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Desalination 285 (2012) 198–204<br />
Contents lists available at SciVerse ScienceDirect<br />
Desalination<br />
journal homepage: www.elsevier.com/locate/desal<br />
<strong>Production</strong> <strong>and</strong> <strong>stability</strong> <strong>studies</strong> <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> <strong>isolated</strong> <strong>from</strong> marine<br />
Nocardiopsis sp. B4<br />
A. Khopade a , R. Biao b , X. Liu b , K. Mahadik a , L. Zhang b , C. Kokare a,c, ⁎<br />
a Department <strong>of</strong> Pharmaceutical Biotechnology, Poona College <strong>of</strong> Pharmacy, Bharati Vidyapeeth Deemed University, Pune, 411 038, India<br />
b Chinese Academy <strong>of</strong> Science Key Laboratory <strong>of</strong> Pathogenic Microbiology <strong>and</strong> Immunology, Institute <strong>of</strong> Microbiology, Chinese Academy <strong>of</strong> Sciences, Beijing 100101, China<br />
c Department <strong>of</strong> Pharmaceutics, STES, Sinhgad Institute <strong>of</strong> Pharmacy, Narhe, Pune, 411 041, India<br />
article<br />
info<br />
abstract<br />
Article history:<br />
Received 7 June 2011<br />
Received in revised form 21 September 2011<br />
Accepted 4 October 2011<br />
Available online 9 November 2011<br />
Keywords:<br />
Biosurfactant<br />
Nocardiopsis<br />
Optimization<br />
Stability<br />
Actinomycetes<br />
A potential <strong>biosurfactant</strong> producing strain, marine Nocardiopsis B4 was <strong>isolated</strong> <strong>from</strong> <strong>the</strong> West coast <strong>of</strong> India.<br />
Culture conditions involving variations in carbon <strong>and</strong> nitrogen sources were examined at constant pH, temperature<br />
<strong>and</strong> revolutions per min (rpm), with <strong>the</strong> aim <strong>of</strong> increasing productivity in <strong>the</strong> process. The <strong>biosurfactant</strong><br />
production was followed by measuring <strong>the</strong> surface tension, emulsification assay <strong>and</strong> emulsifying<br />
index E24. Enhanced <strong>biosurfactant</strong> production was carried out using olive oil as <strong>the</strong> carbon source <strong>and</strong> phenyl<br />
alanine as <strong>the</strong> nitrogen source. The maximum production <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> by Nocardiopsis occurred at a C/<br />
N ratio <strong>of</strong> 2:1 <strong>and</strong> <strong>the</strong> optimized bioprocess condition was pH 7.0, temperature 30°C <strong>and</strong> salt concentration<br />
3%. The production <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> was growth dependent. The surface tension was reduced up to<br />
29 mN/m as well as <strong>the</strong> emulsification index E24 was 80% in 6 to 9 days. Properties <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong><br />
that was separated by acid precipitation were investigated. The <strong>biosurfactant</strong> activity was stable at high temperature,<br />
a wide range <strong>of</strong> pH <strong>and</strong> salt concentrations thus, indicating its application in bioremediation, food,<br />
pharmaceutical <strong>and</strong> cosmetics industries.<br />
© 2011 Elsevier B.V. All rights reserved.<br />
1. Introduction<br />
Research in <strong>the</strong> area <strong>of</strong> <strong>biosurfactant</strong>s has exp<strong>and</strong>ed in recent<br />
years due to its potential use in different areas, such as <strong>the</strong> food industry,<br />
agriculture, pharmaceuticals, oil industry, petro chemistry,<br />
paper <strong>and</strong> pulp industry. The development <strong>of</strong> this line <strong>of</strong> research is<br />
<strong>of</strong> paramount importance, mainly in view <strong>of</strong> <strong>the</strong> present concern regarding<br />
<strong>the</strong> protection <strong>of</strong> <strong>the</strong> environment. They are not only useful<br />
as antibacterial, antifungal <strong>and</strong> antiviral agents, but also have potential<br />
for use as major immunomodulatory molecules, anti-adhesive<br />
agents <strong>and</strong> even in vaccines <strong>and</strong> gene <strong>the</strong>rapy [1–4]. Involvement <strong>of</strong><br />
<strong>biosurfactant</strong>s in microbial adhesion <strong>and</strong> desorption has been widely<br />
described. For example, inhibition <strong>of</strong> uropathogen bi<strong>of</strong>ilm formation<br />
on silicone rubber by protein-like <strong>biosurfactant</strong>s obtained <strong>from</strong><br />
Lactobacillus fermentum RC-14 was reported [1]. Also, exposure to<br />
suspensions <strong>of</strong> active probiotics <strong>and</strong> <strong>the</strong> consumption <strong>of</strong> buttermilk<br />
containing Lactococcus lactis 53 were reported to influence <strong>the</strong><br />
bi<strong>of</strong>ilm formation on silicone rubber voice pros<strong>the</strong>ses [1], possibly<br />
due to <strong>the</strong> release <strong>of</strong> <strong>biosurfactant</strong>s. Biosurfactants, are produced by<br />
bacteria or yeast <strong>from</strong> various substrates including sugars, glycerol,<br />
oils, hydrocarbons <strong>and</strong> agricultural wastes. Biosurfactants are<br />
⁎ Corresponding author at: Department <strong>of</strong> Pharmaceutics, STES, Sinhgad Institute <strong>of</strong><br />
Pharmacy, Narhe, Pune, 411 041, India. Tel.: +91 20 66831806; fax: +91 20 24699051.<br />
E-mail address: kokare71@rediffmail.com (C. Kokare).<br />
classified as glycolipids, lipopeptides, phospholipids, fatty acids, neutral<br />
lipids, <strong>and</strong> polymeric or particulate compounds [2,5]. The hydrophobic<br />
