Abstract
Bacterial communities of Antarctic marine macroalgae remain largely underexplored in terms of diversity and biotechnological applications. In this study, three Antarctic subtidal macroalgae (Himantothallus grandifolius, Pantoneura plocamioides and Plocamium cartilagineum), two of them endemic of Antarctica, were investigated as a source for isolation of agar-degrading bacteria. A total of 21 epiphytic isolates showed agarolytic activity at low temperature on agar plates containing agar as the sole carbon source. 16S rRNA identification showed that the agar-degrading bacteria belonged to the genera Cellulophaga, Colwellia, Lacinutrix, Olleya, Paraglaciecola, Pseudoalteromonas and Winogradskyella. The agarase enzyme from a potential new species of the genus Olleya was selected for further purification. The enzyme was purified from the culture supernatant of Olleya sp. HG G5.3 by ammonium sulfate precipitation and ion-exchange chromatography. Molecular weight of the agarase was estimated to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme exhibited activity at 4 °C, retaining > 50% of its maximum activity at this temperature. This is the first study reporting the phylogeny of agar-degrading bacteria isolated from Antarctic subtidal macroalgae and the results suggest the huge potential of Antarctic algae-associated bacteria as a source of cold-active hydrolytic enzymes of biotechnological interest.
Similar content being viewed by others
References
Alvarado R, Leiva S (2017) Agar-degrading bacteria isolated from Antarctic macroalgae. Folia Microbiol 62:409–416. https://doi.org/10.1007/s12223-017-0511-1
Aoki T, Araki T, Kitamikado M (1990) Purification and characterization of a novel β-agarase from Vibrio sp AP-2. Eur J Biochem 187:461–465
Barreto C, Duarte R, Lima D, de Oliveira Filho E, Absher T, Pellizari V (2017) The cultivable microbiota associated with four antarctic macroalgae. Open Access J Microbiol Biotechnol 2:000122
Barroca M, Santos G, Gerday C, Collins T (2017) Biotechnological aspects of cold-active enzymes. In: Margesin R (ed) Psychrophiles: from biodiversity to biotechnology. Springer, Cham, pp 461–475. https://doi.org/10.1007/978-3-319-57057-0_19
Bech PK, Schultz-Johansen M, Glaring MA, Barbeyron T, Czjzek M, Stougaard P (2017) Paraglaciecola hydrolytica sp. nov., a bacterium with hydrolytic activity against multiple seaweed-derived polysaccharides. Int J Syst Evol Microbiol 67:2242–2247. https://doi.org/10.1099/ijsem.0.001933
Borchert E, Knobloch S, Dwyer E, Flynn S, Jackson SA, Jóhannsson R, Marteinsson VT, O’Gara F, Dobson ADW (2017) Biotechnological potential of cold adapted Pseudoalteromonas spp. isolated from ‘deep sea’ sponges. Mar Drugs 15:184. https://doi.org/10.3390/md15060184
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Brown RE, Jarvis KL, Hyland KJ (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136–139. https://doi.org/10.1016/0003-2697(89)90101-2
Buck JD (1982) Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44:992–993
Chi W, Chang Y, Hong S (2012) Agar degradation by microorganisms and agar-degrading enzymes. Appl Microbiol Biotechnol 94:917–930. https://doi.org/10.1007/s00253-012-4023-2
Chi W-J, Park J-S, Kwak M-J, Kim JF, Chang Y-K, Hong S-K (2013) Isolation and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gen, nov, sp. nov., from the Southern Sea. J Microbiol Biotechnol 23:1509–1518
