10
208 reviews Armin Fiechteer- Biosurfaetants: maving towa industrial apglkation Chemically synthesized surface-active comaunds are widely u:;ed in the pharma- ceutica!, cosmetic, petroieum and food :ndustries. However, with the advantages of biodegradability, and productiofi ‘WI renewable-resource substrates, biosurfac- tants may eventually replace tki: shemically synthesized counterparts. So far, the use of biosurfactants has IV :,I limited to a few specialized applications because biosurfactants have beeit economically uncompetitive. There is a need to gain a greater understanding of the physiology, genetics and biochemistry of biosurfac- tant-producing strains, and to improve process technology to reduce production costs. Surfactants and cmulsificn arc intcgzl to many indus- trial, agricultural and food proccsscs. Mo5.i of the com- pounds arc chemically synthesizd, and it is only in the past t;lw decades that s\t&x-active molc~:~!cs ofbio- logical origin have been dcscrii;eJ. Their su&tant and emulsification propcrtic3 result tioel the prcscnce of both hydrophilic and hydrophobic rqions on the sar,x mol~culc; aggxgates form and accumulate at surface boundarirs, thus separating the two phases. The ix!ustGl demand for surfactants is high: the market wiuc for soaps and detergents rcachcd USS12.8 X lo’ in 1990, with a continuing amn;al incrcasc of 5.9’s. Ofthis market, surt%tants accounted for USS3.9 X 10)‘). It is estimated that the dematld kx surfactants world-wide will increase by 35% by :!le end of the century. Howcvcr, rlearly all tbc surfrce- active compounds currcnely in use arc synthrsized chemically, with petroleum as rhc raw matr~iail. At the moment, biosurfactants arc unable to compete economically with the chemically synthrsizcd com- pounds on the market, due to high production costs. l’hcsc result primarily from ine&ic:tt bioproccssing mcthodolo~~, but also from poor xrain productivief, and the need to USC cxpcnsivc substrates. Prcrequi:itcs CX biosurfactants gaining a si~GLicant share ofthc n;ar- ket are, thcrrforc: (1) an Improved knowledGe and abiliry to manipulat*: the mctabo!ism of the producer strains, such that ch-caper substrates may bc used; and i2) the improvcmcnt of process technology to facili- tate product r~ovcr:_. A broad r.mge o< surfactants is already known (for rc~ KWS scr’Rcfs Z-C), and as the list ofbiosurfactant- produci:lg organisms is extended still further, * J wiIl rhc sptcttum of their physical and chemical properties a c Figure L Surfactants are characterized by an amcnipathlc structure. Hydrophobic and hydrophilic properties depend on the charge of the polar group (anionic, cationic, neutral or amchoterii types). They tend to associate at interfaces or in micelles, favouring a minimal free energy charge of the system. The minimal surfacetension value reached due to the activfly of the slrrfactant, and the critical micelle concentration kmc) needed, give a measure of the efficiency of the s&ace-active compound. (a) Surfactant monomer, denoted by a circle representing the hydroph%c head attached to a hydrocarbon tail; !bl circular rnicelle; Cc)ro&shaped micelle; (dl micellar layer; and (e) vesicle representation. --.____ TIBlECH NINE (B 1992. Ekevler Science PuM;shers Ltd (UK)

Biosurfactants: moving towards industrial application

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Page 1: Biosurfactants: moving towards industrial application

208

reviews

Armin Fiechteer-

Biosurfaetants: maving towa industrial apglkation

Chemically synthesized surface-active comaunds are widely u:;ed in the pharma-

ceutica!, cosmetic, petroieum and food :ndustries. However, with the advantages

of biodegradability, and productiofi ‘WI renewable-resource substrates, biosurfac-

tants may eventually replace tki: shemically synthesized counterparts. So far, the

use of biosurfactants has IV :,I limited to a few specialized applications because

biosurfactants have beeit economically uncompetitive. There is a need to gain a

greater understanding of the physiology, genetics and biochemistry of biosurfac-

tant-producing strains, and to improve process technology to reduce production

costs.

Surfactants and cmulsificn arc intcgzl to many indus- trial, agricultural and food proccsscs. Mo5.i of the com- pounds arc chemically synthesizd, and it is only in the past t;lw decades that s\t&x-active molc~:~!cs ofbio- logical origin have been dcscrii;eJ. Their su&tant and emulsification propcrtic3 result tioel the prcscnce of both hydrophilic and hydrophobic rqions on the sar,x mol~culc; aggxgates form and accumulate at surface boundarirs, thus separating the two phases.

The ix!ustGl demand for surfactants is high: the market wiuc for soaps and detergents rcachcd USS12.8 X lo’ in 1990, with a continuing amn;al incrcasc of 5.9’s. Ofthis market, surt%tants accounted for USS3.9 X 10)‘). It is estimated that the dematld kx surfactants world-wide will increase by 35% by :!le end of the century. Howcvcr, rlearly all tbc surfrce- active compounds currcnely in use arc synthrsized chemically, with petroleum as rhc raw matr~iail. At the moment, biosurfactants arc unable to compete economically with the chemically synthrsizcd com- pounds on the market, due to high production costs. l’hcsc result primarily from ine&ic:tt bioproccssing mcthodolo~~, but also from poor xrain productivief, and the need to USC cxpcnsivc substrates. Prcrequi:itcs CX biosurfactants gaining a si~GLicant share ofthc n;ar- ket are, thcrrforc: (1) an Improved knowledGe and abiliry to manipulat*: the mctabo!ism of the producer strains, such that ch-caper substrates may bc used; and i2) the improvcmcnt of process technology to facili- tate product r~ovcr:_.

