Journal of Applied Phycology

, Volume 31, Issue 1, pp 29–39 | Cite as

Co-cultivation of siderophore-producing bacteria Idiomarina loihiensis RS14 with Chlorella variabilis ATCC 12198, evaluation of micro-algal growth, lipid, and protein content under iron starvation

  • Soundarya Rajapitamahuni
  • Pooja Bachani
  • Raj Kumar Sardar
  • Sandhya MishraEmail author


Co-cultivation systems offer the potential to commercialize microalgae biomass. The key purpose of the study was to understand the relationship between siderophore-producing bacterium Idiomarina loihiensis RS14 and Chlorella variabilis ATCC 12198 strain for Chlorella growth enhancement. After observing growth enhancement in C. variabilis by adding metal chelator deferroxamine mesylate (siderophore standard) and purified siderophore from I. loihiensis (1 mg mL−1), a co-cultivation system was designed where axenic microalgae and co-cultured (microalgae + bacteria) aliquots were grown in (1:9, 9:1, 1:1) volumetric inoculum ratio (mL) under iron-sufficient and iron-deficient conditions. The co-culture volumetric ratio 1:1 (microalgae/bacteria) showed bleaching of microalgae and 1:9 showed less biomass (310 mg L−1) comparatively with 9:1 that increased 35% of biomass, i.e., 650 mg L−1 (axenic) to 1000 mg L−1 (co-cultured) in iron-deficient media. The inoculum ratios were optimized in 100 mL shake flask and 9:1 ratio was further scaled up with the similar conditions, and the co-culture showed 20% increase in biomass, i.e., 285.6 mg L−1 (axenic) to 356 mg L−1 (co-cultured). The co-cultured biomass contains 19.70% lipid content compared with axenic algae, i.e., 18.41% which shows 7% of increase in co-culture. Protein content increased to 30% in co-culture microalgae compared with axenic microalgae. Scanning electron microscope images show crumpled surface of Chlorella cells in co-cultured compared with its axenic cells. This finding is of interest for biofuel production from microalgae, often attained through nutrient-starvation processes leading to oil accumulation.


Co-cultivation Micro-algal growth enhancement Iron starvation Siderophore Lipid 



We are thankful to Ms. Khushbu Bhayani, Dr. Kaumeel Chokshi, and Dr. Sourish Bhattacharya for their timely help. We are also thankful to Mr. Jayesh Chaudhary for SEM analysis.

Funding information

CSIR-CSMCRI Registration Number PRIS 098/2017 has been assigned to the manuscript. RS acknowledges DST for her INSPIRE funding support. PB acknowledges CSIR-SRF for the funding support. RS, PB, and RKS acknowledge AcSIR for their Ph.D. enrolment.

Supplementary material

10811_2018_1591_MOESM1_ESM.docx (2.5 mb)
ESM 1 (DOCX 2553 kb)


