Abstract
Halophilic organisms are a novel attractive option as cell factories for the production of industrially valuable bioproducts. Halomonas elongata is the cell factory of choice for ectoine production, but its levan production has not been well researched. Based on this scientific motivation, in this study, we evaluated the chemical and biological properties of levan produced by the halophilic extremophile Halomonas elongata 153B (HeL). First, the central composite design was used to determine the optimal process variables for maximum levan biosynthesis. Then, the levan produced from HeL was purified, quantified, and chemically characterized with FTIR, 1H-NMR, and GPC analyses. This was followed by antioxidant, anti-inflammatory, antibiofilm, and antimicrobial activity tests to assess its biological activities as well as a cytotoxcity assay. Maximum levan yields of 5.13 ± 0.38 g/L were achieved after dialysis at the optimum levels of process variables. The 1H-NMR spectrum of HeL revealed characteristic signals. It showed a strong antioxidant activity of 67.88% and the best radical scavenger. At a concentration of 400 µg/mL, HeL showed the most anti-inflammatory efficacy. Also, at all indicated concentrations (250, 500, 750, and 1000 μg/mL) HeL, acted against biofilms formed by Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 11778, Candida albicans ATCC 10231. Furthermore, HeL displayed antimicrobial activities against all strains tested. Finally, HeL showed high Cell viability in all dosages and no cytotoxicity was observed. In light of these results, HeL may have high potential in the medical, pharmaceutical and dermo-cosmetics industries.
Graphical Abstract
Similar content being viewed by others
References
Enuh BM, Nural Yaman B, Tarzi C, Aytar Çelik P, Mutlu MB, Angione C (2022) Whole-genome sequencing and genome-scale metabolic modeling of Chromohalobacter canadensis 85B to explore its salt tolerance and biotechnological use. MicrobiologyOpen 11(5):e1328
Kekez BD, Gojgic-Cvijovic GD, Jakovljevic DM, Stefanovic Kojic JR, Markovic MD, Beskoski VP, Vrvic MM (2015) High levan production by Bacillus licheniformis NS032 using ammonium chloride as the sole nitrogen source. Biotechnol Appl Biochem 175:3068–3083
Zhang X, Lin Y, Chen GQ (2018) Halophiles as chassis for bioproduction. Adv Biosyst 2:1800088
Radchenkova N, Erginer Haskoylu M, Vassilev S, Yaşar Yıldız S, Boyadzhieva I, Toksoy Oner E, Kambourova M (2020) Improved exopolymer production by Chromohalobacter canadensis cultures for its potential cosmeceutical applications. Microorganisms 8:1935
Kazak Sarilmiser H, Ates O, Ozdemir G, Arga KY, Toksoy Oner E (2015) Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T. J Biosci Bioeng 119:455–463
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 37:177–183
Silbir S, Dagbagli S, Yegin S, Baysal T, Goksungur Y (2014) Levan production by Zymomonas mobilis in batch and continuous fermentation systems. Carbohydr Polym 99:454–461
Peng J, Xu W, Ni D, Zhang W, Zhang T, Guang C, Mu W (2018) Preparation of a novel water-soluble gel from Erwinia amylovora levan. Int J Biol Macromol 122:469–478
Veerapandiana B, Shanmugama SR, Varadhana S, Sarwareddyb KK, Manib KP, Ponnusamia V (2020) Levan production from sucrose using chicken feather peptone as a low cost supplemental nutrient source. Carbohydr Polym 227:115361
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 113:29–37
Ahmed KBA, Kalla D, Uppuluri KB, Anbazhagan V (2014) Green synthesis of silver and gold nanoparticles employing levan, a biopolymer from Acetobacter xylinum NCIM 2526 as a reducing agent and cap** agent. Carbohydr Polym 112:539–545
Hundschell CS, Jakob F, Wagemans AM (2020) Molecular weight dependent structure of the exopolysaccharide levan. Int J Biol Macromol 161:398–405
Corrigan AJ, Robyt JF (1979) Nature of the fructan of Streptococcus mutans OMZ176. Infect Immun 26:387–389
Jathore NR, Bule MV, Tilay AV, Annapure US (2012) Microbial levan from Pseudomonas fluorescens: characterization and medium optimization for enhanced production. Food Sci Biotechnol 21:1045–1053
Kosarsoy Agceli G, Cihangir N (2020) Nano-sized biopolymer levan: its antimicrobial, anti-biofilm and anti-cancer effects. Carbohydr Res 494:108068
Costa RR, NetoI AI, Calgeris I, Correia CR, Pinho ACM, Fonseca J, Toksoy Oner E, Mano JF (2013) Adhesive nanostructured multilayer films using a bacterial exopolysaccharide for biomedical applications. J Mater Chem B 1:2367–2374