A broad r.mge o< surfactants is already known (for rc~ KWS scr’ Rcfs Z-C), and as the list ofbiosurfactant- produci:lg organisms is extended still further, * J wiIl rhc sptcttum of their physical and chemical properties

a

c

Figure L Surfactants are characterized by an amcnipathlc structure. Hydrophobic and hydrophilic properties depend on the charge of the polar group (anionic, cationic, neutral or amchoterii types). They tend to associate at interfaces or in micelles, favouring a minimal free energy charge of the system. The minimal surfacetension value reached due to the activfly of the slrrfactant, and the critical micelle concentration kmc) needed, give a measure of the efficiency of the s&ace-active compound. (a) Surfactant monomer, denoted by a circle representing the hydroph%c head attached to a hydrocarbon tail; !bl circular rnicelle; Cc) ro&shaped micelle; (dl micellar layer; and (e) vesicle representation.

--.____ TIBlECH NINE (B 1992. Ekevler Science PuM;shers Ltd (UK)

Page 2: Biosurfactants: moving towards industrial application

widen, thus leading to the discovery of surfactants sui:ed for specialty applications. Improved charactcr- i&on of tile strains should open the way for the use of genetic manipulation of the organisms. thus enabling them to bc further tailored towards optimal performance.

In addition to rhc increasing cost and uncertainty in the supply of petroleum, the more readily biodcgrad- able character ofbiosurfictants means that they shouId gcncratc fewer environmental problems - a feature that will doubtlessly gain in importance in industrial processes as more rigorous controls are imposed.

Although many problems remain to he solved and the displacemcnr ofchcap, chemically synthesized sur- factants from the market will not bc easy, these factors bode well for biosurf&tants eventually replacing their &mica1 counterparts for many applications.

Biosurfachnts - definition and classification A biosurfactant is defined 2s a surface-active

molcculc produced by living czlls - in the majority of GWS, by microorganisms. In the literature, Ihe temls sur&tant and cmulsiticr are frequently used inter- changeably. However, whereas the molecular struc- ture of a surfactant ic defined (a sur&ctant has both hydrophilic and hydrophobic moieties present within the same molcculc), the term biocmulsificr is often used in an application-oriented manner to describe the combination of all the surface-active compounds that constitute the emulsion secreted by the cell _ facili:ate uptake of an insoluble substra&. A typical rc-yrcsentative of these bioemulsificrs is Etnuls-:~P~~. currently the only such product on the marker.

The surf&tam character ofmolcculcs is due to their mixed hydrophilicihydrophobic nature. They arc able to form micelle? and reversed micclles, or to aggrcg&e to form rod-shaped micellcs. bilayers and vesicles (Fig. 1). They accumulate at interfaces and ‘mc&ate’ betwee:) phases of different polarity such as oil/water, air/y_vatcr, or water/solid, acting as wetting agents on zolid smt~caces. This dynamic process is base?. on the ability of the su&ctant to reduce the su&c: tension by governing the arrangement of liquid molecules, thus influencing the formation of H-bonds rnd hydrophobic-hydrophilic interxtions. T!le minimum surface tension value reached, and the critical micelle concentration (cmc) nccdr.f, are parameters used CO

measure the efflcicncy of the su&ctant. The be- haviour of contacti!;g molecules at the inter&cc between different phase? constitute: a facused and Fast- gowing field of interest, as evidl:nced by the recent ICSCS meeting in 1991 I’).

Biosurfactants may be classified into three main groups based on the detail of their chemical structure within the basic tiamework: whereas the hydropho- bic moiety consists of the hydrocarbon chain ofa fatty acid, the hydrophilic moiety may consist ofthc ester or alcohol function of neutral lipids, or the carbox~- late group of fatty acids or amino acids, or the phos- pbzr-containiag portions of phospholipids, or the carbohydrate moiety of glycolipids.

0

ii _. C-CCH-cCH;-c-o-CCH-cCn,-coon

I Of:!, I CH:,

Rhamnolipid 1

bn bti CH-. iihamnulipid 2

““gy k.j Rhamnolipid 3

L-f bn on

Rhamnolipid 1

-I Figure 2

Four different forms of rhamnolipids (!?LJ synthesized by Pseudomonas aeruginosa DSM 2659. While RLl and A;3 represent the ty,pical prodticts from liquid cultures. RL2 and RL4 are produced by resting cells only.

??The glycolipid surfactunts

Thcsc include the sophorosc-, rhamnxc-. tx- halos+, sucrose- and fructose-lipids tion~ a range of species such as Tor;r!0fs~‘5~ I: ‘ww~ f mfrom7.i’~ or Arllmhc-

tcr13, and also nx~~~nos~-~ cl?;thritol lipids tionl Crrr&d,x and 4iz~& species, ald cellobiow llptdc tkotn 1_y~i- I<~o ZCW. Among the best investigated biosurktants of this group x-t‘ the rhamuolipids irnm P. W~@WJX and P. _~7mmww, which consist of one or two sugar moieties joiued to one or two caprillic acid moieties via a glycosidic linkage (Fig. 2) (C. Syldatk, l’hl) thesis, Univ. Braunschwcig. FRG, 1984).

