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Applied Microbiology and Biotechnology

, Volume 103, Issue 12, pp 4741–4752 | Cite as

A pH shift induces high-titer liamocin production in Aureobasidium pullulans

  • Katharina Maria Saur
  • Oliver Brumhard
  • Karen Scholz
  • Heiko Hayen
  • Till TisoEmail author
Biotechnological products and process engineering
  • 217 Downloads

Abstract

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.

Keywords

Biosurfactants Liamocin Aureobasidium pullulans Heavy oil Fermentation pH shift 

Notes

Acknowledgements

The authors thank Lars M. Blank for granting lab space and resources for the biological experiments as well as Katja Schröder for experimental support.

Funding

This work was partially funded by the Cluster of Excellence “Tailor-Made Fuels from Biomass” (TMFB), which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities. The Ministry of Innovation, Science and Research financially supported parts of this study, within the framework of the NRW Strategieprojekt Bioeconomy Science Center (BioSC) (No. 313/323–400-002 13). We also acknowledge funding by the Cluster of Excellence “The Fuel Science Center—Adaptive Conversion Systems for Renewable Energy and Carbon Sources,” which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Abdel-Mawgoud AM, Lépine F, Déziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol 86(5):1323–1336CrossRefGoogle Scholar
  2. Abdel-Mawgoud AM, Lépine F, Déziel E (2014) A stereospecific pathway diverts beta-oxidation intermediates to the biosynthesis of rhamnolipid biosurfactants. Chem Biol 21(1):156–164.  https://doi.org/10.1016/j.chembiol.2013.11.010 CrossRefGoogle Scholar
  3. Banat IM, Makkar RS, Cameotra SS (2000) Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol 53(5):495–508.  https://doi.org/10.1007/s002530051648 CrossRefGoogle Scholar
  4. Behrens B, Baune M, Jungkeit J, Tiso T, Blank LM, Hayen H (2016a) High performance liquid chromatography-charged aerosol detection applying an inverse gradient for quantification of rhamnolipid biosurfactants. J Chromatogr A 1455:125–132.  https://doi.org/10.1016/j.chroma.2016.05.079 CrossRefGoogle Scholar
  5. Behrens B, Engelen J, Tiso T, Blank LM, Hayen H (2016b) Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction. Anal Bioanal Chem 408(10):2505–2514.  https://doi.org/10.1007/s00216-016-9353-y CrossRefGoogle Scholar
  6. Behrens B, Helmer PO, Tiso T, Blank LM, Hayen H (2016c) Rhamnolipid biosurfactant analysis using online turbulent flow chromatography-liquid chromatography-tandem mass spectrometry. J Chromatogr A 1465:90–97.  https://doi.org/10.1016/j.chroma.2016.08.044 CrossRefGoogle Scholar
  7. Biely P, Kratky Z, Petrakova E, Bauer S (1979) Growth of Aureobasidium pullulans on waste-water hemicelluloses. Folia Microbiol 24(4):328–333.  https://doi.org/10.1007/Bf02926652 CrossRefGoogle Scholar
  8. Bischoff KM, Leathers TD, Price NP, Manitchotpisit P (2015) Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus. J Antibiot 68(10):642–645.  https://doi.org/10.1038/ja.2015.39 CrossRefGoogle Scholar
  9. Brown RG, Hanic LA, Hsiao M (1973) Structure and chemical composition of yeast chlamydospores of Aureobasidium pullulans. Can J Microbiol 19(2):163–168.  https://doi.org/10.1139/m73-025 CrossRefGoogle Scholar
  10. Brumano LP, Antunes FAF, Souto SG, Dos Santos JC, Venus J, Schneider R, da Silva SS (2017) Biosurfactant production by Aureobasidium pullulans in stirred tank bioreactor: new approach to understand the influence of important variables in the process. Bioresour Technol 243:264–272.  https://doi.org/10.1016/j.biortech.2017.06.088 CrossRefGoogle Scholar
  11. Campbell BS, Siddique A-BM, McDougall BM, Seviour RJ (2004) Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production? FEMS Microbiol Lett 232(2):225–228.  https://doi.org/10.1016/S0378-1097(04)00076-X CrossRefGoogle Scholar
