Microbial Ecology

, Volume 77, Issue 4, pp 980–992 | Cite as

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense

  • Oskar A. Palacios
  • Blanca R. Lopez
  • Yoav Bashan
  • Luz E. de -BashanEmail author
Plant Microbe Interactions


The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth–promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.


PGPB Microalgae Interaction Signal molecules Nutritional effects Starch 



At CIBNOR, Mexico, we thank Manuel Moreno, Francisco Hernandez, and Patricia Hinojosa for technical assistance.

Author Contributions

OAP and BRL designed and executed the study and wrote the initial draft. YB critically revised the article for intellectual content including the final version. LEdeB designed and managed the project and critically revised the manuscript. All the authors read and approved the manuscript.

Funding Information

This work was financially supported by Consejo Nacional de Ciencia y Tecnología of Mexico (CONACYT Basic Science-2015, contract 251102 and CONACYT Basic Science-2017, contract 284562) and the actual drafting of the paper was supported by The Bashan Foundation, USA. This is contribution 2018-027 of the Bashan Institute of Science, USA.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

248_2018_1282_MOESM1_ESM.docx (15 kb)
Table S1 (DOCX 14 kb)


  1. 1.
    Sapp M, Schwaderer AS, Wiltshire KH, Hoppe H-G, Gerdts G, Wichels A (2007) Species-specific bacterial communities in the phycosphere of microalgae? Microb Ecol 53:683–699. CrossRefGoogle Scholar
  2. 2.
    Kouzuma A, Watanabe K (2015) Exploring the potential of algae/bacteria interactions. Curr Opin Biotech 33:125–129. CrossRefGoogle Scholar
  3. 3.
    Harcombe WR, Riehl WJ, Dukovski I, Granger BR, Betts A, Lang AH, Bonilla G, Kar A, Leiby N, Mehta P, Marx CJ, Segrè D (2014) Metabolic resource allocation in individual microbes determines ecosystem interactions and spatial dynamics. Cell Rep 7:1104–1115. CrossRefGoogle Scholar
  4. 4.
    de-Bashan LE, Mayali X, Bebout BM, Weber PK, Detweiler AM, Hernandez J-P, Prufert-Bebout L, Bashan Y (2016) Establishment of stable synthetic mutualism without co-evolution between microalgae and bacteria demonstrated by mutual transfer of metabolites (NanoSIMS isotopic imaging) and persistent physical association (fluorescent in situ hybridization). Algal Res 15:179–186. CrossRefGoogle Scholar
  5. 5.
    Dolinšek J, Goldschmidt F, Johnson DR (2016) Synthetic microbial ecology and the dynamic interplay between microbial genotypes. FEMS Microbiol Rev 40:961–979. CrossRefGoogle Scholar
  6. 6.
    Goers L, Freemont P, Polizzi KM (2014) Co-culture systems and technologies: taking synthetic biology to the next level. J Roy Soc Interface 11:20140065. CrossRefGoogle Scholar
  7. 7.
    Ramanan R, Kim B-H, Cho D-H, Oh H-M, Kim H-S (2016) Algae-bacteria interactions: evolution, ecology and emerging applications. Biotechnol Lett 34:14–29. Google Scholar
  8. 8.
    Kazamia E, Czesnick H, Van Nguyen TT, Croft MT, Sherwood E, Sasso S et al (2012) Mutualistic interactions between vitamin B12-dependent algae and heterotrophic bacteria exhibit regulation. Environ Microbiol 14:1466–1476. CrossRefGoogle Scholar
  9. 9.
    Xie B, Bishop S, Stessman D, Wright D, Spalding MH, Halverson LJ (2013) Chlamydomonas reinhardtii thermal tolerance enhancement mediated by a mutualistic interaction with vitamin B12-producing bacteria. ISME J 7:1544–1555. CrossRefGoogle Scholar
  10. 10.
