World Journal of Microbiology and Biotechnology 7, 53-81
Nutritional requirements and growth characteristics of a biosurfactantproducing Rhodococcus bacterium
A.S. A b u - R u w a i d a ,
The nutritional requirements and growth characteristics of a biosurfactant-producing Rhodococcus bacterium isolated from Kuwaiti soil were determined. Maximum cell yields (6.6 g/I) and biosurfactant production were achieved with a medium containing 2% (v/v) n-paraffin as a carbon and energy source, 0.2% lactose broth, optimal concentrations of nitrogen (nitrate), phosphorus, iron, magnesium and sodium sources, and minimal concentrations of potassium and trace element sources. The optimal pH was 6.8 for surfactant production and optimal temperature was 37°C. The biosurfactant produced after 16 to 33 h growth in a 7 I fermenter decreased both surface tension and interfacial tension of culture broth to below 27 and 1.8 mN/m, respectively, and was effective at critical micelle dilutions of 10 -3. Data on biosurfactant biosynthesis suggest that the product is produced as a primary metabolite and, therefore, could be produced effectively under continuous fermentation conditions. A.S. Abu-Ruwaida, S. Haditirto and A. Khamis are with the Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109 Safat, Kuwait. I,M. Banat is now in Londonderry, Northern Ireland but was at the Kuwait institute for Scientific Research at the time this paper was written. A.S. Abu-Ruwaida is the corresponding author. (continued on next page) @ t991 Rapid Communications of Oxford Ltd.
I . M . B a n a t , S. H a d i t i r t o a n d A. K h a m i s
Biosurfactants are surface-active compounds that can be synthesized by many microorganisms during their growth (Cooper & Zajic 1980; Zajic & Seffens 1984; Singer t985). Their spontaneous release and function is often related to hydrocarbon uptake; therefore, they are synthesized predominantly by hydrocarbondegrading microorganisms (Hisatsuka et al. 1971; Rapp et al. 1979; Kappeli & Finnerty 1980; Ito & Inoue 1982). There have been, however examples of the use of a water-soluble substrate for biosurfactant production by some microorganisms (Guerra-Santos et aL 1986; Cooper & Goldenberg 1987; Palejwala & Desai 1989). Since most synthetic surfactant requirements can be met by biosurfactants, world-wide interest in these has increased. This is particularly due to their potential applications in the oil industries, which include cleaning oil slud I~ein storage tanks and tankers, enhancing oil-recovery processes, mobilizing heavy crude oil, transporting petroleum in pipelines and managing oil spills at sea (Chakrabarty 1984; Haferburg et al. 1986; Rosenberg 1986; Desai 1987). A large variety of biosurfactants have been reported (Cooper & Zajic 1980; Parkinson 1985; Rosenberg 1986; Desai 1987). The type, quantity and quality of biosurfactants produced have been shown to be influenced by the nature of the carbon substrate (Singer et al. 1983), the concentration of N, P, Mg, Fe and Mn ions in the medium (Atlas 1981 ; Cooper et at. 1981a,b; Guerra-Santos et aL 1984, 1986; Haferburg et al. 1986), and culture conditions, such as pH, temperature, agitation and dilution rate in continuous culture (Margaritis et al. 1979; GuerraSantos et al. 1984, 1986; Ratledge 1986). This paper reports on the nutritional requirements, growth characteristics and biosurfactant production by a newlyisolated Rhodococcus bacterium grown on hydrocarbons as the carbon source for potential future use in the oil industry in Kuwait. Materials and Methods Microorganism and Growth Medium
The microorganism used in this study, code name ST-5, is a Gram-positive, non-fermentative, rod-shaped bacterium that exhibits some morphological variations and has been assigned to the bacterial group Rhodococcus. This bacterium was
53
A.S. Abu-Ruwaida et al. tn view of the annexation of Kuwait by Iraq in August 1990, this paper has been accepted without return to the author for attention to minor details. The Editor-in-Chief therefore assumes full responsibility for any errors or omissions.
isolated from an oil-contaminated soil sample in Kuwait and was grown on a mineral salts medium developed earlier at Kuwait Institute for Scientific Research (KISR) for methylotrophic bacteria (Abu-Ruwaida et al. 1990), supplemented with 1 to 4% (v/v) n-paraffin as a carbon and energy source. Initial medium pH was adjusted to 6.8. Bacto agar (1.5% w/v) was added to obtain solid medium for plate cultures. Inoculum Preparation and Culture Cultivation Both inoculum preparation and culture cultivation were carried out in 250 ml Erlenmeyer flasks containing 100 mt of the medium. Generally, the inocula used made up 1 to 2% (v/v) of the medium and were obtained from an 18 to 24 h grown culture. Incubation was carried out at 37°C (except when otherwise specified) on a rotary shaker (New Brunswick Scientific, USA) at 300 rev/min. Batch Fermentation A %Chemap CF-2000 fermenter (Mannedorf, Switzerland) was charged with the mineral medium and sterilized at 121°C for 40 rain. Carbon source (1 to 2% v/v n-paraffin), and growth stimulant (lactose broth) were separately sterilized by filtration and added to the fermenter. An active inoculum (1.0% v/v) was aseptically added to the sterilized medium and grown at 37°C, with the pH controlled at 6.8 and the dissolved oxygen at about 30 to 50% saturation. The pH was controlled by the automatic addition of sterile 0.5 M NaOH or 0.5 M HC1. Samples (ca. 100 ml broth) were withdrawn periodically from the fermenter for measuring surface and interfacial tensions, cell dry weight, emulsification index, optical density and medium composition. The optical density of the broth was measured using a Bausch and Lomb Spectronic 20 spectrophotometer (Berlin, Germany) and 1-cm-diameter glass cuvettes at 620 nm wavelength. The broth was centrifuged using a Beckman J2-21 centrifuge (Beckman RIIC Ltd., UK). A n@tical Procedures Biomass dry weights were determined by filtering the cells from a 5 to 10 ml sample of the culture broth through a pre-washed and weighed 7-cm-diameter Whatman No. 1 filter paper (0.45 #m pore size) and using a pre-weighed aluminum dish. The samples were dried at 105°C overnight and reweighed after 24 h. Surface tension, interracial tension and relative concentration of biosurfactant expressed in terms of CMD were measured as described by Abu-Ruwaida et at. (submitted) using a Kruess K I O T tensiometer (Kruess, Optische-Mechanische Werkstatten, Hamburg, Germany). Emulsifying index (E24) was measured according to the method described by Cooper & Goldenberg (1987). Results and Discussion
Table 1. Effect of different carbon sources on the surface tension of the culture supernatant of Rhodococcus sp. ST-5, Carbon Source