  12. Cheng C, Zhou YP, Lin M, Wei PL, Yang ST (2017) Polymalic acid fermentation by Aureobasidium pullulans for malic acid production from soybean hull and soy molasses: fermentation kinetics and economic analysis. Bioresour Technol 223:166–174.  https://doi.org/10.1016/j.biortech.2016.10.042 CrossRefGoogle Scholar
  13. Chi ZM, Wang F, Chi Z, Yue LX, Liu GL, Zhang T (2009) Bioproducts from Aureobasidium pullulans, a biotechnologically important yeast. Appl Microbiol Biotechnol 82(5):793–804.  https://doi.org/10.1007/s00253-009-1882-2 CrossRefGoogle Scholar
  14. Choudhury AR, Bhattacharjee P, Prasad GS (2013) Development of suitable solvent system for downstream processing of biopolymer pullulan using response surface methodology. PLoS One 8(10):e77071.  https://doi.org/10.1371/journal.pone.0077071 CrossRefGoogle Scholar
  15. de Hoog GS (1993) Evolution of black yeasts - possible adaptation to the human host. Anton Leeuw Int J G 63(2):105–109.  https://doi.org/10.1007/Bf00872386 CrossRefGoogle Scholar
  16. Deshpande MS, Rale VB, Lynch JM (1992) Aureobasidium pullulans in applied microbiology: a status report. Enzym Microb Technol 14(7):514–527.  https://doi.org/10.1016/0141-0229(92)90122-5 CrossRefGoogle Scholar
  17. Ducrey Santopietro LM, Siñeriz F, Castro GR (1992) A spectrophotometric method for the quantitative measurement of pullulan. J Microbiol Methods 16(4):253–258.  https://doi.org/10.1016/0167-7012(92)90015-V CrossRefGoogle Scholar
  18. Elshikh M, Funston S, Chebbi A, Ahmed S, Marchant R, Banat IM (2017) Rhamnolipids from non-pathogenic Burkholderia thailandensis E264: physicochemical characterization, antimicrobial and antibiofilm efficacy against oral hygiene related pathogens. New Biotechnol 36:26–36.  https://doi.org/10.1016/j.nbt.2016.12.009 CrossRefGoogle Scholar
  19. Kim JS, Lee IK, Yun BS (2015) A novel biosurfactant produced by Aureobasidium pullulans L3-GPY from a tiger lily wild flower, Lilium lancifolium Thunb. PLoS One 10(4):e0122917.  https://doi.org/10.1371/journal.pone.0122917 CrossRefGoogle Scholar
  20. Kim JS, Lee IK, Yun BS (2018) Pullusurfactans A-E, new biosurfactants produced by Aureobasidium pullulans A11211-4-57 from a fleabane, Erigeron annus (L.) pers. J Antibiot 71(11):920–926.  https://doi.org/10.1038/s41429-018-0089-0 CrossRefGoogle Scholar
  21. Kurosawa T, Sakai K, Nakahara T, Oshima Y, Tabuch T (1994) Extracellular accumulation of the polyol lipids, 3,5-dihydroxydecanoyl and 5-hydroxy-2-decenoyl esters of arabitol and mannitol, by Aureobasidium sp. Biosci Biotechnol Biochem 58(11):2057–2060.  https://doi.org/10.1271/bbb.58.2057 CrossRefGoogle Scholar
  22. Leathers TD (2002) Pullulan. In: Vandamme E, De Baets S, Steinbüchel A (eds) Biopolymers polysaccharides II: polysaccharides from eukaryotes, vol 6. Wiley-VCH, Weinheim, pp 1–35Google Scholar
  23. Leathers TD (2003) Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol 62(5–6):468–473.  https://doi.org/10.1007/s00253-003-1386-4 CrossRefGoogle Scholar
  24. Leathers TD, Price NPJ, Bischoff KM, Manitchotpisit P, Skory CD (2015) Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media. Biotechnol Lett 37(10):2075–2081.  https://doi.org/10.1007/s10529-015-1892-3 CrossRefGoogle Scholar
  25. Leathers TD, Price NPJ, Manitchotpisit P, Bischoff KM (2016) Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans. World J Microb Biotechnol 32(12):199–205.  https://doi.org/10.1007/s11274-016-2158-5 CrossRefGoogle Scholar
  26. Leathers TD, Skory CD, Price NPJ, Nunnally MS (2018) Medium optimization for production of anti-streptococcal liamocins by Aureobasidium pullulans. Biocatal Agric Biotechnol 13:53–57.  https://doi.org/10.1016/j.bcab.2017.11.008 CrossRefGoogle Scholar
