Chemical Characterization and Biotechnological Applicability of Pigments Isolated from Antarctic Bacteria

  • Tiago R. SilvaEmail author
  • Renata S. N. Tavares
  • Ramon Canela-Garayoa
  • Jordi Eras
  • Marili V. N. Rodrigues
  • Iramaia A. Neri-Numa
  • Glaucia M. Pastore
  • Luiz H. Rosa
  • José A. A. Schultz
  • Hosana M. Debonsi
  • Lorena R. G. Cordeiro
  • Valeria M. Oliveira
Original Article


Considering the global trend in the search for alternative natural compounds with antioxidant and sun protection factor (SPF) boosting properties, bacterial carotenoids represent an opportunity for exploring pigments of natural origin which possess high antioxidant activity, lower toxicity, no residues, and no environmental risk and are readily decomposable. In this work, three pigmented bacteria from the Antarctic continent, named Arthrobacter agilis 50cyt, Zobellia laminarie 465, and Arthrobacter psychrochitiniphilus 366, were able to withstand UV-B and UV-C radiation. The pigments were extracted and tested for UV absorption, antioxidant capacity, photostability, and phototoxicity profile in murine fibroblasts (3T3 NRU PT–OECD TG 432) to evaluate their further potential use as UV filters. Furthermore, the pigments were identified by ultra-high-performance liquid chromatography–photodiode array detector–mass spectrometry (UPLC-PDA-MS/MS). The results showed that all pigments presented a very high antioxidant activity and good stability under exposure to UV light. However, except for a fraction of the A. agilis 50cyt pigment, they were shown to be phototoxic. A total of 18 different carotenoids were identified from 23 that were separated on a C18 column. The C50 carotenes bacterioruberin and decaprenoxanthin (including its variations) were confirmed for A. agilis 50cyt and A. psychrochitiniphilus 366, respectively. All-trans-bacterioruberin was identified as the pigment that did not express phototoxic activity in the 3T3 NRU PT assay (MPE < 0.1). Zeaxanthin, β-cryptoxanthin, β-carotene, and phytoene were detected in Z. laminarie 465. In conclusion, carotenoids identified in this work from Antarctic bacteria open perspectives for their further biotechnological application towards a more sustainable and environmentally friendly way of pigment exploitation.


Antarctic pigments, Psychrophilic bacteria Arthrobacter Zobellia Antioxidant Carotenoids 



The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo–FAPESP for financial funding (process numbers 2014/17936-1, 2016/05640-6, and 2017/21790-0). The MycoAntar Project (CNPq) and the Brazilian Antarctic Program are also acknowledged for making the sampling feasible in the OPERANTAR XXXIII (summer 2014/2015) and OPERANTAR XXXIV (summer 2015/2016). We also would like to thank Dr. Marcos Eberlin and Dr. Fabio Neves from ThoMSon Mass Spectrometry Laboratory in UNICAMP for the analytical chemistry training.


This study was funded by the São Paulo Research Foundation–FAPESP (grant numbers 2014/17936-1, 2016/05640-6, and 2017/21790-0).

Compliance with Ethical Standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10126_2019_9892_MOESM1_ESM.docx (3.6 mb)
ESM 1 (DOCX 3665 kb)


  1. Agogué H, Joux F, Obernosterer I, Lebaron P (2005) Resistance of marine bacterioneuston to solar radiation. Appl Environ Microbiol 71:5282–5289CrossRefGoogle Scholar
  2. Arpin N, Fiasson JL, Norgård S et al (1975) Bacterial carotenoids, XLVI. C50-carotenoids, 14. C50-carotenoids from Arthrobacter glacialis. Acta Chem Scand B 29:921–926CrossRefGoogle Scholar
  3. Augustin C, Collombel C, Damour O (1997) Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro. Photodermatol Photoimmunol Photomed 13:27–36CrossRefGoogle Scholar
  4. Bowman JP, McCammon SA, Brown MV et al (1997) Diversity and association of psychrophilic bacteri in Antarctic sea ice. Appl Environ Microbiol 63:3068–3078Google Scholar