@ fie ahw-acid containing lipid liiusrrr@tiw~s These include the most power&l bilrsur&tant

known today. the so-called surfac~irl produced by Blzc;~lrf_~ s1rbrillis”. Suri&xin is CotU~:3sCd of a W\W~- amino-acid ring structure:

(Glu-Leu-Lcu-Val-Asp-Leu-Lcu)

coupled to one molecuie of3-h~dro_~~-l3-meth~-l- tetradecanoic acidl’. A simnilar hiosu&ctant is

-----.- TklTECH _wE 1992 woe 101

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R,_c(CH2)e-CH-CH,-CO-Glu-Leu-Leu-~~~

1 RI = (CH&&H-

b i

Ps, = CH3_CH2_CH2_

L- I!e - Leu -Asp R3 = (Cti3)~CH-CH2-

I~

R4 = CH3-CH2-CH(CH$-

Ague 3 Structure of the prominent surfaceactive lipopeptide of Bacillus licheniformisr?

produced by Bacillns licl~err~$~n~is, with a lipophiiic fatty acid moiety joined via a lactone linkage to the hydrophilic pepride ring structure (Fig. 3) (K. Jenny,

PhD thesis no. 9163, ETH-Ziirich, Switzerland, 1990). Frequently, biosurfactants in this group have been no:ed for their antibiotic activity with the sur- factant properties having been of only minor interest. Other I ~: -- :_;Y’:; &-&> g&&q of ~~,ll_5:_~~~ac-~,-~~~ in&de :irc orthinine-containing lipid Pseudomonas rubercerrsl6, the lysine-containing lipid of Agrobdctetiunz trmz$kieens, and the orthinine-taurine lipid (Cerilipin) of Chconobuctev cerintts’7. The unusual amino acid taurine makes this last h+c&_r=nt o;.: of thy f&: with a sulphare grottp. The omithine-ccntaming lipid is a zwitterion, with the J3-hydroxy carboxylic acid and the esterified carboxylic acid providing both a iree carboxyl group and a free amino group.

Table 1. L-$apeptide antibl~tics of kc&: spp.:

Name (wow4 Organism Structure

Fatty acid (Wb

Amino Characteristics acid (aa)

ST, IT Antibiotic (mN m-1) a&&y

Cyctic lipopeptides

Win group kurin A 8. silbfilis

fturin C

Uyc! !subtilisin Et. subtilis 8. fliger

Bacillomycin Ld 6. subtilis

Bacillomycin D FA-, !:,:,; f yroAsn+Pror_GIwoSer~Thr

Bacillomycin F FA-LAsnoTyr-oAsMGIMPr~~~Thr

Octapeptine group EM 49 6. circulans 333-25 B. bungoensis

5. subtilis

FAoDab-r.Dabi.DaboLeubPhehLeu(cPhe) LLeu&&Dab

?o!ymyxin group Polvmyxin 8. polymyxa

A-F, K, M, f’, 3 and T (Cotistin~

FAi.DabiThr+DaMoSerhDab-rDaboLeu- (oPhek~r(LLeuhDab-cDa~leu(LThr)

Circulin A, B

5. polymyxa

FA&n-oTyroAsmGlrr+ProoAsh&er

PAOspoTyr-oAsn&w_GlnoSer(ProkThr

@NH C14-,5: 7

iC14 aiC,,

37.5

B-NH C,,,,: 7 -. fPL,6 = 6%

i& = 29% i-C,, = 7% aiC,, = 54%

” .<

$WM: 7 = 38.9%

i-C;; = 25.2% a&C,, 5 i5.4%

i-c,, = 10.1% 2.C‘” .2 Cl%

n.d.

&OH C9-ll: 8 Y!. &I$, Rc,,

i-c, ai-C,

10 n.d.

<a ai-Cs

10 n.d.

