G Model

BIOMAC 5074 1–8 ARTICLE IN PRESS

International Journal of Biological Macromolecules xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

1 Biosurfactant produced from Actinomycetes nocardiopsis A17:

2 Characterization and its biological evaluation
3 Q1 Samrat Chakraborty a , Mandakini Ghosh a , Srijita Chakraborti a , Sougata Jana a,∗ ,
4 Kalyan Kumar Sen a , Chandrakant Kokare b , Lixin Zhang c
a
5 Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol 713301, WB, India
b
6 Department of Pharmaceutics, Sinhgad Institute of Pharmacy, Narhe, Pune 411 041, India
c
7 Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
8

9
21 a r t i c l e i n f o a b s t r a c t
10
11 Article history: This investigation aims to isolate an Actinomycetes strain producing a biosurfactant from the unexplored
12 Received 1 April 2015 region of industrial and coal mine areas. Actinomycetes are selected for this study as their novel chem-
13 Received in revised form 27 April 2015 istry was not exhausted and they have tremendous potential to produce bioactive secondary metabolites.
14 Accepted 28 April 2015
The biosurfactant was characterized and further needed to be utilized for pharmaceutical dosage form.
15 Available online xxx
Isolation, purification, screening, and characterization of the Actinomycetes A17 were done followed by
16
its fermentation in optimized conditions. The cell-free supernatant was used for the extraction of the
17 Keywords:
biosurfactant and precipitated by cold acetone. The dried precipitate was purified by TLC and the emulsi-
18 Actinomycetes A17
19 Biosurfactant
fication index, surface tension and CMC were determined. The isolated strain with preferred results was
20 Critical micelle concentration (CMC) identified as Actinomycetes nocardiopsis A17 with high foam-forming properties. It gives lipase, amylase,
gelatinase, and protease activity. The emulsification index was found to be 93 ± 0.8 with surface tension
66.67 dyne/cm at the lowest concentration and cmc 0.6 ␮g/ml. These biosurfactants were character-
ized by Fourier transform infra red (FT-IR) spectroscopy and liquid chromatography-mass spectrometry
(LC–MS). Therefore, it can be concluded that the biosurfactant produced by Actinomycetes nocardiopsis sp.
strain A17 was found to have satisfactory results with high surface activity and emulsion-forming ability.
© 2015 Elsevier B.V. All rights reserved.

22 1. Introduction and trehalose lipids), lipopeptides and lipoproteins (peptide, vis- 38

cosin, serrawettin, surfactin, subtilisin, gramicidin, and polymyxin), 39

23Q2 The many traditional synthetic surfactants are usually highly fatty acids, neutral lipids, phospholipids, polymeric surfactants 40

24 aquatic toxic due to an insufficient rate of biodegradation [1]. (emulsan, biodispersan, liposan, carbohydrate–lipid–protein, and 41

25 Surfactants are one of the most important and valuable chem- mannan–lipid–protein), and particulate surfactants [5]. They have 42

26 ical products and are consumed in large quantities throughout gained considerable scientific attention due to their low toxicity, 43

27 the world for different purposes. Surfactants (Ss) are amphiphilic higher salinity, and possibility of their production through fermen- 44

28 molecules consisting of a polar head group and a hydrophobic tail. tation using cheap agro-based substrates. Thus they have additional 45

29 They become one of the active ingredients in soaps and detergents. advantages from the view point of resource replacement and recy- 46

30 So in this context recent years, challenges and scientific attention cling. Apart from the potential applications of BSs in environmental 47

31 are drawn in to the production of surfactants (biosurfactants) from protection and management of crude oil recovery, they also have 48

32 microbial resources [2]. emerged as potential agents in health care and food processing 49

33 Biosurfactants (BSs) are microbially produced surface-active industries [6]. Due to an array of interesting features, BSs have 50

34 agents that are heterogeneous group of secondary metabolites [3]. led to a broad range of potential applications in the biomedical 51

35 They are amphiphilic in nature containing at least one hydrophilic field and also have found applications in the food, pharmaceutical, 52

36 and hydrophobic moiety [4]. Biosurfactant occurs in nature as cosmetics, and agriculture industries [7]. They are not only use- 53

37 chemical entities such as glycolipids (rhamnolipids, sophoro lipids, ful as antibacterial, antifungal, and antiviral agents, but also have 54

potential for use as major immune-modulatory molecules, anti- 55

adhesive agent and even in vaccines and gene therapy [8–11]. BSs 56

∗ Corresponding author. Tel.: +91 9434896683. have many advantages over synthetic surfactants due to their high 57

E-mail address: janapharmacy@rediffmail.com (S. Jana). biodegradability, low toxicity, biocompatibility, bio-digestibility 58

http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
0141-8130/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
2 S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

59 (which allows their application in cosmetic and pharmaceutical actinomycetes, bacteria and fungi. Pre-heat treatment at 40 ◦ C for 60 119

60 products and as food additives), and thus being more eco- friendly days showed more number of actinomycetes and very less number 120

61 [5]. They also possess significant stability at different temper- of bacteria and fungi. Starch casein agar and glycerol yeast extract 121