Glucose Kerosene Yeast extract n-Paraffin Peptone Tetradecane
54
Concentration (%) 3.9 2.0 3.5 2.0 3.0 2,0
Surface tension (mNIm)
46.3 29.2 42.0 27.6 36.8 28.0
Nutritional Requirements Table 1 shows the effect of different carbon sources on the biosurfactant production as determined by measuring the broth's surface tension. When the bacterium was grown on hydrocarbons, it produced a broth with lower surface tension measurements and, hence, produced more biosurfactant than when grown on glucose, peptone or yeast extract. This result is not surprising, since many microorganisms have been reported to produce biosurfactants to facilitate metabolism and uptake of water-immiscible hydrocarbons (Cooper & Zajic 1980; Zajic & Seffens 1984; Singer 1985). The effect of different nitrogen sources is shown in Table 2. Highest growth and cell yields were obtained by the culture grown on sodium nitrate as a nitrogen source. Biosurfactant activity, however, in both cultures grown on either sodium nitrate or ammonium sulphate showed better emulsifying activity and CMD values
Biosurfactant-producing Rhodococcus bacterium Table 2. Effect of different nitrogen sources on growth of Rhodococcus sp. ST=5 and biosurfactant activity. Nitrogen
Biosurfactant activity
Source*
Biomass (g/I)
C M D 1t (mN/m)
Emulsification Index E=4 (%)
1.3 2,2 1.0
39.6 34.6 39.0
15 40.5 32.5
NH4OH NaNO 3 (NH4)2SO4
Cultivation temperature 37°C; pH 6.8; duration 24 h. * Each at 0.5 g/I. 1 Critical micelle dilution (broth was diluted 1 : 10 and surface tension was meaured at 25°C),
than the culture grown on ammonia. Based on these results, sodium nitrate w-as selected as the most suitable nitrogen source for this organism. Similar nitrogen source requirements have been reported for other biosurfactant-producing bacteria (Guerra-Santos et al. 1984). Results on the effect of different concentrations of nitrogen, phosphorus, sulphur and magnesium are shown in Figure 1. The results indicated that optimal growth and biosurfactant activity were obtained at N, P, S and Mg concentrations of 330 to 500, 100, 100 and 15 ppm, respectively. The lowest surface tension of 25.5 mN/m was obtained using 500 ppm N as nitrate. At this nitrogen concentration and using 2% (v/v) paraffin, the C: N ratio was about 22 : 1, which is close to the C:N ratio (18:t) reported to enhance biosurfactant production by other bacteria (Guerra-Santos et al. 1984). With phosphate, the highest growth and emulsification activity (E24) were obtained at 100 ppm P. Increasing the concentra-
BM
S.T.
E24
BM
S.T.
E24
5s i
ssi
-50
-50
3454
4sJ
- 40
-40
1
-30
- 30
aaj
-20
- 20 1 -251
25
• 10 J 0 -15 1 .....
o
¢
220 ,4'o d0
i
-15
8ao 11~0
0
Nitrogen Concentration (ppm} BM ST
,
,
200
400
600
800
0 1000
Phosphorus Concentr~ion (ppm) E24
ii"55 f
-10
BM
S.T.
E24
55
-50
50
3
2
45
-40
35
-30
45 2
-40 -30
35
--2o
-20
Figure 1. Effect of different concentrations of nitrogen, PO~a-, SO~- and Mg 2+ on growth and surfactant activity of the isolate ST-5, O - - S T (surface tension mN/m); A - - E 2 4 (emulsification index %); • B M - - ( b i o m a s s g/I).
1'.25_1
1 "25
n0
-10 0:' 15 0
40
80
120
160
Sulfur Concentration (ppm)
0 2OO
0 " 15 0
14
28
42
56
0 7O
Magnesium Concentration (ppm}
55
A . S . Abu-Ruwaida et al.
tion to 800 ppm phosphorus in the growth medium was not associated with remarkable changes in growth, emulsification activity or surface tension of the culture broth (Figure 1). The results also indicated that the culture requirements for Mg 2~ (15 ppm) is low when compared with that for other biosurfactantproducing bacteria (Cooper et al. 1981a; Kaplan et al. 1987). The effects of Fe 3+, Na +, K + and Caz+ are shown in Figure 2. These results indicated that satisfactory growth and biosurfactant production were achieved at high concentrations of Fe (7.5 ppm) and Na (1000 ppm). Salt tolerance is not surprising, since the organism was able to grow in a medium containing up to 5% (w/v) NaC1 (unpublished data). Iron has been reported to have a profound influence on growth and biosurfactant production by other microorganisms. Cooper et at. (1981b) showed that a biosurfactant-producing Bacillus subtillis required large amounts of iron (1.3 x 10 .3 M) as FeSO 4 for higher growth and biosurfactant production. However, higher iron concentration did not improve its biosurfactant yield; on the contrary, the yield decreased. Generally similar findings on the effect of iron were reported by Guerra-Santos et at. (1984) working with Pseudomonas aeruginosa. Our culture had a generally low K requirement (100 ppm) and no Ca requirement ( < 10 ppm) in the growth medium, either for growth or for biosurfactant production. Figure 3 shows the effect of supplementing the medium with different concentrations of yeast extract, glucose and tryptic soy broth as well as of increasing the total trace elements concentration. The addition of yeast extract and tryptic soy broth generally had no effect on biomass production, while glucose addition decreased the biomass concentration. Also, neither yeast extract nor glucose enhanced biosurfactant production, whereas tryptic soy broth addition at between 0.2% and 1.0% (w/v) did increase the concentration as measured by the higher emulsification index values and lower surface tension levels ( < 2 7 mN/m) ob-
BM
S.T.
E24
BM
E24
s5-t
40 3 ~45
S.T.
50
40
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2 ~
30
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"20
1
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0
15
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2
4
6
8
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01.15 I 0
, 480
~ 720
, 960
0 1200
E24
$. T.
554 -50 3 '
45 4 21 3 -J 5 ,J,~k
56
BM
-50
3-
Figure 2. Effect of different concentrations of medium components Fe 3+, Na + K ~ and Ca 2+ on growth and surfactant activity of the isolate ST-5. Q - - S T (surface tension mN/m); A---E24 (emulsification index %); E - - B M (biomass g/I).
24~0
Sodium Concentration (ppm) E24
55H
~ 10
Iron Concentration (ppm) EM
5
:
=
-40
454
-30
2 35 4
-20
-IO
0
240
480
720
960
Potassium Concentration {ppm)
o 1200
.40 .30
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o . 15-[ . . . . . . . . . . . . . . . . . 0 12
24
i 36
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Calcium Concentration (ppm)
6O
Biosurjaetant-producing Rhodococcus bacterium 8M
S.T.
E24
E24 .50
"50
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45-
-
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-20
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-10
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, 0.01
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Yea~ Extract Concentration (%} BM
S.T.
