208
reviews
Biosurfaetants: mavingtowa industrialapglkation Armin FiechteerChemically 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 biosurfactant-producing strains, and to improve process technology to reduce production costs. Surfactants and cmulsificn arc intcgzl to many industrial, agricultural and food proccsscs. Mo5.i of the compounds arc chemically synthesizd, and it is only in the past t;lw decades that s\t&x-active molc~:~!cs ofbiological 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 rcachcd market wiuc for soaps and detergents 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 surfrceactive 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 compounds 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 USCcxpcnsivc substrates. Prcrequi:itcs CX biosurfactants gaining a si~GLicant share ofthc n;arket 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 facilitate product r~ovcr:_. A broad r.mge o< surfactants is already known (for rc~ KWS scr’Rcfs Z-C), and as the list ofbiosurfactantproduci: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)
widen, thus leading to the discovery of surfactants sui:ed for specialty applications. Improved charactcri&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 biodcgradable 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 surfactants 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 In the literature, Ihe temls GWS, by microorganisms. sur&tant and cmulsiticr are frequently used interchangeably. However, whereas the molecular structure 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 behaviour of contacti!;g molecules at the inter&cc between different phase? constitute: a facused and Fastgowing 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 hydrophobic 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 phospbzr-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
““gy
L-f bn
k.j
Rhamnolipid
CH-. iihamnulipid
2
Rhamnolipid
1
3
on
-I Figure 2 Four different forms of rhamnolipids (!?LJ synthesized by Pseudomonas aeruginosa DSM 2659. While RLl and A;3 represent the ty,picalprodticts from liquid cultures. RL2 and RL4 are produced by resting cells only.
??The glycolipid surfactunts
Thcsc include the sophorosc-, rhamnxc-. txtion~ a range of halos+, sucrose- and fructose-lipids species such as Tor;r!0fs~‘5~ I:‘ww~ f mfrom7.i’~ or Arllmhctcr13, and also nx~~~nos~-~ cl?;thritol lipids tionl Crrr&d,x and 4iz~& species, ald cellobiow llptdc tkotn 1_y~iI g&&q of ~~,ll_5:_~~~ac-~,-~~~ in&de :irc orthinine-containing lipid Pseudomonas rubercerrsl6, the lysine-containing lipid of Agrobdctetiunz trmz$kieens, and the orthinine-taurine lipid (Cerilipin) of Chconobuctev cerintts’7. The unusual amino acid taurine makes this last h+c&_r=nt o;.: of thy f&: with a sulphare grottp. The omithine-ccntaming lipid is a zwitterion, with the J3-hydroxy carboxylic acid and the esterified carboxylic acid providing both a iree carboxyl group and a free amino group.
Table 1. L-$apeptideantibl~ticsof kc&: Name (wow4
Organism
Structure
Amino acid (aa)
Fatty acid (Wb
spp.:
Characteristics ST, IT Antibiotic (mN m-1) a&&y
Cycticlipopeptides Win group kurin A
8. silbfilis
FA&n-oTyroAsmGlrr+ProoAsh&er
fturin C Uyc! !subtilisin
@NH C14-,5:
7
37.5
bactericidal frngicidalc
B-NH C,,,,: -. fPL,6 = 6% = 29% i& i-C,, = 7% aiC,, = 54%
7
” .
cdu dition of small amounts of pm&,+ rhamnolipids, the s+hctaseAT: $A is also impor.ant for competence growth of this mutant on !lexadecane could be development and cficient sporulation in B. &rilisi”. restored. Furrhermorc, it was shown that the biosynthesis step affected *was the production ofrhamnosyl transferace. Another mutant, 65El3, was also Az&er group of St&cc-active compounds \vhosc unable to produce extracellular rhamnotipids, or to biology has been studied intenri\-cl! take up hexadecane. A cosmid clone capable of molecular complemcntitlg this mutant defect was isolated and includes the lung surt;?ctmts. which cons:irutc a complex mixture of phospholipids, small amounts ofproallowed to restore production of rhamnolipids and growth on hcxadccanr (A. K. Koch, PhD thesis, teins, carbohydrates and neutral @ids. aud are found
___-__--
TWECH JJtG1492iVCk101
214 reviews
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 prcparations 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 rcduccdstringency 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 CIICOUIItered a numbrr ofprocess difficulties. Among the process 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 and many cases, tGc ~ynthccis of tJJc hydrophilic 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 aynthesis ofhiosurfactants but this is not a prerequisite for all organism@. The carbon source is. however, an important process parameter. Changing the substrate +‘- ,,., r*-.-+..-^ oeen &vc YbLY.C d&c pru&ct, thus altering L CA".*. thr properties of the surf&ant. The choice of the carbon 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 extraceihilar 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 concerttrations: these wcrc investigated success&Jiy to de-ll6TECHJlNE19921'/OL10l
zo!
