Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction

Journal of Microbiological Methods 46 Ž2001. 149–156 www.elsevier.comrlocaterjmicmeth

Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction Maria S. Kuyukina a , Irena B. Ivshina a , Jim C. Philp b,) , Nick Christofi b, Sandra A. Dunbar b, Marina I. Ritchkova a a

Institute of Ecology and Genetics of Microorganisms, Ural Branch of Russian Academy of Sciences, 12 GoleÕ Street, 614081 Perm, Russia b School of Life Sciences, Napier UniÕersity, 10 Colinton Road, Edinburgh, EH10 5DT, Scotland, UK Received 1 February 2001; received in revised form 25 March 2001; accepted 28 March 2001

Abstract In the present study, we proposed methyl tertiary-butyl ether ŽMTBE. as a solvent for extraction of biosurfactants from Rhodococcus bacterial cultures. After comparison with other well known solvent systems used for biosurfactant extraction, it was found that MTBE was able to extract crude surfactant material with high product recovery Ž10 grl., efficiency Žcritical micelle concentration ŽCMC., 130–170 mgrl. and good functional surfactant characteristics Žsurface and interfacial tensions, 29 and 0.9 mNrm, respectively.. The isolated surfactant complex contained 10% polar lipids, mostly glycolipids possessing maximal surface activity. Ultrasonic treatment of the extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. Due to certain characteristics of MTBE, such as relatively low toxicity, biodegradability, ease of downstream recovery, low flammability and explosion safety, the use of this solvent as an extraction agent in industrial scale biosurfactant production is feasible. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Biosurfactants; Extraction; Glycolipids; MTBE; Rhodococcus

1. Introduction Surfactants of biological origin are of increasing interest for many industries due to their chemical diversity, multifunctional characteristics and low toxicity in comparison to synthetic, petrochemical-derived surfactants. Their production from renewable resources is an attraction receiving increasing attention. Fields of application or potential use of biosurfactants are diverse. In the near term, the most

) Corresponding author. Tel.: q44-131-455-2462; fax: q44131-455-2291. E-mail address: [email protected] ŽJ.C. Philp..

promising applications of biosurfactants are the environmental remediation technologies, since product purity is of less concern. At present, however, the industrial use of biosurfactants is not generally competitive with synthetics because of the higher production cost for biosurfactants, mainly due to downstream processing. The recovery and concentration of biosurfactants from fermentation broth largely determines the production cost. Often, low concentration and the amphiphilic nature of microbial surfactants limit their recovery ŽGeorgeou et al., 1992.. Different methods used for biosurfactant isolation include high-speed centrifugation, dia- and ultrafiltration, acid and salt precipitation, solvent extraction and adsorption chro-

0167-7012r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 1 . 0 0 2 5 9 - 7

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matography ŽSyldatk and Wagner, 1987; Bryant, 1990; Mulligan, 1990; Kurane et al., 1994.. Bacteria of the genus Rhodococcus synthesise considerable amounts of glycolipid surfactants, e.g. total product yield reached 20–30 grl for Rhodococcus erythropolis ŽKim et al., 1990., and 9–11 grl for R. ruber Žpresent study.. However, surfactants produced by rhodococci under unrestricted growth conditions are predominantly cell-associated trehalose mycolates, which can be effectively isolated only by organic solvent extraction ŽRapp et al., 1979; Lang and Philp, 1998.. By comparison, the rhamnolipids of Pseudomonas aeruginosa are excreted. A wide variety of organic solvents Že.g. methanol, ethanol, diethyl ether, pentane, acetone, chloroform, dichloromethane. have been used, either singly or in combination, for biosurfactant extraction ŽDesai and Banat, 1997.. Most effective are the mixtures of chloroform and methanol in various ratios, which facilitates adjustment of the polarity of extraction agent to the target extractable material. However, the use of chloroform for preparative extractions demanding large volumes of solvent is not economically warranted. Moreover, chloroform is a highly toxic chloro-organic compound regarded as harmful for the environment and for human health ŽHeitmann et al., 1996; Agency for Toxic Substances and Disease Registry ŽATSDR., 1997; Lilly et al., 1997; Mills et al., 1998.. Thus, there is a need for inexpensive and low toxicity solvents for biosurfactant extraction suitable for industrial applications. To isolate rhodococcal non-ionic glycolipid surfactants having low hydrophilic–lipophilic balance ŽHLB. requires a solvent with low polarity. We suggested that methyl tertiary-butyl ether ŽMTBE., the commonly used octane-enhancing gasoline additive, might be a promising candidate for this application ŽIvshina et al., 1996.. It is relatively non-toxic, less likely to form peroxides and less explosive than other solvents ŽRosenkranz and Klopman, 1991; Gupta and Lin, 1995.. Alkyl ethers, the chemical class to which MTBE belongs, are more polar than hydrocarbons, but less polar than alcohols, ketones, esters and chlorinated hydrocarbons ŽReichardt, 1988.. In this work, we compared MTBE with other well known solvent systems used for biosurfactant extraction. The surface-active properties and chemical

composition of the biosurfactant isolated from the culture of R. ruber by different extraction methods were evaluated.