62Q3 atures and pH levels [12]. Though they are produced by some showed more number of actinomycetes. Thirty-seven actinomycetes 122

63 yeast, filamentous fungi, and bacteria, using several substrates were isolated from the samples. Few research studies have been 123

64 such as carbohydrates, hydrocarbons, oils and fats, industrial and reported on actinomycetes from the marine ecosystem. In the com- 124

65 agricultural residues or a mix of them [13]. BSs have potential prehensive study carried out by Weyland [24], it was described that 125

66 applications in several fields of industrial processes such as agri- the availability of terrestrial actinomycetes were found to be more 126

67 culture, cosmetics, pharmaceuticals, detergents, food processing, with respect to marine actinomycetes. 127

68 textile manufacture, and paint industries [6,14,15].

69 The many BSs produced from the different types of microorgan-
70 ism such as Sphingobacterium detergens [1], Candida lipolytica [2], 2.3. Screening of actinomycetes for biosurfactant production 128

71 Bacillus subtilis [11], Pseudomonas aeruginosa [13], Rhodococcus sp.

72 strain TA6 [15], Persian Gulf [16], Bacillus licheniformis MTCC 5514 Potential biosurfactant producers were screened by different 129

73 [17], Ochrobactrum sp. strain BS-206 (MTCC 5720) [18], Gordonia sp. screening methods. These methods include the oil spread method, 130

74 strain JE-1058 [19], and Acinetobacter calcoaceticus BU03[20] but blue agar plate method, drop collapsing technique, and lipase activ- 131

75 till now Actinomycetes nocardiopsis strain A17 from the region of ity. These methods are described below. 132

76 industrial and coal mine areas have not been reported. The available
77 genome sequence databases of actinomycetes predict a large num- 2.3.1. Oil displacement method 133
78 ber of genes encoding for enzymes of different lipolytic activities. Seven-day-old inoculum grown in a glycerol yeast extract broth 134
79 Lipase acts on water–oil surfaces and therefore it was suggested was used for this screening technique. The petriplate base was filled 135
80 that actinomycetes showed the presence of lipolytic activities and with 50 ml of distilled water. On this water, 50 ␮l of crude oil (in this 136
81 are able to produce BSs [21]. study castor oil was used) was layered uniformly. Further 20 ␮l of 137
82 In this work we have attempted to produce a green Acti- the culture broth was added on the crude oil which was coated on 138
83 nomycetes A17 isolated from industrial and coal mine region, the water surface. The occurrence of a clear zone was the indication 139
84 unexplored resources for the production of BSs, using optimized of a biosurfactant producer [23]. 140
85 growth media and then exploring its biochemical and emulsify-
86 ing activity, surface tension and critical micelle concentration. The
87 isolated BSs were found to have satisfactory results and due to 2.3.2. Blue-agar plate 141

88 their low toxicity and biodegradability their further use as efficient The Glycerol Yeast extract Agar media supplemented with 142

89 excipients in pharmaceutical dosage formulation (emulsion) and carbon sources (2%w/v) and cetyltrimethylammonium bromide 143

90 cosmetics (in shampoo) is possible. (CTAB, 0.5 mg/ml) with methylene blue (MB, 0.2 mg/ml) was pre- 144

91 Actinomycetes are high GC (guanine and cytosine), Gram- pared. A dark blue halo around the culture was considered as 145

92 positive bacteria with fungal morphology. They are rich source of positive for biosurfactant production [24]. 146

93 secondary metabolites with diverse biological activity. Being a large

94 group of microbial resources of wide practical use and high com-
95 mercial value, actinomycetes contribute to around 70% of the source 2.3.3. Drop-collapsing technique 147

96 of antibiotics and also produce numerous non-antibiotic bioactive In the drop-collapse test method, 2 ␮l of mineral oil was added 148

97 metabolites, such as enzymes, enzyme inhibitors, immunological to 96-well microtitre plates. The plate was equilibrated for 1 h at 149

98 regulators, anti-oxidation reagents, and so on. Actinomycetes are 37 ◦ C and 5 ␮l of the culture supernatant was added to the surface 150

99 widely distributed in natural habitats, especially soil and ocean of the oil. The shape of drop on the oil surface was observed after 151

100 [22]. 1 min. The culture supernatant that make the drop collapsed was 152

indicated as a positive result and the drops remain beaded were 153

scored as negative, which was examined with distilled water as a 154

101 2. Materials and methods control [25]. 155

102 2.1. Sample processing

2.3.4. Lipase-activity test 156

103 Six sediment samples were collected from different sites of The isolates that produce lipase were screened using tributyrin 157

104 Rourkela Steel Plant, Odisha and Raniganj coal mine, West Bengal. agar plates. 1% tributyrin was added to Actinomycetes isolation agar. 158

105 All sediment samples were mixed in a plate to ensure uniformity. The pH of the medium was adjusted to 7.3–7.4 using 0.1 N NaOH. A 159

106 Pre-heat treated sample (10 g) was suspended in 100 ml of sterile loopful of inoculum was streaked on to the tributyrin agar plates. 160