Christiansen L, Bech PK, Schultz-Johansen M, Martens HJ, Stougaard P (2018) Colwellia echini sp. nov., an agar- and carrageenan-solubilizing bacterium isolated from sea urchin. Int J Syst Evol Microbiol. https://doi.org/10.1099/ijsem.0.002568
Fu XT, Kim SM (2010) Agarase: review of major sources, categories, purification method, enzyme characteristics and applications. Mar Drugs 8:200–218
Furbino LE, Pellizzari FM, Neto PC, Rosa CA, Rosa LH (2017) Isolation of fungi associated with macroalgae from maritime Antarctica and their production of agarolytic and carrageenolytic activities. Polar Biol 41:527–535. https://doi.org/10.1007/s00300-017-2213-1
Goecke F, Labes A, Wiese J, Imhoff JF (2013) Phylogenetic analysis and antibiotic activity of bacteria isolated from the surface of two co-occurring macroalgae from the Baltic Sea. Eur J Phycol 48:47–60. https://doi.org/10.1080/09670262.2013.767944
Gupta V, Trivedi N, Kumar M, Reddy CRK, Jha B (2013) Purification and characterization of exo-β-agarase from an endophytic marine bacterium and its catalytic potential in bioconversion of red algal cell wall polysaccharides into galactans. Biomass Bioenergy 49:290–298
Hehemann JH, Michel G, Barbeyron T, Czjzek M (2010) Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a βagarase from the flavobacterium Zobellia galactanivorans. Acta Crystallogr F 66:413–417. https://doi.org/10.1107/S174430911000429X
Herrera LM, García-Laviña CX, Marizcurrena JJ, Volonterio O, de León RP, Castro-Sowinski S (2017) Hydrolytic enzyme-producing microbes in the Antarctic oligochaete Grania sp. (Annelida). Polar Biol 40:947–953. https://doi.org/10.1007/s00300-016-2012-0
Higashimura Y, Naito Y, Takagi T, Uchiyama K, Mizushima K, Ushiroda C, Ohnogi H, Kudo Y, Yasui M, Inui S, Hisada T, Honda A, Matsuzaki Y, Yoshikawa T (2016) Protective effect of agaro-oligosaccharides on gut dysbiosis and colon tumorigenesis in high-fat diet-fed mice. Am J Physiol Gastrointest Liver Physiol 310:G367-G375
Hollants J, Leliaert F, De Clerck O, Willems A (2013) What we can learn from sushi: a review on seaweed–bacterial associations. FEMS Microbiol Ecol 83:1–16. https://doi.org/10.1111/j.1574-6941.2012.01446.x
Imran M, Poduval PB, Ghadi SC (2017) Bacterial degradation of algal polysaccharides in marine ecosystem. In: Naik MM, Dubey SK (eds) Marine pollution and microbial remediation. Springer, Singapore, pp 189–203. https://doi.org/10.1007/978-981-10-1044-6_12
Ivanova EP, Ng HJ, Webb HK (2014) The family Pseudoalteromonadaceae. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes: Gammaproteobacteria. Springer, Berlin, pp 575–582. https://doi.org/10.1007/978-3-642-38922-1_229
Khambhaty Y, Mody K, Jha B (2008) Purification, characterization and application of a novel extracellular agarase from a marine Bacillus megaterium. Biotechnol Bioprocess Eng 13:584–591. https://doi.org/10.1007/s12257-008-0026-3
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. Wiley, Chichester, pp 115–175
Lavin P, Gallardo-Cerda J, Torres-Diaz C, Asencio G, Gonzalez M (2013) Antarctic strain of Bacillus sp. with extracellular agarolitic and alginate-lyase activities. Gayana 77:75–82
Lavin P, Atala C, Gallardo-Cerda J, Gonzalez-Aravena M, De La Iglesia R, Oses R, Torres-Díaz C, Trefault N, Molina-Montenegro MA, Laughinghouse IVHD. (2016) Isolation and characterization of an Antarctic Flavobacterium strain with agarase and alginate lyase activities. Pol Polar Res 37:403–419
