Carrageenan Content in Three Species of Hypnea (H. musciformis Wulfen J. V. Lamouroux, H. pannosa J. Agardh and H. valentiae Turner Montagne) of Karachi Coast

Seaweeds are marine algae commonly found in marine environment. The red seaweed (Rhodophyta) is commonly exploited for the production of polysaccharides. Carrageenan is one of the commercial polysaccharide. It is complex of D-galactose 3, 6-anhydro D-galactose and monoesterified sulphuric acid found in cell wall of red seaweeds, used as an emulsifier, a binder. It is also used for suspension and stabilization in pharmaceutical, cosmetic and food processing. Hypnea is a red algal genus and well known carrageenan producing marine plant. Therefore three species of Hypnea: H. musciformis, H. pannosa and H. valentiae were collected from the four different coastal areas (Buleji, Hawks Bay, Manora and Paradise Point) of Karachi coast for the extraction of carrageenan in present study. The highest carrageenan extracted from H. valentiae (18-46.93%) as compared with the other two studied species. The high carrageenan concentration was mostly found in winter season. The total biomass 6846.79 g/m2 of three species of Hypnea was recorded during the study period and the highest biomass was recorded in January and the lowest in August. The highest biomass was obtained from Manora coast as compared to other three coastal areas. There was significant correlation was found in between carrageenan and biomass. From the present investigation it is concluded that studied seaweeds species of Hypnea have good quantity of carrageenan and can be used in industries for the manufacturing and production of carrageenan.


Background
Rhodophyta is the division name for red algae. Most of the world seaweeds, over 10,000 species belong to this group. The cell wall of red algae contains polygalactose esters and cellulose, some time impregnated with calcium carbonate. There are several red species of seaweeds have great demand due to their products as food items and hydrocolloids carrageenan and agar used in manufacturing of industrial products. The family Cystocloniaceae of order Gigartinales has the number of carrageenan producing species.
Carrageenan are a collective family of linear, sulfated galactans found in a number of commercially important species of red seaweeds (Sangha et al., 2011). Carrageenan is commonly used as a thickening, suspending, gelling and stabilizing agents for food products and other industrial applications as gelatin (Renn, 1997). It differs from agar mainly in its higher sulfated fraction and higher ash content (Ramalingam et al., 2003). Carrageenophytes were commercially grown in Philippines significantly for last four decades (Anicia et al., 2012). Pereira et al. (2003) have studied eight species of carrageenophytes collected from west Portuguese coast. Mtolera and Buriyo (2004) studied seasonal variation in content and quality of kappa-carrageenan of Hypnea musciformis from Western Indian Ocean. Seaweeds are still leading producer of carrageenan globally (Anicia et al., 2012). Hoffmann et al. (1995) studied the effect of isolation procedures on the molecular composition and physical properties of Eucheuma cottonii carrageenan.
The studies related to biomass have also been conducted in develop countries such as, Central Puget Sound Washington, USA (Thom, 1980), Dodger channel of Canada (Whyte, 1981) and North Carolina (Peckol, 1982). Chock and Mathieson (1983) quantified the standing crop and biomass variation of estuarine seaweed at Cedar point, Dover New Hampshire Maine, UK whereas Jagtap (1992) described the marine flora of Nicobar group of

Materials and Methods
The monthly fresh samples of Hypnea species (H. musciformis, H. pannosa and H. valentiae) were collected from four exposed shores of Karachi coast Buleji, Hawks Bay, Manora and Paradise Point at low tide by the quadrate method (Chapman, 1964). All three species were placed individually in prelabeled plastic bags and brought to the laboratory where all plants were carefully cleaned from mud debris and other epiphytes with filtered seawater.
The kappa carrageenan was extracted according to a method adopted from Vandermeulen (1988). The gelation temperature, melting point, relative density, viscosity and strength determined by the method of Whyte et al. (1981). Temperature of seawater was read using a mercury thermometer. Water samples of pH were measured with the digital pH meter (Model PM-65) and salinity was measured by hand-hold Refractometer.