107 water. All flasks were kept on a rotary shaker at 150 rpm for 30 min The plates were incubated at 26 ◦ C for 7 days. After incubation, the 161

108 for mixing and the resulting suspension was used for preparation plates were examined for the formation of a clear zone around the 162

109 of serial dilution up to 1012 × 100 ␮l of each diluted suspension colonies [26]. 163

110 was spread over the surface of each agar media with a sterilized
111 bend glass rod and the plates were incubated at 30 ◦ C for 2 to 3
2.3.5. Emulsification index 164
112 weeks. Different mediums had been used for the isolation as well
The ability of the biosurfactant to emulsify some liquid hydro- 165
113 as counting of marine actinomycetes. After 2 to 3 days, total num-
carbons, in this study kerosene, was determined. The microbial 166
114 ber of actinomycetes, bacteria and fungi present on each plate were
culture was inoculated in the production medium broth and incu- 167
115 counted [21].
bated for 14 days. The resultant cell-free supernatant was removed 168

by cold centrifugation at 8000 × g at 4 ◦ C for 20 min and homog- 169

116 2.2. Isolation of actinobacteria from the samples enized with hydrocarbon in a vortex at high speed for 2 min. The 170

emulsification stability was measured after half an hour and the 171

117 Pre-heat treatment of samples at 40 ◦ C for 10, 30, and 60 emulsification index was calculated by the total height of the liq- 172

118 days were used for isolation of actinomycetes and counting of uid layer divided by the total height of the system and multiplying 173

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx 3

174 by 100, keeping the radius of the measuring cylinder constant 2.6. Fermentative production of biosurfactant 229
175
[27].
Among the 15 cultures of cultures of actinomycetes which pro- 230
Total height of the emulsion layer
176 EI(%) = × 100. duce the biosurfactant, the best culture (A 7) with the highest 231
Total height of the system
emulsification index was selected for further study. 232

The seed culture was prepared in a 100 ml conical flask con- 233

177 2.4. Characterization of A17 by16S rDNA sequence analysis taining 50 ml of medium by inoculating loop full of spore and 234

cultivated under agitation at 100 rpm at 28 ◦ C for 2 days. Then 235

178 Genomic DNA was extracted from Nocardiopsis sp. strain A17 50 ml of seed culture was inoculated in the 500 ml of fermenta- 236

179 using a TINAamp Bacteria DNA Kit following the instructions of the tion medium containing glycerol 2%(v/v), yeast extract 0.1%(w/v), 237

180 manufacturer. The primers (P0:5 -GAGAGTTTGATCCTGGCTCAG-3 ; potassium dihydrogen phosphate (KH2 PO4 ), 0.05%(w/v) in distilled 238

181 P6: 5 -CTACGGCTACCTTGTTACGA-3 ) were used to amplify the 16S water and fermentation was carried out 14 days under agitation at 239

182 rDNA. The concentrations for primers, polymerase, and DNA used 100 rpm at 28 ◦ C. The cell-free supernatant containing the biosur- 240

183 in the polymerase chain reaction (PCR) were 25 ␮M, 5.0 U/␮l, and factant harvested by cold centrifugation at 10,000 rpm for 20 min 241

184 10 mg/l, respectively. PCR amplification (25 ␮l final volume: 0.4 ␮l at 4 ◦ C was subjected to partial purification and characterization. 242

185 each primer, 2.5 ␮l 10Xbuffer, 2.5 ␮l 2.5 nM dNTP, 0.4 ␮l Taq DNA
186 polymerase, and 1 ␮l DNA template) of 16S rDNA was achieved 2.7. Isolation and purification of biosurfactant 243
187 using a TaKaRa PCR Thermal Cycler with the initial denaturation at
188 94 ◦ C for 5 min, 30 cycles of denaturation (94 ◦ C for 1 min), anneal- The biosurfactant is an extracellular product, therefore during 244
189 ing (55 ◦ C for 1 min), elongation (72 ◦ C for 1 min 15 s), and a final fermentation it comes out from the cell and present in the fermen- 245
190 elongation at 72 ◦ C for 10 min. Multiple alignments with sequences tation medium along with the cell. So at first centrifugation was 246
191 of the most closely related species and calculations of sequence done to collect the cell-free supernatant. During centrifugation, the 247
192 similarity were carried out using CLUSTAL X. A phylogenetic tree cell will settle at the bottom. Radial centrifugal force (RCF) or rev- 248
193 was constructed using the neighbor-joining method and MEGA 4.0 olutions per minute (rpm) vary according to the size and density 249
194 software. The topology of the phylogenetic tree was evaluated by of the cell. In the present study, the culture was cold centrifuged 250
195 1000 bootstrap re-sampling replicates. at 10,000 rpm for 20 min at 4 ◦ C to remove the cells as well as 251

debris and the supernatant was filtered through a 0.2 ␮m filter. The 252

196 2.5. Optimization of culture conditions for Actinomycetes A17 clear sterile supernatant was used for the extraction of the surface- 253

active compound. After centrifugation, the extraction process was 254

197 Cell growth and the accumulation of the metabolite products performed by cold acetone precipitation [28], where 3 volumes of 255

