Journal of Applied Phycology

, Volume 31, Issue 2, pp 1047–1056 | Cite as

Development of a nitrogen-fixing cyanobacterial consortium for surface stabilization of agricultural soils

  • Xin Peng
  • Mary Ann BrunsEmail author


Cyanobacteria are recognized as important colonizers and protectors of soil surfaces, particularly in biological soil crusts (BSCs) of arid and semiarid regions. Comparatively, little attention has been paid to the growth of cyanobacteria, algae, and moss on agricultural soils in more humid regions like eastern North America. Growth of soil surface consortia (SSCs) in agricultural fields is typically rapid and ephemeral yet recurrent, thereby differing from classical BSCs of semiarid regions and algal mats of aquatic systems. Naturally occurring or intentionally applied cyanobacteria to agricultural soils could thus provide renewable sources of carbon and nitrogen (N) and a means to improve soil resilience. Here, we describe a soil microcosm-based protocol to assess cyanobacteria for their ability to form SSCs using three criteria: reliability of serial transfers in N-free culture media, robust growth in soil microcosms, and resistance to detachment from soil particles subjected to water flushing. Screening of 100 enrichment cultures from local SSCs yielded three that exhibited robust growth on N-free solid media and consistent microscopic appearance as filamentous, heterocystous cyanobacteria. One enrichment (DG1) grew reliably in liquid N-free media and was selected for comparison with pure cultures of commercial strains of other heterocystous cyanobacteria. Growth and biomass density of DG1 and commercial strains on moist, N-limited soils were tracked using chlorophyll a measurements and water flush tests. Anabaena spp. grew faster on soil surfaces, but their 80-day SSCs did not adhere to soil as well as DG1 or Nostoc spp. in water flush tests. The ability of DG1 and Nostoc spp. to produce flocculated growth in liquid culture appeared to be associated with greater soil adherence. While Nostoc spp. formed stable SSCs in soil microcosms, they exhibited lower growth rates and biomass densities than DG1. Attempts to purify the cyanobacterial strain(s) from other bacteria in the DG1 enrichment were unsuccessful. Based on initial metagenomics analysis, the DG1 enrichment was a consortium containing at least six other bacterial genotypes but dominated by one or more closely related strains of Cylindrospermum spp. (Nostocaceae). The presence of bacterial associates did not interfere with rapid growth and high biomass density in soil microcosms, as well as SSC stability in water flush tests. The artificial SSCs formed by DG1 showed good potential for use as a renewable N source for agroecosystems.


Cyanobacteria N2 fixation Agricultural soil Biological soil crust Surface consortium Cylindrospermum Nostocaceae 



Constructive suggestions by two anonymous reviewers are gratefully acknowledged. We thank Dr. Yemin Lan, School of Biomedical Engineering, and Dr. Gail Rosen, Department of Electrical and Computer Engineering, of Drexel University, for the bioinformatics assistance. We thank Dr. Don Bryant, Penn State Dept. of Biochemistry and Molecular Biology, for the advice on culturing, and Dr. Richard Macur, Center for Biofilm Engineering at Montana State University, for his insightful comments.


This research was funded by an internal Research Applications for Innovation grant from the College of Agricultural Sciences of The Pennsylvania State University. The USDA-NIFA Hatch project #1003466 provides salary support for MAB.

Supplementary material

10811_2018_1597_MOESM1_ESM.docx (8.8 mb)
ESM 1 (DOCX 9015 kb)