Trace Elements Concentration (x-fold) E24
55.
BM
S.T.
E24 50
50
345-
40
-30
,30
235,
Figure 3. Effect of addition of different concentrations of y e a s t extract, total trace elements, glucose and tryptic soy broth on growth and surfactant activity of the isolate ST-5. O - - S T (surface tension mN/m); air--E24 (emulsification index % ) ; , - - B M (biomass g/I).
-40
45
'35
J
-10 = 0.25
~ t ~ 0.50 0.75
= 1.0
Glucose Concentration (%)
0
J
_._....--e
"20
1 -25 J
0 "15 . . . . 0
--
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-20 -10
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, 0.2
//,
, 0.6
, 0.8
Tryptlc Soy Broth Concentration (%)
rained. Furthermore, increasing total trace elements depressed growth, cell yield and emulsification activity but had no effect on surface tension. Based on the above results, an improved salt medium was formulated containing (in rag/l): Na2HPO3, 2700; KH2PO4, 1400; NaNO3, 3000; K2804, 360; MgSO4.7H20 , 154; FeSO4.7H2 O, 38; NaC1, 100; and 1.0 ml trace elements solution containing the following (in mg/1): ZnSO4.7H20 , 525; MnSO4.4H20 , 200; CuSO4.5H20 , 705; Na2MoO4.2H20 , 15; COC12.6H20 , 200; H3BO > 15; and NiSO4.6H2 O, 27. The use of this medium in a shake-flask experiment increased broth growth and biosurfactant activity; after 24 h cultivation, cell yield increased from 1.2 to 3.0 g/l, and the emulsification index from 20 to 50%. In addition, surface tension decreased to values below 30 mN/m.
E~ect of CultivationpH The effect of initial pH on growth and biosurfactant production or activity is shown in Table 3. Highest growth was obtained at pH values of 6.5 and 6.8. Biosurfactant production, as determined by surface tension, CMD, interfacial tension (against n-hexane) and emulsification index measurements was less influenced by the cuhivation pH, since the biosurfactant production or activity was highest within a wide pH range (6.5 to 7.2). These findings suggest that the culture, although its highest growth occurs within a narrow pH range, can still produce biosurfactant effectively in a wider pH range, which is useful for large-scale production where unexpected changes in pH can occur. These results compare well with those reported by Cooper & Goldenberg (1987), who found that increasing the pH from 6.5 to 7.0 in the cultivation medium affected neither biosnrfactant synthesis nor yield by a Bacillus sp., whereas lowering the pH to below 5.5 decreased both growth and biosurfactant production. However, growth of P. aeruginosa was less influenced by cultivation pH, whereas biosurfactant
57
/t.S. Abu-Ruwaida et al. Table 3. Grow~ of Rhodococcus sp. ST-5 and biosurfactant activity as a function of cultivation pH. pH*
6,0 6,5 6.8 7.0 7,2 7.5
ST at Oh (mNlm)
55.4 55.4 55,4 55.4 55.4 55.4
After 24 h growth at 37°C ST (mNIm)
CMD -1 (mNIm)
CMD -2 (mN/m)
IFT (mN/m)
E24 (%)
Dry weight (g/I)
33.3 30,4 31.3 30.0 31,6 35.5
58.5 35,1 34.2 33.6 35,1 40.8
ND 55.9 56,4 62.5 61.0 ND
ND 3.7 3.4 2.5 3.7 4.4
18 41 43 40 40 34
0.99 2.0 2.03 1.46 1.25 1.0
ST--surface tension; CMD -1 and CMD-2---critical micelle dilution (broth was diluted 1:10 and 1:100 and surface tension was measured); IFT--interfaciat tension against n-hexane; E24--emulsification index; ND--not determined, * Controlled ±0.05 pH unit by addition of NaOH or H2SO4 (see Materials and Methods).
production dropped when the pH was changed from the optimum 6.2 to 6.4 (Guerra-Santos et al. 1986). Effect of Cultivation Tempep~ture
Optimal growth and biosurfactant production were obtained at 37°C. At this temperature, the highest biomass yield of 2.3 g/l, emulsification activity of 40% and biosurfactant concentration as determined by CMD measurements (31.0 mN/m) were obtained. Lower or higher temperatures generally had a depressing effect on both growth and biosurfactant production as detected by lower cell yields and biosurfactant concentrations (CMD values). This effect was more evident at the higher temperatures; e.g. the biomass yield decreased from 2.3 to 1.1, 1.0, 0.53 and 0.43 g/1 as the cultivation temperature increased from 37°C to 39, 41, 43 and 45°C, respectively, while the yields obtained at 35 and 30°C were about 1.0 and 0.75 g/l, respectively. The biosurfactant properties were not affected within a wide range of temperatures (30 to 41°C). The corresponding measurements for surface tension and interfacial tension of the culture broth ranged from 30 to 31.5 mN/m and from 1.6 to 2.0 mN/m, respectively. These results compare favourably with those reported for other biosurfactant-producing bacteria (Guerra-Santos et al. 1984). Batch Fermentation Experiments
Several fermentation experiments were carried out to evaluate the data obtained in shake-flask experiments using a 7 1 laboratory fermenter. Figure 4 shows typical results when n-paraffin (2% v/v) was used as the sole carbon source. The surface tension of the culture broth rapidly dropped to below 30 mN/m and remained constant to the end of the fermentation (48 h). The CMD plot showed that, initially, the biosurfactant produced was insufficient to form micelles. After about 25 h growth, the biosurfactant concentration started to increase, reached its maximum at the early stationary phase after about 33 h growth, and then stayed constant during the stationary phase up to 40 h growth. At this point, the biosurfactant product was sufficiently effective to form micelles even after the culture broth was diluted to 1:1000 (CMD-3). These findings indicated that high concentrations of biosurfactant were produced by ST-5 culture, comparable with other bacteria reported in the literature (Cooper et at. 1979; Akit et al. 1981; Javaheri et al. 1985; Brown et al. 1985). The emulsification activity values increased with increasing biomass formation, reached their maximum at about 27 h, and remained constant to the end of fermentation. In addition, the interfacial tension
58
Biosurfactant-pro&cing Rhodococcus bacterium 100-
I00
80j
80-
~.~ ''- x ~ x/x
6o- " g 6 o
\
',,
°
g
,
,,,/¢,:-.-
20-
20
0
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- 5
-80
4
x
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•
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----
$ =,
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40- ~ 40 \ Figure 4. Typical batch fermentation of nparaffin by ST-5 culture at controlled temperature of 37°C and pH of 68. O--CMD-1; Y--CMD-2; A--CMD-a: critical micelle dilution (broth was diluted 1:10, 1:100 and 1:1000 and surface tension was measured); x--biomass; II--E24; (}--surface tension.
x
100
2
-40.