  27. Leung S-S, Martin A, Leone RS (2001) Bioadhesive antibacterial wound healing composition US Patent US09511869, 02.26.1999Google Scholar
  28. Manitchotpisit P, Leathers TD, Peterson SW, Kurtzman CP, Li XL, Eveleigh DE, Lotrakul P, Prasongsuk S, Dunlap CA, Vermillion KE, Punnapayak H (2009) Multilocus phylogenetic analyses, pullulan production and xylanase activity of tropical isolates of Aureobasidium pullulans. Mycol Res 113(10):1107–1120.  https://doi.org/10.1016/j.mycres.2009.07.008 CrossRefGoogle Scholar
  29. Manitchotpisit P, Price NP, Leathers TD, Punnapayak H (2011) Heavy oils produced by Aureobasidium pullulans. Biotechnol Lett 33(6):1151–1157.  https://doi.org/10.1007/s10529-011-0548-1 CrossRefGoogle Scholar
  30. Manitchotpisit P, Watanapoksin R, Price NPJ, Bischoff KM, Tayeh M, Teeraworawit S, Kriwong S, Leathers TD (2014) Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity. World J Microbiol Biotechnol 30(8):2199–2204.  https://doi.org/10.1007/s11274-014-1639-7 CrossRefGoogle Scholar
  31. Nagata N, Nakahara T, Tabuchi T (1993) Fermentative production of poly(beta-L-malic acid), a polyelectrolytic biopolyester, by Aureobasidium sp. Biosci Biotechnol Biochem 57(4):638–642.  https://doi.org/10.1271/bbb.57.638 CrossRefGoogle Scholar
  32. Pollock TJ, Thorne L, Armentrout RW (1992) Isolation of new Aureobasidium strains that produce high-molecular-weight pullulan with reduced pigmentation. Appl Environ Microbiol 58(3):877–883Google Scholar
  33. Portilla-Arias J, Patil R, Hu J, Ding H, Black KL, Garcia-Alvarez M, Munoz-Guerra S, Ljubimova JY, Holler E (2010) Nanoconjugate platforms development based in poly(beta,L-malic acid) methyl esters for tumor drug delivery. J Nanotechnol 2010:1–8.  https://doi.org/10.1155/2010/825363 Google Scholar
  34. Prasongsuk S, Ployngam S, Wacharasindhu S, Lotrakul P, Punnapayak H (2013) Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species. Appl Microbiol Biotechnol 97(17):7821–7830.  https://doi.org/10.1007/s00253-013-5069-5 CrossRefGoogle Scholar
  35. Prasongsuk S, Lotrakul P, Ali I, Bankeeree W, Punnapayak H (2018) The current status of Aureobasidium pullulans in biotechnology. Folia Microbiol 63(2):129–140.  https://doi.org/10.1007/s12223-017-0561-4 CrossRefGoogle Scholar
  36. Price NP, Manitchotpisit P, Vermillion KE, Bowman MJ, Leathers TD (2013) Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380. Carbohydr Res 370:24–32.  https://doi.org/10.1016/j.carres.2013.01.014 CrossRefGoogle Scholar
  37. Price NP, Bischoff KM, Leathers TD, Cosse AA, Manitchotpisit P (2017) Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380. J Antibiot 70(2):136–141.  https://doi.org/10.1038/ja.2016.92 CrossRefGoogle Scholar
  38. Qiang N, Yang WH, Li LF, Dong P, Zhu JX, Wang T, Zeng CG, Quan DP (2012) Synthesis of pendent carboxyl-containing poly(epsilon-caprolactone-co-beta-malic acid)-block-poly(L-lactide) copolymers for fabrication of nano-fibrous scaffolds. Polymer 53(22):4993–5001.  https://doi.org/10.1016/j.polymer.2012.09.010 CrossRefGoogle Scholar
  39. Ribeiro IA, Bronze MR, Castro MF, Ribeiro MHL (2012) Optimization and correlation of HPLC-ELSD and HPLC-MS/MS methods for identification and characterization of sophorolipids. J Chromatogr B 899:72–80.  https://doi.org/10.1016/j.jchromb.2012.04.037 CrossRefGoogle Scholar
  40. Schoch CL, Shoemaker RA, Seifert KA, Hambleton S, Spatafora JW, Crous PW (2006) A multigene phylogeny of the Dothideomycetes using four nuclear loci. Mycologia 98(6):1041–1052CrossRefGoogle Scholar
  41. Schoeman MW, Dickinson DJ (1996) Aureobasidium pullulans can utilize simple aromatic compounds as a sole source of carbon in liquid culture. Lett Appl Microbiol 22(2):129–131.  https://doi.org/10.1111/j.1472-765X.1996.tb01125.x CrossRefGoogle Scholar
  42. Schoeman M, Dickinson D (1997) Growth of Aureobasidium pullulans on lignin breakdown products at weathered wood surfaces. Mycologist 11(4):168–172.  https://doi.org/10.1016/S0269-915X(97)80095-X CrossRefGoogle Scholar