198 were strongly influenced by medium composition such as carbon chilled acetone were added and allowed to stand for 24 h at 4 ◦ C. 256

199 sources, nitrogen sources, pH, and temperature. Thus the optimiza- The precipitate was collected by centrifugation and evaporated to 257

200 tion of these factors can result in the high yield of metabolites. dryness to remove the residual acetone. The dried residual was then 258

201 Optimization of biosurfactant production by the strain with high- subjected to purification and thin layer chromatography was per- 259

202 est emulsification index was carried out by search one at a time formed with chloroform:methanol (9:1) as the mobile phase where 260

203 technique. the dried product was loaded. After observing the TLC plate under 261

a UV chamber, a single spot was noted. Both the samples, one at 262

204 2.5.1. pH the loading site and the one which traveled with the mobile phase, 263

205 The importance of characteristics of most of the organisms is were collected separately and dissolved in methanol. The emulsifi- 264

206 their strong dependence on the pH for cell growth and production of cation index was checked and for the detection of the biosurfactant 265

207 secondary metabolites. The emulsification index was measured for among them. 266

208 the biosurfactant produced by the microorganism in the selected

209 medium with different pH values ranging from 4 to 9 and keeping 2.8. Characterization of isolated biosurfactant 267
210 other factors constant.
2.8.1. Emulsification index (EI) 268

211 2.5.2. Temperature The ability of the biosurfactant to emulsify some liquid hydro- 269

212 It was one of the critical parameters that have been controlled in carbons, in this study kerosene, was determined. The microbial 270

213 the bioprocess. In this study, temperature was varied from 10 ◦ C to culture was inoculated in the production medium broth and incu- 271

214 60 ◦ C, with a 10 ◦ C increase. The emulsification index was calculated bated for 14 days. The resultant cell-free supernatant was removed 272

215 as mentioned earlier after 7 days of incubation. by cold centrifugation at 10,000 rpm at 4 ◦ C for 20 min and homog- 273

enized with hydrocarbon in a vortex at high speed for 2 min. The 274

216 2.5.3. Carbon source emulsification stability was measured after half an hour and the 275

217 The different varieties of carbon source such as glucose, fructose, emulsification index [27] was calculated by the total height of the 276

218 and glycerol along with some oils like castor oil, coconut oil, and liquid layer divided by the total height of the system and multiply- 277

219 olive oil with their varying concentration were used. After incuba- ing by 100, keeping the radius of the measuring cylinder constant. 278
279
220 tion for 7 days, the emulsification index was calculated for each of
221 them. Total height of the emulsion layer
EI(%) = × 100. 280
Total height of the system
222 2.5.4. Nitrogen source
223 Nitrogen also plays an important role in the production of bio-
224 surfactants by microorganisms. Nitrogen sources like beef extract, 2.8.2. Surface tension determination 281
225 yeast extract, and peptone with different concentration were used Surface tension determination of the crude as well as the 282
226 in the culture media with all others factors constant. After a week of purified biosurfactant was performed using a stalagmometer. To 283
227 incubation, the emulsification index was measured. All the media increase the accuracy of the surface tension measurements, an 284
228 and reagents used in this study were from Himedia, India. average of triplicates was determined. All the measurements were 285

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
4 S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

286 performed at room temperature, 32 ◦ C. Surface tension of the bio- Table 1

Screening of actinomycetes for biosurfactant production.
287 surfactant was measured by using a stalagmometer. The purified
288 dried sample (0.8 mg) was dissolved in 10 ml of methanol. The vol- Actinomycetes Clear zone in blue Oil spreading Drop collapse
289 ume was up to 50 ml in a volumetric flask using distilled water. By agar plates
290 taking this as the stock solution, 10–60% of biosurfactant concen- A10 + + +
291 tration was prepared. Then, in a clean stalagmometer the solution A11 − + −
292 was filled using a sucker to reach the upper mark. Solution drops A12 − + −
A13 − − +
293 were counted till the solution level reach the lower mark. Next,
294 A17 ++ ++ ++
calculation was made as per the given formula A19 + + −
n1 1 a1 + − +
295 2 = 1 , a2 − − −
n2 2 B6 − − −
B8 + + +
296 where ␥1 = surface tension of water, 72 dyne/cm, ␥2 = surface ten-
B9 + + +
297 sion of the biosurfactant, ␳1 = density of water, ␳2 = density of the B10 − − +
298 biosurfactant, n1 = no of drops of water, and n2 = no of drops of B16 + + −
299 biosurfactant solution.