°
2'4
1;
8
3'2
4'0
m
F
l
-20 L
'
.~
4:8
0
-0
Time (h)
of the broth against n-hexane decreased from about 33 mN/m at 0 h growth to about 1.8 mN/m after 33 h of growth, confirming enhanced biosurfactant production. A similar effect was observed with respect to cell growth and yield. The improved medium composition and optimal cultivation conditions established in this study increased cell yield from about 2 to 4.2 g/1. Cell yields of ST-5 culture were further increased to 6.6 g/1 by the addition of 0.2% (w/v) lactose broth into the medium as a growth stimulant (Figure 5). This increase was accompanied by reduction of the fermentation time required to obtain maximum yield (e.g. a maximum yield of 6.6 g/1 was obtained after 30 h growth, compared with about 40 h without lactose broth addition). Nevertheless, such an increase in biomass yield was not associated with an increase in biosurfactant activity, as determined by measuring the emulsification index and CMD values. The reduction in culture surface tension and interfaciaI tension against n-hexane after the addition of paraffin indicated again, that biosynthesis of the biosurfactant is necessary to aid metabolism of the paraffin. Conclusion The best medium composition for maximum biomass and biosurfactant production by ST-5 bacterial culture was found to be a simple mineral salts solution with 2% v/v n-paraffin and 0.2% w/v lactose broth• The kinetics of both biomass and -10
IO0
1001
2% (v/v) n-Paraffln
3O
8 I20
80
"g = o
10
g T~r
60
,r~v
--T
7-'-
Lts
-6 ~ 0 '°
4
40
~/
20
2
\'~
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Figure 5. Batch fermentation of lactose broth for initial growth of ST-5 culture, followed by n-paraffin after 10 h growth at 37°C and 6.8 pH. x---optical density; T--interfacial tension; O - - C M D - 1 ; i--E24; A--biomass; (}--surface tension.
~ ~ . f'-
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-2
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Time (h}
59
A . S . Abu-Ruwaida et al. biosurfactant production depended appreciably on cultivation pH and temperature. The biosynthesis of biosurfactant from paraffin occurred predominantly during the exponential growth phase, suggesting that this biosurfactant is produced as a primary metabolite and that it could be produced effectively under continuous fermentation conditions. In addition, the high concentration of biosurfactant produced by this culture allows the use of whole broth for product application without the need for product extraction or concentration. Finally, this study opens up the field of biosurfactant production technology for application in the oil-related industries in Kuwait, since it could be adapted for future use in cleaning up oil sludge in storage tanks and tankers, managing oil spills and enhancing oil recovery. However, more work is needed to characterize the product and evaluate its potential applications.
References ABU-RUWAIDA, A.S., BANAT, I.M. & HAMDAN, I.Y. 1990 Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol. Applied Microbiology and Biotechnology 32, 550-555. AKIT, J., COOPER, D.G., MANN1NEN,K.I. & ZAJIC, J.E. 1981 Investigation of potential biosurfactant production among phytopathogenic Corynebacterium and related soil microbes. Current Microbiology 6, 145-150. ATLAS, R.M. 1981 Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiological Reviews 45, 18(}-209. BROWN, M.J., FOSTER, M., MOSES, V., ROBINSON, J.P., SHALES, S.W. &: 8PRINGHAM, D.G. 1985 Novel biosurfactant for EOR. In Proceedings of the Third European Meeting on Improved Oil Recove,y pp. 241-248. Rome: AGIP S.P.A. CHAKRABARTY,A.M. 1984 Genetically-manipulated microorganisms and their products in the oil service industries. Trends in Biolechnology 3, 32-38. COOPER, D.G. & GOLDENBERG,B.G. 1987 Surface-active agents from two Bacillus species. Applied and Environmental Microbiology 83, 224-229. COOPER, D.G. & ZAJIC, J.E. 1980 Surface compounds from microorganisms. AdvaNces in Applied Microbiology 26, 229-256. COOPER, D.G., MACDONALD,C.R., DUFF, J.B. & KOSARIC,N. 1981a Enhanced production of surfactant from Bacillus subtilis by continuous product removal and metal cation addition. Applied and Environmental Microbiology 42, 408-4t 2. COOPER, D.G., ZAJIC,J.E. & DENIS, C. 1981b Surface-active properties of a biosurfactant from CoryNebacterium lepus. Journal of the American Oil Chemists' Society 58, 77-80. COOPER, D.G., ZAJIC, J.E. & GERSON, D.F. 1979 Production of surface-active lipids by Corynebacterium tepus. Applied and Environmenta! Microbiology 37, 4-10. DESAI, J.D. 1987 Microbial surfactants: evaluation, types, production and future application. Journal of Science and Industrial Research 46, 440-449. GUERRA-SANTOS, L., KAPPELI, O. & FIECHTER, A. 1984 Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Applied and Environmental Microbiology 48, 301-305. GUERRA-SANTOS, L., KAPPELI, O. & FIECHTER, A. I986 Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Applied Microbiology and Biotechnology 24, 443--448. HAFERBURG, D., HOMMEL, R., CLAUS, R. & KLEBER, H.P. 1986 Extracellular microbial lipids and biosurfactants. In Advances in Biochemical EngineeriNg/Bioteehnology, Vol. 33, ed. Fiechter, A, pp. 53-93. Berlin: Springer. HISATSUKA,K., NAKAHARA,T., SANO,N. & YAMADA,K. ~971 Formation of rhamnolipid by Pseudomonas aeruginosa and its function in hydrocarbon fermentation. Agricultural and Biological Chemistry 35, 686-692. ITO, S. & INOUE, S. 1982 Sophorolipids from Torulopsis bombicoIa possible relation to atkane uptake. Applied and Environmental Microbiology 43, 1278-1283. JAVAHER~, M., JENNEMAN, G.E., MCINERNEY, M.J. & KNAPP, R.M. t985 Anaerobic production of biosurfactant by Bacillus licheniformis JF-2. Applied and Environmental Microbiology 50, 698-700. KAPLAN, N., ZOSIM, Z. & ROSENBERG,E. 1987 Reconstitution of emulsifying activity of Acinetobacter calcoaceticusBD4 emulsan by using pure polysaccharide and protein. Applied and Environmental Microbiology 53, 440-446. KAPPELI, O. &: FINNERTY, W.R. 1980 Characteristics of bexadecane partition by the growth medium of Acinetobacter sp. Biotecbnology and Bioengineering 22, 495-503.