  5. Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free radical method to evaluate antioxidant activity. LWT -Food Sci Technol 28:25–30Google Scholar
  6. Britton G (1995) Structure and properties of carotenoids in relation to function. FASEB J 9:1551–1558CrossRefGoogle Scholar
  7. Britton G, Pfander H, Liaaen-Jensen S (2004) Carotenoids handbook. Birkhäuser Verlag, Basel BostonGoogle Scholar
  8. Carbonneau MA, Melin AM, Perromat A, Clerc M (1989) The action of free radicals on Deinococcus radiodurans carotenoids. Arch Biochem Biophys 275:244–251CrossRefGoogle Scholar
  9. Caspar JV, Meyer TJ (1983) Application of the energy gap law to excited-state decay. J Phys Chem 87:952–957CrossRefGoogle Scholar
  10. Casteliani AGB, Kavamura VN, Zucchi TD et al (2014) UV-B resistant yeast inhabit the Phyllosphere of strawberry. Br Microbiol Res J 4:1105–1117CrossRefGoogle Scholar
  11. Ceridono M, Tellner P, Bauer D, Barroso J, Alépée N, Corvi R, de Smedt A, Fellows MD, Gibbs NK, Heisler E, Jacobs A, Jirova D, Jones D, Kandárová H, Kasper P, Akunda JK, Krul C, Learn D, Liebsch M, Lynch AM, Muster W, Nakamura K, Nash JF, Pfannenbecker U, Phillips G, Robles C, Rogiers V, van de Water F, Liminga UW, Vohr HW, Wattrelos O, Woods J, Zuang V, Kreysa J, Wilcox P (2012) The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing - the report of an ECVAM-EFPIA workshop. Regul Toxicol Pharmacol 63:480–488CrossRefGoogle Scholar
  12. Cockell CS, Knowland J (1999) Ultraviolet radiation screening compounds. Biol Rev 74:311–345CrossRefGoogle Scholar
  13. Dávalos A, Gómez-Cordovés C, Bartolomé B (2004) Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. J Agric Food Chem 52:48–54CrossRefGoogle Scholar
  14. Delpino-Rius A, Eras J, Marsol-Vall A, Vilaró F, Balcells M, Canela-Garayoa R (2014) Ultra performance liquid chromatography analysis to study the changes in the carotenoid profile of commercial monovarietal fruit juices. J Chromatogr A 1331:90–99CrossRefGoogle Scholar
  15. Dieser M, Greenwood M, Foreman CM (2010) Carotenoid pigmentation in Antarctic heterotrophic bacteria as a strategy to withstand environmental stresses. Arct Antarct Alp Res 42:396–405CrossRefGoogle Scholar
  16. ECVAM DB-ALM (2008) 3T3 Neutral Red Uptake (NRU) phototoxicity assay. DB-ALM Protocol n ° 78, pp 1–19Google Scholar
  17. Ehling-Schulz M, Scherer S (1999) UV protection in cyanobacteria. Eur J Phycol 34(4):329–323Google Scholar
  18. Ehling-Schulz M, Bilger W, Scherer S (1997) UV-B-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium Nostoc commune. J Bacteriol 179:1940–1945CrossRefGoogle Scholar
  19. Fong NJC, Burgess ML, Barrow KD, Glenn DR (2001) Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress. Appl Microbiol Biotechnol 56:750–756CrossRefGoogle Scholar
  20. Freitas JV, Gaspar LR (2016) In vitro photosafety and efficacy screening of apigenin, chrysin and beta-carotene for UVA and VIS protection. Eur J Pharm Sci 89:146–153CrossRefGoogle Scholar
  21. Freitas JV, Lopes NP, Gaspar LR (2015) Photostability evaluation of five UV-filters, trans-resveratrol and beta-carotene in sunscreens. Eur J Pharm Sci 78:79–89CrossRefGoogle Scholar
  22. Gammone MA, Riccioni G, D’Orazio N (2015) Marine carotenoids against oxidative stress: effects on human health. Mar Drugs 13:6226–6246CrossRefGoogle Scholar
  23. Gaspar LR, Maia Campos PMBG (2006) Evaluation of the photostability of different UV filter combinations in a sunscreen. Int J Pharm 307:123–128CrossRefGoogle Scholar
  24. Ghiselli A, Nardini M, Baldi A, Scaccini C (1998) Antioxidant activity of different phenolic fractions separated from an Italian red wine. J Agric Food Chem 46:361–367CrossRefGoogle Scholar
  25. Giuffrida D, Sutthiwong N, Dugo P et al (2016) Characterisation of the C50 carotenoids produced by strains of the cheese-ripening bacterium Arthrobacter arilaitensis. Int Dairy J 55:10–16Google Scholar