300 2.8.3. Critical micelle concentration (CMC) Meso-diaminopimelic acid (meso-DAP), arabinose, and galactose 343

301 The surface tension of the biosurfactant was measured by the were found to be major constituents of the cell wall. 16S rDNA 344

302 drop count method. It is important for several biosurfactant appli- sequencing showed that strain A17 was closely related to the genus 345

303 cations to establish their cmc, because above this concentration Nocardiopsis (Fig. 1). The culture was deposited in Gene Bank and 346

304 no further effect is expected in the surface activity. The CMC was the accession number is HQ 419063. 347

305 determined by plotting the surface tension as a function of the

306 biosurfactant concentration (% w/v). 3.2. Screening for biosurfactant producing actinomycetes strains 348

307 2.8.4. TLC analysis Satpute et al. [29] reported that the single screening method is 349

308 Thin layer chromatography was performed for purification of not suitable to identify all types of BSs and recommended more than 350

309 the crude biosurfactant. Plates were prepared using silica gel G one screening methods should be included in the primary screening 351

310 as the stationary phase. The mobile phase was taken as chloro- as to identify potential biosurfactant producers (Fig. 2). 352

311 form:methanol (9:1 v/v). After the saturation of the TLC chamber, Therefore, in this study, lipase (Fig. 2a), blue-agar plate drop 353

312 the dried sample was loaded over the plate and the mobile phase (Fig. 2b), collapsing test, oil displacement test, and emulsification 354

313 was allowed to run. TLC plates were then exposed to a UV cham- activity were used to screen the biosurfactant producer (Table 1). 355

314 ber to visualize the spot. The spots so developed on the plate were According to Kokare et al. [30], activity was shown by lipase over the 356

315 scraped out and dissolved in fresh mobile phase and filtered. The water–oil surfaces and hence it was suggested that actinomycetes 357

316 purified samples were tested for the emulsification activity. TLC showed the presence of lipases are able to produce bioemulsifiers. 358

317 was performed for carbohydrates (chloroform:acetic acid:water In the current work, the strain A10, A17, B8, and B9 showed posi- 359

318 60:30:10) and lipids (chloroform:methanol:water 65:25:4). The tive results in all the four screening methods used. But strain A17 360

319 resultant spots on the TLC plates were visualized by spraying of had the highest activity among all the four strains with the highest 361

320 50% H2 SO4 for carbohydrates. The TLC plates were exposed in an emulsification index of 93.2 ± 0.84. 362

321 iodine chamber to visualize the lipid fraction.

3.2.1. Emulsification index for biosurfactant 363

322 2.8.5. LC–MS Emulsification activity is one of the criteria to support the 364

323 The purified sample was analyzed on a Micromass Q-TOF spec- selection of potential biosurfactant producers. Emulsifying activ- 365

324 trometer. The result was determined by Electroscopy Ionization ities determine productivity of the biosurfactant. Ellaiah et al. [31] 366

325 (ESI) in positive mode. screened 68 bacterial isolates from soil and found only 6% of isolates 367

with good emulsification activity of up to 61%. During this study, 368

326 2.8.6. Fourier transform-infrared (FTIR) spectroscopy emulsification of kerosene by A17 was up to 92%. This observa- 369

327 The BSs obtained from Actenomycetes sp A17 were analyzed as tion is important to suggest that potent biosurfactant cultures can 370

328 KBr pellets by using a Fourier transform-infrared (FTIR) spectro- be detected through such assays. Cultures like A10, A13, A19, B8, 371

329 scope (Perkin-Elmer Spectrum RX I, USA). The pellet was placed B9, and B10 showing >60% emulsification activity were also pos- 372

330 in the sample holder. Spectral scanning was taken in the wave- itive for biosurfactant production. Measurement of emulsification 373

331 length region between 4000 cm−1 and 400 cm−1 with a scan speed units help to choose the carbon and energy source for biosurfactant 374

332 of 2 mm/s. production. Patil and Chopade [32] introduced emulsification assay 375

based on emulsification units of the tested oils. Thus by examining 376

333 3. Results and discussion emulsification units, it is possible to select a potent biosurfactant. 377

So this process was recommended to be important and effective 378

334 3.1. Isolation and identification of actinomycetes strain of A17 assay for screening the biosurfactant producers. 379

335 The pre-heat treatment of sediment samples yielded more num- 3.3. Optimization of nutritional and physiological requirement 380

336 ber of actinomycetes and less number of contaminated organisms.

337 Heat treatment at 40 ◦ C for 30 days resulted in slight reduction 3.3.1. Effect of pH and temperature 381

338 of bacterial (non-actinomycetes) and fungal count. Cell culture has The biosurfactant production from strain A17 was found to be 382

339 yellow colored pigmentation with excessive foam-forming prop- the highest at pH 6.8 though it was having biosurfactant activity at 383

340 erties. Aerial and substrate mycelium were observed and both the higher and lower pH (Fig. 3a). It clearly shows almost neutral nature 384

341 mycelia fragmented into short rods. The spore chain was found of the biosurfactant was obtained from this isolate. Almost similar 385

342 to be short and spiral; and it was observed on aerial mycelium. reports have been reported for B.subtilis which was able to produce 386

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx 5

Fig. 1. Phylogenetic tree (16S rDNA sequencing) of strain A17.