60
Nutritional requirements and growth characteristics of a biosurfactantproducing Rhodococcus bacterium
A.S. A b u - R u w a i d a ,
The nutritional requirements and growth characteristics of a biosurfactant-producing Rhodococcus bacterium isolated from Kuwaiti soil were determined. Maximum cell yields (6.6 g/I) and biosurfactant production were achieved with a medium containing 2% (v/v) n-paraffin as a carbon and energy source, 0.2% lactose broth, optimal concentrations of nitrogen (nitrate), phosphorus, iron, magnesium and sodium sources, and minimal concentrations of potassium and trace element sources. The optimal pH was 6.8 for surfactant production and optimal temperature was 37°C. The biosurfactant produced after 16 to 33 h growth in a 7 I fermenter decreased both surface tension and interfacial tension of culture broth to below 27 and 1.8 mN/m, respectively, and was effective at critical micelle dilutions of 10 -3. Data on biosurfactant biosynthesis suggest that the product is produced as a primary metabolite and, therefore, could be produced effectively under continuous fermentation conditions. A.S. Abu-Ruwaida, S. Haditirto and A. Khamis are with the Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109 Safat, Kuwait. I,M. Banat is now in Londonderry, Northern Ireland but was at the Kuwait institute for Scientific Research at the time this paper was written. A.S. Abu-Ruwaida is the corresponding author. (continued on next page) @ t991 Rapid Communications of Oxford Ltd.
I . M . B a n a t , S. H a d i t i r t o a n d A. K h a m i s
Biosurfactants are surface-active compounds that can be synthesized by many microorganisms during their growth (Cooper & Zajic 1980; Zajic & Seffens 1984; Singer t985). Their spontaneous release and function is often related to hydrocarbon uptake; therefore, they are synthesized predominantly by hydrocarbondegrading microorganisms (Hisatsuka et al. 1971; Rapp et al. 1979; Kappeli & Finnerty 1980; Ito & Inoue 1982). There have been, however examples of the use of a water-soluble substrate for biosurfactant production by some microorganisms (Guerra-Santos et aL 1986; Cooper & Goldenberg 1987; Palejwala & Desai 1989). Since most synthetic surfactant requirements can be met by biosurfactants, world-wide interest in these has increased. This is particularly due to their potential applications in the oil industries, which include cleaning oil slud I~ein storage tanks and tankers, enhancing oil-recovery processes, mobilizing heavy crude oil, transporting petroleum in pipelines and managing oil spills at sea (Chakrabarty 1984; Haferburg et al. 1986; Rosenberg 1986; Desai 1987). A large variety of biosurfactants have been reported (Cooper & Zajic 1980; Parkinson 1985; Rosenberg 1986; Desai 1987). The type, quantity and quality of biosurfactants produced have been shown to be influenced by the nature of the carbon substrate (Singer et al. 1983), the concentration of N, P, Mg, Fe and Mn ions in the medium (Atlas 1981 ; Cooper et at. 1981a,b; Guerra-Santos et aL 1984, 1986; Haferburg et al. 1986), and culture conditions, such as pH, temperature, agitation and dilution rate in continuous culture (Margaritis et al. 1979; GuerraSantos et al. 1984, 1986; Ratledge 1986). This paper reports on the nutritional requirements, growth characteristics and biosurfactant production by a newlyisolated Rhodococcus bacterium grown on hydrocarbons as the carbon source for potential future use in the oil industry in Kuwait. Materials and Methods Microorganism and Growth Medium
The microorganism used in this study, code name ST-5, is a Gram-positive, non-fermentative, rod-shaped bacterium that exhibits some morphological variations and has been assigned to the bacterial group Rhodococcus. This bacterium was
53
A.S. Abu-Ruwaida et al. tn view of the annexation of Kuwait by Iraq in August 1990, this paper has been accepted without return to the author for attention to minor details. The Editor-in-Chief therefore assumes full responsibility for any errors or omissions.
isolated from an oil-contaminated soil sample in Kuwait and was grown on a mineral salts medium developed earlier at Kuwait Institute for Scientific Research (KISR) for methylotrophic bacteria (Abu-Ruwaida et al. 1990), supplemented with 1 to 4% (v/v) n-paraffin as a carbon and energy source. Initial medium pH was adjusted to 6.8. Bacto agar (1.5% w/v) was added to obtain solid medium for plate cultures. Inoculum Preparation and Culture Cultivation Both inoculum preparation and culture cultivation were carried out in 250 ml Erlenmeyer flasks containing 100 mt of the medium. Generally, the inocula used made up 1 to 2% (v/v) of the medium and were obtained from an 18 to 24 h grown culture. Incubation was carried out at 37°C (except when otherwise specified) on a rotary shaker (New Brunswick Scientific, USA) at 300 rev/min. Batch Fermentation A %Chemap CF-2000 fermenter (Mannedorf, Switzerland) was charged with the mineral medium and sterilized at 121°C for 40 rain. Carbon source (1 to 2% v/v n-paraffin), and growth stimulant (lactose broth) were separately sterilized by filtration and added to the fermenter. An active inoculum (1.0% v/v) was aseptically added to the sterilized medium and grown at 37°C, with the pH controlled at 6.8 and the dissolved oxygen at about 30 to 50% saturation. The pH was controlled by the automatic addition of sterile 0.5 M NaOH or 0.5 M HC1. Samples (ca. 100 ml broth) were withdrawn periodically from the fermenter for measuring surface and interfacial tensions, cell dry weight, emulsification index, optical density and medium composition. The optical density of the broth was measured using a Bausch and Lomb Spectronic 20 spectrophotometer (Berlin, Germany) and 1-cm-diameter glass cuvettes at 620 nm wavelength. The broth was centrifuged using a Beckman J2-21 centrifuge (Beckman RIIC Ltd., UK). A n@tical Procedures Biomass dry weights were determined by filtering the cells from a 5 to 10 ml sample of the culture broth through a pre-washed and weighed 7-cm-diameter Whatman No. 1 filter paper (0.45 #m pore size) and using a pre-weighed aluminum dish. The samples were dried at 105°C overnight and reweighed after 24 h. Surface tension, interracial tension and relative concentration of biosurfactant expressed in terms of CMD were measured as described by Abu-Ruwaida et at. (submitted) using a Kruess K I O T tensiometer (Kruess, Optische-Mechanische Werkstatten, Hamburg, Germany). Emulsifying index (E24) was measured according to the method described by Cooper & Goldenberg (1987). Results and Discussion