portion <strong>of</strong> <strong>the</strong> molecule is long-chain fatty acids, hydroxyl<br />
fatty acids or a-alkyl-b-hydroxyl fatty acids. The hydrophilic moiety<br />
can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic<br />
acid or alcohol. Biosurfactants have been receiving increasing attention<br />
as a result <strong>of</strong> <strong>the</strong>ir unique properties, i.e. mild production<br />
conditions, lower toxicity <strong>and</strong> higher biodegradability, compared to<br />
<strong>the</strong>ir syn<strong>the</strong>tic chemical counterparts [3]. Even though interest in <strong>biosurfactant</strong>s<br />
is increasing, <strong>the</strong>se compounds do not compete economically<br />
with syn<strong>the</strong>tic surfactants. To reduce production costs, different<br />
routes could be investigated such as <strong>the</strong> increase <strong>of</strong> yields <strong>and</strong> product<br />
accumulation; <strong>the</strong> development <strong>of</strong> economical engineering<br />
processes <strong>and</strong> <strong>the</strong> use <strong>of</strong> cost-free or cost credit feedstock for microorganism<br />
growth <strong>and</strong> surfactant production. The choice <strong>of</strong> inexpensive<br />
raw materials is important to overall economy <strong>of</strong> <strong>the</strong> process because<br />
<strong>the</strong>y account for 50% <strong>of</strong> <strong>the</strong> final product cost <strong>and</strong> also reduce <strong>the</strong><br />
expenses with wastes treatment [1,6,7]. Glycolipid <strong>from</strong><br />
Rhodococcus erythropolis, R. aurantiacus <strong>and</strong> surface active lipid <strong>from</strong><br />
Nocardia erythropolis were studied in <strong>the</strong> literature survey. There<br />
are very few reports on <strong>biosurfactant</strong>s production <strong>from</strong> marine<br />
actinomycetes [6].<br />
The objective <strong>of</strong> present study was to isolate marine actinomycetes<br />
<strong>and</strong> <strong>the</strong> identification <strong>of</strong> strain for potent <strong>biosurfactant</strong><br />
production. The cultural conditions for maximum production <strong>of</strong><br />
<strong>biosurfactant</strong> <strong>and</strong> <strong>the</strong> <strong>stability</strong> study <strong>of</strong> <strong>the</strong> product were investigated.<br />
0011-9164/$ – see front matter © 2011 Elsevier B.V. All rights reserved.<br />
doi:10.1016/j.desal.2011.10.002
A. Khopade et al. / Desalination 285 (2012) 198–204<br />
199<br />
2. Material <strong>and</strong> methods<br />
2.1. Isolation <strong>of</strong> actinomycetes strain B4<br />
Marine sediment sample was collected <strong>from</strong> Mumbai coastal region<br />
<strong>of</strong> India at <strong>the</strong> time <strong>of</strong> low tide [8]. Collected sediment sample<br />
was suspended in sterile water <strong>and</strong> mixed on rotary incubator shaker<br />
(New Brunswick Scientific, Model Excella E24, USA), at 150 rpm for<br />
20 min. Different marine actinomycete species were <strong>isolated</strong> by<br />
using selective media such as glycerol yeast extract agar, starch casein<br />
agar, maltose yeast extract agar <strong>and</strong> glucose aspargine agar (Himedia,<br />
India). The <strong>isolated</strong> strains were screened for <strong>biosurfactant</strong> production<br />
by using different techniques [9–14]. Maximum <strong>biosurfactant</strong><br />
producing marine sp. B4 was maintained on glycerol yeast extract<br />
agar medium [15].<br />
2.2. Identification <strong>of</strong> strain B4<br />
Identification <strong>of</strong> strain was done by scanning electron microscopy<br />
(SEM) (Pune, India), 16S r-DNA sequencing, biochemical <strong>and</strong> cultural<br />
characterizations. The method adopted for preparation <strong>of</strong> slide culture<br />
for SEM analysis was used, as described by Williams <strong>and</strong> Davies<br />
[12,15–17].<br />
2.3. Inoculum preparation <strong>and</strong> culture condition<br />
The glycerol yeast extract medium prepared in artificial seawater<br />
(ASW) was used for development <strong>of</strong> inoculum. The seed culture<br />
was prepared in 100 ml conical flasks containing 50 ml <strong>of</strong> medium<br />
by inoculating 2.0 ml <strong>of</strong> spore suspension containing 2.5 to<br />
3.0×10 6 CFU ml −1 <strong>and</strong> cultivated with agitation (150 rpm) at 28 °C<br />
for 4 days. The seed culture (50 ml) was inoculated in <strong>the</strong> 1 l fermentation<br />
medium prepared in artificial sea water supplemented with<br />
0.1 ml trace element solution. The pH <strong>of</strong> <strong>the</strong> medium was adjusted<br />
to 7.0. Fermentation was carried out in 2 l bench scale fermenter<br />
(New Brunswick Scientific, USA) for 12 days. The operating conditions<br />
during batch fermentation were temperature 28 °C, agitation<br />
rate 150 rpm <strong>and</strong> <strong>the</strong> aeration rate 1.0 VVM [8].<br />
2.4. Medium optimization<br />
The medium optimization was conducted in a series <strong>of</strong> experiments<br />
changing one variable at a time, keeping o<strong>the</strong>r factors<br />
unchanged. The production <strong>of</strong> <strong>biosurfactant</strong> was growth dependent.<br />
Cell growth <strong>and</strong> <strong>the</strong> accumulation <strong>of</strong> metabolic products were strongly<br />
influenced by medium composition such as carbon sources, nitrogen<br />
sources. Three factors were chosen aiming to obtain higher<br />
productivity <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong>: carbon source (C), nitrogen source<br />
(N) <strong>and</strong> C/N ratio. The carbon sources used were n-hexadecane (2%<br />
