3 percent for potassium and 1.1-3.0 percent for
calcium in other edible seaweeds such as Undaria pinnatifida and Ulva sp. Amongst the
micronutrient, Iron is maximum followed by zinc and copper. Santoso et al., (2006)
reported Iron as the maximum content followed by zinc and copper in K. alvarezii. The
highest iron content in the present study also correlates with earlier reports of Fayaz et
al., (2005) and Rajasulochana et al., (2010) in Kappaphycus sp. This significant amount
of iron in K. alvarezii may be due to its metabolic system in which it is capable of
directly absorbing elements from the sea water. The overall mineral content (both
macro and micronutrients) in the present study is very low in infected thalli compared
to the healthy thalli. During stress conditions the thalli are prone to infection and the
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production of hydrogen peroxide (H2O2) and volatile hydrocarbons increases leading to
biochemical and physiological damage of the cells (Fridovich, 1986; Keppler &
Novacky, 1987; Devlin & Geutine, 1992; Collén et al., 1995; Mtolera et al., 1995a, b
and Imsande, 1999). Once the infection starts, the mineral content decreases and the
plant defense mechanism such as enzyme superoxide dismutase reduce their activity
because the functioning of this enzyme depends on the mineral content of the plant
(Lidon & Teixeira, 2000). Mtolera, (2003) also reported that the infected K. alvarezii
thalli by epiphytes in Zanzibar reduce the metal content by 35-99%.
Carrageenan Analysis
Carrageenans are sulfated galactans composed of D-galactose residues linked
alternately in α-1, 3 and β-1, 4 bonds. They are classified as kappa, iota and lambda
according to their sulfate substitution pattern and 3, 6-anhydrogalactose content (Freile-
Pelegrín et al., 2006). The hydrocolloid extracted from Kappaphycus is almost pure
Kappa carrageenan with minimal amount of Iota, while the Eucheuma extract is pure
iota carrageenan. kappa and iota carrageenan are gel forming whereas lambda
carrageenan do not form gel and are used as thickeners (Prado-Fernández et al., 2003
and Tojo & Prado, 2003).
Chemically, Kappa-carrageenan consists of disaccharide-repeating units of 3-
linked β-D-galactopyranosyl-4-sulfate and 4-linked 3, 6-anhydro-α-D-galactopyranosyl
residues, while Iota-carrageenan consists of disaccharide repeating units of 3-linked βD-galactopyranosyl-4-sulfate and 4-linked 3, 6-anhydro-α-D-galactopyranosyl-2-sulfate
residues. The carrageenan yield and quality depends on several factors such as the algal
species (Craigie, 1990), seasonal fluctuations (Dawes et al., 1977 and Yakovleva et al.,
2001) and extraction method (Dawes et al., 1977, McCandless et al., 1977, Hoffmann
et al., 1995 and Piculell, 1995). In the present work Kappaphycus showed positive
methylene blue test, milk reactivity test, solubility in KCl solution and water, strongly
supports that the phycocolloid contain is carrageenan only. This observation is also
supported by the histochemical studies of Kappaphycus where the cells show positive
staining with TBO and PAS indicating the presence of sulphated polysaccharides
(present study). The chemical nature and its composition of carrageenan are further
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confirmed by Fourier Transformed Infrared Spectroscopy (FTIR) and High Pressure
Liquid Chromatography (HPLC).