  43. Shabtai Y, Mukmenev I (1995) Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC42023) in a two-stage fermentation with shift from soy bean oil to sucrose. Appl Microbiol Biotechnol 43(4):595–603CrossRefGoogle Scholar
  44. Sharma RR, Singh D, Singh R (2009) Biological control of postharvest diseases of fruits and vegetables by microbial antagonists: a review. Biol Control 50(3):205–221.  https://doi.org/10.1016/j.biocontrol.2009.05.001 CrossRefGoogle Scholar
  45. Simon L, Bouchet B, Caye-Vaugien C, Gallant DJ (1995) Pullulan elaboration and differentiation of the resting forms in Aureobasidium pullulans. Can J Microbiol 41(1):35–45.  https://doi.org/10.1139/m95-005 CrossRefGoogle Scholar
  46. Singh RS, Saini GK, Kennedy JF (2008) Pullulan: microbial sources, production and applications. Carbohydr Polym 73(4):515–531.  https://doi.org/10.1016/j.carbpol.2008.01.003 CrossRefGoogle Scholar
  47. Tang RR, Chi Z, Jiang H, Liu GL, Xue SJ, Hu Z, Chi ZM (2018) Overexpression of a pyruvate carboxylase gene enhances extracellular liamocin and intracellular lipid biosynthesis by Aureobasidium melanogenum M39. Process Biochem 69:64–74.  https://doi.org/10.1016/j.procbio.2018.03.008 CrossRefGoogle Scholar
  48. Terán Hilares R, Orsi CA, Ahmed MA, Marcelino PF, Menegatti CR, da Silva SS, dos Santos JC (2017) Low-melanin containing pullulan production from sugarcane bagasse hydrolysate by Aureobasidium pullulans in fermentations assisted by light-emitting diode. Bioresour Technol 230:76–81.  https://doi.org/10.1016/j.biortech.2017.01.052 CrossRefGoogle Scholar
  49. Tiso T, Sabelhaus P, Behrens B, Wittgens A, Rosenau F, Hayen H, Blank LM (2016) Creating metabolic demand as an engineering strategy in Pseudomonas putida – rhamnolipid synthesis as an example. Metab Eng Commun 3:234–244.  https://doi.org/10.1016/j.meteno.2016.08.002 CrossRefGoogle Scholar
  50. Torzilli AP (1997) Tolerance to high temperature and salt stress by a salt marsh isolate of Aureobasidium pullulans. Mycologia 89(5):786–792.  https://doi.org/10.2307/3761135 CrossRefGoogle Scholar
  51. Tsujisaka Y, Mitsuhashi M (1993) Pullulan. In: BeMiller J, Whistler R (eds) Industrial gums. Academic Press, London, pp 447–460CrossRefGoogle Scholar
  52. Wang WL, Chi ZM, Chi Z, Li J, Wang XH (2009) Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity. Bioresour Technol 100(9):2639–2641.  https://doi.org/10.1016/j.biortech.2008.12.010 CrossRefGoogle Scholar
  53. Wang D, Yu X, Gongyuan W (2013) Pullulan production and physiological characteristics of Aureobasidium pullulans under acid stress. Appl Microbiol Biotechnol 97(18):8069–8077.  https://doi.org/10.1007/s00253-013-5094-4 CrossRefGoogle Scholar
  54. Wang Y, Song X, Zhang Y, Wang B, Zou X (2016) Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus Aureobasidium pullulans. Microb Cell Factories 15(1):146.  https://doi.org/10.1186/s12934-016-0547-y CrossRefGoogle Scholar
  55. Wittgens A, Kovacic F, Müller MM, Gerlitzki M, Santiago-Schübel B, Hofmann D, Tiso T, Blank LM, Henkel M, Hausmann R, Syldatk C, Wilhelm S, Rosenau F (2017) Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Appl Microbiol Biotechnol 101:2865–2878.  https://doi.org/10.1007/s00253-016-8041-3 CrossRefGoogle Scholar
  56. Wu S, Jin Z, Kim JM, Tong Q, Chen H (2009) Downstream processing of pullulan from fermentation broth. Carbohydr Polym 77(4):750–753.  https://doi.org/10.1016/j.carbpol.2009.02.023 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.iAMB—Institute of Applied Microbiology, ABBt—Aachen Biology and BiotechnologyRWTH Aachen UniversityAachenGermany
  2. 2.Institute of Inorganic and Analytical ChemistryUniversity of MünsterMünsterGermany

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