w/v) (Himedia, India), olive oil (2% w/v) (commercial type), sucrose<br />
(Himedia, India), trehalose (Himedia, India), maltose (Himedia,<br />
India), dextrose <strong>and</strong> glucose (Himedia, India) (20 g/l), with ammonium<br />
chloride (NH 4 Cl) (Himedia, India) as nitrogen source. For evaluation<br />
<strong>of</strong> <strong>the</strong> most appropriate nitrogen sources for <strong>the</strong> production <strong>of</strong><br />
<strong>biosurfactant</strong>s, phenyl alanine (Himedia, India), urea (Himedia,<br />
India), ammonium sulfate (Merck, India), NH 4 Cl <strong>and</strong> sodium nitrate<br />
(NaNO 3 )(Merck, India), were employed at a concentration <strong>of</strong> 1 g/l<br />
with <strong>the</strong> optimum carbon source. The C/N ratio (with optimized<br />
carbon <strong>and</strong> nitrogen sources) was varied <strong>from</strong> 10 to 40 by keeping a<br />
constant nitrogen source concentration 1 g/l [18–21].<br />
2.5. Biosurfactant production kinetics<br />
The kinetics <strong>of</strong> <strong>biosurfactant</strong> production was followed in batch<br />
cultures during 12 days at optimum conditions by measuring surface<br />
tension <strong>and</strong> emulsification assay <strong>of</strong> supernatant samples obtained<br />
after cell separation [18,19].<br />
2.6. Effect <strong>of</strong> pH, temperature, sodium chloride <strong>and</strong> aeration on<br />
<strong>biosurfactant</strong> production <strong>and</strong> activity<br />
In order to evaluate <strong>the</strong> effect <strong>of</strong> pH <strong>and</strong> temperature on <strong>the</strong> <strong>biosurfactant</strong><br />
production, <strong>the</strong> pH <strong>of</strong> medium was adjusted in <strong>the</strong> range<br />
between 4 <strong>and</strong> 12 <strong>and</strong> <strong>the</strong> temperature was set at 4, 15, 25, 30, 35<br />
40, 45 <strong>and</strong> 60 °C. The pH <strong>of</strong> <strong>the</strong> medium was measured with a digital<br />
pH-meter (Systronics, India). To examine <strong>the</strong> effect <strong>of</strong> sodium chloride<br />
on <strong>biosurfactant</strong> production in optimized medium, <strong>the</strong> sodium<br />
chloride was added in medium to achieve final concentrations <strong>of</strong><br />
1–10% (w/v). Effect <strong>of</strong> aeration on production <strong>of</strong> <strong>biosurfactant</strong> was<br />
detected by incubating inoculated fermentation media at different<br />
aeration conditions such as 50, 75, 100, 125, 150, 175, 200, 225 <strong>and</strong><br />
300 rpm. Biosurfactant production was measured by emulsification<br />
assay <strong>and</strong> absorbance was measured at 400 nm [21].<br />
2.7. Effect <strong>of</strong> oils, surfactants <strong>and</strong> hydrocarbon on <strong>biosurfactant</strong><br />
production<br />
The effect <strong>of</strong> crude oil <strong>and</strong> surfactant was evaluated for <strong>biosurfactant</strong><br />
production. The different oils were used such as castor oil, codliver<br />
oil, eucalyptus oil, sesame oil, mustard oil <strong>and</strong> surfactants such<br />
as ethylene diamine tetra acetic acid (EDTA) (Himedia, India), cetyl<br />
trimethyl ammonium bromide (CTAB) (Himedia, India), sodium<br />
dodicyl sulfate (SDS) (Loba Chemie, India), tweens 20, 40, 80 (Loba<br />
Chemie, India) were added separately in 1% (v/v) <strong>and</strong> emulsification<br />
activity <strong>of</strong> medium was measured. The hydrocarbons such as diesel,<br />
petrol, toluene, xylene, n-hexane <strong>and</strong> kerosene (commercial grade,<br />
India) were added [1% (v/v)] separately in optimized medium <strong>and</strong><br />
<strong>the</strong>ir effect was observed. The surface tension measurement was carried<br />
out using <strong>the</strong> du Nouy ring method [6,8].<br />
2.8. Surface tension measurement<br />
The surface tension measurement <strong>of</strong> cell free supernatant was<br />
determined in a K6 tensiometer, using <strong>the</strong> du Nouy ring method.<br />
The values reported were <strong>the</strong> mean <strong>of</strong> three measurements. All measurements<br />
were made on cell-free broth obtained by centrifuging <strong>the</strong><br />
cultures at 10,000 rpm for 20 min [18].<br />
2.9. Bioemulsifier production assay<br />
Actinomycetes species were grown for 12 to 15 days in glycerol<br />
yeast extract (GYE) broth. The microbial cells were separated by centrifugation<br />
(Eppendorff, model 5810R, Germany) at 10,000 rpm for<br />
15 min at 30 °C. Cell free culture broth (3 ml) was added in 0.5 ml<br />
test oil, mixed vigorously for 2 min <strong>and</strong> incubated at 30 °C for 1 h<br />
for phase separation. Aqueous phase was removed carefully <strong>and</strong> absorbance<br />
<strong>of</strong> aqueous phase was recorded at 400 nm. The absorbance<br />
maxima arrived after scanning at entire visible light spectrum. The<br />
blank was prepared with sterile medium. An absorbance <strong>of</strong><br />
0.01 units at 400 nm multiplied by dilution factor, if any, was considered<br />
as one unit <strong>of</strong> emulsification activity per ml (EU/ml) [6].<br />
2.10. Emulsification index (E24)<br />
Emulsification index <strong>of</strong> culture samples was determined by adding<br />
2 ml <strong>of</strong> a hydrocarbon to <strong>the</strong> same amount <strong>of</strong> culture, mixing with a<br />
vortex for 2 min, <strong>and</strong> left st<strong>and</strong>ing for 24 h. The E24 index is given<br />
as percentage <strong>of</strong> height <strong>of</strong> emulsified layer (mm) divided by total<br />
height <strong>of</strong> <strong>the</strong> liquid column (mm) [9–11].