Table 1. Effect of different carbon sources on the surface tension of the culture supernatant of Rhodococcus sp. ST-5, Carbon Source
Glucose Kerosene Yeast extract n-Paraffin Peptone Tetradecane
54
Concentration (%) 3.9 2.0 3.5 2.0 3.0 2,0
Surface tension (mNIm)
46.3 29.2 42.0 27.6 36.8 28.0
Nutritional Requirements Table 1 shows the effect of different carbon sources on the biosurfactant production as determined by measuring the broth's surface tension. When the bacterium was grown on hydrocarbons, it produced a broth with lower surface tension measurements and, hence, produced more biosurfactant than when grown on glucose, peptone or yeast extract. This result is not surprising, since many microorganisms have been reported to produce biosurfactants to facilitate metabolism and uptake of water-immiscible hydrocarbons (Cooper & Zajic 1980; Zajic & Seffens 1984; Singer 1985). The effect of different nitrogen sources is shown in Table 2. Highest growth and cell yields were obtained by the culture grown on sodium nitrate as a nitrogen source. Biosurfactant activity, however, in both cultures grown on either sodium nitrate or ammonium sulphate showed better emulsifying activity and CMD values
Biosurfactant-producing Rhodococcus bacterium Table 2. Effect of different nitrogen sources on growth of Rhodococcus sp. ST=5 and biosurfactant activity. Nitrogen
Biosurfactant activity
Source*
Biomass (g/I)
C M D 1t (mN/m)
Emulsification Index E=4 (%)
1.3 2,2 1.0
39.6 34.6 39.0
15 40.5 32.5
NH4OH NaNO 3 (NH4)2SO4
Cultivation temperature 37°C; pH 6.8; duration 24 h. * Each at 0.5 g/I. 1 Critical micelle dilution (broth was diluted 1 : 10 and surface tension was meaured at 25°C),
than the culture grown on ammonia. Based on these results, sodium nitrate w-as selected as the most suitable nitrogen source for this organism. Similar nitrogen source requirements have been reported for other biosurfactant-producing bacteria (Guerra-Santos et al. 1984). Results on the effect of different concentrations of nitrogen, phosphorus, sulphur and magnesium are shown in Figure 1. The results indicated that optimal growth and biosurfactant activity were obtained at N, P, S and Mg concentrations of 330 to 500, 100, 100 and 15 ppm, respectively. The lowest surface tension of 25.5 mN/m was obtained using 500 ppm N as nitrate. At this nitrogen concentration and using 2% (v/v) paraffin, the C: N ratio was about 22 : 1, which is close to the C:N ratio (18:t) reported to enhance biosurfactant production by other bacteria (Guerra-Santos et al. 1984). With phosphate, the highest growth and emulsification activity (E24) were obtained at 100 ppm P. Increasing the concentra-
BM
S.T.
E24
BM
S.T.
E24
5s i
ssi
-50
-50
3454
4sJ
- 40
-40
1
-30
- 30
aaj
-20
- 20 1 -251
25
• 10 J 0 -15 1 .....
o
¢
220 ,4'o d0
i
-15
8ao 11~0
0
Nitrogen Concentration (ppm} BM ST
,
,
200
400
600
800
0 1000
Phosphorus Concentr~ion (ppm) E24
ii"55 f
-10
BM
S.T.
E24
55
-50
50
3
2
45
-40
35
-30
45 2
-40 -30
35
--2o
-20
Figure 1. Effect of different concentrations of nitrogen, PO~a-, SO~- and Mg 2+ on growth and surfactant activity of the isolate ST-5, O - - S T (surface tension mN/m); A - - E 2 4 (emulsification index %); • B M - - ( b i o m a s s g/I).
1'.25_1
1 "25
n0
-10 0:' 15 0
40
80
120
160
Sulfur Concentration (ppm)
0 2OO
0 " 15 0
14
28
42
56
0 7O
Magnesium Concentration (ppm}
55
A . S . Abu-Ruwaida et al.
tion to 800 ppm phosphorus in the growth medium was not associated with remarkable changes in growth, emulsification activity or surface tension of the culture broth (Figure 1). The results also indicated that the culture requirements for Mg 2~ (15 ppm) is low when compared with that for other biosurfactantproducing bacteria (Cooper et al. 1981a; Kaplan et al. 1987). The effects of Fe 3+, Na +, K + and Caz+ are shown in Figure 2. These results indicated that satisfactory growth and biosurfactant production were achieved at high concentrations of Fe (7.5 ppm) and Na (1000 ppm). Salt tolerance is not surprising, since the organism was able to grow in a medium containing up to 5% (w/v) NaC1 (unpublished data). Iron has been reported to have a profound influence on growth and biosurfactant production by other microorganisms. Cooper et at. (1981b) showed that a biosurfactant-producing Bacillus subtillis required large amounts of iron (1.3 x 10 .3 M) as FeSO 4 for higher growth and biosurfactant production. However, higher iron concentration did not improve its biosurfactant yield; on the contrary, the yield decreased. Generally similar findings on the effect of iron were reported by Guerra-Santos et at. (1984) working with Pseudomonas aeruginosa. Our culture had a generally low K requirement (100 ppm) and no Ca requirement ( < 10 ppm) in the growth medium, either for growth or for biosurfactant production. Figure 3 shows the effect of supplementing the medium with different concentrations of yeast extract, glucose and tryptic soy broth as well as of increasing the total trace elements concentration. The addition of yeast extract and tryptic soy broth generally had no effect on biomass production, while glucose addition decreased the biomass concentration. Also, neither yeast extract nor glucose enhanced biosurfactant production, whereas tryptic soy broth addition at between 0.2% and 1.0% (w/v) did increase the concentration as measured by the higher emulsification index values and lower surface tension levels ( < 2 7 mN/m) ob-
BM
S.T.
E24
BM
E24
s5-t
40 3 ~45
S.T.
50
40
45
4O
3O
2 ~
30
2 - 35
"20
1
"25 -10
0
15
0
2
4
6
8
10
0 12
-20 1 -
2
s_T.
01.15 I 0
, 480
~ 720
, 960
0 1200
E24
$. T.
554 -50 3 '
45 4 21 3 -J 5 ,J,~k
56
BM
-50
3-
Figure 2. Effect of different concentrations of medium components Fe 3+, Na + K ~ and Ca 2+ on growth and surfactant activity of the isolate ST-5. Q - - S T (surface tension mN/m); A---E24 (emulsification index %); E - - B M (biomass g/I).
24~0
Sodium Concentration (ppm) E24
55H
~ 10
Iron Concentration (ppm) EM
5
:
=
-40
454
-30
2 35 4
-20
-IO
0
240
480
720
960
Potassium Concentration {ppm)
o 1200
.40 .30
•
--_
'0
i "2s-I
.20 .10
o . 15-[ . . . . . . . . . . . . . . . . . 0 12
24
i 36
,o 48
Calcium Concentration (ppm)
6O
Biosurjaetant-producing Rhodococcus bacterium 8M
S.T.
E24
E24 .50
"50
!-
3 -40
45-
-
S.T. 55-
3 "55-
2
8M
35-
_...._=,......~.---~-"'~ --..-----~"--"-=
~
-30
45 2
-40
-
-30
35-20
-20
1
1 "25-
-10
0 -15
0
, 0.01
0.05
"25 -10
0
-15,,,
0,1
Yea~ Extract Concentration (%} BM
S.T.