  26. González MT, Fumagalli F, Benevenuto CG et al (2017) Novel benzophenone-3 derivatives with promising potential as UV filters: relationship between structure, photoprotective potential and phototoxicity. Eur J Pharm Sci 101:200–210CrossRefGoogle Scholar
  27. González-Toril E, Amils R, Delmas RJ, Petit JR, Komárek J, Elster J (2008) Diversity of bacteria producing pigmented colonies in aerosol, snow and soil samples from remote glacial areas (Antarctica, Alps and Andes). Biogeosci Discuss 5:1607–1630CrossRefGoogle Scholar
  28. Harm W (1980) Biological effects of ultraviolet radiation. Cambridge University Press, LondonGoogle Scholar
  29. Hartwig VG, Brumovsky LA, Fretes RM, Boado LS (2012) A novel procedure to measure the antioxidant capacity of Yerba maté extracts. Food Sci Technol 32:126–133CrossRefGoogle Scholar
  30. Heider SAE, Peters-Wendisch P, Netzer R, Stafnes M, Brautaset T, Wendisch VF (2014) Production and glucosylation of C50 and C40 carotenoids by metabolically engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 98:1223–1235CrossRefGoogle Scholar
  31. Hertzberg S, Jensen SL (1966) The carotenoids of blue-green algae—II. Phytochemistry 5:565–570CrossRefGoogle Scholar
  32. Hojerová J, Medovcíková A, Mikula M (2011) Photoprotective efficacy and photostability of fifteen sunscreen products having the same label SPF subjected to natural sunlight. Int J Pharm 408:27–38CrossRefGoogle Scholar
  33. Holzhütter HG (1997) A general measure of in vitro phototoxicity derived from pairs of dose-response curves and its use for predicting the in vivo phototoxicity of chemicals. ATLA Altern Lab Anim 25:445–462Google Scholar
  34. Horneck G (1995) Exobiology, the study of the origin, evolution and distribution of life within the context of cosmic evolution: a review. Planet Space Sci 43:189–217CrossRefGoogle Scholar
  35. ICH (1996) Photostability Testing of New Drug Substances and Products Q1B, International Conference on Harmonisation, IFPMA, GenevaGoogle Scholar
  36. Jagannadham MV, Narayanan K, Mohan Rao C, Shivaji S (1996) In vivo characteristics and localisation of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic spectroscopy. Biochem Biophys Res Commun 227:221–226CrossRefGoogle Scholar
  37. Jagannadham MV, Chattopadhyay MK, Subbalakshmi C, Vairamani M, Narayanan K, Mohan Rao C, Shivaji S (2000) Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum. Arch Microbiol 173:418–424CrossRefGoogle Scholar
  38. Karentz D (1991) Ecological considerations of antarctic ozone depletion. Antarct Sci 3:3–11CrossRefGoogle Scholar
  39. Kejlová K, Jírová D, Bendová H, Kandárová H, Weidenhoffer Z, Kolářová H, Liebsch M (2007) Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests. Toxicol In Vitro 21:1298–1303CrossRefGoogle Scholar
  40. Kleinig H, Heumann W, Meister W, Englert G (1977) Carotenoids of rhizobia. I. New carotenoids from rhizobium lupini. Helv Chim Acta 60:254–258. Google Scholar
  41. Kottemann M, Kish A, Iloanusi C, Bjork S, DiRuggiero J (2005) Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation. Extremophiles 9:219–227CrossRefGoogle Scholar
  42. Kuhlman KR, Allenbach LB, Ball CL, Fusco WG, la Duc MT, Kuhlman GM, Anderson RC, Stuecker T, Erickson IK, Benardini J, Crawford RL (2005) Enumeration, isolation, and characterization of ultraviolet (UV-C) resistant bacteria from rock varnish in the Whipple Mountains, California. Icarus 174:585–595CrossRefGoogle Scholar
  43. Kushwaha SC, Pugh EL, Kramer JKG, Kates M (1972) Isolation and identification of dehydrosqualene and C40-carotenoid pigments in Halobacterium cutirubrum. Biochim Biophys Acta Lipids Lipid Metab 260:492–506CrossRefGoogle Scholar
  44. Le K, Chiu F, Ng K (2007) Identification and quantification of antioxidants in Fructus lycii. Food Chem 105:353–363CrossRefGoogle Scholar
  45. Link L, Sawyer J, Venkateswaran K, Nicholson W (2004) Extreme spore UV resistance of Bacillus pumilus isolates obtained from an ultraclean spacecraft assembly facility. Microb Ecol:159–163Google Scholar