  1. Adams DG (2000) Heterocyst formation in cyanobacteria. Curr Opin Microbiol 3:618–624CrossRefGoogle Scholar
  2. Barak P, Jobe B, Krueger A, Peterson L, Laird D (1997) Effects of long-term soil acidification due to nitrogen fertilizer inputs in Wisconsin. Plant Soil 197:61–69CrossRefGoogle Scholar
  3. Belnap J, Lange O (2001) Biological soil crusts: structure, function and management. Springer, New YorkGoogle Scholar
  4. Casida LE (1982) Ensifer adhaerens gen. nov., sp. nov.: a bacterial predator of bacteria in soil. Int J Systemat Bacteriol 32:339–345CrossRefGoogle Scholar
  5. Castillo-Gonzalez H, Bruns MA (2001) Cyanobacterial diversity in agricultural soils, Ninth International Symposium in Microbial Ecology, Amsterdam, The NetherlandsGoogle Scholar
  6. Chen L, Xie Z, Hu C, Li D, Wang G, Liu Y (2006) Man-made desert algal crusts as affected by environmental factors in Inner Mongolia, China. J Arid Environ 67:521–527CrossRefGoogle Scholar
  7. Chien, S.H., Prochnow, L.I., Cantarella, H. 2009. Recent developments of fertilizer production and use to improve nutrient efficiency and minimize environmental impacts, In: Donald, L.S. (Ed.), Advances in agronomy. Academic Press, NY pp. 267–322Google Scholar
  8. Chittapun S, Limbipichai S, Amnuaysin N, Boonkerd R, Charoensook M (2018) Effects of using cyanobacteria and fertilizer on growth and yield of rice, Pathum Thani I: a pot experiment. J Appl Phycol 30:79–85CrossRefGoogle Scholar
  9. Choudhury ATMA, Kennedy IR (2004) Prospects and potentials for systems of biological nitrogen fixation in sustainable rice production. Biol Fertil Soils 39:219–227CrossRefGoogle Scholar
  10. Cohen Y, Gurevitz M (2006) The cyanobacteria—ecology, physiology, and molecular genetics, in: M. Dworkin et al. (eds.), The prokaryotes: an evolving electronic resource for the microbiological community, 3rd ed., [1st online ed.] Vol. 4, Springer, New York. pp. 1074–1098Google Scholar
  11. Dexter J, Dziga D, Lv J, Zhu J, Strzalka W, Maksylewicz A, Maroszek M, Marek S, Fu P (2018) Heterologous expression of mlrA in a phototrophic host—engineering cyanobacteria to degrade microcystins. Environ Pollut 237:926–935CrossRefGoogle Scholar
  12. Elbert W, Weber B, Burrows S, Steinkamp J, Budel B, Andreae MO, Poschl U (2012) Contribution of cryptogamic covers to the global cycles of carbon and nitrogen. Nat Geosci 5:459–462CrossRefGoogle Scholar
  13. Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai Z, Freney JR, Martinelli LA, Seitzinger SP, Sutton MA (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320:889–892CrossRefGoogle Scholar
  14. Garcia-Pichel F, Wojciechowski MF (2009) The evolution of a capacity to build supra-cellular ropes enabled filamentous cyanobacteria to colonize highly erodible substrates. PLoS One 4:e7801CrossRefGoogle Scholar
  15. Gardner JB, Drinkwater LE (2009) The fate of nitrogen in grain cropping systems: a meta-analysis of 15N field experiments. Ecol Appl 19:2167–2184CrossRefGoogle Scholar
  16. Gruber N, Galloway JN (2008) An earth-system perspective of the global nitrogen cycle. Nature 451:293–296CrossRefGoogle Scholar
  17. Hashem MA (2001) Problems and prospects of cyanobacterial biofertilizer for rice cultivation. Funct Plant Biol 28:881–888CrossRefGoogle Scholar
  18. Hegazi AZ, Mostafa SSM, Ahmed HMI (2010) Influence of different cyanobacterial application methods on growth and seed production of common bean under various levels of mineral nitrogen fertilization. Nat Sci 8:183–194Google Scholar
  19. Jha MN, Prasad AN (2006) Efficacy of new inexpensive cyanobacterial biofertilizer including its shelf-life. World J Microbiol Biotechnol 22:73–79CrossRefGoogle Scholar
  20. Johansen, J.R., Bohunická, M., Lukešová, A., Hrčková, K., Vaccarino, M.A., Chesarino, N.M 2014. Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae, Cyanobacteria), with descriptions of three new species. J Phycol 50:187–202Google Scholar
  21. Karthikeyan N, Prasanna R, Nain L, Kaushik BD (2007) Evaluating the potential of plant growth promoting cyanobacteria as inoculants for wheat. Eur J Soil Biol 43:23–30CrossRefGoogle Scholar
  22. KleinJan H, Jeanthon C, Boyen C, Dittami S (2017) Exploring the cultivable Ectocarpus microbiome. Front Microbiol 8:2456CrossRefGoogle Scholar