    Zhou K, Qiao K, Edgar S, Stephanopoulos G (2015) Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat Biotechnol 33:377–385. CrossRefGoogle Scholar
  11. 11.
    Gonzalez LE, Bashan Y (2000) Growth promotion of the microalgae Chlorella vulgaris when coimmobilized and cocultured in alginated beads with the plant growth-promoting bacteria Azospirillum brasilense. Appl Environ Microbiol 66:1537–1541. Google Scholar
  12. 12.
    de-Bashan LE, Bashan Y (2008) Joint immobilization of plant growth-promoting bacteria and green microalgae in alginate beads as an experimental model for studying plant-bacterium interactions. Appl Environ Microb 74:6797–6802. CrossRefGoogle Scholar
  13. 13.
    de-Bashan LE, Schmid M, Rothballer M, Hartmann A, Bashan Y (2011) Cell-cell interaction in the eukaryote–prokaryote model of the microalgae Chlorella vulgaris and the bacterium Azospirillum brasilense immobilized in polymer beads. J Phycol 47:1350–1359. CrossRefGoogle Scholar
  14. 14.
    de-Bashan LE, Hernandez J-P, Bashan Y (2015) Interaction of Azospirillum spp. with microalgae; a basic eukaryotic–prokaryotic model and its biotechnological applications. In: Cassán FD, Okon Y, Creus CM (Eds) Handbook for Azospirillum. Technical issued and protocols, Springer International Publishing, Switzerland, pp 367–388Google Scholar
  15. 15.
    Lebsky VK, Gonzalez-Bashan LE, Bashan Y (2001) Ultrastructure of coinmmobilization of the microalgae Chlorella vulgaris with the plant growth-promoting bacterium Azospirillum brasilense and with its natural associative bacterium Phyllobacterium myrsinacearum in alginate beads. Can J Microbiol 47:1–8. CrossRefGoogle Scholar
  16. 16.
    de-Bashan LE, Bashan Y, Moreno M, Lebsky VK, Bustillos JJ (2002) Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlorella spp. when co-immobilized in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense. Can J Microbiol 48:514–521. CrossRefGoogle Scholar
  17. 17.
    de-Bashan LE, Magallon P, Antoun H, Bashan Y (2008) Role of glutamate dehydrogenase and glutamine synthetase in Chlorella vulgaris during assimilation of ammonium when jointly immobilized with the microalgae-growth-promoting bacterium Azospirillum brasilense. J Phycol 44:1188–1196. CrossRefGoogle Scholar
  18. 18.
    Choix FJ, de-Bashan LE, Bashan Y (2012) Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: I. autotrophic conditions. Enzyme Microb Tech 51:294–299. CrossRefGoogle Scholar
  19. 19.
    Leyva LA, Bashan Y, Mendoza A, de-Bashan LE (2014) Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense. Naturewissenschaften 101:819–830. CrossRefGoogle Scholar
  20. 20.
    Palacios OA, Bashan Y, Schmid M, Hartmann A, de-Bashan LE (2016a) Enhancement of thiamine release during synthetic mutualism between Chlorella sorokiniana and Azospirillum brasilense growing under stress conditions. J Appl Phycol 28:1521–1531. CrossRefGoogle Scholar
  21. 21.
    Palacios OA, Choix FC, Bashan Y, de-Bashan LE (2016b) Influence of tryptophan and indole-3-acetic acid on starch accumulation in synthetic mutualistic Chlorella sorokinianaAzospirillum brasilense system under heterotrophic conditions. Res Microbiol 167:367–379. CrossRefGoogle Scholar
  22. 22.
    de-Bashan LE, Moreno M, Hernandez JP, Bashan Y (2002) Removal of ammonium and phosphorous ions from synthetic wastewater by the microalgae Chlorella vulgaris coimmobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense. Water Res 36:2941–2948. CrossRefGoogle Scholar
  23. 23.