Trace Elements Concentration (x-fold) E24
55.
BM
S.T.
E24 50
50
345-
40
-30
,30
235,
Figure 3. Effect of addition of different concentrations of y e a s t extract, total trace elements, glucose and tryptic soy broth on growth and surfactant activity of the isolate ST-5. O - - S T (surface tension mN/m); air--E24 (emulsification index % ) ; , - - B M (biomass g/I).
-40
45
'35
J
-10 = 0.25
~ t ~ 0.50 0.75
= 1.0
Glucose Concentration (%)
0
J
_._....--e
"20
1 -25 J
0 "15 . . . . 0
--
"25] 0 "15 I 0
-20 -10
~ 0.1
, 0.2
//,
, 0.6
, 0.8
Tryptlc Soy Broth Concentration (%)
rained. Furthermore, increasing total trace elements depressed growth, cell yield and emulsification activity but had no effect on surface tension. Based on the above results, an improved salt medium was formulated containing (in rag/l): Na2HPO3, 2700; KH2PO4, 1400; NaNO3, 3000; K2804, 360; MgSO4.7H20 , 154; FeSO4.7H2 O, 38; NaC1, 100; and 1.0 ml trace elements solution containing the following (in mg/1): ZnSO4.7H20 , 525; MnSO4.4H20 , 200; CuSO4.5H20 , 705; Na2MoO4.2H20 , 15; COC12.6H20 , 200; H3BO > 15; and NiSO4.6H2 O, 27. The use of this medium in a shake-flask experiment increased broth growth and biosurfactant activity; after 24 h cultivation, cell yield increased from 1.2 to 3.0 g/l, and the emulsification index from 20 to 50%. In addition, surface tension decreased to values below 30 mN/m.
E~ect of CultivationpH The effect of initial pH on growth and biosurfactant production or activity is shown in Table 3. Highest growth was obtained at pH values of 6.5 and 6.8. Biosurfactant production, as determined by surface tension, CMD, interfacial tension (against n-hexane) and emulsification index measurements was less influenced by the cuhivation pH, since the biosurfactant production or activity was highest within a wide pH range (6.5 to 7.2). These findings suggest that the culture, although its highest growth occurs within a narrow pH range, can still produce biosurfactant effectively in a wider pH range, which is useful for large-scale production where unexpected changes in pH can occur. These results compare well with those reported by Cooper & Goldenberg (1987), who found that increasing the pH from 6.5 to 7.0 in the cultivation medium affected neither biosnrfactant synthesis nor yield by a Bacillus sp., whereas lowering the pH to below 5.5 decreased both growth and biosurfactant production. However, growth of P. aeruginosa was less influenced by cultivation pH, whereas biosurfactant
57
/t.S. Abu-Ruwaida et al. Table 3. Grow~ of Rhodococcus sp. ST-5 and biosurfactant activity as a function of cultivation pH. pH*
6,0 6,5 6.8 7.0 7,2 7.5
ST at Oh (mNlm)
55.4 55.4 55,4 55.4 55.4 55.4
After 24 h growth at 37°C ST (mNIm)
CMD -1 (mNIm)
CMD -2 (mN/m)
IFT (mN/m)
E24 (%)
Dry weight (g/I)
33.3 30,4 31.3 30.0 31,6 35.5
58.5 35,1 34.2 33.6 35,1 40.8
ND 55.9 56,4 62.5 61.0 ND
ND 3.7 3.4 2.5 3.7 4.4
18 41 43 40 40 34
0.99 2.0 2.03 1.46 1.25 1.0
ST--surface tension; CMD -1 and CMD-2---critical micelle dilution (broth was diluted 1:10 and 1:100 and surface tension was measured); IFT--interfaciat tension against n-hexane; E24--emulsification index; ND--not determined, * Controlled ±0.05 pH unit by addition of NaOH or H2SO4 (see Materials and Methods).
production dropped when the pH was changed from the optimum 6.2 to 6.4 (Guerra-Santos et al. 1986). Effect of Cultivation Tempep~ture
Optimal growth and biosurfactant production were obtained at 37°C. At this temperature, the highest biomass yield of 2.3 g/l, emulsification activity of 40% and biosurfactant concentration as determined by CMD measurements (31.0 mN/m) were obtained. Lower or higher temperatures generally had a depressing effect on both growth and biosurfactant production as detected by lower cell yields and biosurfactant concentrations (CMD values). This effect was more evident at the higher temperatures; e.g. the biomass yield decreased from 2.3 to 1.1, 1.0, 0.53 and 0.43 g/1 as the cultivation temperature increased from 37°C to 39, 41, 43 and 45°C, respectively, while the yields obtained at 35 and 30°C were about 1.0 and 0.75 g/l, respectively. The biosurfactant properties were not affected within a wide range of temperatures (30 to 41°C). The corresponding measurements for surface tension and interfacial tension of the culture broth ranged from 30 to 31.5 mN/m and from 1.6 to 2.0 mN/m, respectively. These results compare favourably with those reported for other biosurfactant-producing bacteria (Guerra-Santos et al. 1984). Batch Fermentation Experiments
Several fermentation experiments were carried out to evaluate the data obtained in shake-flask experiments using a 7 1 laboratory fermenter. Figure 4 shows typical results when n-paraffin (2% v/v) was used as the sole carbon source. The surface tension of the culture broth rapidly dropped to below 30 mN/m and remained constant to the end of the fermentation (48 h). The CMD plot showed that, initially, the biosurfactant produced was insufficient to form micelles. After about 25 h growth, the biosurfactant concentration started to increase, reached its maximum at the early stationary phase after about 33 h growth, and then stayed constant during the stationary phase up to 40 h growth. At this point, the biosurfactant product was sufficiently effective to form micelles even after the culture broth was diluted to 1:1000 (CMD-3). These findings indicated that high concentrations of biosurfactant were produced by ST-5 culture, comparable with other bacteria reported in the literature (Cooper et at. 1979; Akit et al. 1981; Javaheri et al. 1985; Brown et al. 1985). The emulsification activity values increased with increasing biomass formation, reached their maximum at about 27 h, and remained constant to the end of fermentation. In addition, the interfacial tension
58
Biosurfactant-pro&cing Rhodococcus bacterium 100-
I00
80j
80-
~.~ ''- x ~ x/x
6o- " g 6 o
\
',,
°
g
,
,,,/¢,:-.-
20-
20
0
0
- 5
-80
4
x
.40 :
•
3
,I t
----
$ =,
•
,/
40- ~ 40 \ Figure 4. Typical batch fermentation of nparaffin by ST-5 culture at controlled temperature of 37°C and pH of 68. O--CMD-1; Y--CMD-2; A--CMD-a: critical micelle dilution (broth was diluted 1:10, 1:100 and 1:1000 and surface tension was measured); x--biomass; II--E24; (}--surface tension.
x
100
2
-40.