2. Materials and methods 2.1. Bacterial strain and culture conditions The strain R. ruber IEGM 231 was from the Regional Specialised Alkanotrophic Microorganisms Collection of the Institute of Ecology and Genetics of Microorganisms, Perm, Russia. The organism was maintained on nutrient agar slants. A mineral salts medium contained Žper litre of distilled water.: KH 2 PO4 , 2.0 g; K 2 HPO4 , 2.0 g; KNO 3 , 1.0 g; ŽNH 4 . 2 SO4 , 2.0 g; NaCl, 1.0 g; MgSO4 P 7H 2 O, 0.2 g; CaCl 2 P 2H 2 O, 0.02 g; FeCl 3 P 7H 2 O, 0.01 g; trace elements solution, 1.0 ml; thiamine, 4 mg. n-Hexadecane was used as carbon source and added at a concentration of 3% Žvrv.. All chemicals were analytical grade reagents unless specified. n-Hexadecane was obtained 99% pure from Aldrich ŽGillingham, Dorset.. Cultivation was carried out at 288C on a rotary shaker at 170 rpm for 3 days. 2.2. Biosurfactant isolation Broth culture was centrifuged at 6000 rpm and 158C for 15 min, and the hydrophobic layer located at the surface was extracted using different solvent systems at 278C on a rotary shaker Ž200 rpm. for 3 h. Solvent extraction was also carried out on the whole culture broth, and in some cases, the extraction was performed using sonication ŽIvshina et al., 1998.. The solvent systems used were: MTBE, dichloromethane, chloroform–methanol Ž1:2 or 2:1. and MTBE–chloroform Ž1:1.. The solvent layer was separated from the aqueous phase, and solvent was removed by rotary evaporation at 508C under reduced pressure. Resulting crude extracts were freeze-dried and stored under nitrogen. 2.3. Surface and interfacial properties Surface and interfacial tensions were measured by the ring de Nuoy method using a White digital

M.S. Kuyukina et al.r Journal of Microbiological Methods 46 (2001) 149–156

surface tension balance. As crude biosurfactant was sparingly soluble in water, its surface and interfacial tension measurements were performed immediately after emulsification by ultrasonic treatment in water Ž23 kHz, 1 min. ŽKim et al., 1990.. Interfacial tension was determined against n-hexadecane. Critical micelle concentration ŽCMC. was calculated as the lowest concentration of biosurfactant at which the surface tension value was minimal. 2.4. Determination of surfactant composition Protein was determined by the Coomassie blue method, with bovine serum albumin ŽBSA. as a standard ŽBradford, 1976.. Extractable hexoses were estimated by the phenol–sulphuric acid method, using trehalose as a standard ŽDubois et al., 1956.. Separation of lipids was performed by chromatography on a silica gel Ž60–140 mesh, Merck. column using solvent systems with increasing polarity ŽKretschmer et al., 1982.. The eluates from the column were collected and dried under nitrogen. Different column fractions were analysed by TLC after dissolving in chloroform. Lipids were subject to chromatography with the following solvent systems: saturated hydrocarbons with n-hexane; unsaturated hydrocarbons with n-hexane–dichloromethane Ž9:1.; acylglycerols and fatty acids with n-hexane–chloroform–acetic acid Ž20:80:0.5.; fatty alcohols with chloroform–methanol Ž95:5.; glycolipids with chloroform–methanol–water Ž85:15:2.; phospholipids with chloroform–methanol–water Ž65:25:4.. Detection of lipids was performed using the following spray reagents: 50% sulphuric acid for n-alkanes; 4-methoxy benzaldehyde Ž p-anisaldehyde. for unsaturated hydrocarbons and fatty alcohols; hydroxylamine–ferric chloride for acylglycerols; 2,7-dichlorofluorescein–aluminium chloride–ferric chloride for fatty acids; anthrone and phenol–sulphuric acid for glycolipids; ammonium molybdate–perchloric acid and ninhydrin for phospholipids. Corresponding lipids, purchased from Sigma, St. Louis, MO, USA, were used as reference standards. The fatty acids composition of surfactant extracts was analysed by GC–FID ŽChrom-5, Laboratorni Pristroje, Prague, Czech Republic. of fatty acids methyl ethers. Methylation of organic extracts was carried out after alkaline hydrolysis Ž708C for 1 h. at