387 the biosurfactant in a pH range of 6.0–9.0, although maximum yield carbon source and yeast extract (Fig. 3e) as the nitrogen source. The 405

388 of the biosurfactant was obtained at pH 7 [33] and the strain MSF3 optimum concentration of glycerol for biosurfactant production 406

389 produced the highest yield of the biosurfactant at pH 7, even though was found to be 2% w/v followed by a decline in higher concentra- 407

390 the growth was higher in pH 8 and 9 [34]. tion (Fig. 3d). This may be due to the increase in viscosity of broth 408

391 Optimum temperature for biosurfactant production from strain at such concentrations and that high concentration interferes with 409

392 A17 was found out to be at 28 ◦ C and the biosurfactant retained O2 transfer leading to limitation of dissolved O2 for the growth of 410

393 its activity almost 83%, 53%, 42%, and 33% at higher tempera- the organisms [39]. 411

394 tures 30 ◦ C, 40 ◦ C, 50 ◦ C, and 60 ◦ C, respectively, as well (Fig. 3b). Complex nitrogenous sources were required for growth, forma- 412

395 A change in temperature caused alteration in the composition of tion of cell constituents, and production of bioactive secondary 413

396 biosurfactant in case of A. paraffineus [35] and Pseudomonas sp. metabolites [40]. The enhancement in enzyme production from 414

397 [36]. Despite from being terrestrial actinomycetes, the optimum both these strains was observed in the presence of inorganic 415

398 biosurfactant production by A17 in this study was found to be at phosphate in the medium and optimum concentration was found 416

399 28 ◦ C. to be 0.1% w/v. It acts as an important constituent of cellular 417

biomolecules such as cAMP, nucleic acid, phospholipids, and coen- 418

400 3.3.2. Carbon and nitrogen sources zymes. In addition to that, inorganic phosphate is known to play a 419

401 Biosurfactant production from these strains was found to be regulatory role in the synthesis of primary and secondary metabo- 420

402 constitutive, since it took place not only in starch but also in simple lites in microorganisms [41]. Desai and Banat [6] reported that the 421

403 carbon sources [37,38]. In fact, highest amount of biosurfactant pro- limitation of nitrogen source increases the biosurfactant produc- 422

404 duction was obtained in a medium having glycerol (Fig. 3c) as the tion. 423

Fig. 2. Screening of actinomycetes for bosurfactant production: (a) lipase activity test showing a clear zone and the blue-agar plate showing a clear zone (b).

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
6 S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

Fig. 3. Comparison of emulsification index of A17 for various pH ranges (a), various temperature ranges (b), carbon sources (c), % of glycerol (d), nitrogen sources (e) and %
of yeast extract (f).

424 In the present study it can be demonstrated that the emulsifi-

425 cation by the biosurfactant is substrate specific, similar to the one
426 reported study by Falatko and Novak [42]. They explained that BSs
427 produced from growth on glucose or vegetable oil could not emul-
428 sify gasoline hydrocarbons, while BSs produced from growth on
429 gasoline could emulsify. Some microbes have been reported to be
430 capable of producing surfactants with the ability to emulsify vari-
431 ous hydrocarbons, irrespective of the substrates used as the carbon
432 source [43].

433 3.4. Fermentative production of biosurfactant

434 The isolated Actinomycetes sp. A17 produces the biosurfactant

435 (Fig. 4) when grown in various nutrients. The amount of biosur-
436 factant production was varied with respect to media composition.
437 However, the maximum biosurfactant production was observed
438 in glycerol yeast extract. The production of biosurfactant was not
439 observed in minimal salt medium and potato dextrose medium. A
440 similar result was observed by Desai and Banat. Therefore, glycerol
441 yeast extract was selected as the biosurfactant production medium Fig. 4. Biosurfactant production by A17 in the optimized culture medium.
442 for the strain at 28 ◦ C for 9 days [44].

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx 7

Fig. 6. Critical micelle concentration (CMC) of the biosurfactant produced by A17.

Fig. 5. LC–MS of the purified biosurfactant A17.

443 3.5. Extraction and purification of biosurfactant

444 As the biosurfactant was an extracellular product, it comes out of

445 the cell into the medium along with the cell. So, during centrifuga-
446 tion, when the cell settles down, the cell-free supernatant contains
447 the biosurfactant within it. This product was then precipitated with
448 chilled acetone. The precipitate was collected by centrifugation and
449 the remaining acetone was allowed to evaporate.
450 The dried crude biosurfactant was not soluble in any polar sol- Fig. 7. FTIR spectra of SLS (sodium lauryl sulphate) and the biosurfactant produced
451 vent or water, hence was purified in TLC plates where the impurity by A17.
452 traveled with the mobile phase (methanol:chloroform; 1:9). The
453 purified biosurfactant was soluble in methanol. This method used (CH band: CH2 CH3 stretching), 1561 cm−1 (C C ring stretch), 481
454 in the recovery of the biosurfactant gives an approximate yield of 1463 cm−1 (CH bend of CH2 /CH3 ), and 1297 cm−1 (C O stretch). 482
455 6 ␮g/ml. Also, peaks were shown between 900 and 500 cm−1 . The peak at 483