°
2'4
1;
8
3'2
4'0
m
F
l
-20 L
'
.~
4:8
0
-0
Time (h)
of the broth against n-hexane decreased from about 33 mN/m at 0 h growth to about 1.8 mN/m after 33 h of growth, confirming enhanced biosurfactant production. A similar effect was observed with respect to cell growth and yield. The improved medium composition and optimal cultivation conditions established in this study increased cell yield from about 2 to 4.2 g/1. Cell yields of ST-5 culture were further increased to 6.6 g/1 by the addition of 0.2% (w/v) lactose broth into the medium as a growth stimulant (Figure 5). This increase was accompanied by reduction of the fermentation time required to obtain maximum yield (e.g. a maximum yield of 6.6 g/1 was obtained after 30 h growth, compared with about 40 h without lactose broth addition). Nevertheless, such an increase in biomass yield was not associated with an increase in biosurfactant activity, as determined by measuring the emulsification index and CMD values. The reduction in culture surface tension and interfaciaI tension against n-hexane after the addition of paraffin indicated again, that biosynthesis of the biosurfactant is necessary to aid metabolism of the paraffin. Conclusion The best medium composition for maximum biomass and biosurfactant production by ST-5 bacterial culture was found to be a simple mineral salts solution with 2% v/v n-paraffin and 0.2% w/v lactose broth• The kinetics of both biomass and -10
IO0
1001
2% (v/v) n-Paraffln
3O
8 I20
80
"g = o
10
g T~r
60
,r~v
--T
7-'-
Lts
-6 ~ 0 '°
4
40
~/
20
2
\'~
"E
Figure 5. Batch fermentation of lactose broth for initial growth of ST-5 culture, followed by n-paraffin after 10 h growth at 37°C and 6.8 pH. x---optical density; T--interfacial tension; O - - C M D - 1 ; i--E24; A--biomass; (}--surface tension.
~ ~ . f'-
".
/'
J"
t
01
-4
- 20
-2
.4
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~
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.~ m
i A ...--------~
o
-4o~
~
A
1'6
2'4
:i2
,~
,~
~o
Time (h}
59
A . S . Abu-Ruwaida et al. biosurfactant production depended appreciably on cultivation pH and temperature. The biosynthesis of biosurfactant from paraffin occurred predominantly during the exponential growth phase, suggesting that this biosurfactant is produced as a primary metabolite and that it could be produced effectively under continuous fermentation conditions. In addition, the high concentration of biosurfactant produced by this culture allows the use of whole broth for product application without the need for product extraction or concentration. Finally, this study opens up the field of biosurfactant production technology for application in the oil-related industries in Kuwait, since it could be adapted for future use in cleaning up oil sludge in storage tanks and tankers, managing oil spills and enhancing oil recovery. However, more work is needed to characterize the product and evaluate its potential applications.
References ABU-RUWAIDA, A.S., BANAT, I.M. & HAMDAN, I.Y. 1990 Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol. Applied Microbiology and Biotechnology 32, 550-555. AKIT, J., COOPER, D.G., MANN1NEN,K.I. & ZAJIC, J.E. 1981 Investigation of potential biosurfactant production among phytopathogenic Corynebacterium and related soil microbes. Current Microbiology 6, 145-150. ATLAS, R.M. 1981 Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiological Reviews 45, 18(}-209. BROWN, M.J., FOSTER, M., MOSES, V., ROBINSON, J.P., SHALES, S.W. &: 8PRINGHAM, D.G. 1985 Novel biosurfactant for EOR. In Proceedings of the Third European Meeting on Improved Oil Recove,y pp. 241-248. Rome: AGIP S.P.A. CHAKRABARTY,A.M. 1984 Genetically-manipulated microorganisms and their products in the oil service industries. Trends in Biolechnology 3, 32-38. COOPER, D.G. & GOLDENBERG,B.G. 1987 Surface-active agents from two Bacillus species. Applied and Environmental Microbiology 83, 224-229. COOPER, D.G. & ZAJIC, J.E. 1980 Surface compounds from microorganisms. AdvaNces in Applied Microbiology 26, 229-256. COOPER, D.G., MACDONALD,C.R., DUFF, J.B. & KOSARIC,N. 1981a Enhanced production of surfactant from Bacillus subtilis by continuous product removal and metal cation addition. Applied and Environmental Microbiology 42, 408-4t 2. COOPER, D.G., ZAJIC,J.E. & DENIS, C. 1981b Surface-active properties of a biosurfactant from CoryNebacterium lepus. Journal of the American Oil Chemists' Society 58, 77-80. COOPER, D.G., ZAJIC, J.E. & GERSON, D.F. 1979 Production of surface-active lipids by Corynebacterium tepus. Applied and Environmenta! Microbiology 37, 4-10. DESAI, J.D. 1987 Microbial surfactants: evaluation, types, production and future application. Journal of Science and Industrial Research 46, 440-449. GUERRA-SANTOS, L., KAPPELI, O. & FIECHTER, A. 1984 Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Applied and Environmental Microbiology 48, 301-305. GUERRA-SANTOS, L., KAPPELI, O. & FIECHTER, A. I986 Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Applied Microbiology and Biotechnology 24, 443--448. HAFERBURG, D., HOMMEL, R., CLAUS, R. & KLEBER, H.P. 1986 Extracellular microbial lipids and biosurfactants. In Advances in Biochemical EngineeriNg/Bioteehnology, Vol. 33, ed. Fiechter, A, pp. 53-93. Berlin: Springer. HISATSUKA,K., NAKAHARA,T., SANO,N. & YAMADA,K. ~971 Formation of rhamnolipid by Pseudomonas aeruginosa and its function in hydrocarbon fermentation. Agricultural and Biological Chemistry 35, 686-692. ITO, S. & INOUE, S. 1982 Sophorolipids from Torulopsis bombicoIa possible relation to atkane uptake. Applied and Environmental Microbiology 43, 1278-1283. JAVAHER~, M., JENNEMAN, G.E., MCINERNEY, M.J. & KNAPP, R.M. t985 Anaerobic production of biosurfactant by Bacillus licheniformis JF-2. Applied and Environmental Microbiology 50, 698-700. KAPLAN, N., ZOSIM, Z. & ROSENBERG,E. 1987 Reconstitution of emulsifying activity of Acinetobacter calcoaceticusBD4 emulsan by using pure polysaccharide and protein. Applied and Environmental Microbiology 53, 440-446. KAPPELI, O. &: FINNERTY, W.R. 1980 Characteristics of bexadecane partition by the growth medium of Acinetobacter sp. Biotecbnology and Bioengineering 22, 495-503.
60