Results and Discussion
During the study period the total one hundred four samples (104) were collected from four sites: Buleji (31 samples), Hawks Bay (19 samples), Manora (29 samples) and Paradise Point (25 samples). Total 39 numbers of individuals of Hypnea musciformis, 28 H. pannosa and 37 H. valentiae were recorded in the whole study period. The present data reveals high variability in the content of carrageenan in between all three species, sampling sites and collection time (Figure 1; Figure 2; Figure 3). The concentration of carrageenan was mostly decreasing from January to August and then again increased till December (Figure 1). The high carrageenan concentration was mostly found in winter season. The highest content of carrageenan was obtained from Buleji samples as compared to other three coasts samples ( Figure 2). The highest carrageenan extracted from H. valentiae (18-46.93%) as compared to other two species H. musciformis (18-31%) and H. pannosa (20-30.4%) (Figure 3).
The total biomass 6846.79 g/m 2 was recorded during the study period and the highest biomass was recorded in January (919.38g/m 2 ) and the lowest (97.8 g/m 2 ) recorded in August (Figure 4). The highest biomass was obtained from Manora coast as compared to other three coastal areas ( Figure 5). The highest contribution to the total biomass was observed by H. musciformis 37.63% as compared to H. pannosa (34.87%) and H. valentiae (27.49%) ( Figure 6). The range of carrageenan content in Hypnea musciformis was 18-31% with mean concentration value of 23.72%. In H. musciformis Carrageenan concentration varied throughout the years. At Buleji it was high (31%) in May and August whereas at Hawks Bay and Paradise Point in February (28% and 24% respectively) and at Manora (29.7%) in May (Figure 7). In H. musciformis the range of biomass value were 20-120 g/m 2 with mean value of 63.45 g/m 2 for the four sites Buleji, Hawks Bay, Manora and Paradise Point (Figure 8). At Buleji biomass was high (112 g/m 2 ) in May whereas at Hawks Bay and Manora in January (58 g/m 2 and 120 g/m 2 respectively) and at Manora (91.32 g/m 2 ) in November ( Figure 8). The extract of carrageenan in H. musciformis had density in a range of 0.95-1.34 g/cm 3 in all sites samples. In Buleji samples it was high in May (1.34 g/cm 3 ) and in Hawks Bay and Manora samples it was high in December (1.2 g/cm 3 ) whereas in Paradise Point samples it was high (1.1 g/cm 3 ) in November (Table 1). The gel viscosity of H. musciformis carrageenan extract was in the range of 7-18 cP. In Buleji samples viscosity was high in January and September (16.57 cP and 16.0 cP respectively) and in Hawks Bay samples it was high (18 cP ) in March. Whereas in Manora samples viscosity was high in January and September (17 cP) and in Paradise Point samples it was high (18 cP) in December ( Table 2). The gel boiling point of H. musciformis carrageenan was in the range of 30-56.5°C. In Buleji, Hawks Bay and Manora samples boiling point was high in June (55°C, 56.5°C and 52°C respectively) whereas in Paradise Point samples it was high (43°C) in March (Table 3). The gel melting point in H. musciformis carrageenan samples were in the range of (42-77°C). The highest melting point was found in January in both Buleji (67°C) and Paradise Point (68°C) samples. Whereas in Hawks Bay samples it was high in March (77°C) and in Manora samples it was high in May (71°C) ( Table 4). The gel strength of H. musciformis carrageenan extract was 100 g/cm 2 in all sites samples except Buleji sample has high gel strength (105 g/cm 2 ) ( Table 5).       Figure 9). The range of biomass value was 51.7-121 g/m 2 with mean value of 84.4 g/m 2 for the four sites Buleji, Hawks Bay, Manora and Paradise Point in H. pannosa (Figure 7). At Buleji biomass was high (112 g/m 2 ) in March whereas at Hawks Bay, Manora and Paradise Point in January (96 g/m 2 , 121 g/m 2 and 100 g/m 2 respectively) ( Figure 10).   Figure 11). In H. valentiae the range of biomass value was 31.8-64.75 g/m 2 with mean value of 50.86 g/m 2 for the four sites Buleji, Hawks Bay, Manora and Paradise Point (Figure 7). At Buleji and Manora biomass was high in October (62.8 g/m 2 and 60.4 g/m 2 , respectively) and at Hawks Bay and Paradise Point in January (61.46 g/m 2 and 64.75 g/m 2 respectively) ( Figure 12). Manora samples it was high in January (1.03 g/cm 3 ) and in Paradise Point samples it was 1.2 g/cm 3 in July (Table  1). The gel viscosity of H. valentiae carrageenan extract was in the range of 8.8-19 cP. In both Buleji (19 cP) and Hawks Bay (14 cP) samples viscosity was high in January and in Manora samples it was high (17 cP) in July whereas in samples of Paradise Point viscosity was high (16 cP) in February ( Table 2). The gel boiling point of H. valentiae carrageenan was in the range of 40-53.5°C. In Buleji samples boiling point was high in July (52°C) and in both Hawks Bay (53.5°C) and Manora (52.5°C) samples it was high in October. In Paradise Point samples it was high (51°C) in February (Table 3). The gel melting