[J. Ferment. Technol., Vol. 62, No. 4, p. 311-314, 1984]
`
`Selection of Microbes Producing Biosurfactants in
`Media without Hydrocarbons
`
`CATHERINE N. MULLIGAN, DAVID G. COOPER, and RONALD J. NEUFELD
`
`Biochemical Engineering Research Unit, McGill University, 3480 University Street,
`Department of Chemical Engineering, Montreal, PQ_, Canada H3A 2A7
`
`Microorganisms capable of synthesizing biosurfactants on non-hydrocarbon sub­
`strates were screened by their hemolytic activity on blood agar plates. Hemolytic zones
`around colonies could be related to the ability of the microbes to produce surfactants.
`The degree of clearing caused by surfactin, a biosurfactant from Bacillus subtilis, was
`proportional to the concentration applied. Microorganisms capable of producing
`biosurfactants on hydrocarbon-containing media only did not demonstrate lytic activity.
`The technique of using blood agar plates to screen for biosurfactant production on soluble
`substrates is shown to be quick and reliable.
`
`Materials and Methods
`
`Most of the known microbial surface active been shown that surfactin, although it is not
`agents originate from bacteria growing on a hemolytic enzyme, can rupture erythrocytes
`hydrocarbons or related substrates. 1-6> There
`in liquid media.8'
`are a few exceptions, such as the lipopeptide
`surfactin from Bacillus subtilis, produced on
`water-soluble substrates. 7~9>
`In spite of the
`The organisms were grown in media which have
`been described elsewhere. These included Bacillus
`temporary reduction in the price of crude oil,
`subtilis,9>
`Corynebacterium
`lepus,2>
`Corynebacterium
`it is essential to isolate organisms which can
`fascians10) Corynebacterium
`insidiosum1' and Torulopsis
`assemble biosurfactants from cheap, renewable
`bombicolafl
`substrates if production is to be commercially
`In the screening studies, bacteria were isolated from
`feasible.
`environmental sources by inoculating a medium con­
`Attempts have been made to screen for
`sisting of 4% glucose, 0.1% yeast extract, 0.1% nutrient
`organisms which synthesize surfactants during broth, 0.4% NH4NO3,0.4% KH2PO4,0.4% Na2HP04,
`growth on soluble substrates or to induce
`and trace amounts of MgSOs, CaCl2, FeSOi, and Na2
`surfactant production, on these substrates,
`EDTA. After 2 days growth, these mixed cultures
`from microbes known to synthesize surfactants
`were used to inoculate sheep blood agar plates obtained
`from the Institute Armand Frappier, Laval, Quebec.
`from hydrocarbons.3'10'11) This work has
`The plates were incubated for 2 days at 25°C. Single
`been slow because there was no quick method
`colonies were used to inoculate fresh media containing
`to evaluate which organisms were potentially
`4% glucose, 0.1% yeast extract, and the mineral salts.
`useful for this purpose. It was necessary to
`Surface tensions were determined using a Fisher
`grow each microbe for several days on the
`Autotensiomat. Surfactin was isolated from B. subtilis
`chosen medium and then test for surface
`and purified using previously published methods."
`activity.
`Similarly, experiments for Strain Diffusion of surfactin through the agar was studied by
`improvement of biosurfactant producers are placing aqueous solutions into a well in the blood agar
`not feasible until a method is developed to made with a 10 fil micropipette.
`select
`improved clones.
`The use of blood agar plates to characterize
`certain hemolytic bacteria is well known.12'13'
`These plates also provide a rich growth
`medium for many organisms.
`It has already
`
`Results
`Spreading dilute cultures of B. subtilis on
`blood agar and incubating for 2 days resulted
`in colorless rings around the colonies. Typ-
`
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`312
`
`MULLIGAN, COOPER, and NEOFELD
`
`[J. Ferment. Technol.,
`
`ically these were about twice as wide as the
`diameter of the colonies.
`Figure 1 illustrates the results of adding
`various concentrations of aqueous solutions
`of surfactin to narrow wells punched into the
`agar. The maximum diameter was reached
`4 h after the addition of the solution. As the
`concentration of surfactin
`increased
`the
`diameter of the resulting cleared area also
`increased.
`Three other bacteria, C. fascians, C. insidio-
`
`sum, and C. lepus, could all be induced to
`grow on the blood agar but caused no lysis
`of the red blood cells. All three bacteria
`produce significant quantities of biosurfactant
`when grown on hydrocarbon media but do
`not produce these products on carbohydrate
`media.
`The yeast T. bombicola was also unable to
`clear the agar. This organism produced
`copious amounts of glycolipid surfactant on
`media containing carbohydrate and vegetable
`
`Table 1 Results of experiments to isolate biosurfactant-producing organisms.