bactericidal frngicidalc

fungicidal

fungicidal

fungicidal bactericEdale

~~~_~ ~~

bactericidal (G-1

bactericidal (G-1

-&ECH JUNE 10% \VOL l$

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0 Biosu@ants containing poiysacchanut4yi.i compfewos

This third group ofbiosurfactants contains the po!y- sacsharide-lipid complexes of Ca?ndido tropicR/is and :-~‘-l~~z:~i :I’& “l,ii.;__:; I-- ---+!c RAG-l. This latter organism is the somie of Em&an, an excr.+&.z!ar lipo- hcteropolysaccharide polyanionic bioemulsirier’-‘. ‘The majority ofthe polysaccharides which are located pre- dominantly in the outer membrane of Gram-negative bacteria exert remarkable surfactant activities. The ceti walls of yeasts such as the alkane-assimilating Cur&& tropicnlic have been shown to contam a ~o~;~Lc!w- ide-lipid Lomplex related to alkzr,c n-ansfer through the cellular membrane’s. TLr biosynthesis ofthe pdy-

sacch~ride-lipid complex is induced by growing the yeas; tii, t$--n+;ions. and the subsequent incrcascd substrate-uptake rate is taken as indirect prsof nf rhc surfaccant activity of the complex. No applications have originated as yer from this observation.

??Protein-like substances Finally, there arc a number of protein-like sub-

stances with biosurfactant activity which can be clasri- fied a~ a fourth group of biosuriactants. A 70 kDa protein called serraphobin, iso!ated from both the cell surface and culture supematants of Serrtlplr~birr I~JUK~X~W~~, is able to bind to hcxadecanc drop&. In the yeast Tontlapsis petr~p/plri/rrrr~, a glycoli?id

strMi~e, fiinction and biosynthesisa

Name (synonym1 Qrgadrm Structure

Fatty acid (Wb

Amino Characteristics acrd (aa)

ST, IT Antibiotic (mNm-1) activity

La&one group

Esperin 8. rnesentericus FA+Glu-Le~eu+VahAsp-Le~euWal)OH

Surfactin 6. subtilis (subtilisin, streptolysin, ADP I-lid e

Surfactin analogue

Polypeptin

B. subtilis

B. circulans

Brevistin B. brevis

FADablleDab-oPheLeuDaboVaH.eu+Phe

FkThrilDatKAspGly-oAsnoAsflbTrp LPh~DabilleNal)

C19; 4 AASP, 2-4 oAsp, 2 LGIu, 2 oTyr, 2oSer k&l, 2 LPro

FA+Asn-oVaWaUoPl-tehAsrkoAs~~ kLys~all~Trp&e&GfykaThr

P4J+Ll5: ci3 = 20% Cl, = 35% c,, = 45%

BQ+L,,: i-C,, and ai-&, = 10% n-C,, = 40% X,, and ai& = 50%

7

7

7

9

11

linear Iipopeptides

Subsporin A-C

Cerexin A-D

6. subtiiis

6. cereus

n.d. fungicidal

n.d. bactericidal !G+)

Tridecaptin A-C

B. POMW n.d. bactericidal

“Adapted from K. Jenny, PhD thesis no. 9263, ETHZikfch, Swifzeknd, 1990. V&se a differeM atnina acid may be present in a structwe, the a&ma live is presented in parenthesis. Abbreviations: ST = surface tension; IT = i~&tie:~al tension; n.d. = not detectable: Dab = 2,4diamk&@rk acid; Hyl = hydroxylysine; G- = Gramnegative bacteria, G+ = Gram-posifive bacti. bNomenclature for branched ca&n chains: i = iso: ai = .&i&o <Has clinical application against dermatomycosis. *Biosynthesis pathway is W Yias tic&#o~s application against G- . %osyiiifiesir pathway involves thio template. ggiosmesis pa&way is nonfibosomal. Acts as hemkylic, afdkholesterine iorm:w, inhibitor of the cyclic 3’.5 monophasphate phosp!?te dies&are. inhibitor of fibrin formation.

n.d. bactericidal

27.2, bactericidal 0.1 fungicidal

n.d.

n.d. bactericidal

n.d. bactericidal (G-1

___- 1 1 1 _ _

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Table 2. Lipepeptides and lipoproteins produced by microorganisms other than Bacillus spp.=

Organism Composition

Lipophific part HydrophiRc part Mycobacterium fortuitum CzO or Cz2 FA 9aa (3 &al; 2 LThr; tAla; LPro; 2 MeLeu) Mycobacterium paratubercufosis C20 FA 3aa Wne, DPhe, Ala, L:eu, Llle) Nocardia asteroides @OK CZF FA 7aa (2 LThr, LVal, LPro, LAla. DAla, oallolle) Corynebacterium iepus C,,-C,, FA (25%); 13 different aa

Corynomycolic acid (75%) Streptomyces canus 10 aa

Streptomyces violaceus 3-ai C,,, 3-i Cl2 FA

Serrafia marcescens 2 p-OH C,, FA 2 LSer Pjeudomonas Ruorescens P-0~10 LLeu-oGlu oalloThr oval-

&y&-gqyK ;;f -r-IIcs _ -.. .I..“““” WoSer+Leu-oSer-LLeu Pseudomonas rubescens P-OH FA Orn

Thiobacillus thiooxidans Rhodopseudomonas spheroides Strepfomyces sioyaensis

Agrobacferium tumefaciens $2

LYS Gluconobacter cerhus Taurine and Om Cark!ida peirophilum nonidentiiied FA Peptide (Glti, Asp, Ala and Leu)

Acinetobacfer calcoaceticus Protein-lipid Pseudomonas ffuorescens Corynebacterium hydrocarboclastus

Carbohydrate-protein complex with a minor lipid part (300 kDa) Protein-lipid-carbohydrate complex

Pseudomonas aeruginosa Protein-like activator (147 aa) (-!4.3 kDa) Candida Iipolytica Carbohydrate-protein complex (-27.6 kDa)

aAdapted from K. Jenny, PhD thesis na. 9263, ETHAirich, Switzerland~990.

sur&tant and a protein emulsifier able to stabilize water/oil emulsions have- beei) identified”‘. Cti&rlrT liyc~/~rM produces liposan, a 27.6 kDa complex protein-like substance composed of83’% carbohydrate which is capable of stabilizing water/oil emulsions”. Sinliiar protein-carbohydrate complrxes which act as cmulsific:rs arc found in P. /IIIO~~SWKP and P. rwry~im~~~~LS, the complex found-in the latter being notable for its high serinc content.

Biosynthesis of biosurfactants Org.misms which product biosurfactants include

many of the yeasts, bacteria and filamentous fungi. A survey oflipopeptidcs and lipoproteins produced by &~ci/Ilr~ spp. is prcscntcd in Table 1, while those pro- ducrd by other microorganisms arc listed in Table 2. An extensive listing has been compiled by Haferburg cl ,tl.s, to which a couple of new cxamplcs presented at the ICSCS 1991 meeting’” may bc added.