    de-Bashan LE, Hernandez JP, Morey T, Bashan Y (2004) Microalgae growth-promoting bacteria as “helpers” for microalgae: a novel approach for removing ammonium and phosphorous for municipal wastewater. Water Res 38:466–474. CrossRefGoogle Scholar
  24. 24.
    Choix FJ, López-Cisneros GC, Méndez-Acosta HO (2018) Azospirillum brasilense increase CO2 fixation on microalgae Scenedesmus obliquus, Chlorella vulgaris, and Chlamydomonas reinhardtii cultured on high CO2 concentrations. Microb Ecol.
  25. 25.
    Bashan Y (1986) Alginate beads as synthetic inoculant carriers for the slow release of bacteria that affect plant growth. Appl Environ Microb 51:1089–1098Google Scholar
  26. 26.
    Bashan Y, Lopez BR, Volker AR, Amavizca E, de-Bashan LE (2016) Chlorella sorokiniana (formerly C. vulgaris) UTEX 2714, a non-thermotolerant microalgae useful for biotechnological applications and as a reference strain. J Appl Phycol 28:113–121. CrossRefGoogle Scholar
  27. 27.
    Gonzalez LE, Cañizares RO, Baena S (1997) Efficiency of ammonia and phosphorous removal from a Colombian agroindustrial wastewater by the microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresour Technol 60:259–262. CrossRefGoogle Scholar
  28. 28.
    Bashan Y, Trejo A, de-Bashan LE (2011) Development of two culture media for mass cultivation of Azospirillum spp. and for production of inoculants to enhance plant growth. Biol Fertil Soils 47:963–969. CrossRefGoogle Scholar
  29. 29.
    de-Bashan LE, Bashan Y (2010) Immobilized microalgae for removing pollutants: review of practical aspects. Bioresour Technol 101:1611–1627. CrossRefGoogle Scholar
  30. 30.
    Bashan Y, Holguin G, Lifshitz R (1993) Isolation and characterization of plant growth-promoting rhizobacteria. In: Glick BR, Thompson JE (eds) Methods in plant molecular biology and biotechnology. CRC Press, Boca Raton, pp 331–345Google Scholar
  31. 31.
    Chrzanowski TH, Crotty RD, Hubbard JG, Welch RP (1984) Applicability of fluorescein diacetate method of detecting active bacteria in freshwater. Microb Ecol 10:179–185. CrossRefGoogle Scholar
  32. 32.
    Zakharova EA, Shcherbakov AA, Brudnik VV, Skipko NG, Bulkhin NS, Ignatov VV (1999) Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry. Eur J Biochem 259:572–576. CrossRefGoogle Scholar
  33. 33.
    Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356. CrossRefGoogle Scholar
  34. 34.
    Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microb 56:1919–1925Google Scholar
  35. 35.
    Daims H, Brühl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444. CrossRefGoogle Scholar
  36. 36.
    Stoffels M, Castellanos T, Hartmann A (2001) Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster. Syst Appl Microbiol 24:83–97CrossRefGoogle Scholar
  37. 37.
    Bronstein JL (1994) Conditional outcomes in mutualistic interactions. Trends Ecol Evol 9:214–217. CrossRefGoogle Scholar
  38. 38.
    Krediet CJ, Ritchie KB, Paul VJ, Teplitski M (2013) Coral-associated micro-organisms and their roles in promoting coral health and thwarting diseases. P Roy Soc B 280:20122328. CrossRefGoogle Scholar
  39. 39.
    de Mazancourt C, Loreau M, Dieckmann U (2005) Understanding mutualism when there is adaptation to the partner. J Ecol 93:305–314. CrossRefGoogle Scholar
  40. 40.
    Wäckers FL, Alberola JS, Garcia-Marí F, Pekas A (2017) Attract and distract: manipulation of a food-mediated protective mutualism enhances natural pest control. Agric Ecosyst Environ 246:168–174. CrossRefGoogle Scholar
  41. 41.
    Zuroff TR, Xiques SB, Curtis WR (2013) Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture. Biotechnol Biofuels 6:59. CrossRefGoogle Scholar