Sima F, Mutlu EC, Eroglu MS, Sima LE, Serban N, Ristoscu C, Petrescu SM, Toksoy Oner E, Mihailescu IN (2011) Levan nanostructured thin films by MAPLE assembling. Biomacromolecules 12:2251–2256
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(21):1–10
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):e0144042
Porras-Domínguez JR, Ávila-Fernández Á, Miranda-Molina A, Rodríguez-Alegría ME, López-Munguía A (2015) Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight. Carbohydr Polym 132:338–344
Visnapuu T, Mardo K, Alamäe T (2015) Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides. New Biotechnol 32:597–605
Belghith KS, Dahech I, Hamden K, Feki A, Mejdoub H, Belghith H (2012) Hypolipidemic effect of diet supplementation with bacterial levan in cholesterol-fed rats. Int J Biol Macromol 50:1070–1074
Combie J, Toksoy Oner E (2018) From healing wounds to resorbable electronics, levan can fill bioadhesive roles in scores of markets. Bioinspir Biomim 14:011001
Tohme S, Hacıosmanoglu GG, Eroglu MS, Kasavi C, Genc S, Can ZS, Toksoy Oner E (2018) Halomonas smyrnensis as a cell factory for co-production of PHB and levan. Int J Biol Macromol 118:1238–1246
Erdal O, Kaplan-Turkoz B, Taştan O, Goksungur Y (2017) Levansucrase production by Zymomonas mobilis: optimization of process parameters and fructooligosaccharide production. J Food Biochem 41:3
Güngör G, Gedikli S, Toptaş Y, Akgün DE, Demirbilek M, Yazıhan N, Aytar Çelik P, Denkbaş EB, Çabuk A (2019) Bacterial hyaluronic acid production through an alternative extraction method and its characterization. J Chem Technol Biotechnol 94:1843–1852
Gedikli S, Çelik PA, Demirbilek M, Mutlu MB, Denkbaş EB, Çabuk A (2019) Experimental exploration of thermostable poly(β-hydroxybutyrates) by Geobacillus kaustophilus using Box–Behnken design. J Polym Environ 27:245–255
Gedikli S, Güngör G, Toptaş Y, Sezgin DE, Demirbilek M, Yazıhan N, Aytar Çelik P, Denkbaş EB, Bütün V, Çabuk A (2018) Optimization of hyaluronic acid production and its cytotoxicity and degradability characteristics. Prep Biochem Biotechnol 48:610–618
Goksungur Y, Uzunoğulları P, Dağbağlı S (2011) Optimization of pullulan production from hydrolysed potato starch waste by response surface methodology. Carbohydr Polym 83:1330–1337
Yoon S, Hong E, Kim S, Lee P, Kim M, Yang H, Ryu Y (2012) Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans. Bioprocess Biosyst Eng 35:167–172
Majumder A, Singh A, Goyal A (2009) Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization. Carbohydr Polym 75:150–156
Melo IR, Pimentel MF, Lopes CE, Calazans GMT (2007) Application of fractional factorial design to levan production by Zymomonas mobilis. Braz J Microbiol 38:45–51
Daoud L, Ben Ali M (2020) Chapter 5—halophilic microorganisms: ınteresting group of extremophiles with important applications in biotechnology and environment. In: Salwan R, Sharma V (eds) Physiological and biotechnological aspects of extremophiles. Elsevier, Amsterdam, pp 51–64
Cakmak H, Aytar Çelik P, Çınar S, Hosgün EZ, Mutlu MB, Çabuk A (2020) Levan production potentials from different hypersaline environments in Turkey. J Microbiol Biotechnol Food Sci 10:61–64
Cınar S, Mutlu MB (2016) Comparative analysis of prokaryotic diversity in solar satern in eastern Anatolia (Turkey). Extremophile 20:589–601
Kucukasik F, Kazak H, Guney D, Fınore I, Poli A, Yenıgun O, Nicolaus B, Toksoy Oner E (2011) Molasses as fermentation substrate for levan production by Halomonas sp. Appl Microbiol Biotechnol 89:1729–1740
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
Viikari L (1984) Formation of levan and sorbitol from sucrose by Zymomonas mobilis. Appl Microbiol Biotechnol 19:252–255
Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:695–702
Poli A, Kazak H, Gurleyendag B, Tommonaro G, Pieretti G, Toksoy Oner E, Nicolaus B (2009) High level synthesis of levan by a novel Halomonas species growing on defined media. Carbohydr Polym 78:651–657
Myers RH, Montgomery DC (1995) Response surface methodology, process and product optimization using designed experiments, 4th edn. Wiley, New York
Erkorkmaz BA, Kırtel O, Ates Duru O, Toksoy Oner E (2018) Development of a cost-effective production process for Halomonas levan. Bioprocess Biosyst Eng 41:1247–1259