FTIR is a technique which is based on the analysis of absorption peaks at certain
wave numbers (expressed in cm-1). It requires minute amount of sample (milligrams)
and it is non-aggressive method with reliable accuracy and is not a time consuming
method thus avoiding lengthy sample extraction (Chopin & Whalen, 1993; Pereira et
al., 2003; Pereira & Mesquita, 2004 and Pereira, 2006). In the present work a broad
band of spectra at 1200-1264 cm-1 was observed in carrageenan extracted from both the
healthy and infected Kappaphycus thalli indicating the presence of sulphate esters
group. Similar range of broad band spectra (1210-1270 cm-1) were also reported by
Villanueva & Montaño, (2003); Pereira et al., (2009) 1240-1260 cm-1 and Ordóñez &
Rupérez, (2011) 1210-1260 cm-1. Strong bands both at 845 cm-1 and at 930 cm-1
indicating both D-galactose-4-sulphates and 3, 6-anhydro-D-galactose respectively
observed in the present work is coupled with the work of Villanueva & Montaño,
(2003), Pereira & Mesquita, (2004), Campo et al., (2009), Pereira et al., (2009) and
Ordóñez & Rupérez, (2011). These strong bands (845 cm-1 and 930 cm-1) indicate that
the hydrocolloid extract is kappa carrageenan, but the intensity of these bands is
reduced in the carrageenan extracted from fish grazed and ice-ice infected thalli (present
work). This decrease in the intensity of spectra is also supported by the histochemical
studies where the transverse section of the infected thalli shows degradation of the cell
layers (present work). When the plant is either infested by grazing or by epiphytes, the
cell walls are degraded first leading to decrease in the carrageenan content thus
lowering the intensity of the spectra in FTIR analysis of infected thallus. Another slight
shoulder at 805 cm-1 which indicates the presence of C2-sulphated 3, 6-anhydro-D-
galactose was found (present study). This band is the characteristic feature of Iota
carrageenan and the intensity of this band is very low. They are almost absent in the
commercial carrageenan and in the carrageenan extracted from the infected thalli of
Kappaphycus. The occurrence of iota carrageenan at 805 cm-1 indicates that the
carrageenan produced from seaweed is not pure. Similar observation was also reported
in previous studies (Bellion et al., 1983; Rochas et al., 1989; Santos, 1989; Chopin &
Whalen, 1993; Van De Velde et al., 2001; Mendoza et al., 2002; Aguilan et al., 2003;
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Pereira & Mesquita, 2003; Villanueva & Montaño, 2003; Villanueva et al., 2004,
Pereira et al., 2009 and Ordóñez & Rupérez, 2011).
Another method for the analysis of the presence of the type of sugar in the plant
is High Pressure Liquid Chromatography (HPLC). In the present study D-galactose is
the main sugar observed in Kappaphycus indicating that the hydrocolloid content is
carrageenan. The presence of D-galactose is confirmed by the retention time which
ranges between 5.035-5.089 min in all the tested samples. In the present study
carrageenan yield is maximum in 45 days old thalli and minimum in 120 days old thalli
which are also supported by the quantification of D-galactose by HPLC where the
highest concentration was in 45 days old thalli and least in 120 days old thalli. Similar
range of carrageenan yield were also found in the earlier studies of Kappaphycus
(Mendoza et al., 2002; Hayashi et al., 2007 b; Hurtado et al., 2008 and Góes & Reis,
2012). The decreased carrageenan content quantitively within the healthy thalli
coincided with a decreasing growth rate as the duration of culture progressed (present
study). This indicates that as the duration of cultivation extended from 45 days, the
growth rate decreased in coordination with a decrease in carrageenan content. It was
also reported earlier by Mendoza et al., (2002) in Kappaphycus and in other red
seaweeds such as Gelidium pristoides (Carter & Anderson, 1986) and Gracilaria
chilensis (Hemmingson & Furneaux, 1997) that younger thalli or tissues may indeed
have a higher yield of the galactans than the fully matured thalli. Therefore, it is
suggested that even if the growth rate of Kappaphycus reaches maximum at 30 days of
culture, harvesting is ideal at the onset of stationary phase (45 days of culture) as
minimal increment in biomass is expected beyond this point. In case of infected thalli
carrageenan yield was found to be very low in ice-ice infected thalli than the fish grazed
thalli (present study). Since bacterial attack is the nature of ice-ice infection (Largo et
al., 1995a), the decrease in the yield of carrageenan is due to depolymerization of the
seaweed polysaccharide in the carrageenolytic activity of enzymes secreted by the
bacteria. Another possibility is that the bacterial metabolic by-products may also be
responsible for the hydrolysis of carrageenan polymers leading to decrease in
carrageenan yield (Mendoza et al., 2002).
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Other qualities of carrageenan are determined by viscosity and gel strength.
Viscosity ranges from 2-10 cps while the gel strength ranged from 22.5-135 g/cm2 in all
the healthy and infected thalli. In contrast to carrageenan yield viscosity and gel
strength increased with increased in culture period upto 90 days and decreased after 90
days of culture (present work). As the seaweed thalli become more mature with age in
culture, higher molecular weight structural polysaccharides build up producing stronger
gels (Mendoza et al., 2002). The gel strength however decreased at latter stage of the
culture which coincides with the decrease in biomass due to fragmentation of the thalli
and herbivory which leads to infection of the thallus (present study). When the thalli get
infected carrageenan degradation occurred due to the naturally occurring carrageenan
degrading marine bacteria (Sarwar et al., 1983 and Villanueva et al., 2011). Thalli
fragmentation leading to diminished molecular weight and reduced gel strength may be
the signs of senescence in cultured seaweeds (Villanueva et al., 2011). This may be
another reason where harvesting is usually done at 30-45 days of culture even if the gel
strength is very high as the culture period increased.