200 A. Khopade et al. / Desalination 285 (2012) 198–204<br />
2.11. Biosurfactant recovery<br />
Culture broth was centrifuged at 10,000 rpm for 20 min to get cell<br />
free broth. Biosurfactant was precipitated by adjusting <strong>the</strong> pH <strong>of</strong> <strong>the</strong><br />
cell free broth to 2.0 using 6 N hydrochloric acid (HCl) (Merck,<br />
India) <strong>and</strong> keeping it overnight at 4 °C. The precipitate thus formed<br />
was collected by centrifugation (10,000 rpm, 20 min; 4 °C) <strong>and</strong> dissolved<br />
in distilled water. Its pH was adjusted to 8.0 with 1 N sodium<br />
hydroxide (NaOH) (Qualigens, India), <strong>and</strong> <strong>the</strong> solution was lyophilized<br />
[18].<br />
2.12. Stability <strong>studies</strong><br />
To determine <strong>the</strong> <strong>the</strong>rmal <strong>stability</strong> <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong>, cell-free<br />
broth was maintained at a constant temperature range <strong>of</strong> 20–100 °C<br />
for 15 min, <strong>the</strong>n cooled to room temperature <strong>and</strong> activity <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong><br />
was investigated. To determine <strong>the</strong> effect <strong>of</strong> pH on activity,<br />
<strong>the</strong> pH <strong>of</strong> <strong>the</strong> cell free broth was adjusted to different values using 1 N<br />
NaOH or 1 N HCl. The effect <strong>of</strong> addition <strong>of</strong> different concentration <strong>of</strong><br />
NaCl on <strong>the</strong> activity <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> was investigated. The <strong>biosurfactant</strong><br />
was re-dissolved after purification with distilled water containing<br />
<strong>the</strong> specific concentration <strong>of</strong> NaCl (0–9%, w/v) [22,23].<br />
3. Results <strong>and</strong> discussions<br />
The advantages <strong>of</strong> <strong>biosurfactant</strong>s over syn<strong>the</strong>tic ones include<br />
lower toxicity, biodegradability, selectivity, specific activity at extreme<br />
temperatures, pH <strong>and</strong> salinity. In our laboratory, we have<br />
<strong>isolated</strong> <strong>the</strong> strain <strong>of</strong> marine Nocardiopsis <strong>from</strong> sediments.<br />
3.1. Characterization <strong>of</strong> strain B4<br />
The strain B4 showed good growth in <strong>the</strong> temperature range<br />
25–45 °C in 7 days on glycerol yeast extract agar medium. Outer surface<br />
<strong>of</strong> colonies was perfectly round initially, but later <strong>the</strong>y developed<br />
aerial mycelium that may appear velvety <strong>and</strong> spore formation started<br />
after <strong>the</strong> 4th day <strong>of</strong> incubation. Spore chain was long <strong>and</strong> sporulating<br />
hyphae were straight. Spores were oval <strong>and</strong> warty, appeared like<br />
hairy <strong>and</strong> were 1–2.5 mm in size (Fig. 1A <strong>and</strong> 1B). Good growth was<br />
observed at neutral pH. By morphology, SEM <strong>and</strong> 16S DNA sequencing<br />
(Fig. 2), <strong>the</strong> <strong>isolated</strong> strain was found to be a member <strong>of</strong><br />
Nocardiopsis genus [8].<br />
3.2. Growth characteristics <strong>and</strong> <strong>biosurfactant</strong> production <strong>from</strong> strain B4<br />
Most <strong>of</strong> <strong>the</strong> actinomycetes species are slow-growing. Biosurfactant<br />
production started in early log phase but <strong>the</strong>re was drastic<br />
increase in production at late growth phase <strong>and</strong> early stationary<br />
phase; <strong>and</strong> <strong>biosurfactant</strong> production continued up to late stationary<br />
phase <strong>and</strong> after that it declined (Fig. 3). It clearly indicates that <strong>biosurfactant</strong><br />
production was dependent on <strong>the</strong> growth phase [8].<br />
3.3. Optimization <strong>of</strong> cultivation medium<br />
3.3.1. Effect <strong>of</strong> carbon source<br />
The production <strong>of</strong> <strong>biosurfactant</strong> was studied by using carbon<br />
sources such as n-hexadecane, olive oil, trehalose, sucrose, fructose,<br />
maltose <strong>and</strong> glucose (Fig. 4A). The use <strong>of</strong> vegetable oil as carbon<br />
sources to produce <strong>biosurfactant</strong>s seems to be an interesting <strong>and</strong><br />
low cost alternative [18]. Screening <strong>of</strong> nutrient substrates showed<br />
that Nocardiopsis supported growth on all substrates although <strong>the</strong><br />
yield was limited with galactose or starch as carbon sources because<br />
<strong>of</strong> inhibition due to <strong>the</strong> decrease in pH is probably caused by <strong>the</strong><br />
production <strong>of</strong> secondary acid such as uronic acid [18]. The maximum<br />
<strong>biosurfactant</strong> production was occurring only with trehalose, hexadecane<br />
<strong>and</strong> olive oil. The strain grew on starch but did not produce<br />
Fig. 1. Scanning electron microscopy <strong>of</strong> strain B4 [A] ×3000 magnification, <strong>and</strong><br />
[B] ×6000 magnification.<br />
<strong>the</strong> surfactant under <strong>the</strong>se conditions. Olive oil was <strong>the</strong> best carbon<br />