0.0
.
I
0.1
*
I
.
c.2
I
0.3
.
1
0.4
.
I
0.5
D(W) Figure4 Produc?ionof 6. ficheniformrsblosurfactant in continuous culture under aerobic (0). semi.aerobic ki) and anaerobic (01 conditions. The !Dwcst surface-tension ISi) values, and thus the highest biosurf&ant p:odXtion rates were obtained whencells 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 linucation, 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 modules (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 responsible fbr gas cxchangc and thus helps to avoid foam formation. The control of foam formation in surfactant production processes by antifoam agents is inadequate, and requires either such gas exchange filters and/or mechanical foam breakers. Under true steadystate 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 speculate that optimizing the design of moduhr production 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 productivities 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
215 mkews
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 Liosynthetic pathways is known, the genes encoding the enzymes involved may be expressed in hosts to allow the use of cheapersubstrates (e.g. for P. r~en@r~~~~‘l grown on lactose or cheese whey; see above), co f;cilitate 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’bioprocesses 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 application 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 petrochemical industries”. The &king advantages of biosurfactants over chemically synthesized sur&e-active compounds include their broad range of novel s~rtictural characteristics and physical properties, their production 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 petroleum 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 integrated 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. expensive Biosurfactants could replace the relatixly 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 faculcarire 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 biosurfJctant 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~ulapplication 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 application 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 ikotlValdez oil spill) is not uniformly acccptcd. The shortterm 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+.
111tire 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. 111ag.icukarr, surface-nctivc CoIllpounds 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 COSmcriss 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 biz2dcgradahlc 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.
._....- __.“_ IBTECH JUPjE 1
L’I I
reviews
.-
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 iarges&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 ??
??
TtBTECHJUlw 1?92 (VOL 101
reviews
Biosurfaetants: mavingtowa industrialapglkation Armin FiechteerChemically 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 biosurfactant-producing strains, and to improve process technology to reduce production costs. Surfactants and cmulsificn arc intcgzl to many industrial, agricultural and food proccsscs. Mo5.i of the compounds arc chemically synthesizd, and it is only in the past t;lw decades that s\t&x-active molc~:~!cs ofbiological 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 rcachcd market wiuc for soaps and detergents 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 surfrceactive 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 compounds 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 USCcxpcnsivc substrates. Prcrequi:itcs CX biosurfactants gaining a si~GLicant share ofthc n;arket 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 facilitate product r~ovcr:_. A broad r.mge o< surfactants is already known (for rc~ KWS scr’Rcfs Z-C), and as the list ofbiosurfactantproduci: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)
widen, thus leading to the discovery of surfactants sui:ed for specialty applications. Improved charactcri&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 biodcgradable 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 surfactants 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 In the literature, Ihe temls GWS, by microorganisms. sur&tant and cmulsiticr are frequently used interchangeably. However, whereas the molecular structure 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 behaviour of contacti!;g molecules at the inter&cc between different phase? constitute: a facused and Fastgowing 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 hydrophobic 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 phospbzr-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
““gy
L-f bn
k.j
Rhamnolipid
CH-. iihamnulipid
2
Rhamnolipid
1
3
on
-I Figure 2 Four different forms of rhamnolipids (!?LJ synthesized by Pseudomonas aeruginosa DSM 2659. While RLl and A;3 represent the ty,picalprodticts from liquid cultures. RL2 and RL4 are produced by resting cells only.
??The glycolipid surfactunts
Thcsc include the sophorosc-, rhamnxc-. txtion~ a range of halos+, sucrose- and fructose-lipids species such as Tor;r!0fs~‘5~ I:‘ww~ f mfrom7.i’~ or Arllmhctcr13, and also nx~~~nos~-~ cl?;thritol lipids tionl Crrr&d,x and 4iz~& species, ald cellobiow llptdc tkotn 1_y~iI g&&q of ~~,ll_5:_~~~ac-~,-~~~ in&de :irc orthinine-containing lipid Pseudomonas rubercerrsl6, the lysine-containing lipid of Agrobdctetiunz trmz$kieens, and the orthinine-taurine lipid (Cerilipin) of Chconobuctev cerintts’7. The unusual amino acid taurine makes this last h+c&_r=nt o;.: of thy f&: with a sulphare grottp. The omithine-ccntaming lipid is a zwitterion, with the J3-hydroxy carboxylic acid and the esterified carboxylic acid providing both a iree carboxyl group and a free amino group.