But in case of ice-ice infected thalli the viscosity is found to be only 4 cps which
is supported by the low yield and low gel strength (present study). The present finding
also coincides with the previous study by Mendoza et al., (2002 and 2006). This severe
loss in carrageenan yield and quality in infected thalli may be due to the loss of
structural integrity which leads to fragmentation of the thalli. The short depolymerized
chain could not form sufficient intra and inter strand hydrogen bonds required to form
stable helical aggregates leading to easy penetration of the gel strength measuring probe
(Rey & Labuza, 1981). But the present observation of viscosity and gel strength is
found to be low when compared to the earlier work of Góes & Reis, (2012) and
Villanueva et al., (2011) in Kappaphycus. This variation may be due to the differences
in their geographical position of the cultivation site.
In Vitro Culture Studies of Endophytes and ice-ice Disease
Laboratory culture of economically important seaweeds will allow development
of outdoor cultivation programme (Polne et al., 1980). In fact, problems such as
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decreasing productivity and vigour of the stocks, seasonality in production, low quality
of product, susceptibility of various strains of Kappaphycus to ice-ice and other diseases
in various cultivation farms can be attributed to a variety of biological and economic
factors (Trono & Ohno, 1989). These problems are believed to result from the
vegetative propagation of a single clone (Paula et al., 1999). Traditionally hybridisation
methods have been suggested as the logical solution to produce new strains while the in
vitro work is limited due to large size of thalli that requires large volumes of water and
adequate facilities (Doty, 1987; Azanza-Corrales, 1990 and Trono, 1994).
Ice-ice disease can also be induced in the laboratory if the thalli are kept in the
environment which is favourable for the disease. In vitro culture of the untreated
explants with disinfectant series showed rigorous growth of endophytes Acrochaetium
sp. within two weeks which finally leads to ice-ice disease symptoms such as whitening
of the thalli, while other explants treated with various disinfectants show regeneration
of branchlets (Present work). Similarly, Dawes & Koch, (1991) reported that limited
tolerances to low temperature and salinity and low acclimation rate in Eucheuma
isiforme culture and reduction of bacterial contamination was accomplished by dipping
the explants in antibiotic solution respectively. Largo et al., (1995a) also confirmed that
ice-ice could be triggered artificially by sub-optimal levels of temperature, light
intensity and salinity on K. alvarezii. But the level of ice-ice infection is more in the
natural seawater than in the laboratory. In the natural field the incidence of different
epiphytes and many herbivores could also be another reason leading to ice-ice disease
whereas such conditions are absent in laboratory. In case of explants treated with
various disinfectants series, complete regeneration of branchlets up to 2 cm was
observed during 90 days of culture without any infection (present study). The present
observation is also correlated with the earlier work of Dawes et al., (1994) where they
studied laboratory culture of both Eucheuma denticulatum and K. alvarezii and
observed a complete regeneration of branchlets upto 1-2 cm during four months culture
period. Thus, laboratory culture study will helps in maintenance of seed stocks for
mariculture and to determine acclimation ability of K. alvarezii for manipulation and
selection of desirable strains.