source for <strong>biosurfactant</strong> syn<strong>the</strong>sis. The <strong>isolated</strong> <strong>biosurfactant</strong> decreased<br />
<strong>the</strong> surface tension to 30 mN/m <strong>and</strong> <strong>the</strong> emulsifying activity<br />
was 80%. Similar results were found with <strong>biosurfactant</strong> production<br />
form P. aeruginosa 44T1 [24,25].<br />
3.3.2. Effect <strong>of</strong> nitrogen source<br />
The effect <strong>of</strong> nitrogen source affects <strong>the</strong> <strong>biosurfactant</strong> production<br />
as shown in Fig. 4B. Nocardiopsis sp. was able to use nitrogen sources<br />
such as ammonia <strong>and</strong> urea for <strong>biosurfactant</strong> production. However, in<br />
order to obtain high concentrations <strong>of</strong> <strong>biosurfactant</strong> it is necessary to<br />
have restrained conditions <strong>of</strong> <strong>the</strong>se macro-nutrients. Phenyl alanine<br />
was <strong>the</strong> best source <strong>of</strong> nitrogen for growth <strong>and</strong> <strong>biosurfactant</strong> syn<strong>the</strong>sis.<br />
Ammonium salts in <strong>the</strong> form <strong>of</strong> ammonium chloride were used<br />
for growth but not for <strong>biosurfactant</strong> production <strong>and</strong> caused a significant<br />
decrease in pH (4.03) [24,26,27]. The maximum emulsifying<br />
activity <strong>and</strong> minimal surface tension (30 mN/m) were reached in<br />
media with phenyl alanine. No significant change in pH was observed<br />
in this case. A similar result was reported in <strong>biosurfactant</strong> <strong>isolated</strong><br />
<strong>from</strong> Pseudomonas fluorescens by Abouseoud et al. [18].<br />
3.3.3. Effect <strong>of</strong> carbon/nitrogen ratio<br />
The fundamental aspect to <strong>the</strong> improvement <strong>of</strong> <strong>biosurfactant</strong> productivity<br />
was <strong>the</strong> ratio <strong>of</strong> C/N. These results were obtained using olive<br />
oil <strong>and</strong> phenyl alanine as carbon <strong>and</strong> nitrogen source respectively<br />
since <strong>the</strong> best results were attained with lower values <strong>of</strong> this parameter<br />
(C/N=20 ) (ST=30; EA=265 U/ml) (Fig. 4C). There were no<br />
significant differences between C/N ratios <strong>of</strong> 30, 35 <strong>and</strong> 40 in relation<br />
to emulsification index, but a C/N ratio <strong>of</strong> 20 presented a significant<br />
difference in relation to <strong>the</strong> emulsification index. These results are<br />
similar with those found using waste frying oil <strong>and</strong> sodium nitrate<br />
as carbon <strong>and</strong> nitrogen sources respectively [25–28]. Guerra-Santos<br />
et al. [29] observed that <strong>biosurfactant</strong> production was poor with
A. Khopade et al. / Desalination 285 (2012) 198–204<br />
201<br />
76<br />
76<br />
95<br />
99 Nocardiopsis exhalans ES10.1 T (AY036000)<br />
Nocardiopsis valliformis 20028 T (AY336503)<br />
Nocardiopsis metallicus KBS6 T (AJ420769)<br />
Nocardiopsis ganjiahuensis HBUM 20038 T (AY336513)<br />
Nocardiopsis prasina DSM 43845 T (X97884)<br />
Nocardiopsis listeri DSM40297 T (X97887)<br />
76<br />
Nocardiopsis alkaliphila DSM 44657 T (AY230848)<br />
B4<br />
Nocardiopsis alba DSM 43377 T (X97883)<br />
Nocardiopsis umidischolae 66/93 T (AY036001)<br />
100<br />
Nocardiopsis tropica VKM Ac -1457 T (AF105971)<br />
Marinactinospora <strong>the</strong>rmotoleransSCSIO 00652 T (EU698029)<br />
0.005<br />
Fig. 2. Neighbor-joining phylogenetic tree <strong>of</strong> strain B4 made by MEGA 4.0. Numbers at nodes indicate levels <strong>of</strong> bootstrap support (%) based on a neighbor-joining analysis <strong>of</strong> 1000<br />
resampled datasets; only values >50% are given. NCBI accession numbers are given in paren<strong>the</strong>ses. Bar, 0.005 nucleotide substitutions per site.<br />
both yeast extract <strong>and</strong> nitrate as nitrogen sources. When <strong>the</strong> yeast extract<br />
was omitted, <strong>the</strong> biomass concentration decreased, rhamnolipid<br />
increased <strong>and</strong> a moderate accumulation <strong>of</strong> glucose occurred, indicating<br />
a nitrogen-limiting medium.<br />
3.4. Kinetics <strong>of</strong> <strong>biosurfactant</strong> production<br />
The <strong>biosurfactant</strong> production <strong>and</strong> surface tension were dependent<br />
on growth <strong>of</strong> culture in <strong>the</strong> fermentation medium. The surface tension<br />
dropped rapidly after inoculation, reaching its lowest value<br />
(29 mN/m) during exponential phase after about 9 days <strong>of</strong> growth<br />
(Fig. 3A <strong>and</strong> Fig. 3B). The emulsification activity plot, a measure <strong>of</strong><br />
<strong>biosurfactant</strong> concentration, showed that insufficient surfactant was<br />
initially present to form micelles. At about 6th day <strong>of</strong> growth, <strong>the</strong> surfactant<br />
concentration started to increase, reaching its maximum after<br />
about 9th day. The increase in surface tension <strong>and</strong> <strong>the</strong> decrease in E24<br />
after 12th <strong>of</strong> incubation showed that <strong>biosurfactant</strong> biosyn<strong>the</strong>sis<br />
