Bioprocess and Biosystems Engineering

, Volume 41, Issue 9, pp 1247–1259 | Cite as

Development of a cost-effective production process for Halomonas levan

  • Burak Adnan Erkorkmaz
  • Onur Kırtel
  • Özlem Ateş Duru
  • Ebru Toksoy Öner
Research Paper


Levan polysaccharide is an industrially important natural polymer with unique properties and diverse high-value applications. However, current bottlenecks associated with its large-scale production need to be overcome by innovative approaches leading to economically viable processes. Besides many mesophilic levan producers, halophilic Halomonas smyrnensis cultures hold distinctive industrial potential and, for the first time with this study, the advantage of halophilicity is used and conditions for non-sterile levan production were optimized. Levan productivity of Halomonas cultures in medium containing industrial sucrose from sugar beet and food industry by-product syrup, a total of ten sea, lake and rock salt samples from four natural salterns, as well as three different industrial-grade boron compounds were compared and the most suitable low-cost substitutes for sucrose, salt and boron were specified. Then, the effects of pH control, non-sterile conditions and different bioreactor modes (batch and fed-batch) were investigated. The development of a cost-effective production process was achieved with the highest yield (18.06 g/L) reported so far on this microbial system, as well as the highest theoretical bioconversion efficiency ever reported for levan-producing suspension cultures. Structural integrity and biocompatibility of the final product were also verified in vitro.


Exopolysaccharide Levan Halomonas smyrnensis Microbial bioprocess Cost-effective production 