`
`Source ofa
`organism
`
`Ability to
`cause
`clearing
`
`Surface1"
`tension
`(mN/m)
`
`Colony
`morphology
`
`Gram
`stain
`
`Microscopic0
`observations
`
`A
`A
`
`B
`C
`
`D
`
`B
`
`B
`E
`
`F
`
`D
`A
`
`G
`
`G
`D
`
`D
`
`C
`B
`
`+
`
`+
`+
`+
`
`4-
`+
`+
`+
`+
`+
`
`72
`68
`
`68
`68
`
`67
`
`63
`
`63
`56
`
`55
`
`55
`55
`
`48
`
`45
`43
`
`40
`
`38
`37
`
`smooth, yellow
`smooth, beige,
`small
`white, small
`smooth, bright
`yellow
`rough, beige,
`irregular
`dark yellow,
`small
`flat, clear
`rough, beigh,
`small
`
`smooth, yellow
`
`white, large
`pale-yellow,
`small
`
`dull, beige
`
`biege, irregular
`dull, white, flat
`
`large, white,
`irregular
`smooth, yellow
`smooth, white,
`small
`
`+
`
`+
`+
`
`+
`+
`+
`
`+
`+
`+
`
`+
`+
`+
`+
`
`-f-
`
`+
`
`motile, spores
`non-motile
`
`non-motile, cocci
`motile, short
`chains
`motile
`
`small, non-motile
`
`non-motile
`non-motile,
`spores, short
`chains
`non-motile,
`short chains
`motile
`non-motile,
`spores, short
`chain
`non-motile,
`spores, paired
`non-motile, spores
`motile, spores
`small
`motile, irregular
`shape
`motile, small
`non-motile,
`spores, short
`chains
`motile, spores
`short chains
`motile
`
`+
`
`D
`
`A
`
`+
`+
`
`32
`
`29
`
`smooth, beige,
`flat
`irregular, white
`large
`a Sources include; A, water from a peat bog; B, neopeptone solution exposed
`to air in laboratory; C, industrial sewage sludge; D, Athabasca tar sand;
`E, diseased leaf from Crassula species; F, oil from aeration basin; G, bark
`used as potting medium for orchid species.
`b Surface tension of liquid media inoculated with the colony.
`c All rod-shaped unless noted otherwise.
`
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`

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`Vol. 62, 1984]
`
`Biosurfactant-Producing Microbes
`
`313
`
`O
`
`diluted 200-fold before an appreciable
`increase was observed in the surface tension,
`suggesting biosurfactant concentrations great­
`ly in excess of the CMC.
`
`Ur
`
`1.2 -
`
`•O'
`
`2:
`
`o-
`
`1 <>•'-
`2
`O 0.2-
`—o
`
`Discussion
`
`It has been shown previously that the
`lipopeptide surfactin isolated from B. subtilis
`can cause the lysis of red blood cells.8>
`Inoculating blood agar plates with B. subtilis
`resulted in clear rings of lysed erythrocytes
`around each colony.
`If
`surfactin was
`added to wells in the blood agar the degree
`of clearing was proportional to the con­
`centration of surfactin in the solution. Thus
`the lysis of the red blood cells was related to
`the production of a highly surface active com­
`pound by B. subtilis.
`The Gorynebacteria C. lepus, C. fascians,
`and C. insidiosum all require the presence of a
`hydrocarbon to produce significant quantities
`of extracellular surfactants.J'2'10'11) Thus
`it was not surprising that none of these
`bacteria caused clearing of the blood agar
`plates. T. bombicola produces large amounts
`of glycolipid only if there is vegetable oil or
`related compounds in the growth media, and
`colonies on the blood agar did not cause
`clearing.
`If the surfactant was isolated from
`an appropriate fermentation of T. bombicola
`and put on the blood agar, this did result in
`clearing.
`Thus clearing of the agar around a colony
`could be an indication that the organism was
`capable of producing appreciable quantities
`of biosurfactant on carbon sources other than
`hydrocarbons. It could also
`indicate a
`lytic enzyme, however the surfactant pro­
`ducers are easily verified by inoculating
`liquid media and measuring the surface
`tension after a few days.
`This method was used for preliminary
`screening of a number of microorganisms from
`a variety of sources for the ability to produce
`biosurfactants on non-hydrocarbon media.
`Non-clearing colonies were
`incapable of
`biosurfactant production in liquid media
`while clearing isolates produced biosurfac­
`tants, some greatly in excess of the CMC.
`
`10
`SURFACTIN CONC. (g/l)
`Fig. 1. A plot of the diameter of the clear zone vs
`the concentration of the surfactin solution added
`to the well in the blood agar.