stopped <strong>and</strong> is probably due to <strong>the</strong> production <strong>of</strong> secondary metabolites<br />
which could interfere with emulsion formation <strong>and</strong> <strong>the</strong> adsorption<br />
<strong>of</strong> surfactant molecules at <strong>the</strong> oil–water interface [30]. These<br />
results indicate that <strong>the</strong> <strong>biosurfactant</strong> biosyn<strong>the</strong>sis <strong>from</strong> olive oil occurred<br />
predominantly during <strong>the</strong> exponential growth phase, suggesting<br />
that <strong>the</strong> <strong>biosurfactant</strong> is produced as primary metabolite<br />
accompanying cellular biomass formation (growth-associated kinetics)<br />
[31]. This property suggests that <strong>biosurfactant</strong> could be effectively<br />
produced under chemostat conditions or by immobilized cells<br />
[18,19,32,33].<br />
3.5. Effect <strong>of</strong> sodium chloride, pH, temperature <strong>and</strong> aeration on<br />
<strong>biosurfactant</strong> production<br />
The strain B4 was found to be moderately halophilic in nature as<br />
maximum <strong>biosurfactant</strong> production was obtained in presence <strong>of</strong> 3%<br />
Fig. 3. Growth kinetics <strong>and</strong> <strong>biosurfactant</strong> production <strong>of</strong> Nocardiopsis B4 sp. (OD600).<br />
(w/v) <strong>of</strong> NaCl <strong>and</strong> it retained almost 80% <strong>of</strong> its activity in presence<br />
<strong>of</strong> 12% (w/v) <strong>of</strong> NaCl. (Fig. 5A). The strain B4 showed gradual increase<br />
in <strong>biosurfactant</strong> production <strong>and</strong> optimum pH for <strong>biosurfactant</strong> production<br />
was found to be 7 (Fig. 5B). The research was focused on<br />
<strong>the</strong> isolation <strong>of</strong> alkaline <strong>biosurfactant</strong> <strong>from</strong> microbes because <strong>the</strong>re<br />
is tremendous potentiality <strong>of</strong> <strong>biosurfactant</strong> in detergent industry.<br />
The strain B4 showed good growth in <strong>the</strong> temperature range <strong>of</strong><br />
25–45 °C but optimum growth was observed at 30 °C (Fig. 5C). This<br />
clearly indicates <strong>the</strong> moderately <strong>the</strong>rmostable nature <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong>.<br />
The maximum <strong>biosurfactant</strong> production was obtained at<br />
150 rpm. Until today, bacteria belonging to genus Bacillus have been<br />
exploited for commercial production <strong>of</strong> <strong>biosurfactant</strong> [21,24]. There<br />
is no such report on isolation <strong>of</strong> moderately <strong>the</strong>rmostable surfactant<br />
<strong>from</strong> marine Nocardiopsis sp.<br />
3.6. Effect <strong>of</strong> oils, surfactants <strong>and</strong> hydrocarbons on production <strong>of</strong><br />
<strong>biosurfactant</strong><br />
Fermentation was carried out with addition <strong>of</strong> different concentrations<br />
<strong>of</strong> oils, surfactant <strong>and</strong> hydrocarbons in <strong>the</strong> fermentation medium.<br />
It was observed that olive oil, tween 80 <strong>and</strong> hexane as a<br />
substrate showed maximum activity against all test oils, surfactants<br />
<strong>and</strong> hydrocarbons respectively. Olive oil <strong>and</strong> tween 80 showed emulsification<br />
activity at 198 EU/ml <strong>and</strong> 225 EU/ml respectively (Fig. 6A<br />
<strong>and</strong> 6B). Hexane was used [6] as a substrate for <strong>biosurfactant</strong> production<br />
<strong>and</strong> it was observed that 1% v/v showed maximum <strong>biosurfactant</strong><br />
production activity (Fig. 6C).<br />
3.7. Stability study<br />
3.7.1. Temperature <strong>stability</strong><br />
The applicability <strong>of</strong> <strong>biosurfactant</strong>s in several fields depends on<br />
<strong>the</strong>ir <strong>stability</strong> at different temperatures <strong>and</strong> pH values. The <strong>stability</strong><br />
<strong>of</strong> <strong>biosurfactant</strong> was tested over a wide range <strong>of</strong> temperature. The<br />
<strong>biosurfactant</strong> produced by Nocardiopsis sp. was shown to be <strong>the</strong>rmostable<br />
(Fig. 7A). Heating <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> to 100 °C caused no significant<br />
effect on <strong>the</strong> <strong>biosurfactant</strong> performance. The emulsification<br />
activity was quite stable at <strong>the</strong> temperatures used (E24=66%) in<br />
comparison with syn<strong>the</strong>tic surfactants such as SDS which exhibits a<br />
significant loss <strong>of</strong> emulsification activity beginning at 70 °C [31].<br />
Therefore, it can be concluded that this <strong>biosurfactant</strong> maintains its<br />
surface properties unaffected in <strong>the</strong> range <strong>of</strong> temperatures between<br />
30 <strong>and</strong> 100 °C. This activity was discovered indicating <strong>the</strong> usefulness<br />
<strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong> in food, pharmaceutical <strong>and</strong> cosmetics industries<br />
where heating to achieve sterility is <strong>of</strong> paramount importance<br />
[18,25].