151

808C for 4 h, under nitrogen, with methanol–benzene Ž1:5.. Concentrated sulphuric acid was used as a catalyst. The GC capillary column was 3.7 m = 4.0 mm containing PEGA Ž10%. in Intertone NAWDMCS. The carrier gas was helium at 24 mlrmin; evaporator temperature was 3108C; column temperature was 1808C; detector temperature was 2108C. The sample volume was 10 ml. The chromatographic peaks were identified by comparing with the chromatogram of the standard fatty acid methyl ester ŽFAME. mixture ŽSigma.. The presence of 10-methyl branched fatty acids was indirectly indicated by bromination of FAME in diethyl ether ŽKates, 1988. and by comparison with the retention time data published for Rhodococcus species. Residual n-hexadecane concentration in crude surfactant was measured by gas chromatography of n-hexane ŽSigma GC grade. extracts using capillary GC-FID ŽPerkin-Elmer 8320.. The column was 50 m = 0.25 mm containing 0.22 mm CPSIL-8. The carrier gas was helium at 7.5 psi pressure. Oven temperature ramp rate was 40–3008C at 48Crmin. The sample Ž1 ml. was injected on column at 608C. The column temperature regime was 608Cr0.1 min at 200–3008Crmin. 3. Results and discussion 3.1. Extraction of crude biosurfactant The ability of solvent systems to isolate surfaceactive components from 60-h cultures of R. ruber differed under various extraction conditions. Although differences in the amount of biosurfactant extracted by tested solvent systems were small, use of MTBE and chloroform–methanol resulted in greater crude extract yield than the use of dichloromethane ŽTable 1.. When extraction was performed with MTBE or dichloromethane, the solvent and water fractions produced two clearly separate layers after 10-min settling of the extracts. The surfactant-containing fraction was either the top layer ŽMTBE extract. or the lower layer Ždichloromethane extract. of the extraction mixture. Therefore, the surfactant material was easily removed from the extraction mixture. In the case of chloroform– methanol systems, the extraction mixture did not produce distinct layers, and the separation of ex-

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Table 1 Yield and surface-active properties of crude surfactants extracted using different solvent systems Solvent system

Surfactant Surface Interfacial CMC Žmgrl. concentration tension tension Žgrl. ŽmNrm. ŽmNrm.

MTBE MTBE a MTBE b CH 2 Cl 2 CH 2 Cl a2 CH 2 Cl b2 CHCl 3 rCH 3 OH Ž1:2. CHCl 3 rCH 3 OH Ž1:2. a CHCl 3 rCH 3 OH Ž1:1. b CHCl 3 rCH 3 OH Ž2:1. MTBErCHCl 3 Ž1:1. MTBErCHCl 3 Ž1:1. b

10.1 8.6 9.1 9.4 8.1 9.3 9.8

29.2 30.1 29.5 35.0 29.9 32.4 28.9

0.9 1.5 1.5 0.5 1.4 1.3 1.0

173 135 444 180 86 463 171

9.6

30.0

1.6

90

12.2

30.9

2.2

119

10.7

28.5

0.3

97

10.1

29.2

1.2

140

10.7

30.3

7.6

320

a Extraction was performed by ultrasonic treatment Ž23 kHz, 10 min.. b Extraction was performed from the whole culture broth.

tracted surfactant required 15-min centrifugation at 3000 rpm. 3.2. Surface and interfacial actiÕe properties All tested crude extracts isolated by different solvent systems were able to reduce surface and interfacial tensions of water. Minimal surface tension values were Žin mNrm.: chloroform–methanol Ž2:1. extract, 28.5; chloroform–methanol Ž1:2. extract, 28.9; MTBE and MTBE–chloroform Ž1:1. extracts, 29.2; and dichloromethane extract, 29.9. The interfacial tensions of all extracts were near or below 1 ŽTable 1.. Most solvent systems produced similar results for the CMC of extracted material Ž140–180 mgrl., but the efficiency of surface-active products recovered by chloroform–methanol Ž2:1. under the same conditions was greater ŽCMC, 97 mgrl.. Repeated extraction with any solvents was checked by surface tension measurement Ždata not shown. and did not result in detectable amounts of surface-active material. Thus, most of the surfactant