Table 1. L-$apeptideantibl~ticsof kc&: Name (wow4
Organism
Structure
Amino acid (aa)
Fatty acid (Wb
spp.:
Characteristics ST, IT Antibiotic (mN m-1) a&&y
Cycticlipopeptides Win group kurin A
8. silbfilis
FA&n-oTyroAsmGlrr+ProoAsh&er
fturin C Uyc! !subtilisin
@NH C14-,5:
7
37.5
bactericidal frngicidalc
B-NH C,,,,: -. fPL,6 = 6% = 29% i& i-C,, = 7% aiC,, = 54%
7
” .
cdu dition of small amounts of pm&,+ rhamnolipids, the s+hctaseAT: $A is also impor.ant for competence growth of this mutant on !lexadecane could be development and cficient sporulation in B. &rilisi”. restored. Furrhermorc, it was shown that the biosynthesis step affected *was the production ofrhamnosyl transferace. Another mutant, 65El3, was also Az&er group of St&cc-active compounds \vhosc unable to produce extracellular rhamnotipids, or to biology has been studied intenri\-cl! take up hexadecane. A cosmid clone capable of molecular complemcntitlg this mutant defect was isolated and includes the lung surt;?ctmts. which cons:irutc a complex mixture of phospholipids, small amounts ofproallowed to restore production of rhamnolipids and growth on hcxadccanr (A. K. Koch, PhD thesis, teins, carbohydrates and neutral @ids. aud are found
___-__--
TWECH JJtG1492iVCk101
214 reviews
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 prcparations 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 rcduccdstringency 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 CIICOUIItered a numbrr ofprocess difficulties. Among the process 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 and many cases, tGc ~ynthccis of tJJc hydrophilic 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 aynthesis ofhiosurfactants but this is not a prerequisite for all organism@. The carbon source is. however, an important process parameter. Changing the substrate +‘- ,,., r*-.-+..-^ oeen &vc YbLY.C d&c pru&ct, thus altering L CA".*. thr properties of the surf&ant. The choice of the carbon 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 extraceihilar 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 concerttrations: these wcrc investigated success&Jiy to de-ll6TECHJlNE19921'/OL10l
zo!
0.0
.
I
0.1
*
I
.
c.2
I
0.3
.
1
0.4
.
I
0.5
D(W) Figure4 Produc?ionof 6. ficheniformrsblosurfactant in continuous culture under aerobic (0). semi.aerobic ki) and anaerobic (01 conditions. The !Dwcst surface-tension ISi) values, and thus the highest biosurf&ant p:odXtion rates were obtained whencells 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 linucation, 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 modules (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 responsible fbr gas cxchangc and thus helps to avoid foam formation. The control of foam formation in surfactant production processes by antifoam agents is inadequate, and requires either such gas exchange filters and/or mechanical foam breakers. Under true steadystate 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 speculate that optimizing the design of moduhr production 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 productivities 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
215 mkews
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 Liosynthetic pathways is known, the genes encoding the enzymes involved may be expressed in hosts to allow the use of cheapersubstrates (e.g. for P. r~en@r~~~~‘l grown on lactose or cheese whey; see above), co f;cilitate 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’bioprocesses 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 application 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 petrochemical industries”. The &king advantages of biosurfactants over chemically synthesized sur&e-active compounds include their broad range of novel s~rtictural characteristics and physical properties, their production 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 petroleum 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 integrated 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. expensive Biosurfactants could replace the relatixly 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 faculcarire 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 biosurfJctant 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~ulapplication 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 application 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 ikotlValdez oil spill) is not uniformly acccptcd. The shortterm 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+.
111tire 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. 111ag.icukarr, surface-nctivc CoIllpounds 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 COSmcriss 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 biz2dcgradahlc 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.
._....- __.“_ IBTECH JUPjE 1
L’I I
reviews
.-
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 iarges&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 ??
??
TtBTECHJUlw 1?92 (VOL 101