816 cm−1 explains a possibility of the presence of cyclohexylamine 484

456 3.6. Characterization of biosurfactants and 880 cm−1 explains the phenyl ring substitution band. Due to 485

the presence of CH2 CH3 stretch and C O stretch, the biosurfactant 486
457 3.6.1. TLC analysis probably may be a fatty acid. 487
458 From the TLC analysis, it was observed that the surface-active
459 extract of Actinomycetes sp. A17 do not contain any carbohydrate,
4. Conclusion 488
460 but have shown lipid fragments in an iodine chamber, with a Rf
Q4 value 05. Hence, it can be established as lipid-based BSs.
461
In this study, it was proved that biosurfactant-producing acti- 489

nomycetes were isolated from industrial regions of Rourkela Steel 490

462 3.6.2. LC–MS spectroscopy study Plant and coal mine regions of Raniganj successfully. After biochem- 491
463 The LC–MS reveals an idea of the molecular weight of the sur- ical screening the strain Actinomycetes nocardiopsis(strain A17), 492
464 face-active agent of A17 (Fig. 5). According to the Electrospray isolated from soil collected from Raniganj, showed the satisfac- 493
465 Ionization in positive mode, the major peaks 437, 415, and 452 tory results for surface activities and emulsifying abilities with high 494
466 were explained and it was concluded that the molecular weight of emulsification index. The metabolites obtained from this strain 495
467 the A17 biosurfactant could be 414 with a hydrogen, sodium, or showed positive response for amylase, protease, gelatinase, and 496
468 potassium molecule in it. It was also explained that there no traces lipase activity. From the optimization study it has been observed 497
469 of protein were found in the product. In another study previously that the fermentation procedure can be performed in the following 498
470 reported, a fraction of the biosurfactant with higher surface activ- optimum conditions: glycerol 2% (carbon source), yeast extract 0.1% 499
471 ity was partially purified by S. thermophilus with no protein content (nitrogen source), KH2 PO4 0.05% w/v, at temperature 28 ◦ C and pH 500
472 [44]. 6.8, to get maximum biosurfactant production. The purified product 501

obtained from the cell-free supernatant contains lipid but have no 502
473 3.6.3. Critical micelle concentration (CMC) traces for protein and carbohydrate. So it was established as a lipid- 503
474 It was found to reduce the surface tension of the water from 72 based biosurfactant. This new biosurfactant from A17 strain may 504
475 to 51.43 dyne/cm. Critical micelle concentration (CMC) determined be potentially used as an alternative to chemical surfactant-active 505
476 with the aid of a series of concentrations was around 6 mg/L (Fig. 6). excipients of pharmaceutical dosage form and cosmetics. 506

477 3.6.4. Fourier transform infra red (FT-IR) spectroscopy References Q5 507
478 The molecular composition of the crude biosurfactant along
479 with a sodium lauryl sulphate was evaluated by FTIR spectroscopy [1] C. Burgos-Díaz, R. Pons, J.A. Teruel, F.J. Aranda, A. Ortiz, A. Manresa, A.M. Mar- 508
480 (Fig. 7). The most important band was located at 2935 cm−1 qués, J. Colloid Interface Sci. 394 (2013) 368–379. 509

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068
 G Model
BIOMAC 5074 1–8 ARTICLE IN PRESS
8 S. Chakraborty et al. / International Journal of Biological Macromolecules xxx (2015) xxx–xxx