202 A. Khopade et al. / Desalination 285 (2012) 198–204<br />
Surface tension (mN/m)<br />
AEmulsification activity (EU/ml)<br />
E24 (%)<br />
B<br />
Surface tension (mN/m)<br />
C<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Olive oil<br />
Galato se<br />
Ammo. sulphate<br />
300<br />
200<br />
100<br />
0<br />
Phenyl alanine<br />
Dextrose<br />
Strac h<br />
Mannitol<br />
Sucrose<br />
Trehalose<br />
Carbon sources<br />
Alanine<br />
Ammo. chloride<br />
Urea<br />
Nitrogen source<br />
Hexadecne<br />
Fructo se<br />
Maltose<br />
Aspargine<br />
Sodium nitrate<br />
Emlusification activity<br />
Surface tension<br />
10 15 20 25 30 35 40<br />
C:N ratio<br />
A<br />
Surface tension (mN/m)<br />
0 20 40 60 80<br />
B<br />
Surface tension (mN/m)<br />
C<br />
Surface tension (mN/m)<br />
80<br />
60<br />
40<br />
20<br />
0<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
1 2 3 4 5 6 7 8 9 10<br />
NaCl (% w/v)<br />
4<br />
5<br />
6<br />
7<br />
4<br />
15<br />
25<br />
30<br />
8<br />
pH<br />
35<br />
9<br />
10<br />
11<br />
12<br />
40<br />
Temperature (°C)<br />
45<br />
60<br />
Fig. 4. [A] Effect <strong>of</strong> carbon source on <strong>biosurfactant</strong> production, [B] effect <strong>of</strong> nitrogen<br />
source on <strong>biosurfactant</strong> production, <strong>and</strong> [C] effect <strong>of</strong> C:N ratio on <strong>biosurfactant</strong><br />
production.<br />
Fig. 5. [A] Effect <strong>of</strong> NaCl on <strong>biosurfactant</strong> production, [B] effect <strong>of</strong> pH on <strong>biosurfactant</strong><br />
production, <strong>and</strong> [C] effect <strong>of</strong> temperature on <strong>biosurfactant</strong> production.<br />
3.7.2. pH <strong>stability</strong><br />
The surface activity <strong>of</strong> <strong>the</strong> crude <strong>biosurfactant</strong> remained relatively<br />
stable to pH changes between pH 8 <strong>and</strong> 12, showing higher <strong>stability</strong><br />
at alkaline pH 9 than acidic conditions. At pH 12, <strong>the</strong> value in emulsification<br />
activity (E24) showed almost 66% activity, whereas below pH<br />
7 activity was decreased up to 55%. In addition, for pH values lower<br />
than 6, <strong>the</strong> samples become turbid, due to partial precipitation <strong>of</strong><br />
<strong>the</strong> <strong>biosurfactant</strong>. Fig. 7B shows <strong>the</strong> effect <strong>of</strong> pH on <strong>the</strong> <strong>biosurfactant</strong><br />
properties. These results indicate that increase pH has a positive effect<br />
on emulsification activity <strong>and</strong> emulsion <strong>stability</strong>. This could be<br />
caused by a better <strong>stability</strong> <strong>of</strong> fatty acid surfactant micelles in <strong>the</strong><br />
presence <strong>of</strong> NaOH <strong>and</strong> <strong>the</strong> precipitation <strong>of</strong> secondary metabolites at<br />
higher pH values. The effect <strong>of</strong> pH on surface activity has been<br />
reported for <strong>biosurfactant</strong>s for different microorganisms [18,34].<br />
3.7.3. Effect <strong>of</strong> salinity<br />
The effect <strong>of</strong> sodium chloride addition on <strong>biosurfactant</strong> produced<br />
<strong>from</strong> Nocardiopsis was studied. Optimum <strong>stability</strong> <strong>of</strong> <strong>biosurfactant</strong><br />
was observed at 3% NaCl concentration. Little changes were observed<br />
in increased concentration <strong>of</strong> NaCl up to 8% (w/v) (Fig. 7C). At higher<br />
concentration <strong>of</strong> NaCl <strong>the</strong> <strong>biosurfactant</strong> retains 50% <strong>of</strong> <strong>the</strong> emulsification<br />
activity. The <strong>biosurfactant</strong> has <strong>stability</strong> at alkaline pH <strong>and</strong> high<br />
salinity; such a <strong>biosurfactant</strong> may be useful for bioremediation <strong>of</strong><br />
spills in marine environment because <strong>of</strong> its <strong>stability</strong> in alkaline condition<br />
<strong>and</strong> in <strong>the</strong> presence <strong>of</strong> salt. Stability <strong>of</strong> emulsion in <strong>the</strong> presence
A. Khopade et al. / Desalination 285 (2012) 198–204<br />