  1. Akbarnezhad M, Mehrgan MS, Kamali A, Baboli MJ (2016) Bioaccumulation of Fe+2 and its effects on growth and pigment content of Spirulina (Arthrospira platensis). AACL Bioflux 9:227–238Google Scholar
  2. Amin SA, Hmelo LR, Van Tol HM, Durham BP, Carlson LT, Heal KR, Morales RL, Berthiaume CT, Parker MS, Djunaedi B, Ingalls AE (2015) Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria. Nature 7554:98–101CrossRefGoogle Scholar
  3. Andersen RA (ed) (2005) Algal Culturing Techniques. Academic Press, West Boothbay HarborGoogle Scholar
  4. Avendaño-Herrera R, Riquelmes C, Silva F, Avendañod M, Irgang R (2003) Optimization of settlement of larval Argopecten purpuratus using natural diatom biofilms. J Shellfish Res 22:393–399Google Scholar
  5. Bendale MS, Chaudhari BL, Chincholkar SB (2010) Influence of environmental factors on siderophore production by Streptomyces fulvissimus ATCC 27431. Curr Trends Biotechnol Pharm 3:362–371Google Scholar
  6. Benderliev KM, Ivanova NI (1994) High-affinity siderophore-mediated iron-transport system in the green alga Scenedesmus incrassatulus. Planta 193:163–166CrossRefGoogle Scholar
  7. Bhattacharya S, Soundarya R, Mishra S (2016) Ammonium bicarbonate as nutrient substitute for improving biomass productivity of Chlorella variabilis. Chem Eng Technol 39:1738–1742CrossRefGoogle Scholar
  8. Biondi N, Cheloni G, Tatti E, Decorosi F, Rodolfi L, Giovannetti L, Viti C, Tredici MR (2017) The bacterial community associated with Tetraselmis suecica outdoor mass cultures. J Appl Phycol 29:67–78CrossRefGoogle Scholar
  9. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Phys 37:911–917CrossRefGoogle Scholar
  10. Brettar I, Christen R, Höfle MG (2003) Idiomarina baltica sp. nov., a marine bacterium with a high optimum growth temperature isolated from surface water of the Central Baltic Sea. Int J Syst Evol Microbiol 53:407–413CrossRefGoogle Scholar
  11. Bruland KW, Donat JR, Hutchins DA (1991) Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnol Oceanogr 368:1555–1577CrossRefGoogle Scholar
  12. Cho DH, Ramanan R, Kim BH, Lee J, Kim S, Yoo C, Choi GG, Oh HM, Kim HS (2013) Novel approach for the development of axenic microalgal cultures from environmental samples. J Phycol 494:802–810CrossRefGoogle Scholar
  13. Cho DH, Ramanan R, Heo J, Lee J, Kim BH, Oh HM, Kim HS (2015) Enhancing microalgal biomass productivity by engineering a microalgal–bacterial community. Bioresour Technol 175:578–585CrossRefGoogle Scholar
  14. Cooper MB, Smith AG (2015) Exploring mutualistic interactions between microalgae and bacteria in the omics age. Curr Opin Plant Biol 26:147–153CrossRefGoogle Scholar
  15. Croft MT, Lawrence AD, Raux Deery E, Warren MJ, Smith AG (2005) Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438-7064:90CrossRefGoogle Scholar
  16. Davey M, Geider RJ (2001) Impact of iron limitation on the photosynthetic apparatus of the diatom Chaetoceros muelleri (Bacillariophyceae). J Phycol 376:987–1000CrossRefGoogle Scholar
  17. Estevez MS, Malanga G, Puntarulo S (2001) Iron-dependent oxidative stress in Chlorella vulgaris. Plant Sci 1611:9–17CrossRefGoogle Scholar
  18. Fidler MC, Walczyk T, Davidsson L, Zeder C, Sakaguchi N, Juneja LR, Hurrell RF (2004) A micronised, dispersible ferric pyrophosphate with high relative bioavailability in man. Br J Nutr 911:107–112CrossRefGoogle Scholar
  19. Fujita MJ, Nakano K, Sakai R (2013) Bisucaberin B, a linear hydroxamate class siderophore from the marine bacterium Tenacibaculum mesophilum. Molecules 184:3917–26Google Scholar
  20. Geng H, Belas R (2010) Molecular mechanisms underlying Roseobacter–phytoplankton symbioses. Curr Opin Biotechnol 21:332–338Google Scholar
  21. Glaesener AG, Merchant SS, BlabyHaas CE (2013) Iron economy in Chlamydomonas reinhardtii. Front Plant Sci 4:337CrossRefGoogle Scholar
  22. Goecke F, Thiel V, Wiese J, Labes A, Imhoff JF (2013) Algae as an important environment for bacteria–phylogenetic relationships among new bacterial species isolated from algae. Phycologia 52:14–24CrossRefGoogle Scholar
  23. Gonzalez LE, Bashan Y (2000) Increased growth of the microalga Chlorella vulgaris when coimmobilized and cocultured in alginate beads with the plant-growth-promoting bacterium Azospirillum brasilense. Appl Environ Microbiol 664:1527–1531CrossRefGoogle Scholar
  24. Guo Z, Tong YW (2014) The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions. J Appl Phycol 26:1483–1492CrossRefGoogle Scholar
  25. Kean MA, Delgado EB, Mensink BP, Bugter MH (2015) Iron chelating agents and their effects on the growth of Pseudokirchneriella subcapitata, Chlorella vulgaris, Phaeodactylum tricornutum and Spirulina platensis in comparison to Fe-EDTA. J Algal Biomass Util 6:56–73Google Scholar
  26. Keshtacher-Liebso E, Hadar Y, Chen Y (1995) Oligotrophic bacteria enhance algal growth under iron-deficient conditions. Appl Environ Microbiol 616:2439–2441Google Scholar