  46. Maciel OMC, Tavares RSN, Caluz DRE, Gaspar LR, Debonsi HM (2018) Photoprotective potential of metabolites isolated from algae-associated fungi Annulohypoxylon stygium. J Photochem Photobiol B Biol 178:316–322CrossRefGoogle Scholar
  47. Margesin R, Miteva V (2011) Diversity and ecology of psychrophilic microorganisms. Res Microbiol 162:346–361CrossRefGoogle Scholar
  48. Margesin R, Schinner F, Marx JC, Gerday C (2008) Psychrophiles, from Biodiversity to Biotechnology. Springer, BerlinGoogle Scholar
  49. Mathews MM, Sistrom WR (1959) Function of carotenoid pigments in non-photosynthetic bacteria. Nature 184:1892–1893CrossRefGoogle Scholar
  50. Mathews-Roth MM (1987) Photoprotection by carotenoids. Fed Proc 46:1890–1893Google Scholar
  51. Mohana D, Thippeswamy S, Abhishek R (2013) Antioxidant, antibacterial, and ultraviolet-protective properties of carotenoids isolated from Micrococcus spp. Radiat Prot Environ 36:168CrossRefGoogle Scholar
  52. Myers JA, Curtis BS, Curtis WR (2013) Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophys 6:4CrossRefGoogle Scholar
  53. Nguyen K-H, Chollet-Krugler M, Gouault N, Tomasi S (2013) UV-protectant metabolites from lichens and their symbiotic partners. Nat Prod Rep 30:1490–1508CrossRefGoogle Scholar
  54. Nupur LNU, Vats A, Dhanda SK, Raghava GPS, Pinnaka AK, Kumar A (2016) ProCarDB: a database of bacterial carotenoids. BMC Microbiol 16:96CrossRefGoogle Scholar
  55. OECD (2004) OECD guidelines for the testing of chemicals. Test:1–21.
  56. Pettijohn D, Hanawalt P (1964) Evidence for repair-replication of ultraviolet damaged DNA in bacteria. J Mol Biol 9:395–410CrossRefGoogle Scholar
  57. Prior RL, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M, Huang D, Ou B, Jacob R (2003) Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. J Agric Food Chem 51:3273–3279CrossRefGoogle Scholar
  58. Rattray FP, Fox PF (1999) Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review. J Dairy Sci 82:891–909CrossRefGoogle Scholar
  59. Roos JC, Vincent WF (1998) Temperature dependence of UV radiation effects on Antarctic cyanobacteria. J Phycol 34:118–125CrossRefGoogle Scholar
  60. Saito T, Miyabe Y, Ide H, Yamamoto O (1997) Hydroxyl radical scavenging ability of bacterioruberin. Radiat Phys Chem 50:267–269CrossRefGoogle Scholar
  61. Sajilata MG, Singhal RS, Kamat MY (2008) The carotenoid pigment zeaxanthin - a review. Compr Rev Food Sci Food Saf:29–49Google Scholar
  62. Scherer S, Chen TW, Böger P (1988) A new UV-A/B protecting pigment in the terrestrial cyanobacterium Nostoc commune. Plant Physiol 88:1055–1057CrossRefGoogle Scholar
  63. Schwender J, Seemann M, Lichtenthaler HK, Rohmer M (1996) Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus. Biochem J 316(Pt 1):73–80CrossRefGoogle Scholar
  64. Shaath N (2007) SPF boosters & photostability of ultraviolet filters. Happi 10:77–83Google Scholar
  65. Shahmohammadi HR, Asgarani E, Terato H et al (1997) Effects of co-60 gamma-rays, ultraviolet-light, and mitomycin-c on halobacterium-salinarium and thiobacillus-intermedius. J Radiat Res 38:37–43CrossRefGoogle Scholar
  66. Shahmohammadi HR, Asgarani E, Terato H et al (1998) Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents. J Radiat Res 39:251–262CrossRefGoogle Scholar
  67. Silva TR, Duarte AWF, Passarini MRZ, Ruiz ALTG, Franco CH, Moraes CB, de Melo IS, Rodrigues RA, Fantinatti-Garboggini F, Oliveira VM (2018) Bacteria from Antarctic environments: diversity and detection of antimicrobial, antiproliferative, and antiparasitic activities. Polar Biol 41:1505–1519CrossRefGoogle Scholar
  68. Spielmann H, Balls M, Dupuis J, Pape WJ, Pechovitch G, de Silva O, Holzhütter HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W, Brantom P (1998) The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial). Part 1: the 3T3 NRU phototoxicity test. Toxicol In Vitro 12:305–327CrossRefGoogle Scholar