The authors greatly appreciate the technical support provided by Prof Mehmet S. Eroglu (Marmara University, Turkey) for the chemical characterization studies. This work was financially supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (Grant number: 114M239).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Öner ET (2013) Microbial production of extracellular polysaccharides from biomass. Pretreatment techniques for biofuels and biorefineries. Springer, Berlin, pp 35–56CrossRefGoogle Scholar
  2. 2.
    Adamberg K, Tomson K, Talve T, Pudova K, Puurand M, Visnapuu T, Alamäe T, Adamberg S (2015) Levan enhances associated growth of Bacteroides, Escherichia, Streptococcus and Faecalibacterium in fecal microbiota. PLoS One 10(12):e0144042CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Visnapuu T, Mardo K, Alamaee T (2015) Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo-and polysaccharides. New Biotechnol 32(6):597–605CrossRefGoogle Scholar
  4. 4.
    Adamberg S, Tomson K, Vija H, Puurand M, Kabanova N, Visnapuu T, Jõgi E, Alamäe T, Adamberg K (2014) Degradation of fructans and production of propionic acid by Bacteroides thetaiotaomicron are enhanced by the shortage of amino acids. Front Nutr 1:21CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hamdy AA, Elattal NA, Amin MA, Ali AE, Mansour NM, Awad GE, Farrag ARH, Esawy MA (2018) In vivo assessment of possible probiotic properties of Bacillus subtilis and prebiotic properties of levan. Biocatal Agric Biotechnol 13:190–197Google Scholar
  6. 6.
    Feng J, Gu Y, Quan Y, Zhang W, Cao M, Gao W, Song C, Yang C, Wang S (2015) Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Sci Rep 5:13814CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Feng J, Gu Y, Han L, Bi K, Quan Y, Yang C, Zhang W, Cao M, Wang S, Gao W (2015) Construction of a Bacillus amyloliquefaciens strain for high purity levan production. FEMS Microbiol Lett 362(11):fnv079CrossRefPubMedGoogle Scholar
  8. 8.
    Kucukasik F, Kazak H, Guney D, Finore I, Poli A, Yenigun O, Nicolaus B, Oner ET (2011) Molasses as fermentation substrate for levan production by Halomonas sp. Appl Microbiol Biotechnol 89(6):1729–1740. CrossRefPubMedGoogle Scholar
  9. 9.
    Sarilmiser HK, Ates O, Ozdemir G, Arga KY, Oner ET (2015) Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6 T. J Biosci Bioeng 119(4):455–463CrossRefPubMedGoogle Scholar
  10. 10.
    Poli A, Kazak H, Gürleyendağ B, Tommonaro G, Pieretti G, Öner ET, Nicolaus B (2009) High level synthesis of levan by a novel Halomonas species growing on defined media. Carbohyd Polym 78(4):651–657. CrossRefGoogle Scholar
  11. 11.
    Sarilmiser HK, Oner ET (2014) Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis AAD6 T. Biochem Eng J 92:28–34CrossRefGoogle Scholar
  12. 12.
    Erginer M, Akcay A, Coskunkan B, Morova T, Rende D, Bucak S, Baysal N, Ozisik R, Eroglu MS, Agirbasli M (2016) Sulfated levan from Halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications. Carbohyd Polym 149:289–296CrossRefGoogle Scholar
  13. 13.
    Sam S, Kucukasik F, Yenigun O, Nicolaus B, Oner ET, Yukselen MA (2011) Flocculating performances of exopolysaccharides produced by a halophilic bacterial strain cultivated on agro-industrial waste. Bioresour Technol 102(2):1788–1794CrossRefPubMedGoogle Scholar
  14. 14.
    Sezer AD, Kazak H, Öner ET, Akbuğa J (2011) Levan-based nanocarrier system for peptide and protein drug delivery: optimization and influence of experimental parameters on the nanoparticle characteristics. Carbohyd Polym 84(1):358–363CrossRefGoogle Scholar
  15. 15.
    Sezer AD, Kazak Sarilmiser H, Rayaman E, Cevikbas A, Oner ET, Akbuga J (2015) Development and characterization of vancomycin-loaded levan-based microparticular system for drug delivery. Pharm Dev Technol 22:1–8. CrossRefGoogle Scholar
  16. 16.
    Axente E, Sima F, Sima LE, Erginer M, Eroglu MS, Serban N, Ristoscu C, Petrescu SM, Oner ET, Mihailescu IN (2014) Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts. Biofabrication 6(3):035010CrossRefPubMedGoogle Scholar
  17. 17.
    Sima F, Mutlu EC, Eroglu MS, Sima LE, Serban N, Ristoscu C, Petrescu SM, Oner ET, Mihailescu IN (2011) Levan nanostructured thin films by MAPLE assembling. Biomacromolecules 12(6):2251–2256. CrossRefPubMedGoogle Scholar
  18. 18.
    Sima F, Axente E, Sima L, Tuyel U, Eroglu M, Serban N, Ristoscu C, Petrescu S, Oner ET, Mihailescu I (2012) Combinatorial matrix-assisted pulsed laser evaporation: single-step synthesis of biopolymer compositional gradient thin film assemblies. Appl Phys Lett 101(23):233705CrossRefGoogle Scholar
  19. 19.
    Costa RR, Neto AI, Calgeris I, Correia CR, Pinho AC, Fonseca J, Öner ET, Mano JF (2013) Adhesive nanostructured multilayer films using a bacterial exopolysaccharide for biomedical applications. J Mater Chem B 1(18):2367–2374CrossRefGoogle Scholar
  20. 20.
    Bostan MS, Mutlu EC, Kazak H, Keskin SS, Oner ET, Eroglu MS (2014) Comprehensive characterization of chitosan/PEO/levan ternary blend films. Carbohyd Polym 102:993–1000CrossRefGoogle Scholar