  42. 42.
    Palacios OA, Gomez-Anduro G, Bashan Y, de-Bashan LE (2016c) Tryptophan, thiamine and indole-3-acetic acid exchange between Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense. FEMS Microbiol Ecol 92:fiw077. CrossRefGoogle Scholar
  43. 43.
    Tarrand JJ, Krieg NR, Döbereiner J (1978) A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerink) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 24:967–980. CrossRefGoogle Scholar
  44. 44.
    Bashan Y, Levanony H, Klein E (1986) Evidence for a weak active external adsorption of Azospirillum brasilense Cd to wheat roots. J Gen Microbiol 132:3069–3073. Google Scholar
  45. 45.
    Pereg L, de-Bashan LE, Bashan Y (2016) Assessment of affinity and specificity of Azospirillum for plants. Plant Soil 399:389–414. CrossRefGoogle Scholar
  46. 46.
    Michiels KW, Croes CL, Vanderleyden J (1991) Two different modes of attachment of Azospirillum brasilense Sp7 to wheat roots. J Gen Microbiol 137:2241–2246. CrossRefGoogle Scholar
  47. 47.
    Schnurr PJ, Allen DG (2015) Factors affecting algae biofilm growth and lipid production: a review. Renew Sust Energ Rev 52:418–429. CrossRefGoogle Scholar
  48. 48.
    Shen Y, Zhang H, Xu X, Lin X (2015) Biofilm formation and lipid accumulation of attached culture of Botryococcus braunii. Bioprocess Biosyst Eng 38:481–488. CrossRefGoogle Scholar
  49. 49.
    Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant-growth? – a critical assessment. Adv Agron 108:77–136. CrossRefGoogle Scholar
  50. 50.
    Zhu S, Huang W, Xu J, Wang Z, Xu J, Yuan Z (2014) Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga Chlorella zofingiensis. Bioresour Technol 152:292–298. CrossRefGoogle Scholar
  51. 51.
    Park J-J, Wang H, Gargouri M, Deshpande RR, Skepper JN, Holguin O et al (2015) The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis. Plant Biol 81:611–624Google Scholar
  52. 52.
    Zhang X, Reed JL (2014) Adaptive evolution of synthetic cooperating communities improves growth performance. PLoS One 9:e108297. CrossRefGoogle Scholar
  53. 53.
    Klitgord N, Segrè D (2010) Environments that induce synthetic microbial ecosystems. PLoS Comput Biol 6:e1001002. CrossRefGoogle Scholar
  54. 54.
    Morris BEL, Henneberger R, Huber H, Moissl-Eichinger C (2013) Microbial syntrophy: interaction for the common good. FEMS Microbiol Rev 37:384–406. CrossRefGoogle Scholar
  55. 55.
    Choix FJ, Bashan Y, Mendoza A, de-Bashan LE (2014) Enhanced activity of ADP glucose pyrophosphorylase and formation of starch induced by Azospirillum brasilense in Chlorella vulgaris. J Biotechnol 177:22–34. CrossRefGoogle Scholar
  56. 56.
    Wu H, Miao X (2014) Biodiesel quality and biochemical changes of microalgae Chlorella pyrenoidosa and Scenedesmus obliquus in response to nitrate levels. Bioresour Technol 170:421–427. CrossRefGoogle Scholar
  57. 57.
    González-Fernández C, Ballesteros M (2012) Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnol Adv 30:1655–1661. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Oskar A. Palacios
    • 1
    • 2
  • Blanca R. Lopez
    • 1
    • 2
  • Yoav Bashan
    • 1
    • 2
    • 3
  • Luz E. de -Bashan
    • 1
    • 2
    • 3
    Email author
  1. 1.Environmental Microbiology GroupNorthwestern Center for Biological Research (CIBNOR)La PazMexico
  2. 2.The Bashan Institute of ScienceAuburnUSA
  3. 3.Department of Entomology and Plant PathologyAuburn UniversityAuburnUSA

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