203<br />
A<br />
250<br />
A<br />
150<br />
Emulsification activity<br />
(EU/ml)<br />
200<br />
150<br />
100<br />
50<br />
E24 (%)<br />
100<br />
50<br />
0<br />
Caster oil<br />
Olive oil<br />
Ecualyptus oil<br />
Clove oil<br />
Coconut oil<br />
Oils<br />
Cod liver oil<br />
Senamom oil<br />
B<br />
0<br />
150<br />
30<br />
40<br />
50<br />
60<br />
70<br />
80<br />
Temperature (°C)<br />
90<br />
100<br />
B<br />
Emulsification activity (EU/ml)<br />
300<br />
200<br />
100<br />
0<br />
EDTA<br />
CTAB<br />
SDS<br />
Tween 20<br />
Tween 40<br />
Surfactants<br />
Tween 80<br />
Triton X-100<br />
E24 (%)<br />
C<br />
100<br />
50<br />
0<br />
150<br />
4<br />
5<br />
6<br />
7<br />
8<br />
pH<br />
9<br />
10<br />
11<br />
12<br />
C<br />
Surface tension (mN/m)<br />
80<br />
60<br />
40<br />
20<br />
E24 (%)<br />
100<br />
50<br />
0<br />
1<br />
2<br />
3<br />
4<br />
5<br />
6<br />
NaCl (% w/v)<br />
7<br />
8<br />
9<br />
<strong>of</strong> salt has been reported as one <strong>of</strong> <strong>the</strong> properties <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong><br />
produced by Bacillus licheniformis strain JF-2 [21].<br />
4. Conclusions<br />
0<br />
Tolune<br />
n Hexane<br />
Xylene<br />
Kerosens<br />
Hydrocarbons<br />
Petrol<br />
Diesel<br />
Fig. 6. [A] Effect <strong>of</strong> oils on <strong>biosurfactant</strong> production, [B] effect <strong>of</strong> surfactants on <strong>biosurfactant</strong><br />
production, <strong>and</strong> [C] effect <strong>of</strong> hydrocarbons on <strong>biosurfactant</strong> production.<br />
In <strong>the</strong> present study <strong>the</strong> <strong>biosurfactant</strong> <strong>isolated</strong> <strong>from</strong> marine<br />
Nocardiopsis sp. showed good <strong>stability</strong> at high temperature, a wide<br />
range <strong>of</strong> pH <strong>and</strong> salt concentrations <strong>and</strong> <strong>the</strong> maximum <strong>biosurfactant</strong><br />
production was observed with olive oil as a carbon source <strong>and</strong> phenyl<br />
alanine as <strong>the</strong> nitrogen source. The important finding was <strong>the</strong>rmo<br />
<strong>stability</strong> <strong>of</strong> <strong>biosurfactant</strong>; <strong>isolated</strong> <strong>biosurfactant</strong> was extreme <strong>stability</strong><br />
Fig. 7. Effect <strong>of</strong> [A] temperature, [B] pH <strong>and</strong> [C] salinity on <strong>biosurfactant</strong> <strong>stability</strong>.<br />
at high temperature (100°). The <strong>the</strong>rmal <strong>stability</strong> <strong>of</strong> <strong>the</strong> <strong>biosurfactant</strong>s<br />
increases its scope <strong>of</strong> application in a broader perspective<br />
including at conditions where high temperatures prevail as in microbial<br />
enhanced oil recovery. Considering <strong>the</strong> potential need <strong>of</strong> halotolerant<br />
strains <strong>and</strong> <strong>biosurfactant</strong>s for <strong>the</strong> bioremediation <strong>of</strong> oil<br />
contaminated sites (oil spills), it is m<strong>and</strong>atory to screen <strong>and</strong> develop<br />
potential <strong>biosurfactant</strong> producers <strong>from</strong> <strong>the</strong> marine environment.<br />
It was found that <strong>the</strong> <strong>biosurfactant</strong> produced by <strong>the</strong> marine<br />
Nocardiopsis was stable up to 8% NaCl; however <strong>the</strong> chemical surfactants<br />
are deactivated by 2–3% salt concentration. The <strong>biosurfactant</strong><br />
was <strong>isolated</strong> <strong>from</strong> natural sources thus, indicating <strong>the</strong> application <strong>of</strong><br />
<strong>the</strong> <strong>biosurfactant</strong> in food, pharmaceutical <strong>and</strong> cosmetics industries.<br />
Acknowledgments<br />
The authors would like to acknowledge All India Council for Technical<br />
Education (AICTE), HRD Ministry, New Delhi, Govt. <strong>of</strong> India for
204 A. Khopade et al. / Desalination 285 (2012) 198–204<br />
financial support to this research project under National Doctoral<br />
Fellowship (NDF), 2010–2013.<br />
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