The main physiological role of biosurfactants is to permit microorganisms to grow on water-immiscible substrates by reducing the surface tension at the phase boundary, thus making the substrate more readily available for uptake and metabolism. The molecular mechanisms of the npr;l.ke process of these substrates (e.g. alkmcs) are, however, still not clear. nircct uptake of hydrocarbons dissolved in the aqueous phase, direct contact of cells with large hydrocarbon droplets as we11 as interaction with ‘solubilized’ droplets (emulsion) have been described’+. The basic and applied aspects ofmicrobial adhesion at the hydro-

carbon-water mtcrface have recently been revicwcd by Roscnberg2j.

In addition to emulsification of the ._arbon source, biosurfactants are also involved in the adhesion of microbial cells to the hydrocarbon. The cellular adsorption of the hydrocarbon-degrading microor- ganisms to water-immiscible substrates’“, and the excretion of surface-active compouuds together allow growth on such carbon sources. In experiments with A. c&m-rim RAG-I cells on crude oilZ7, the loca- tion of Emulsan (cell-bound or excreted) was related to growth rate: the exponentially @owing cells were found to be attached to the hydrocarbon whereas the slowly growing cells were free with Emulsan capsules being present in the extracellular space. In general, negatively charged biosurfactants inhibit, whereas positively charged biosurfactants promote microbial adhesion to hydrophobic phases.

Another physiological role of biosurfactants is their antibiotic effect on various microbes. The antibiotic activity of B. h&m$mnir biosurfactants against yeast, bacteria and funsi in agar diffusion tests is summarized in Table 3.

Biosurfactant genes Keiser et al.‘” have recently reviewed what is

known of the genetics of expression of surface-active compounds. Although the molecular bioiogy of this field is still in its infancy, the generation of mutant strains and subsequent isolation of genes involved in the biosynthesis of biosurfactants will become increas- ingly important in the attempt to influence regulation mechanisms and modit;/ production pathways. In

_ _ _ _ “ “ _ _

TfBTECH JUNE 1992 NOL 10)

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Gram-negative bacteria, the best-studied cases include ‘4, C&CWCC~~CIIS, Pscr~rl,:r;~urr~l,: and Scrn~io i,lrlrstjltlf,ls. When the strain A. c~lcoocericrrs RA57 war gown on cm& oii sludgcZJ, it was found to harbour four plasmids. Screening mutants that had lost the ability to grow on this substrate enabled the physiological intcr- action of the cells with the hydrocarbon substrate to bc assigned as a function of one of these plasmids, pSRi. This 20 kb plasmid is assumed to encode a factor tightly associated with the cell surface, rather than a factor influencing the production of ehe extra- cellular emulsifying activity. Experiments invceti- ga:ating the production of Emulsan, using mutancs and revcrtants of A. &~ncetictrs RAG-I, showed chat Em&an is not the only estraccllular facto: for emulsification27: mutants with enhanced Em&an production were found to contain mutations affecting an emutsifier-synthesis step leading eo enhanced capsule production3”.

In Pscr&~,rriur~ns &~t~~rcllls, the alkane-utilizir;g sys- tcm is cncodcd by the ol/&AC gcncs located on the OCT plasmid. The gcncs are csscntial for the alkane terminal hydroxylation and dehydrogcnation”‘. It is not yet known whether addicial-rl host-cncz!e? EC- tars arc also involved in alkanc uptake. Witholt cf irl.“’ suggcstcd that the cell-wall lipopolysaccharidcs arc involved in the emulsification process of hydrophobic substrates: the hetcrologous expression of the regulat- ory and structural genes of the n/&AC opcron in P. p&Id al\d E. cdi enabled rhcse organisms to grow on rr-octane as sole carbon and energy source33.

The biosynthetic pathway of rhamnohpld produc- tion in P. c~cn!~i~~~s~ during late exponential-growth phase has been clucidatcd3+-j’ as scqucntial glycosyl transfer catalysed by specific rhamnosyl transferascs, with TDP rhamnose as an efficient rhamnosyl donor. Twr rliffcrcnt transfcrascs lead to the formation of the four tliffcrcnt thamnolipids (RLs) shown in Fig. 2. WhiJr RLl and RL3 represent the typical products isolated from liquid culture, RL2 and RL4 are pro- duced exclusively in rta:ing cells. The contrJ of rhamnolipid synthesis is linked to the nitrogen metabolism of the cell”.

A chemically induced mutant of the strain KY-4025, displaying a tenfold reduction of rhamno- lipid production when grown on n-paraffin”‘, was iso- lated, though the lesion in rhamnolipid biosynthesis has not yet been described. Koch et RI.+” conscructcd transposon TN5-GM induced muants of P. ctew.yirww PG201 that were unable to grow on minimal medium with hexadecane as the sole carbo:l source. By ad- dition of small amounts of pm&,+ rhamnolipids, the growth of this mutant on !lexadecane could be restored. Furrhermorc, it was shown that the bio- synthesis step affected *was the production ofrhamno- syl transferace. Another mutant, 65El3, was also unable to produce extracellular rhamnotipids, or to

take up hexadecane. A cosmid clone capable of complemcntitlg this mutant defect was isolated and allowed to restore production of rhamnolipids and growth on hcxadccanr (A. K. Koch, PhD thesis,

Table 3. Antibietic a&vii of the surface-active lipopeptides from f3. lfcfien~if

Pseudomonas aeruginosa 1 +++ Escherichia co\; +-t-k+ Candida utilis : :.: +++ Candida trockalis 1 0:1 +++ frichosporch cufaneum i0 Saccharomyces cerevisiae 5 Trichoderma reesei 0.5 Penicillium 0;ialicum 0.1

f ++ ++

++++

aAntibiotic actkity against yeasts, bacteria and fungi in agardiffusion tesls. No inhibition of grow?h was found for Aspergiitus nidulafls, Phanerochaetae chrvsosporium, Bjerkandera adMa, Ustifaga maydts, Sordaria fimicob, BotryGs cinerea, Fusarkm fycopersici, Phytophthora infestans. Phytium debyryanum77. ! Abbreviatir;ns: M!CY, minimal inhibitory concentration against microorganism during the exponential growth phase; MKFd, minimum inhibitory concentration against microorganism during stationary and death phase. blnhibiion zone diameters: + = 7-iCI mm; ++ = 11-14 mm; +++ = 15-18 mm; ++++ = >18 mm.