  23. Komárek J, Zapomelova E, Hindak F (2010) Cronbergia gen. nov., a new cyanobacterial genus (Cyanophyta) with a special strategy of heterocyte formation. Cryptogam Algol 31:321–334Google Scholar
  24. Lan S, Wu L, Zhang D, Hu C (2012) Successional stages of biological soil crusts and their microstructure variability in Shapotou region (China). Environ Earth Sci 65:77–88CrossRefGoogle Scholar
  25. Langhans TM, Storm C, Schwabe A (2009) Community assembly of biological soil crusts of different successional stages in a temperate sand ecosystem, as assessed by direct determination and enrichment techniques. Microb Ecol 58:394–407CrossRefGoogle Scholar
  26. Lupton FS, Marshall KC (1981) Specific adhesion of bacteria to heterocysts of Anabaena spp. and its ecological significance. Appl Environ Microbiol 42:1085–1092Google Scholar
  27. Maqubela M, Mnkeni P, Issa OM, Pardo M, D’Acqui L (2009) Nostoc cyanobacterial inoculation in South African agricultural soils enhances soil structure, fertility, and maize growth. Plant Soil 315:79–92CrossRefGoogle Scholar
  28. Metting FB Jr (1996) Biodiversity and application of microalgae. J Indust Microbiol 17:477–489Google Scholar
  29. Mi-Kyeong K, Park H, Oh T-J (2013) Antioxidant properties of various microorganisms isolated from Arctic lichen Stereocaulon spp. Korean J Microbiol Biotechnol 41:350–357CrossRefGoogle Scholar
  30. Mishra U, Pabbi S (2004) Cyanobacteria: a potential biofertilizer for rice. Resonance 9:6–10CrossRefGoogle Scholar
  31. Nausch M (1996) Microbial activities on Trichdesmium colonies. Marine Ecol Prog Series 141:173–181Google Scholar
  32. Nayak S, Prasanna R, Pabby A, Dominic TK, Singh PK (2004) Effect of urea, blue green algae and Azolla on nitrogen fixation and chlorophyll accumulation in soil under rice. Biol Fertil Soils 40:67–72CrossRefGoogle Scholar
  33. Paerl HW (1977) Role of heterotrophic bacteria in promoting N2 fixation by Anabaena in aquatic habitats. Microb Ecol 4:215–231Google Scholar
  34. Paerl HW, Priscu JC, Brawner DL (1989) Immunochemical localization of nitrogenase in marine Trichodesmium aggregates: relationship to N2 fixation potential. Appl Environ Microbiol 55:2965–2975Google Scholar
  35. Pagnier I, Croce O, Robert C, Raoult D, La Scola B (2012) Genome sequence of Reyranella massiliensis, a bacterium associated with amoebae. J Bacteriol 194:5608Google Scholar
  36. Pankratova E (2006) Functioning of cyanobacteria in soil ecosystems. Eurasian Soil Sci 39:S118–S127CrossRefGoogle Scholar
  37. Peng Y, Leung HC, Yiu S-M, Chin FY (2012) IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28:1420–1428CrossRefGoogle Scholar
  38. Pereira I, Ortega R, Barrientos L, Moya M, Reyes G, Kramm V (2008) Development of a biofertilizer based on filamentous nitrogen-fixing cyanobacteria for rice crops in Chile. J Appl Phycol 21:135–144CrossRefGoogle Scholar
  39. Pointing SB, Belnap J (2012) Microbial colonization and controls in dryland systems. Nat Rev Microbiol 10:551–562CrossRefGoogle Scholar
  40. Prasanna R, Chaudhary V, Gupta V, Babu S, Kumar A, Singh R, Shivay YS, Nain L (2013) Cyanobacteria mediated plant growth promotion and bioprotection against Fusarium wilt in tomato. Eur J Plant Pathol 136:337–353CrossRefGoogle Scholar
  41. Prasanna R, Jaiswal P, Nayak S, Sood A, Kaushik BD (2009) Cyanobacterial diversity in the rhizosphere of rice and its ecological significance. Indian J Microbiol 49:89–97CrossRefGoogle Scholar
  42. Prasanna R, Nayak S (2007) Influence of diverse rice soil ecologies on cyanobacterial diversity and abundance. Wetl Ecol Manag 15:127–134CrossRefGoogle Scholar
  43. Prasanna R, Singh RN, Joshi M, Madhan K, Pal RK, Nain L (2011) Monitoring the biofertilizing potential and establishment of inoculated cyanobacteria in soil using physiological and molecular markers. J Appl Phycol 23:301–308CrossRefGoogle Scholar
  44. Roger PA, Ladha JK (1992) Biological N2 fixation in wetland rice fields: estimation and contribution to nitrogen balance. In: Ladha JK, George T, Bohlool BB (eds) Biological nitrogen fixation for sustainable agriculture. Springer, Dordrecht, pp 41–55CrossRefGoogle Scholar