Lee S-Y, Park S, Oh T-K, Yoon J-H (2010) Description of Olleya aquimaris sp. nov., isolated from seawater, and emended description of the genus Olleya Mancuso Nichols et al. 2005. Int J Syst Evol Microbiol 60:887–891. https://doi.org/10.1099/ijs.0.014563-0
Lee M-H, Jung Y-T, Park S, Yoon J-H (2013) Olleya namhaensis sp. nov., isolated from wood falls, and emended description of the genus Olleya Mancuso Nichols et al. 2005 emend. Lee et al. 2010. Int J Syst Evol Microbiol 63:1610–1615. https://doi.org/10.1099/ijs.0.044131-0
Li J, Sha Y (2015) Expression and enzymatic characterization of a cold-adapted β-agarase from Antarctic bacterium Pseudoalteromonas sp. NJ21. Chin J Oceanol Limnol 33:319–327. https://doi.org/10.1007/s00343-015-4072-3
Li G, Sun MM, Wu J, Ye M, Ge XC, Wei W, Li HX, Hu F (2015a) Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp strain LGH. Appl Microbiol Biotechnol 99:10019–10029
Li J, Hu Q, Li Y, Xu Y (2015b) Purification and characterization of cold-adapted β-agarase from an Antarctic psychrophilic strain. Braz J Microbiol 46:683–690
Martin M, Portetelle D, Michel G, Vandenbol M (2014) Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications. Appl Microbiol Biotechnol 98:2917–2935. https://doi.org/10.1007/s00253-014-5557-2
Martin M, Barbeyron T, Martin R, Portetelle D, Michel G, Vandenbol M (2015) The cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharide-degrading bacteria. Front Microbiol 6:1487. https://doi.org/10.3389/fmicb.2015.01487
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Morrice LM, McLean MW, Williamson FB, Long WF (1983) β-agarases I and II from Pseudomonas atlantica. Eur J Biochem 135:553–558. https://doi.org/10.1111/j.1432-1033.1983.tb07688.x
Murielle J, Flament D, Allouch J, Potin P, Thion L, Kloareg B, Czjzek M, Helbert W, Michel G, Barbeyron T (2005) The endo-β-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem J 385:703–713
Nedashkovskaya OI, Kim S-G, Zhukova NV, Mikhailov VV (2017) Olleya algicola sp. nov., a marine bacterium isolated from the green alga Ulva fenestrata. Int J Syst Evol Microbiol 67:2205–2210. https://doi.org/10.1099/ijsem.0.001926
Nichols CM, Bowman JP, Guezennec J (2005) Olleya marilimosa gen. nov., sp. nov., an exopolysaccharide-producing marine bacterium from the family Flavobacteriaceae, isolated from the Southern Ocean. Int J Syst Evol Microbiol 55:1557–1561. https://doi.org/10.1099/ijs.0.63642-0
Oh C, Nikapitiya C, Lee Y, Whang I, Kim S-J, Kang D-H, Lee J (2010) Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4. J Ind Microbiol Biotechnol 37:483–494. https://doi.org/10.1007/s10295-010-0694-9
Park S, Jung Y-T, Yoon J-H (2016) Colwellia sediminilitoris sp. nov., isolated from a tidal flat. Int J Syst Evol Microbiol 66:3258–3263. https://doi.org/10.1099/ijsem.0.001183
Potin P, Richard C, Rochas C, Kloareg B (1993) Purification and characterization of the α-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur J Biochem 214:599–607. https://doi.org/10.1111/j.1432-1033.1993.tb17959.x
Ramos KRM, Valdehuesa KNG, Nisola GM, Lee W-K, Chung W-J (2018) Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C. New Biotechnol 40:261–267. https://doi.org/10.1016/j.nbt.2017.09.006
Schroeder DC, Jaffer MA, Coyne VE (2003) Investigation of the role of a β(1–4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis. Microbiology 149:2919–2929. https://doi.org/10.1099/mic.0.26513-0
Schultz-Johansen M, Glaring MA, Bech PK, Stougaard P (2016) Draft genome sequence of a novel marine bacterium, Paraglaciecola sp. strain S66, with hydrolytic activity against seaweed polysaccharides. Genome Announc 4:e00304–e00316. https://doi.org/10.1128/genomeA.00304-16