ETH-Ziirich, Switzerland, 199?), In a17 zttcmpr zo USC cheap substrates for the production of biosu&ctants. Koch et 01. succeeded in constructing a lactorc-utiliz- ing strain of P. oc*n<@~t>sn by ins&on of the E. n& /(7~-2Y gcncs into the chromosome”.

In Sermdd ~~~d~ri(w~~~, spontaneously occutriu,g. non-

hydrophobic mutants were ana!ysed and classified in two groups% (1) mut:.nts showing a reduced adhcr- cncc tc hcsadccanc or polystyrene surf~ics; and (2) the pigmented mutants that formed translucent colonies as compared with wild-Tpc cells. T\vo moduiarcrs ot‘ cell-su&cc hydrophobicity were isollced and desctibed irl this organism: (I) sc~~phobin (see above). and (3j serratamolidc, an anlkjlipid asting as a wetting agent that increases the ccl1 surface hydrophilicity. presumably b>- blocking hydrophobic sit&“.

The cnzymcs catalysing chc ATl’-P,-cschange re- action in the biosynthesis of suriarrin in B. slrlrrilis were isolated and partialiy purified from cell-free extracr+4j. This exchange reaction was shoivn to be mediated by the amino acid COIIlpOIlclltS 0f

surt‘actiu. The substrate amino acids arc activated simultaneously as reactive a minnacyl phosphates by a multienzyme complex_ The ge~tic loci invol\-cd ill the production of surf&in ha\-= been identified and characterizedJ”-‘7: the 23 kb locus jrf;4 encodes wo

proteins that may constitute subuni& of rhe surt>cdu s+hctaseAT: $A is also impor.ant for competence development and cficient sporulation in B. &rilisi”.

Az&er group of St&cc-active compounds \vhosc molecular biology has been studied intenri\-cl! includes the lung surt;?ctmts. which cons:irutc a com- plex mixture of phospholipids, small amounts ofpro- teins, carbohydrates and neutral @ids. aud are found

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at the air-liquid interface in the lung alveoli. They are essential for normal respiration and their deficiency can lead to an ah-colar collapse in premature babies. Small (j-18 kDa) hydrophobic proteins are isolated as the only protein components from bovine prcp- arations of lung w&tams. In addition, hydrophilic, sialoglycoprotcins of 35 kDa calkd W-A or SAP-35 have also been found to be present in other lung surfaccants~~. SP-A shows remarkable similarity to Clq, a subunit of the first componm; of tix classical complement pathway and contains a collagen-like amino-terminal domain. Two proteins. SP-B (6-8 kDa) and SP-C (4.5 kDa). have been identified as human lung surfactants, and their primaly amino acid sequences have been derived from cDJVAS~‘~V. Cr.nstedt et al. have shown that native SP-C is a lipopeptidc with two palmitoyl groups attached to the polypeptide chain by covalent linkages-7. The humtn SP-A gene WIS cloned in 1985 by applying rcduccd- stringency hybridization tcchmques 50 the screening of cDNA 1ibratiessJ. Recently the entire human SP-I) gene was isolated and scq~~mced, and mapped to chromosome 2s’. III the case of human SP-C, two distinct genes WEX identified and sequenccds~ and both wcrc shown ~3 encode an active hydrophobic region in the protein thar could bc responsible for the low solubility of 9-C and its lipid association upon

isolation f?om the lung.

Production of biosurfactants Attempts to produce biosurfactants have CIICOUII-

tered a numbrr ofprocess difficulties. Among the pro- cess parameters influencing the type and amounts of biosurfactant groduced are the nature‘ of the carbon source, possible mltritional limitations, and physical and chemical parameter., su-h as aeration, temperatl;rr and pH. In addition, a major factor is the identity of organism or stmin used for the productic’n prorcas. In many cases, tGc ~ynthccis of tJJc hydrophilic and lipophilic surfactanr moieties derives tier- 1 the primary metabolism but relates to two different degradation pathways for carbohydrates and hydrocarbonP7. In most cases, growth on hydrocarbons induces the ayn- thesis ofhiosurfactants but this is not a prerequisite for all organism@. The carbon source is. however, an

important process parameter. Changing the substrate oeen &vc +‘- r*-.-+..-^ L CA".*. ,,., YbLY.C d&c pru&ct, thus altering thr properties of the surf&ant. The choice of the car- bon source is, therefore, determined by the intended specific application. One such good example is A&r&a&v: in Arthrobac/erculturcs, the trehalosc lipids formed were substituted by sucrose lipids when grown on sucrose54. In addition, the carbon soutcc also seems to determine whether the biosurfactant is extra- ceihilar or intracellular@‘. The nitrogen source and concentration as weJJ as the C:N ratio are also reported to have a major effect on biosutictant synthesisG’.Q. Other factors !eading to pronounced effects on the rhamnolipid production by P. aemgiltosa are the iron, magnesium, calcium and potassium salt concert- trations: these wcrc investigated success&Jiy to de-

-- ll6TECHJlNE19921'/OL10l

zo! . I * I . I . 1 . I

0.0 0.1 c.2 0.3 0.4 0.5 D(W)

Figure 4 Produc?ion of 6. ficheniformrs blosurfactant in continuous culture under aerobic (0). semi.aerobic ki) and anaerobic (01 conditions. The !Dwcst surface-tension ISi) values, and thus the highest biosur- f&ant p:odXtion rates were obtained when cells were zrown under semkanaerobic (2 conditions at dlkrtion rates (0) befrreen 0.1 and G.4: D is calcu!ated by dividing flux (I h-1) by reactor volume (1) IK. Jenny, PhD thesis no. 9263, ETWZijrich. Switzerland, 1990).

v&p dctincd media”‘.“‘. The influence ofaeratIon on the production of biosu&zmts bt B. liiilc,rri~~l;,;,rri.~ is shown in Fig. 4. The best results are obtained under scnii-anaerobic conditions (K. jenny. op. cit.).