point in H. valentiae carrageenan samples were in the range of (41-55°C). The highest melting point (55°C) was found in September, November, October and March in all studied sites samples i.e., Buleji, Hawks Bay, Manora and Paradise Point. The gel strength of H. valentiae carrageenan extracts was in the range of 98-105 g/cm 2 and the high value was found in Manora sample (105 g/cm 2 ) ( Table 5). Table 6 showed the temperature of seawater ranged from 18-34°C and the mean was 25.44°C during the study period. The high temperature in seawater was recorded in summer (June to August) and the lowest temperature was recorded in winter (December to February). The salinity of seawater ranged from 34.5-38‰ with the mean of 36.33‰ The high salinity (38‰) was recorded in April and January in the sample of Buleji and Hawks Bay respectively ( Table 6). The pH of surface seawater ranged from 6.5-7.5 with the mean of 7.0 (Table 6). In the present study it is noted that physical factor temperature affect on the biomass and carrageenan content. The results of two way analysis of variance (ANOVA) in carrageenan of three different species of Hypnea collected from Karachi coast show that there were highly significant variations observed between species (P<0.001), month (P<0.01) and sites (P<0.01). Analysis of variance ANOVA showed that there were highly significant variations observed in biomass between species (P<0.05) and in between months and sites (P<0.001). The differences in species, sites and month in present results reveal that biomass and carrageenan content was different in different species at different sites in different times (Table 7; Table 8). The data for biomass and carrageenan concentrations in different species were analyzed by looking at the relationship in between carrageenan of three different species of Hypnea at four different sites found; particular positive significant correlation such as at Hawks Bay in carrageenan of Hypnea musciformis and H. valentiae correlation was r 2 =0.617 and at Paradise Point in carrageenan of Hypnea musciformis and H. pannosa correlation was r 2 =0.717. There was significant correlation was found in between carrageenan and biomass (r 2 =0.671). The hydrographic conditions showed insignificant correlation with carrageenan content and biomass of all three studied species. The results for yield of carrageenan extracted from Hypnea musciformis in present work was similar to the values reported by Tuvikene et al. (2006). Dass et al. (1980) obtained high concentration of carrageenan from Hypnea musciformis (51.6%) in December when compared with present study. Rodrigueza and Montano (2007) discover in their stud that carrageenan yield and biomass were inversely related to each other whereas in present study both carrageenan yield and biomass were found high in the same time i.e., winter season ( Figure 1; Figure 4). Qasim and Qari (1988;1994), Qari et al. (2014), Qari (2017) reported that winter (December to February) was the period of optimal growth and would be the best period for optimum harvesting of seaweeds due to winter period with low air temperature and sun light i.e., low intensity and small duration and high dissolve nutrients are present that affect seaweeds growth, biomass and biochemical composition that results in high biomass of seaweed in winter compared to other seasons. Juneja et al. (2013) described in their study that the relative amounts of carbohydrate product are linked to environmental conditions (temperature, pH and salinity). The present data also showed that seasonal differences between locations and years in the magnitude and timing of the environmental parameters affecting algal growth and polysaccharides content.
The carrageenan is actually natural carbohydrate obtained from red seaweeds (Necas and Bartosikova, 2013). All Hypnea species and other carrageenophytes available at Karachi coastal areas in abundant quantities can be utilized for obtaining carrageenan in Pakistan (Qari et al., 2014;Qari, 2017). Today desired and need is the utilization of seaweed in food and medicine. Considering these facts, the present data contributes baseline information about the exploitation of seaweeds as a source of carrageenan for bright future of marine algae or seaweeds.

Conclusions
The studied shores have rich vegetation of red seaweed. The data of present study reveals high variability in the content of carrageenan in between species, sampling sites and collection time. The highest carrageenan extracted from H. valentiae (18-46.93%) as compared with the other two studied species. The highest concentrations of carrageenan were found during the winter season and it was concluded that environmental conditions may play a vital role in the development of these polymers in situ in the red seaweeds. The viscosity measurement showed high molecular weight polysaccharides. The high yield of carrageenan from the above mention species of seaweeds proves their increased market demand and global exploitation. There are also seasonal differences