  27. Kim BH, Ramanan R, Cho DH, Oh HM, Kim HS (2014) Role of Rhizobium, a plant growth promoting bacterium, in enhancing algal biomass through mutualistic interaction. Biomass Bioenergy 69:95–105CrossRefGoogle Scholar
  28. Kobayashi T (1993) Filtering rates of Daphnia carinata King (Crustacea: Cladocera) on the blue green algae Microcystis aeruginosa Kutz and Anabaena cylindrica (Lemm.). Aust J Ecol 18:1–4CrossRefGoogle Scholar
  29. Kolber ZS, Barber RT, Coale KH, Fitzwateri SE, Greene RM, Johnson KS, Lindley S, Falkowski PG (1994) Iron limitation of phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature 371:145–149CrossRefGoogle Scholar
  30. Kropat J, Gallaher SD, Urzica EI, Nakamoto SS, Strenkert D, Tottey S, Mason AZ, Merchant SS (2015) Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis. Proc Natl Acad Sci 112:2644–2651CrossRefGoogle Scholar
  31. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382Google Scholar
  32. Liu ZY, Wang GC, Zhou BC (2008) Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol 9911:4717–4722CrossRefGoogle Scholar
  33. Magdouli S, Brar SK, Blais JF (2016) Co-culture for lipid production: advances and challenges. Biomass Bioenergy 92:20–30CrossRefGoogle Scholar
  34. Malasarn D, Kropat J, Hsieh SI, Finazzi G, Casero D, Loo JA, Pelligrini M, Wollman FA, Merchant SS (2013) Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii. J Biol Chem 288:10672–10683CrossRefGoogle Scholar
  35. Mishra SK, Shrivastav A, Maurya RR, Patidar SK, Haldar S, Mishra S (2012) Effect of light quality on the C-phycoerythrin production in marine cyanobacteria Pseudanabaena sp. isolated from Gujarat coast, India. Protein Expr Purif 81:5–10CrossRefGoogle Scholar
  36. Mouget JL, Dakhama A, Lavoie MC, de la Noüe J (1995) Algal growth enhancement by bacteria: is consumption of photosynthetic oxygen involved? FEMS Microbiol Ecol 18:35–43CrossRefGoogle Scholar
  37. Muñoz R, Guieysse B (2006) Algal–bacterial processes for the treatment of hazardous contaminants: a review. Water Res 40:2799–2815CrossRefGoogle Scholar
  38. Park Y, Je KW, Lee K, Jung SE, Choi TJ (2008) Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga. Hydrobiologia 598:219–228CrossRefGoogle Scholar
  39. Ramanan R, Kang Z, Kim BH, Cho DH, Jin L, Oh HM, Kim HS (2015) Phycosphere bacterial diversity in green algae reveals an apparent similarity across habitats. Algal Res 8:140–144CrossRefGoogle Scholar
  40. Safi C, Charton M, Pignolet O, Silvestre F, Vaca-Garcia C, Pontalier PY (2013) Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen to protein conversion factors. J Appl Phycol 25:523–529CrossRefGoogle Scholar
  41. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefGoogle Scholar
  42. Segev E, Wyche TP, Kim KH, Petersen J, Ellebrandt C, Vlamakis H, Barteneva N, Paulson JN, Chai L, Clardy J, Kolter R (2016) Dynamic metabolic exchange governs a marine algal-bacterial interaction. Elife 5:e17473CrossRefGoogle Scholar
  43. Seyedsayamdost MR, Case RJ, Kolter R, Clardy J (2011) The Jekyll and Hyde chemistry of Phaeobacter gallaeciensis. Nat Chem 3:331–335CrossRefGoogle Scholar
  44. Smriga S, Fernandez VI, Mitchell JG, Stocker R (2016) Chemotaxis toward phytoplankton drives organic matter partitioning among marine bacteria. Proc Natl Acad Sci-Biol:1131576–1131581Google Scholar
  45. Soria-Dengg S, Horstmann U (1995) Ferrioxamines B and E as iron sources for the marine diatom Phaeodactylum tricornutum. Mar Ecol Prog Ser 127:269–277CrossRefGoogle Scholar
  46. Subashchandrabose SR, Ramakrishnan B, Megharaj M, Venkateswarlu K, Naidu R (2011) Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential. Biotechnol Adv 29:896–907CrossRefGoogle Scholar
  47. Sunda WG, Price NM, Morel FM (2005) Trace metal ion buffers and their use in culture studies. In: Andersen RA (ed) Algal culturing techniques. Academic Press, London, pp 35–63Google Scholar
  48. Terry N, Abadía J (1986) Function of iron in chloroplasts. J Plant Nutr 9:609–646CrossRefGoogle Scholar
  49. Vraspir JM, Holt PD, Butler A (2011) Identification of new members within suites of amphiphilic marine siderophores. Biometals 241:85–92Google Scholar
  50. Wells ML, Price NM, Bruland KW (1994) Iron limitation and the cyanobacterium Synechococcus in equatorial Pacific waters. Limnol Oceanogr 39:1481–1486CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Soundarya Rajapitamahuni
    • 1
    • 2
  • Pooja Bachani
    • 1
    • 2
  • Raj Kumar Sardar
    • 1
    • 2
  • Sandhya Mishra
    • 1
    • 2
    Email author
  1. 1.Division of Biotechnology and PhycologyCentral Salt and Marine Chemicals Research Institute (CSIR)BhavnagarIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)Central Salt and Marine Chemicals Research Institute (CSIR)BhavnagarIndia

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