`
`K
`
`16
`
`oil but not on carbohydrate alone. The
`glycolipid was isolated from a fermentation
`using the appropriate medium and streaked
`on the blood agar. This compound was
`able to lyse the erythrocytes and cause clear­
`ing.
`This method was used for a preliminary
`screening of a number of organisms from a
`variety of sources for the ability to produce
`biosurfactants on non-hydrocarbon media.
`Table 1 contains data for a number of typical
`isolates. The table includes results for all
`of the clearing colonies and some of the non-
`clearing colonies. Seven of
`the colonies
`which did not cause clearing were tested for
`surfactant production. In each case, there
`was no surfactant produced and the surface
`tension remained above 60 mNm-1. Twelve
`isolates, from a variety of sources, lysed the
`erythrocytes. Half of these were not promis­
`ing as the surface tension measurements when
`cultured in liquid media ranged from 45 to
`55 mNm-1. In most cases, diluting
`the
`broth samples several fold caused the initial,
`low surface tension to increase. This sug­
`gests that the surfactant concentration was
`less than or near the critical micelle concentra­
`tion (CMC). The other half of the isolates
`which caused clearing, produced effective
`biosurfactants and two of these resulted in
`very low surface tensions of 32 and 29 mNm-1.
`Samples from the culture broth could be
`
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`EXHIBIT NO. 1026 Page 3 of 4
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`

`
`314
`
`MULLIGAN, COOPER, and NEUFELD
`
`Appl.
`
`and by the Natural Sciences and Engineering Research
`The CMC is the minimum amount of
`surfactant required to cause the maximum Council of Canada,
`decrease in surface tension, and excess sur­
`factant allows proportional dilution of the
`mixture without an
`increase
`in surface
`tension. Most of the biosurfactants were
`produced in low yields and diluting the
`samples showed that in most cases the con­
`centration present varied from a few-fold
`excess of the CMC to insufficient biosurfactant
`to reach the CMC. The most promising
`exception to this was the organism which
`produced
`the most
`effective surfactant
`(29 mNm-i). In this case the concentration
`produced was 200 times the CMC.
`The use of blood agar provides a quick
`preliminary screening
`technique for
`the
`selection of microorganisms capable of pro­
`ducing biosurfactants on renewable, water-
`soluble substrates. There is a correlation
`between the amount of surfactant and the
`amount of lysis (Fig. 1) and potentially
`this method could also be used to select for
`superior clones in mutation studies. This
`technique may also select organisms produc­
`ing lytic enzymes but these are quickly
`eliminated by growing the microbes in liquid
`culture and measuring surface tension.
`
`References
`^ Akit' J'' GooPer> D' G-> Mannmen, J. I., Zajic,
`J. E.: Curr. Microbiol., 6, 145 (1981).
`2) Cooper, D. G., Zajic, J. E., Gerson, D. F.:
`Environ. Microbiol., 37, 4 (1979).
`3) Cooper, D. G., Liss, S. N., Longay, R., Zajic,
`J. E.: J. Ferment. Technol., 59, 97 (1981).
`4) Cooper, D. G., Zajic, J. E.: Adv. Appl. Microbiol.,
`26, 229 (1980).
`5) Gorin, P. A. J., Spencer, J. F. T., Tullock, A. P.:
`Can. J. Chem., 39, 846 (1961).
`6) RaPP> P-> Bock' H-> Wra>'' V" WaSner> F-: J-
`Gen. Microbiol., 115, 491 (1979).
`7) Arima, K., Kakinuma, A., Tamura, G.: Bio-
`chem. Biophys. Res. Commun., 31, 488 (1970).
`8) Bernheimer, A. W., Avigad, L. S.: J. Gen. Micro-
`biol., 61, 361 (1970).
`9) Cooper, D. G., Macdonald, C. R., Duff, S.J. B.,
`Kosaric, N.: Appl. Environ. Microbiol., 42, 408
`(1981).
`10) Cooper, D. G., Akit, J., Kosaric, N.: J. Ferment.
`Technol, 60, 19 (1982).
`11) Gerson, D. F., Cooper, D. G., Ramsay, B. A.,
`Zajic, J. E.: Can. J. Microbiol., 26, 1498 (1980).
`12) Snavely, J. G., Brahier, J.: Amer. J. Clin. Path.,
`33, 511 (1960).
`13) Stainsby, W.J., Nicholls, E. E.: J. Lab. Clin.
`Med., 17, 530 (1932).
`
`Acknowledgement
`
`This research was supported by the program Forma­
`tion de Chercheurs et Action Concertee (Quebec)
`
`(Received January 18, 1984)
`
`v'>
`
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