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Impact of grazing, epiphytism and lower pH on biomass production of Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
Cultivation of Kappaphycus alvarezii under high CO2 and low pH
Increase in atmospheric CO2 has become a major concern. Due to rapid increase
of atmospheric CO2 concentration since the industrial revolution, the ocean is absorbing
greater amount of CO2 at increasingly rapid rates. As a result the ocean becomes more
acidic in nature which is known as “Ocean acidification”. Ocean acidification is a
reduction in seawater pH throughout the world’s oceans due to the absorption of
anthropogenically produced atmospheric CO2 (Sabine et al., 2004). Average surface
oceanic pH has dropped by 0.09 units since the industrial revolution and depending on
future CO2 emission levels, seawater pH may decrease by another 0.3–0.7 units by 2300
(Caldeira & Wickett, 2003). Recently, there has been a good deal of interest in the
potential of marine vegetation as a sink for anthropogenic Carbon emissions (Blue
Carbon - Nellemann et al., 2009 and Chung et al., 2011). Seaweeds act as a major
scrubber for the CO2 removal from ocean as it can easily remove the excess CO2 from
the sea through photosynthesis. These predicted changes in pH and the seawater
carbonate system may have negative impacts on coral reef ecosystems (HoeghGuldberg et al., 2007). A wide range of marine invertebrates are expected to be severely
affected by ocean acidification due to the dissolution of their calcite skeleton with a
decrease in seawater pH (Anthony et al., 2011). Ocean acidification can potentially
affect all marine primary producers, phytoplanktons and seaweeds (Hinga, 2002; Hurd
et al., 2009, 2011; Hepburn et al., 2011 and Cornwall et al., 2012) because their growth
and productivity are dependent on the supply of inorganic carbon for photosynthesis
(Beardall et al., 1998).
Seaweeds play an important role in the coastal carbon cycle (Reiskind et al.,
1989) and contribute remarkably to sea-farming activities. The rate of primary
production of some species is comparable with those of the most productive land plants,
therefore seaweeds have a great potential for CO2 bioremediation (Gao & McKinley,
1994 and Chung et al., 2011). The present work is intended to examine how macroalgal
species K. alvarezii responds and acclimates to lower pH levels in response to ocean
acidification. The seawater pH in the present study were lowered either by using acid
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(in field) or by increasing CO2 concentration (in laboratory). It was observed that
explants at pH 7.9 showed rapid morphological changes in terms of susceptibility,
acclimatization, and depigmentation while the explants at pH 8.1 and 8.2 lowered by
acid showed considerably slow process of depigmentation but they acclimatized with
the ambient pH level showing slow growth rate. On the other hand the explants at pH
8.1 and 8.2 altered using enriched CO2 showed better response to the medium in terms
of regeneration rate of branchlets as compared to explants grown at pH 8.3 (control).
Similar findings were reported in other seaweeds like Gracilaria sp., Gracilaria
chilensis and Hizikia fusiforme (Gao et al., 1993 and Zou, 2005) respectively. Juveniles
of Porphyra yezoensis germinated from the chonchospores when grown at enriched
CO2 gas showed significantly enhanced regeneration (Gao et al., 1991). On the other
hand, a decrease in growth rate caused by elevated CO2 has been also reported in
Gracilaria tenuistipitata (Garcìa-Sánchez et al., 1994), Porphyra leucostica (Mercado
et al., 1999) and Porphyra linearis (Israel et al., 1999). Such an inhibition of growth is
associated with lower photosynthetic activity at high CO2 concentrations (Garcìa-
Sánchez et al., 1994) and acidification of the medium (Israel et al., 1999). Lower pH
(using acid) caused a significant reduction in regeneration whereas bubbling enriched
CO2 had the opposite effect, indicating that increased CO2 at lower pH ameliorates
physiological stress condition (Roleda et al., 2012). The H+ ion effect observed in case
of lower pH by acid supports that of Sorokin, (1964) who showed the inhibition of cell
division in Chlorella sp. in acidified medium which illustrates that H+ and dissolved
inorganic carbon (DIC) have separate effects on algal physiology. However, higher
photosynthetic rate at a critical high CO2 concentration may not be directly translated to
a higher growth rate. Protective mechanisms to minimize H+ ion effects on cellular
metabolism may operate at the expense of growth (Roleda et al., 2007).
The present finding concludes that elevated CO2 upto certain level may provide
advantages to the growth of macroalgae. Macroalgae have the capability of reducing the
CO2 level in the atmosphere thereby reducing global warming. Macroalgae have great
potential for biomass production and CO2 bioremediation. They have high productivity
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and do not compete with terrestrial crops for farm land. Biomass from macroalgae
promises to provide environmentally and economically feasible alternatives to fossil
fuels. Nevertheless the techniques and technologies for growing macroalgae on a large
scale and for converting feedstocks to energy carriers must be more fully developed for
the potential to be realized. More research efforts on biochemical and physiological
aspects for a wider range of species grown at high CO2/low pH conditions are needed to
further evaluate the impacts of increasing atmospheric CO2 concentrations on seaweeds.
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