Identification of The Potential of Degrading Carrageenan in Red Algae Kappaphycus alvarezii Symbiotic Bacteria

— Kappahycus alvarezii is a red alga contained large amount of bioactive material, such as carrageenan. Carrageenan is useful as a raw material for several industries and can be degraded by marine bacteria through breaking the linkages in polysaccharide carrageenan into oligosaccharide carrageenan. The aim of this study is identification of degrading carrageenan in K. alvarezii symbiotic bacteria. The results showed there was 14 isolate bacteria, and all of the isolates have clear zone on congo red staining activity. The isolate bacteria were 7 genera as K. alvarezii symbiotic bacteria, such as Labrenzia sp., Alteromonas sp., Vibrio sp., Celeribacter sp., Pseudoalteromonas sp., Phaeobacter sp. and Cobetia sp. Labrenzia sp., Alteromonas sp., Vibrio sp., Pseudoalteromonas sp., Phaeobacter sp. were recognized to have strong interactions with carrageenan in red algae, while the other Celeribacter sp. and Cobetia sp. have strong interactions with alginate in brown algae.


INTRODUCTION
Kappahycus alvarezii is red algae that generally has cylindric thallus, smooth surface, cartilaginous and consists of several types based on the color, such as green, yellowish green, gray, brown and red (Parenrengi et al., 2010). K. alvarezii was lived in tidal habitats, coral reef flats and attached to hard substrates (Erlania, 2013). K. alvarezii was contained large amount of carrageenan and used as stabilizer and gelling agent in processed meat, ice cream, chocolate, pudding, pet food, shampoo, toothpaste and cleaning products industrial (Hotchkiss et al., 2016;Barret, 2018).
In the ecosystem, bacteria were played an important role because of its ability to degrade organic matters to inorganic matters (Ginting et al., 2019). The existence of symbiont bacteria was to protect their host and produce secondary metabolites (Funty, 2015). The use of secondary metabolites in algae, for example as bioactive materials (Nurhaedar, 2008  Algae K. alvarezii were collected from USA Marine Biological Institute, Kochi University, Japan and floated in Uranouchi Bay, Tosa, Kochi Prefecture, Japan for one week to collect the bacteria. While several commercial carrageenan was used in this study, κ-carrageenan and λcarrageenan purchased from Wako.

SYMBIOTIC-BACTERIA
Arificial water agar medium was made with adding 0.5% carrageenan and incubated with red algae K. alvarezii for 3 days. After 3 days, the artificial seawater bacteria were dropped around 0.1 ml in marine broth agar. Moreover, bacteria were incubated at 25C for 2 days to get the pure bacteria cultured.

DEGRADING CARRAGEENAN SCREENING BY CONGO RED STAINING
Bacteria in marine broth agar medium was inoculated to marine broth medium and incubated at 25C for 2 days. Congo red agar medium was made from 4 gr gar medium, 25 ml 0.5% carrageenan and 1 ml congo red. Bacteria colonies from marine broth medium was dropped into congo red agar medium for 0.1 ml. The bacteria were incubated at 25C for 2 days to identify the clear zone. Formed clear zone was an indicator of the carrageenan degrading activity existence. Bacteria with clear zone was inoculated and analyzed by 16S rDNA.

16S rDNA ANALYSIS OF K. alvarezii SYMBIOTIC-BACTERIA
A colony of 14 isolate bacteria were used as a template for PCR. Isolate bacteria were amplified by using universal primer pr0R2 PCR products were applied to agarose gel electrophoresis and purified using Wizard® SV Gel and PCR Clean-Up System (Promega). The purified DNA were sequenced by ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems Japan) using BigDye® Terminator v3.1 and analyzed with BLAST on NCBI.

III. RESULT AND DISCUSSION
Bacteria isolation found 14 isolate bacteria with big size colonies. Congo red staining activity showed that there was clear zone in 14 isolate bacteria (Fig 1). Congo red was used for degrading carrageenans activity because congo red have strong interaction with polysaccharide which contained cellulose linked by -1,4-glycosidic linkages (Teather & Wood, 1982). FAO (2003) was explained that red algae contain carrageenan and cellulose which insoluble in water and alkali. The clear zone was formed because of the reaction between congo red and -1,4-glycosidic linkages in cellulose polymer (Missa et al., 2016).

Fig 1: Congo red staining activity on 14 isolate bacteria
Electrophoresis showed that the measurement of bacteria DNA fragment was about 500 bp and it was compared to the marker 1 kbp DNA (Fig 2). The purification of DNA was measured by absorbance 260 nm and 280 nm. It showed that the absorbance of purified DNA was about 1.73-2.07 (Table 1). Thermo Fisher Scientific (2010) explained that the ratio of absorbance 260 nm and 280 nm was about 1.8 and it was "pure" for DNA purification.   Moreover, Pseudoalteromonas sp. had an ability to utilize carrageenan and -carrageenan for their energy source (Hettle et al., 2019). Pseudoalteromonas sp. was also degraded -carrageenan (Liu et al., 2011) and carrageenan (Guibet et al., 2007). Furthermore, Phaeobacter inhibens was found in red algae Tichocarpus crinitus to degrade the carrageenan (Kalitnik et al., 2017). Based on the several studies, Labrenzia sp., Alteromonas sp., Vibrio sp., Pseudoalteromonas sp. and Phaeobacter sp. were recognized to have strong interactions with red algae through the utilization of red algae carrageenan. Whereas Celeribacter sp. and Cobetia sp. was found in brown algae through the utilization of brown algae alginate. Ihua et al. (2020) showed that Celeribacter sp. was found on brown algae thallus Laminaria digitata and Yagi et al. (2016) explained that Cobetia sp. was isolated from brown algae Padina arborescens with alginate degrading enzyme.

IV. CONCLUSION
In this study, we found 7 genera of Kappahycus alvarezii symbiotic bacteria, such as Labrenzia sp., Alteromonas sp., Vibrio sp., Celeribacter sp., Pseudoalteromonas sp., Phaeobacter sp. and Cobetia sp. All of the bacteria showed an activity on congo red staining based on the formed clear zone. The clear zone was indicated the carrageenan degrading activity.