The cffcienry of J range of production svstems, including varsrllls re:.ctor systems and culdvatioo modes such as f?d-batch, continuous cuiture. classic;: stirred tank reactor (STR} and immobilized cells on calcium alginate has rcccntly been cvaluatcd (Th. Grubcr, I’hD thesis, Univ. Stuttgart, FRG, 1991)). However. only low production rates wcrc achirvcd (gcnt~rally below 500 mg 1-I ‘n- :). Mass-transfer linu- cation, feedback inhibition and competition bctwce:: growth and production are the main reasons for thcsr rather disappointing producr yields. Grubcr (op. cit.) developed an iutegraced chcmostat production system combining a STR with two specific membrane mod- ules (Fig. 5). The first module retains the P. r~crr$rrr!ta cells and removes the rhamnolipid from the ctilturc brct!:, -.;-li& the second membrane module is respon- sible fbr gas cxchangc and thus helps to avoid foam formation. The control of foam formation in surfac- tant production processes by antifoam agents is in- adequate, and requires either such gas exchange filters and/or mechanical foam breakers. Under true steady- state conditions at optimal bleeding streams, a specific volumetric productivity af 545 mg 1-I h-l could bc rcachcd in a first attempt. It is therefore safe to spec- ulate that optimizing the design of moduhr pro- duction bystems (e.g. by cffic~~~t separation ofproducts from cr!!s to prevent possible feedback inhibition, by improving production media and by introducing oxygen limitation to redirect the ener&T f$ix into product formation) will eventually permit pro- ductivities ofbetween 5 and 8 g 1-l h- ’ This integrated chcmostat system equipped wisith modern me,mbrane technology and coupled to powerful on-line analysis is suited not only for the production of biosur&ants but also for the enrichment and selection of producer

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strains. Beside5 the progress in process development in the engineering Geld, a further contribution towards achieving higher yields is expected co arise 6om the genetic engineering of producer strains as an alternative approach. Once the molecular bio!ogy of the Lio- synthetic pathways is known, the genes encoding the enzymes involved may be expressed in hosts to allow the use of cheaper substrates (e.g. for P. r~en@r~~~~‘l grown on lactose or cheese whey; see above), co f;cili- tate product recovery and that could replace pathogenic producers such as P. arncgirt~rcn.

Much of the effort co date has been directed at the itnprovement of the economics and efficiency o’bio- processes in order to 410~ biosurfactants to compctc successfully with chhcmically synthesized surface-active compounds. So hr, few economic production systems for biosurfactants have been reportrd and t5c expected breakthrough in biosurfactant application has been hampered by the high production costs, the lack of pubhc acccptaacc OZ producer strains (e.g. P. rlm@w~z), and the required high purification for apphcatlons m the cosmetics, food and phannaccutica: industries.

Applications of biosurfactants and future potential

The increasing interest in the potcntiai applications of microbial surface-a&v; compounds is based on their broad nnge offunctional properties that includes emulsitcation, phase separation, wetting, foaming, solubilization, de-emulsification, corrosion-inhibition and viscosity-reduction (e.g. of heavy crude oils). There are, therefore, many areas of industrial appli- cation where chemical surfactants rculd be substituted by biosurfactants in fields as diverse as ahticulture, building and construction, the food and beverage industries, industrial cleaging, leather, paper and metal industries, textiles, cosmetics, the pharmaceutical industry and, last but not least, petroleum and petro- chemical industries”. The &king advantages of bio- surfactants over chemically synthesized sur&e-active compounds include their broad range of novel s~rtic- tural characteristics and physical properties, their pro- duction on renewable substrates, their capacity to be modified (by genetic engineerGg, biological or biochemical techniques) and thus tailored to meet specific requirements, and (probably most important) their biodegradability. Many chemical surfactants cause environmental problems due to their resistance to biodegradation and their toxicity when allowed to accu.mulate in natural ecosystems.

In spite of all these advantages, the industrial use of biosurfactants is limited, as yet, to applications in the petroleum indusF. Much effort has been put into the application of biosurfactancs for the microbial enhanced oil recovery (MEOR)a. It is estimated that only 3#-50% of the oil can be recovered from pet- roleum resour-es by conventional pumping techniques. Much higher values may be reached by applying the enhanced oil recovery by steam and fire flooding. A major factor here is to dccrese the surface- and intcr-

___zl> Bleed srream 10 proces6ng

Flow scheme of continuous produckw of P. aeru~mosa rharwoliptds by an inte- grated process iih. Gr&r. PRD the%., WV. %uttgart, FRG, 199i)). Two n-e&w fiitratkw mod&5 are respwsWe fortbe removal d prcdwt and for the gas erchange whkh avoids excessive foaming. A foam centiifuge instakd on top of the reactor containment inot SICIW) leads to acHit& separation capac~!~ of ga: and iiqmd.

facial iensiou between ;\ratcr and oil in the groutId. Biosurfactants could replace the relatixly expensive petroleum sulfonatcs or lignosulfonates that arc currently used for this purpose. It wouid. of course. bc panic&rly desirable to produce the biosurfacrant irt si~zr by simply introdncing .J producer culture undergrcnnd. Th:s, however, would require faculcar- ire anaerobic, thermophile and baxphilc producer strains. The descbpment c ‘MEOR is being pursued by most North-A!llcnc:lr-+ J d petroleum companies.