Song T, Zhang WJ, Wei CC, Jiang TF, Xu H, Cao Y, Cao Y, Qiao DR (2015) Isolation and characterization of agar-degrading endophytic bacteria from plants. Curr Microbiol 70:275–281
Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 8:6–9
Tropeano M, Vázquez S, Coria S, Turjanski A, Cicero D, Bercovich A, Mac Cormack W (2013) Extracellular hydrolytic enzyme production by proteolytic bacteria from the Antarctic. Pol Polar Res 34:253–267. https://doi.org/10.2478/popore-2013-0014
Vera J, Alvarez R, Murano E, Slebe JC, Leon O (1998) Identification of a marine agarolytic Pseudoalteromonas isolate and characterization of its extracellular agarase. Appl Environ Microbiol 64:4378–4383
Wang JX, Mou HJ, Jiang XL, Guan HS (2006) Characterization of a novel β-agarase from marine Alteromonas sp SY37-12 and its degrading products. Appl Microbiol Biotechnol 71:833–839
Wiencke C, Amsler C, Clayton M (2014) Macroalgae. In: Biogeographic atlas of the Southern Ocean. Scientific Committee on Antarctic Research, Cambridge, pp 66–73
Wiese J, Thiel V, Nagel K, Staufenberger T, Imhoff JF (2009) Diversity of antibiotic-active bacteria associated with the brown alga Laminaria saccharina from the Baltic Sea. Mar Biotechnol 11:287–300. https://doi.org/10.1007/s10126-008-9143-4
Wu H, Liu M, Zhang W, Xiao T (2014) Phylogenetic analysis of epibacterial communities on the surfaces of four red macroalgae. J Ocean Univ China 13:1025–1032. https://doi.org/10.1007/s11802-014-2325-y
Xu Z-X, Zhang H-X, Han J-R, Dunlap CA, Rooney AP, Mu D-S, Du Z-J (2017) Colwellia agarivorans sp. nov., an agar-digesting marine bacterium isolated from coastal seawater. Int J Syst Evol Microbiol 67:1969–1974. https://doi.org/10.1099/ijsem.0.001897
Yang M, Mao XZ, Liu N, Qiu YQ, Xue CH (2014) Purification and characterization of two agarases from Agarivorans albus OAY02. Process Biochem 49:905–912
Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. https://doi.org/10.1099/ijsem.0.001755
Youngdeuk L, Oh C, De Zoysa M, Kim H, Wickramaarachchi WDN, Whang I, Kang DH, Lee J (2013) Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-agarase from marine bacterium Saccharophagus sp AG21 in Escherichia coli. J Microbiol Biotechnol 23:913–922
Yun EJ, Yu S, Kim KH (2017) Current knowledge on agarolytic enzymes and the industrial potential of agar-derived sugars. Appl Microbiol Biotechnol 101:5581–5589. https://doi.org/10.1007/s00253-017-8383-5
Zhang D-C, Yu Y, Chen B, Wang H-X, Liu H-C, Dong X-Z, Zhou P-J (2006) Glaciecola psychrophila sp. nov., a novel psychrophilic bacterium isolated from the Arctic. Int J Syst Evol Microbiol 56:2867–2869. https://doi.org/10.1099/ijs.0.64575-0
Acknowledgements
We thank the INACH staff at Station Prof. Julio Escudero for logistic support. Special thanks to Dr. Iván Gómez and his group (Project Anillo ART1101) for its valuable support during the field work. Verónica Sánchez Hinojosa was supported by a scholarship of the Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT) of Ecuador. This study was supported by Grant RT_06-13 from the Instituto Antártico Chileno (INACH).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Sánchez Hinojosa, V., Asenjo, J. & Leiva, S. Agarolytic culturable bacteria associated with three antarctic subtidal macroalgae. World J Microbiol Biotechnol 34, 73 (2018). https://doi.org/10.1007/s11274-018-2456-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-018-2456-1