Domżał-Kędzia M, Lewińska A, Jaromin A, Weselski M, Pluskota R, Łukaszewicz M (2019) Fermentation parameters and conditions affecting levan production and its potential applications in cosmetics. Bioorg Chem 93:102787
Garcıa EJ, Oldoni TLC, Alencar SM, Reis A, Loguercio AD, Grande RHM (2012) Antioxidant activity by DPPH assay of potential solutions to be applied on bleached teeth. Braz Dent J 23:22–27
Vidhyalakshmi R, Valli NC, Narendra KG, Sunkar S (2016) Bacillus circulans exopolysaccharide: production, characterization and bioactivities. Int J Biol Macromol 87:405–414
Mummaleti G, Sarma C, Kalakandan SK, Gazula H, Vignesh Sivanandham V, Anandharaj A (2022) Characterization of levan produced from coconut inflorescence sap using Bacillus subtilis and its application as a sweetener. J Food Sci Technol 154:112697
Tang HJ, Chen CC, Ko WC, Yu WL, Chiang SR, Chuang YC (2011) In vitro efficacy of antimicrobial agents against high-inoculum or biofilm embedded methicillin-resistant Staphylococcus aureus with vancomycin minimal inhibitory concentrations equal to 2 μg/ml (VA2-MRSA). Int J Antimicrob Agents 38:46–55
Teanpaisan R, Senapong S, Puripattanavong J (2014) In vitro antimicrobial and antibiofilm activity of Artocarpus lakoocha (Moraceae) extract against some oral pathogens. Trop J Pharm 13:1149–1155
CLSI Guidelines (2016) Performance standards for antimicrobial standards institute susceptibility testing, 26th edn. CLSI supplement M100S. Clinical and Laboratory, Wayne
Kumar P, Nagarajan A, Pradeep DU (2018) Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot095505
Mata JA, Béjar V, LIamas I, Arias S, Bressollier P, Tallon R (2006) Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res Microbiol 157:827–835
Permatasari NU, Ratnaningsih E, Hertad R (2018) The use of response surface method in optimization of levan production by heterologous expressed levansucrase from halophilic bacteria Bacillus licheniformis BK2. IOP Conf Ser Earth Environ Sci 209:012015
Nicolaus B, Kambourova M, Toksoy Oner E (2010) Exopolysaccharides from extremophiles: from fundamentals to biotechnology. Environ Technol 31:1145–1158
Erginer M, Akcay A, Coskunkan B, Morova T, Rende D, Bucak S, Baysal N, Ozisik R, Eroglu MS, Agirbasli M, Toksoy Oner E (2016) Sulfated levan from Halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications. Carbohydr Polym 149:289–296
Vu THY, Quach NT, Nguyen NA, Nguyen HT, Ngo CC, Nguyen TD, Ho PH, Hoang H, Chu HH, Phi QT (2021) Genome mining associated with analysis of structure, antioxidant activity reveals the potential production of levan-rich exopolysaccharides by food-derived Bacillus velezensis VTX20. Appl Sci 11:7055
Bouallegue A, Casillo A, Chaari F, La Gatta A, Lanzetta R, Corsaro MM, Bachoual R, Ellouz-Chaabouni S (2020) Levan from a new isolated Bacillus subtilis AF17: purification, structural analysis and antioxidant activities. Int J Biol 144:316–324
Hertadi R, Permatasari NU, Ratnaningsih E (2021) Box-Wilson design for optimization of in vitro levan production and levan application as antioxidant and antibacterial agents. Iran Biomed J 25:202–212
Lee JK, Kim DB, Kim JI, Kim PY (2000) In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants. Toxicol In Vitro 14:345–349
Soltan-Dallal MM, Validi M, Douraghi M, Fallah-Mehrabadi J, Lormohammadi L (2017) Evaluation the cytotoxic effect of cytotoxin-producing Klebsiella oxytoca isolates on the HEp-2 cell line by MTT assay. Microb Pathog 113:416–420
Kim KH, Chung CB, Kim YH, Kim KS, Han CS, Kim CH (2005) Cosmeceutical properties of levan produced by Zymomonas mobilis. Int J Cosmet Sci 56:395–406
Acknowledgements
The study was supported by Eskisehir Osmangazi University Scientific Research Projects Coordination Unit under grand number #FDK-2021-2174. Besides, we thank Prof. Mutlu for providing the strain. This study is based partly on the Ph.D. thesis of O. Erdal-Altintas who is one of the co-authors.
Funding
The study was supported by Eskişehir Osmangazi Üniversitesi (Grant No. FDK-2021-2174).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing Interests
The authors have not disclosed any competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Erdal Altıntaş, Ö., Toksoy Öner, E., Çabuk, A. et al. Biosynthesis of Levan by Halomonas elongata 153B: Optimization for Enhanced Production and Potential Biological Activities for Pharmaceutical Field. J Polym Environ 31, 1440–1455 (2023). https://doi.org/10.1007/s10924-022-02681-1
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10924-022-02681-1