  45. Rossi F, Li H, Liu Y, De Philippis R (2017) Cyanobacterial inoculation (cyanobacterisation): perspectives for the development of a standardized multifunctional technology for soil fertilization and desertification reversal. Earth-Sci Rev 171:28–43CrossRefGoogle Scholar
  46. Saadatnia H, Riahi H (2009) Cyanobacteria from paddy fields in Iran as a biofertilizer in rice plants. Plant Soil Environ 55:207–212CrossRefGoogle Scholar
  47. Salomon PS, Janson S, Granéli E (2003) Molecular identification of bacteria associated with filaments of Nodularia spumigena and their effect on the cyanobacterial growth. Harmful Algae 2:261–272Google Scholar
  48. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt M, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87CrossRefGoogle Scholar
  49. Schindler D, Hecky R (2009) Eutrophication: more nitrogen data needed. Science 324:721–722CrossRefGoogle Scholar
  50. Segata N, Börnigen D, Morgan XC, Huttenhower C (2013) PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nature Commn 4:1–11Google Scholar
  51. Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla C,A, Cai F, Tandeau de Marsac N, Rippka R, Herdman M, Sivonen K, Coursin T, Laurent T, Goodwin L, Nolan M, Davenport KW, Han CS, Rubin EM, Eisen JA, Woyke T, Gugger M, Kerfeld CA (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci 110:1053–1058CrossRefGoogle Scholar
  52. Silva PG, Silva dJH (2013) Biomass production of Tolypothrix tenuis as a basic component of a cyanobacterial biofertilizer. J Appl Phycol 25:1729–1736CrossRefGoogle Scholar
  53. Singh JS, Pandey VC, Singh D (2011) Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agricult Ecosyst Environ 140:339–353CrossRefGoogle Scholar
  54. Singh, R.N., 1961. Role of blue-green algae in nitrogen economy of Indian agriculture. Indian Council of Agricultural Research, New DelhiGoogle Scholar
  55. Soil Survey Staff, 2010. Keys to soil taxonomy, 11th ed. USDA-Natural Resources Conservation Service, Washington, DCGoogle Scholar
  56. Starks TL, Shubert LE (1982) Colonization and succession of algae and soil-algal interactions associated with disturbed areas. J Phycol 18:99–107CrossRefGoogle Scholar
  57. Swarnalakshmi K, Prasanna R, Kumar A, Pattnaik S, Chakravarty K, Shivay YS, Singh R, Saxena AK (2013) Evaluating the influence of novel cyanobacterial biofilmed biofertilizers on soil fertility and plant nutrition in wheat. Eur J Soil Biol 55:107–116CrossRefGoogle Scholar
  58. Tindall BJ (2008) The genus name Sinorhizobium Chen et al. 1988 is a later synonym of Ensifer Casida 1982 and is not conserved over the latter genus name, and the species name ‘Sinorhizobium adhaerens’ is not validly published. Opinion 84. Int J Systemat Evol Microbiol 58:1973CrossRefGoogle Scholar
  59. Toledo G, Bashan Y, Soeldner A (1995) In vitro colonization and increase in nitrogen fixation of seedling roots of black mangrove inoculated by a filamentous cyanobacteria. Can J Microbiol 41:1012–1020CrossRefGoogle Scholar
  60. UNESCO (1966) Determination of photosynthetic pigments in seawater. In: Monographs on oceanographic methodology. United Nations Educational, Scientific and Cultural Organization, Place de Fontenoy, France pp. 15–16Google Scholar
  61. UTEX (2016) BG-11(-N) Medium .
  62. Vaishampayan A, Sinha R, Hader D-P, Dey T, Gupta A, Bhan U, Rao A (2001) Cyanobacterial biofertilizers in rice agriculture. Bot Rev 67:453–516CrossRefGoogle Scholar
  63. Wang W, Liu Y, Li D, Hu C, Rao B (2009) Feasibility of cyanobacterial inoculation for biological soil crusts formation in desert area. Soil Biol Biochem 41:926–929CrossRefGoogle Scholar
  64. Weber B, Wu D, Tamm A, Ruckteschler N, Rodríguez-Caballero E, Steinkamp J, Meusel H, Elbert W, Behrendt T, Sörgel M (2015) Biological soil crusts accelerate the nitrogen cycle through large NO and HONO emissions in drylands. Proc Nat Acad Sci 112:15384–15389CrossRefGoogle Scholar
  65. Wu Y-W, Tang Y-H, Tringe SG, Simmons BA, Singer SW (2014) MaxBin: an automated binning method to recover individual genomes from metagenomes using an expectation-maximization algorithm. Microbiome 2:1CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Intercollege Graduate Degree Program in EcologyThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of Ecosystem Science and ManagementThe Pennsylvania State UniversityUniversity ParkUSA

Personalised recommendations