  69. Stafsnes MH, Josefsen KD, Kildahl-Andersen G, Valla S, Ellingsen TE, Bruheim P (2010) Isolation and characterization of marine pigmented bacteria from Norwegian coastal waters and screening for carotenoids with UVA-blue light absorbing properties. J Microbiol 48:16–23CrossRefGoogle Scholar
  70. Suryawanshi RK, Patil CD, Borase HP, Narkhede CP, Stevenson A, Hallsworth JE, Patil SV (2015) Towards an understanding of bacterial metabolites prodigiosin and violacein and their potential for use in commercial sunscreens. Int J Cosmet Sci 37:98–107CrossRefGoogle Scholar
  71. Takaichi S (2014) General methods for identification of carotenoids. Biotechnol Lett 36:1127–1128CrossRefGoogle Scholar
  72. Takaichi S, Shimada K, Ishidsu J (1990) Carotenoids from the aerobic photosynthetic bacterium, Erythrobacter longus: β-carotene and its hydroxyl derivatives. Arch Microbiol 153:118–122CrossRefGoogle Scholar
  73. Uchino O, Bojkov RD, Balis DS, Akagi K, Hayashi M, Kajihara R (1999) Essential characteristics of the Antarctic-spring ozone decline: update to 1998. Geophys Res Lett 26:1377–1380CrossRefGoogle Scholar
  74. Venil CK, Zakaria ZA, Ahmad WA (2013) Bacterial pigments and their applications. Process Biochem 48:1065–1079CrossRefGoogle Scholar
  75. Wagener S, Völker T, De Spirt S et al (2012) 3,3′-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts. Free Radic Biol Med 53:457–463CrossRefGoogle Scholar
  76. Whitehead K, Hedges JI (2005) Photodegradation and photosensitization of mycosporine-like amino acids. J Photochem Photobiol B Biol 80:115–121CrossRefGoogle Scholar
  77. Wright DL, Whitehead CR, Sessions EH, Ghiviriga I, Frey DA (1999) Studies on inducers of nerve growth factor: synthesis of the cyathin core. Org Lett 1:1535–1538CrossRefGoogle Scholar
  78. Wynn-Williams DD, Edwards HGM (2002) Environmental UV radiation: biological strategies for protection and avoidance. Astrobiol Quest Cond Life:245–260Google Scholar
  79. Wynn-Williams DD, Newton EM, Edwards HGM (2001) The role of habitat structure for biomolecule integrity and microbial survival under extreme environmental stress in Antarctica (and Mars?): ecology and technology. In Exo-/astro-biology (vol 496. pp. 225-237)Google Scholar
  80. Yokoyama A, Shizuri Y, Hoshino T, Sandmann G (1996) Thermocryptoxanthins: novel intermediates in the carotenoid biosynthetic pathway of Thermus thermophilus. Arch Microbiol 165:342–345CrossRefGoogle Scholar
  81. Zhang G, Zhu B, Nakamura Y et al (2008) Structure-dependent photodegradation of carotenoids accelerated by dimethyl tetrasulfide under UVA irradiation. Biosci Biotechnol Biochem 72(8):2176–2183CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Tiago R. Silva
    • 1
    • 2
    Email author
  • Renata S. N. Tavares
    • 3
  • Ramon Canela-Garayoa
    • 4
  • Jordi Eras
    • 4
  • Marili V. N. Rodrigues
    • 5
  • Iramaia A. Neri-Numa
    • 6
  • Glaucia M. Pastore
    • 6
  • Luiz H. Rosa
    • 7
  • José A. A. Schultz
    • 8
  • Hosana M. Debonsi
    • 3
  • Lorena R. G. Cordeiro
    • 3
  • Valeria M. Oliveira
    • 2
  1. 1.Institute of BiologyCampinas State University (UNICAMP)CampinasBrazil
  2. 2.Division of Microbial Resources, Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA)Campinas State UniversityCampinasBrazil
  3. 3.School of Pharmaceutical Sciences of Ribeirão PretoUniversity of Sao Paulo (USP)Sao PauloBrazil
  4. 4.Department of Chemistry, ETSEAUniversity of Lleida-Agrotecnio CenterLleidaSpain
  5. 5.Department of Organic Chemistry; Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA)Campinas State UniversityCampinasBrazil
  6. 6.Department of Food Science, School of Food EngineeringUniversity of Campinas (UNICAMP)CampinasBrazil
  7. 7.Department of Microbiology, Institute of Biological ScienceFederal University of Minas GeraisBelo HorizonteBrazil
  8. 8.Department of Data ScienceTelcelMexico CityMexico

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