  21. 21.
    Osman A, Oner ET, Eroglu MS (2017) Novel levan and pNIPA temperature sensitive hydrogels for 5-ASA controlled release. Carbohyd Polym 165:61–70CrossRefGoogle Scholar
  22. 22.
    Gomes TD, Caridade SG, Sousa MP, Azevedo S, Kandur MY, Öner ET, Alves NM, Mano JF (2018) Adhesive free-standing multilayer films containing sulfated levan for biomedical applications. Acta Biomater 69:183–195CrossRefPubMedGoogle Scholar
  23. 23.
    Avsar G, Agirbasli D, Agirbasli MA, Gunduz O, Oner ET (2018) Levan based fibrous scaffolds electrospun via co-axial and, single-needle techniques for tissue engineering applications. Carbohyd Polym 193:316–325CrossRefGoogle Scholar
  24. 24.
    Öner ET, Hernández L, Combie J (2016) Review of levan polysaccharide: from a century of past experiences to future prospects. Biotechnol Adv 34(5):827–844CrossRefPubMedGoogle Scholar
  25. 25.
    Meyer H-P, Minas W, Schmidhalter D (2017) Industrial-scale fermentation. In: Wittmann C, Liao JC (eds) Industrial biotechnology. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 1–53. CrossRefGoogle Scholar
  26. 26.
    Moussa TA, Al-Qaysi SA, Thabit ZA, Kadhem SB (2017) Microbial levan from Brachybacterium phenoliresistens: characterization and enhancement of production. Process Biochem 57:9–15CrossRefGoogle Scholar
  27. 27.
    Abou-Taleb KA, Abdel-Monem MO, Yassin MH, Draz AA (2015) Production, purification and characterization of levan polymer from Bacillus lentus V8 strain. Br Microbiol Res J 5(1):22–32CrossRefGoogle Scholar
  28. 28.
    Moosavi-Nasab M, Layegh B, Aminlari L, Hashemi MB (2010) Microbial production of levan using date syrup and investigation of its properties. World Acad Sci Eng Technol 44:1248–1254Google Scholar
  29. 29.
    Han Y, Watson M (1992) Production of microbial levan from sucrose, sugarcane juice and beet molasses. J Ind Microbiol Biotechnol 9(3):257–260Google Scholar
  30. 30.
    de Oliveira MR, da Silva RSSF., Buzato JB, Celligoi MAPC. (2007) Study of levan production by Zymomonas mobilis using regional low-cost carbohydrate sources. Biochem Eng J 37(2):177–183CrossRefGoogle Scholar
  31. 31.
    Ates O (2015) Systems biology of microbial exopolysaccharides production. Front Bioeng Biotechnol 3:200CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Ates O, Arga KY, Oner ET (2013) The stimulatory effect of mannitol on levan biosynthesis: lessons from metabolic systems analysis of Halomonas smyrnensis AAD6(T.). Biotechnol Prog 29(6):1386–1397. CrossRefPubMedGoogle Scholar
  33. 33.
    Aydin B, Ozer T, Oner ET, Arga KY (2018) The Genome-based metabolic systems engineering to boost levan production in a halophilic bacterial model. OMICS 22(3):198–209CrossRefPubMedGoogle Scholar
  34. 34.
    Katsube T, Tsurunaga Y, Sugiyama M, Furuno T, Yamasaki Y (2009) Effect of air-drying temperature on antioxidant capacity and stability of polyphenolic compounds in mulberry (Morus alba L.) leaves. Food Chem 113(4):964–969CrossRefGoogle Scholar
  35. 35.
    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(1–2):248–254CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhang Y, Zhang Z, Suzuki K, Maekawa T (2003) Uptake and mass balance of trace metals for methane producing bacteria. Biomass Bioenerg 25(4):427–433CrossRefGoogle Scholar
  37. 37.
    Patidar S, Tare V (2006) Effect of nutrients on biomass activity in degradation of sulfate laden organics. Process Biochem 41(2):489–495CrossRefGoogle Scholar
  38. 38.
    Müller B (2009) Impact of the bacterium Pseudomonas fluorescens and its genetic derivatives on vermiculite: effects on trace metals contents and clay mineralogical properties. Geoderma 153(1):94–103CrossRefGoogle Scholar
  39. 39.
    Liu Q, Yu S, Zhang T, Jiang B, Mu W (2017) Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii. Carbohyd Polym 157:1732–1740CrossRefGoogle Scholar
  40. 40.
    Ni D, Xu W, Bai Y, Zhang W, Zhang T, Mu W (2018) Biosynthesis of levan from sucrose using a thermostable levansucrase from Lactobacillus reuteri LTH5448. Int J Biol Macromol 113:29–37CrossRefPubMedGoogle Scholar
  41. 41.
    Saum SH, Müller V (2008) Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies. Saline Syst 4(1):4CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Da Silva JF, Williams RJP (2001) The biological chemistry of the elements: the inorganic chemistry of life. Oxford University Press, OxfordGoogle Scholar
  43. 43.
    Belghith KS, Dahech I, Belghith H, Mejdoub H (2012) Microbial production of levansucrase for synthesis of fructooligosaccharides and levan. Int J Biol Macromol 50(2):451–458CrossRefPubMedGoogle Scholar
  44. 44.
    González-Garcinuño Á, Tabernero A, Sánchez-Álvarez JM, Galán MA, del Valle EMM (2017) Effect of bacteria type and sucrose concentration on levan yield and its molecular weight. Microb Cell Fact 16(1):91CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Srikanth R, Siddartha G, Reddy CHS, Harish B, Ramaiah MJ, Uppuluri KB (2015) Antioxidant and anti-inflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization. Carbohyd Polym 123:8–16CrossRefGoogle Scholar