510 [2] R.D. Rufino, J.M. Luna, L.A. Sarubbo, L.R.M. Rodrigues, J.A.C. Teixeira, G.M. [22] M. Goodfellow, S.T. Williams, Ann. Rev. Microbiol. 73 (1983) 189–216. 543
511 Campos-Takaki, Colloids Surf., B 84 (2011) 1–5. [23] M. Morikawa, H. Daido, T. Takao, S. Marato, Y. Shimonishi, T. Imanaka, J. Bacte- 544
512 [3] E.V. Chandrasekaran, J.N. Bemiller, R.L. Whistler (Eds.), Methods in Carbohy- riol. 175 (1993) 6459–6466. 545
513 drate Chemistry, Academic Press Inc., New York, 1980, p. 89. [24] I. Siegmund, F. Wagner, Biotechnol. Tech. 5 (1991) 265–268. 546
514 [4] M. Givskov, J. Ostling, I.L. Eber, P. Lindum, A.B. Christensen, G. Christensen, S. [25] N.H. Youssef, K.E. Dunacn, D.P. Nagle, K.N. Savage, R.M. Knapp, M.J. McInerney, 547
515 Molin, S. Kjellerberg, J. Bacteriol. 180 (1998) 742–745. J. Microbiol. Methods 56 (2004) 339–347. 548
516 [5] M.P.S. Pirôllo, Estudo da Produção de Biossurfactantes utilizando Hidrocar- [26] G.S. Kiran, T.A. Thomas, J. Selvin, Colloids Surf., B 78 (2010) 8–16. 549
517 bonetos. Dissertação, Master’s thesis, Universidade Estadual Paulista (UNESP), [27] D.G. Cooper, B.G. Goldenberg, Appl. Environ. Microbiol. 53 (1987) 224–229. 550
518 Rio Claro, São Paulo, 2006, pp. 61. [28] V. Pruthi, S.S. Cameotra, J. Surfactants Deterg. 6 (2003) 65–68. 551
519 [6] J.D. Desai, I.M. Banat, Microbiol. Mol. Biol. Rev. 61 (1997) 47–64. [29] S.K. Satpute, B.D. Bhawsar, P.K. Dhakephalkar, B.A. Chopade, Indian J. Mar. Sci. 552
520 [7] I.M. Banat, R.S. Makkar, S.S. Cameotra, Appl. Microbiol. Biotechnol. 53 (2000) 37 (2008) (2008) 243–250. 553
521 495–508. [30] C.R. Kokare, S.S. Kadam, K.R. Mahadik, B.A. Chopade, Indian J. Biotechnol. 6 554
522 [8] R.M. Maier, G. Soberon-Chavez, Appl. Microbiol. Biotechnol. 54 (2000) 625–633. (2007) 78–84. 555
523 [9] F. Ahimou, P. Jacques, M. Deleu, Enz. Microb. Technol. 27 (2000) 749–754. [31] P. Ellaiah, T. Prabhakar, M. Sreekanth, A.T. Taleb, P. Bhima, V. Saisha, Indian J. 556
524 [10] L.R. Rodrigues, H.C. Van der Mei, J.A. Teixeira, R. Oliveira, Appl. Environ. Micro- Exp. Biol. 40 (2002) 1083–1086. 557
525 biol. 70 (2004) 4408–4410. [32] J.R. Patil, B.A. Chopade, J. Appl. Microbiol. 91 (2001) 290–298. 558
526 [11] D. Vollenbroich, M. ÖZel, J. Vater, R.M. Kamp, G. Pauli, Biologicals 25 (1997) [33] M. Abouseoud, R. Maachi, A. Amrane, S. Boudergua, A. Nabi, Desalination 223 559
527 289–297. (2008) 143–151. 560
528 [12] C.N. Mulligan, Environ. Pollut. 133 (2005) 183–198. [34] G. SehgalKiran, T.A. Hema, R. Ganghimathi, J. Selvin, T. Anto Thomas, T. 561
529 [13] M.P. Pirôllo, A.P. Mariano, R.B. Lovaglio, S.G. Costa, V. Walter, R. Hausmann, J. RajeethaRajvi, K. Natarajaseenivasan, Colloids Surf., B 73 (2009) 250–256. 562
530 Contiero, J. Appl. Microbiol. 105 (2008) 1484–1490. [35] R.S. Makkar, S.S. Cameotra, J. Surfactants Deterg. 2 (1999) 237–241. 563
531 [14] B. Angelova, H.P. Schmauder, J. Biotechnol. 67 (1999) 13–32. [36] Z. Duvnjak, D.G. Cooper, N. Kosaric, Biotechnol. Bioeng. 24 (1982) 165–175. 564
532 [15] M. Shavandi, G. Mohebali, A. Haddadi, H. Shakarami, A. Nuhi, Colloids Surf., B [37] R. Malhotra, S.M. Noorwez, T. Satyanarayana, Lett. Appl. Microbiol. 31 (2000) 565
533 82 (2011) 477–482. 378–384. 566
534 [16] M. Hassanshahian, Mar. Pollut. Bull. 86 (2014) 361–366. [38] G. Mamo, A. Gessesse, Lett. Appl. Microbiol. 29 (1999) 61–65. 567
535 [17] V. Kavitha, A.B. Mandal, A. Gnanamani, Int. Biodeterior. Biodegrad. 94 (2014) [39] R. Rukhaiyar, S.K. Srivastava, World J. Microbiol. Biotechnol. 10 (1995) 568
536 24–30. 76–82. 569
537 [18] C.G. Kumara, P. Sujithaa, S.K. Mamidyala, P. Usharani, B. Das, C.R. Reddy [40] A.K. Chandra, S. Medda, A.K. Bhadra, J Ferment. Technol. 58 (1980) 1–10. 570
538 Ochrosin, Process Biochem. 49 (2014) 1708–1717. [41] A.C.R. Demain, J. Appl. Chem. Biotechnol. 22 (1972) 245–259. 571
539 [19] H. Saeki, M. Sasak, K. Komatsu, A. Miura, H. Matsuda, Bioresour. Technol. 100 [42] D.F. Falatko, J.T. Novak, Water Environ. Res. 64 (1992) 163–169. 572
540 (2009) 572–577. [43] Y. Prabhu, PhaleF P.S., Appl. Microbiol. Biotechnol. 61 (2003) 342–351. 573
541 [20] Z. Zhao, A. Selvam, J. Woon-Chung Wong, J. Hazard. Mater. 190 (2011) 345–350. [44] L.R. Rodrigues, J.A. Teixeira, H.C. Van der Mei, R. Oliveira, Colloids Surf., B 53 574
542 [21] C.R. Kokare, K.R. Mahadik, S.S. Kadam, B.A. Chopade, Indian J. Mar. Sci. 33 (2004) (2006) 105–112. 575
248–256.

Please cite this article in press as: S. Chakraborty, et al., Int. J. Biol. Macromol. (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.068