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    ARTICLE IN PRESSG ModelRBI-10893; No. of Pages 10Process Biochemistry xxx (2016) xxxxxx

    Contents lists available at ScienceDirect

    Process Biochemistry

    journa l homepage: www.e lsev ier .com/ locate /procbio

    ntegrated sophorolipid production and gravity separation

    en M. Dolman, Candice Kaisermann, Peter J. Martin, James B. Winterburn

    chool of Chemical Engineering and Analytical Science, The Mill, The University of Manchester, Manchester, M13 9PL, UK

    r t i c l e i n f o

    rticle history:eceived 9 November 2016eceived in revised form3 December 2016ccepted 21 December 2016vailable online xxx

    hemical compounds:ophorolipid (PubChem CID: 11856871)

    a b s t r a c t

    A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has beendeveloped, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fer-mentation broth, while cells and other media components are recirculated and returned to the bioreactor.The capability of the separation system to recover an enriched sophorolipid product phase was demon-strated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11%reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recov-eries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichmentof up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phaseenabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of theeywords:ntegrated separationophorolipidettlingandida bombicolaiosurfactant

    fermentation, with a 9% average reduction over the course of the fermentation. Fermentation durationcould be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume.These benefits could lead to a substantial decrease in the cost of sophorolipid production, making highvolume applications such as enhanced oil recovery economically feasible.

    2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY licenselycolipid

    . Introduction

    Sophorolipids are microbially produced glycolipid biosurfac-ants, which are rapidly increasing their market share of the 27illion USD global surfactant market [1]. While several yeast strainsre able to synthesize sophorolipids, most research and industrialse is focused on Candida bombicola ATCC 22214, the organism usedn this study [2]. Sophorolipids consist of a hydrophilic sophoroseisaccharide bound to a hydrophobic fatty acid with a typical chainength of 1618 carbon atoms. The fatty acid may be joined byn ester bond to the second glucose monomer, giving a lactonicophorolipid, or joined to only one glucose monomer, giving ancidic sophorolipid due to the unbound fatty acid. These and otherifferences in the fatty acid chain and acetylation of the sophoroseolecules give a range of different structures and properties. Twoommon structures representing lactonic and acidic sophorolipidsre shown in Fig. 1 [2].Sophorolipids are produced industrially by a number of compa-

    ies, who often utilize sophorolipids detergent and low foamingPlease cite this article in press as: B.M. Dolman, et al., Integrated so(2016),

    roperties in a variety of formulated cleaning products [3]. Theherapeutic properties of sophorolipids have allowed them to beommercialized in anti-dermatitis soap and other body washes,

    Corresponding author.E-mail address: [email protected] (J.B. Winterburn).

    ttp:// 2017 The Authors. Published by Elsevier Ltd. This is an open access article u(

    and in a cream to reduce oily skin by MG Intobio Co and Soliance.There is ongoing research into potential medical applications ofsophorolipid, with anti-cancer, anti-HIV, antimicrobial and anti-biofilm activity being investigated [46]. Sophorolipids also havepotential for use in low cost, high volume applications such asbioremediation and enhanced oil recovery if production costs canbe significantly reduced [7,8].

    In sophorolipid producing fermentations, product concentra-tions of over 300 g l1, with productivities of over 1 g l1 h1 areroutinely achieved [911]. Sophorolipid producing fermentationsbegin with a cell growth phase, which typically lasts until thenitrogen in the media is depleted, at which point the sophorolipidproduction rate increases significantly, if both a hydrophilic and ahydrophobic carbon source are present [12]. The sophorolipid pro-duction phase normally lasts for around 200 h, at which point thedissolved oxygen level in the fermenter cannot be maintained dueto oxygen mass transfer limitation.

    This dissolved oxygen reduction is caused by the high viscosityof the sophorolipid produced, meaning the fermentation must bestopped and the sophorolipid recovered [12,13]. It is well knownthat the presence of a separate sophorolipid phase in the bioreactorsignificantly reduces the oxygen mass transfer coefficient, kLa, byphorolipid production and gravity separation, Process Biochem

    both providing a resistance to mass transfer across the air/liquidinterface and increasing the viscosity of the medium, which resultsin oxygen limitation, increased stirring power requirements andnon-homogeneity in the bioreactor [1214].

    nder the CC BY license ([email protected]/10.1016/j.procbio.2016.12.021

  • ARTICLE IN PRESSG ModelPRBI-10893; No. of Pages 102 B.M. Dolman et al. / Process Biochemistry xxx (2016) xxxxxx

    F s. A lao ree car






    ig. 1. Molecular structure of common lactonic (left) and acidic (right) sophorolipidf the sophorose in the lactonic sophorolipid, where the acidic sophorolipid has a f

    The physical form of sophorolipids is dependent on the con-itions under which they are produced, which directly affecthe proportions of acidic and lactonic sophorolipids produced.ophorolipids typically separate from the fermentation broth as

    crystalline material if the lactonic to acidic ratio is high and theydrophobic carbon source concentration is low. The sophorolipidstherwise form a viscous second phase of around 50% sophorolipidnd 50% water, which may sit below residual oil at the surface ofhe broth or sink to the bottom of the bioreactor when agitation istopped [10,15,16].

    These properties are commonly exploited at the end of a fermen-ation to give an easy, crude separation of the sophorolipid from theermentation broth, either by crystal decantation, crystal filtrationr decantation of the sophorolipid gel [11,16,17]. These techniquesave not previously been used effectively to recover sophorolipidsuring fermentation.Industrially, there are a number of costs associated with

    epeated batch cycles for sophorolipid fermentation, in terms ofowntime between cycles, cleaning costs and the lengthy inocu-um preparation required for large scale production [18,19]. Therere numerous proposed partial solutions to this problem in the lit-rature. For example, a portion of the broth can be removed andeplaced with fresh media, allowing high productivity to be main-ained for seven 80130 h cycles, nevertheless removing biomassroportionally to other components [19]. Sophorolipid settling byravity within a fermentation vessel or shake flask has previouslyeen demonstrated for small scale sophorolipid production. Signif-cant benefits of sophorolipid separation have been shown, with aoubling of the duration of sophorolipid production and little effectn production after 15 min without agitation or aeration demon-trated by Guilmanov et al. [20], and a productivity increase from.38 to 1.89 g l1 h1 shown by Marchal et al. [19]. Both studies relyn gravity settling within the fermentation vessel or shake flask,owever, making scale up impractical due to the excessive settlingistances present if this technique were applied at industrial scale.Effective integrated separation techniques have been developed

    or other biosurfactant systems, notably foam fractionation forydrophobin proteins, surfactin and rhamnolipids, but there havePlease cite this article in press as: B.M. Dolman, et al., Integrated so(2016),

    een no successful scalable attempts at integrated separation forophorolipid production [2123].

    This paper details a novel technique, based on an

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