In 1987, the only comt 7~: Gal industrial biosurfJc- tant on the market was EC: &an. patented by Fucnick er n/.QJ and markcccd by Petroleum Fcrmenr~rions (Petro&ml) for uce ir. &aning orl-cor;taminaccl vessels, oil spills and MEOR. Em&an is also used t<> facilitate pipefine transportation of heat? crude oil because its ability to reduce viscosity contributes to reduced transportation costs. l’hc ~uccctsfi~ul appli- cation of 5iosu&ctanti 6 the cleaning ofoil tmks has recently been described”‘. Bacterial biosurfactants were used to clean an oil storage tauk of the Kuwait Oil Company by removal of the oil sludge from the tank bottom. Ofthe hydrocarbon found to be trapped in the slude, !XHt could bc easily recovcrcd 2nd cvcn sold aficr being blended with fresh crude. The appli- cation of biosu&ctants for the clean-up of oil spills is widely debated. A model system for the microbial soil decontamination has been developed and presenccd by Oberbremer and MiilIer-Rmig~l? tk cotnpirtc degradation of oil in a stirred-tank reactor by the original microbial soil population waz achieved, On the other hand, the use of biosurt~ccanh or chcir producer strains in the cleaning of oil-poilatcd ronval areas (such as the Yrincc Williain Bay dim the ikotl- Valdez oil spill) is not uniformly acccptcd. The short- term apparent succcss of such rre.mnent73 stands in sharp contrast to thr long-term &kcts. and the corresponding findings that uncrcatcd areas recovered much fastt+.

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111 tire food industry, surf&x-active compounds :KC used as emulsif:xs in food additives for the processing of raw matcrinls. Emi~lsificatiou plqs an importuit r& in forming the right conaistcrlcy :~nd tcxturc ;IS ucll as in phase dispersion. Other ~pplickms of surface-active comp~mnds ;~rc’ in h&cry atd IIIGI~

lmxiiicts whrrc they i&uct~cc the rh4ogical ~)l:~r;mcristics oiflour or tlic cr:~t*!sitkation ofp;uKi:llly broken bt ti\sttc. 111 ag. icukarr, surface-nctivc CoIll-

pounds arc nccdcd for the hyGrp!lilization of heavy soils to obtain good wcttability, :md also to achieve cqual distribution of fertilizers in the soil. A broad potential application 3rca is the cosmetics industry where surf&c-active substnnccs arc found in shampoos and many skin-cnrc products. The US market for COS- mcriss nnd toiletry raw matcri;:ls, which directly rcflccts thr world-wide dcnlnnd for surf&c-active compounds, rcnchcd USS I .6 X IIP in 198’3, and is prcdictcd to kr.rcase considcmbly7”. A rcvicw of this important market potential is prcsc-ntcd by t$rown7”.

Many of- the potential applications that hzvc hccn

considered for biosurfktants drpcnd on whcthcr they can bc produced cco~~omically. Much effort IS still nccdcd tkr process optimization at the engineering and biological lcvcls. Legal aspects such 3s stricter rcgulntions concerning the cnvironnrcntal pc!!ution by industrial activities, 3s well as health regulations, will also stronglv influcncc the rh:lnccc 4 biz2- dcgradahlc biofu;factar,ts rcplatring their chemical connterprts.

Acknowledgements The assistance by 12 Isabella Berctta and Wolfgang

Scghczzj in the prcparntion ofthc manuscript is highly apprcciatcd.

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.-

European Commission Biotechnology ?992-MS4

Deadline for propasals: 23 July 1992

Proposals from EC and EFTA members for financial support under the EC Biotechnology initiative (budget 143 MloECU) should address more basic research than those for the BRIDGE programme that was launched in Jan 1990. The current programme seeks to support research in three main areas:

(1) Molecular approaches (including Membrane proteins and catalysis, Design and application of antigen-binding sites, Receptor ?ructure/function and signal transduction, Biocatalysis, and Genome structure of Bacillus subfilk Saccharomyces cerevisiae and Arabidupsis fhaiiana). (2) Cellular and organism approaches (including Control of development, Bovine developmenUgenome mapping, Mechanism of action of plant growth factors, In vitro development, Toxicology, Metabolism of animals, plants and microbes, Human and animal vaccines, In V&I human responses, in vifro toxicology, and Neurobiology). (3) Ecology. and population biology approaches (inc/&ing Ecological impkations of biotechnology, Rapid molecular-screening methods, Taxonomy, preservation and exploratiorr of biodiversity).

Funds are also available to help integrate existing national projects, to remove ‘technology bottlenecks’ in the research areas addressed, and to support associated activities (e.9. travel. seminars, co-ordination etc. j.

Associated cost-shared research actions * Basic research: integrating projects where progres, is hindered through gaps in knowledge. * Generic research: directed toward removing ‘bottlenecks’ ;Nhich arise through limitation of scale or

structure. ?? ?rc#ects of fechnologkal priorify: c&ordinatot% may apply for funds to implement and manage a iarge-

s&ie project) combining national and EC Community resources. * Concerted acfions: co-ordinators may apply to ‘add v&Y to projects adequatel)‘Cunded in different EC

member states, through meetings, reports and short visits to laboratories. *Accompanying measures: application for support of workshops, co-ordination or the dissemination of

information and promotion of results:

Contact details ??For research proposakx Directorate General for Science Research and L%e!opment, Division

Biotechnology DGXII-F2, Rue de la Ini 200, B-1049 Brussels, Belgium. ??Programme mana&?r: D. de Nettancourt

Tef: +32 2 2354044/2356491 Fax: +32 2 2355385 -

TtBTECH JUlw 1?92 (VOL 101