  46. 46.
    Han W-C, Byun S-H, Kim M-H, Sohn EH, Lim JD, Um BH, Kim CH, Kang SA, Jang K-H (2009) Production of lactosucrose from sucrose and lactose by a levansucrase from Zymomonas mobilis. J Microbiol Biotechnol 19(10):1153–1160PubMedGoogle Scholar
  47. 47.
    Szwengiel A, Czarnecka M, Czarnecki Z (2007) Levan synthesis during associated action of levansucrase and Candida cacaoi DSM 2226 yeast. Pol J Food Nutr Sci 57(4):433–440Google Scholar
  48. 48.
    Papenfort K, Bassler BL (2016) Quorum sensing signal-response systems in Gram-negative bacteria. Nat Rev Microbiol 14(9):576–588CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Giuseppina T, Roberto AG, Ebru TO, Barbara N (2015) Investigating the quorum sensing system in halophilic bacteria in halophiles. Springer, Cham, pp 189–207Google Scholar
  50. 50.
    Abbamondi GR, Suner S, Cutignano A, Grauso L, Nicolaus B, Oner ET, Tommonaro G (2016) Identification of N-hexadecanoyl-l-homoserine lactone (C16-AHL) as signal molecule in halophilic bacterium Halomonas smyrnensis AAD6. Ann Microbiol 66(3):1329–1333CrossRefGoogle Scholar
  51. 51.
    Şahin B, Çöl B, Güneş H (2017) Bacillus thuringiensis isolation from the environments of boron mines and effects of boric acid on bioactivity. Gazi Univ J Sci 30(1):223–234Google Scholar
  52. 52.
    Wu F-C, Chou S-Z, Shih L (2013) Factors affecting the production and molecular weight of levan of Bacillus subtilis natto in batch and fed-batch culture in fermenter. J Taiwan Inst Chem Eng 44(6):846–853CrossRefGoogle Scholar
  53. 53.
    Santos-Moriano P, Fernandez-Arrojo L, Poveda A, Jimenez-Barbero J, Ballesteros AO, Plou FJ (2015) Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions. J Mol Catal B Enzymat 119:18–25CrossRefGoogle Scholar
  54. 54.
    Ua-Arak T, Jakob F, Vogel RF (2017) Fermentation pH modulates the size distributions and functional properties of Gluconobacter albidus TMW 2.1191 levan. Front Microbiol 8(807):807. CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Runyon JR, Nilsson L, Ulmius M, Castro A, Ionescu R, Andersson C, Schmidt C (2014) Characterizing changes in levan physicochemical properties in different pH environments using asymmetric flow field-flow fractionation. Anal Bioanal Chem 406(6):1597–1605CrossRefPubMedGoogle Scholar
  56. 56.
    Kekez B, Gojgic-Cvijovic G, Jakovljevic D, Kojic JS, Markovic M, Beskoski V, Vrvic M (2015) High levan production by Bacillus licheniformis NS032 using ammonium chloride as the sole nitrogen source. Appl Biochem Biotechnol 175(6):3068–3083CrossRefPubMedGoogle Scholar
  57. 57.
    Arvidson SA, Rinehart BT, Gadala-Maria F (2006) Concentration regimes of solutions of levan polysaccharide from Bacillus sp. Carbohyd Polym 65(2):144–149CrossRefGoogle Scholar
  58. 58.
    Yoo S-H, Yoon EJ, Cha J, Lee HG (2004) Antitumor activity of levan polysaccharides from selected microorganisms. Int J Biol Macromol 34(1):37–41CrossRefPubMedGoogle Scholar
  59. 59.
    Chen Z, Wan C (2017) Non-sterile fermentations for the economical biochemical conversion of renewable feedstocks. Biotech Lett 39(12):1765–1777CrossRefGoogle Scholar
  60. 60.
    Litchfield CD (2011) Potential for industrial products from the halophilic Archaea. J Ind Microbiol Biotechnol 38(10):1635CrossRefPubMedGoogle Scholar
  61. 61.
    Yin J, Chen J-C, Wu Q, Chen G-Q (2015) Halophiles, coming stars for industrial biotechnology. Biotechnol Adv 33(7):1433–1442CrossRefPubMedGoogle Scholar
  62. 62.
    Chen G-Q, Jiang X-R (2018) Next generation industrial biotechnology based on extremophilic bacteria. Curr Opin Biotechnol 50:94–100CrossRefPubMedGoogle Scholar
  63. 63.
    Tan D, Xue Y-S, Aibaidula G, Chen G-Q (2011) Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour Technol 102(17):8130–8136CrossRefPubMedGoogle Scholar
  64. 64.
    Chen X, Yin J, Ye J, Zhang H, Che X, Ma Y, Li M, Wu L-P, Chen G-Q (2017) Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Bioresour Technol 244:534–541CrossRefPubMedGoogle Scholar
  65. 65.
    Yue H, Ling C, Yang T, Chen X, Chen Y, Deng H, Wu Q, Chen J, Chen G-Q (2014) A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates. Biotechnol Biofuels 7(1):1CrossRefGoogle Scholar
  66. 66.
    Assavasirijinda N, Ge D, Yu B, Xue Y, Ma Y (2016) Efficient fermentative production of polymer-grade d-lactate by an engineered alkaliphilic Bacillus sp. strain under non-sterile conditions. Microb Cell Fact 15(1):3CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Silbir S, Dagbagli S, Yegin S, Baysal T, Goksungur Y (2014) Levan production by Zymomonas mobilis in batch and continuous fermentation systems. Carbohyd Polym 99:454–461CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.IBSB-Industrial Biotechnology and Systems Biology Research Group, Department of BioengineeringMarmara UniversityIstanbulTurkey
  2. 2.Nişantaşı UniversityIstanbulTurkey

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