was recovered from bacterial culture after the first extraction, eliminating the need for repeated extraction. Although ultrasonic treatment did not increase the amount of extracted material ŽTable 1., this treatment resulted in better efficiency Žlower CMC values. of surfactants isolated, perhaps due to additional release of cell-bound surface-active glycolipids from rhodococcal cells destroyed by sonication. Experimental data showed, with all solvents used, that extraction from whole culture broth produced less efficient surfactants, with CMC values more than two times those produced by extraction from hydrophobic layers of culture broths. However, the results of surface and interfacial tension measurement of products obtained by both extraction procedures were comparable. This suggests that in industrial scale applications, where extraction from the whole fermentation broth is economically and technically preferable because it avoids the centrifugation stage, this procedure could be used for biosurfactant recovery. 3.3. Composition of crude surfactant extract Table 2 shows the chemical composition of crude extracts obtained using different solvent systems. Extracts contained between 24% and 63% lipids, 1% and 16% proteins, 0.01% and 0.4% extractable Žfree. hexoses and 36% and 60% residual substrate Ž nhexadecane., depending on solvents used. The lipid fraction consisted of non-polar and polar components. Amounts of non-polar lipids in crude extracts ranged from 16.5% to 50.3%. Polar lipid content was between 7.2% and 15.4%. Slightly higher concentrations of polar lipids were found in surfactant extracts obtained by sonication. Considerably more protein was detected in extracts made from whole culture broth ŽTable 2.. The TLC analysis of crude extract lipid fractions separated by column chromatography indicated the presence of neutral and polar lipids, identified on the basis of their Rf values. Non-polar components of crude surfactants isolated by different solvent systems were represented by saturated hydrocarbons Žmostly n-hexadecane., acylglycerols and free fatty acids Žfour major and several minor fatty acids were found in all extracts.. No detectable amount of unsaturated hydrocarbons and fatty alcohols were found in

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Table 2 Chemical composition of crude surfactant extracts Ž%wrw. Solvent system

Polar lipids

Non-polar lipids

Protein

Extractable hexoses

Residual n-hexadecane

MTBE MTBE a MTBE b CH 2 Cl 2 CH 2 Cl a2 CH 2 Cl b2 CHCl 3 rCH 3 OH Ž1:2. CHCl 3 rCH 3 OH Ž1:2. a CHCl 3 rCH 3 OH Ž1:2. b CHCl 3 rCH 3 OH Ž2:1. CHCl 3 rCH 3 OH Ž2:1. a CHCl 3 rCH 3 OH Ž2:1. b MTBErCHCl 3 Ž1:1. MTBErCHCl 3 Ž1:1. a MTBErCHCl 3 Ž1:1. b

8.5 9.8 7.9 8.2 10.0 7.2 8.9 9.5 8.4 15.4 10.4 9.3 8.3 9.7 7.3

48.0 44.3 35.3 48.8 44.0 28.1 49.8 50.3 31.1 47.7 47.4 38.8 32.3 25.9 16.5

1.8 4.3 7.5 2.0 3.5 13.6 2.7 2.4 11.4 0.5 ND 10.7 2.5 ND 15.8

0.01 0.03 0.02 0.05 0.10 0.02 0.09 0.35 0.07 0.12 ND 0.08 0.01 ND 0.02

41.5 41.6 49.1 39.2 42.3 50.2 38.2 36.7 46.2 36.0 40.2 40.6 55.2 59.3 60.1

a b

Extraction was performed by ultrasonic treatment Ž23 kHz, 10 min.. Extraction was performed from the whole culture broth.

the biosurfactant. Also, no qualitative differences in neutral lipid content of all extracts tested were detected. Polar lipids in crude surfactant were glyco- and phospholipids. The analysis of polar lipids extracted by MTBE showed three glycolipid fractions at Rf values of 0.18, 0.39 and 0.75 ŽFig. 1.. Dichloromethane extraction produced glycolipids with the same Rf values, but corresponding spots were much fainter than those produced by MTBE, suggesting that dichloromethane extracted smaller amount of glycolipids. Chloroform–methanol solvent systems were able to extract, apart from three glycolipid components characteristic for MTBE and dichloromethane extracts, another glycolipid with an Rf value of 0.10. The structural elucidation of purified glycolipid components of R. ruber surfactant is currently under study. Phospholipid content of surfactant extracts obtained using different solvents varied from a faint single spot detected in MTBE and dichloromethane extracts, and tentatively identified according to Rf value as cardiolipin, to three phospholipid spots, detected in chloroform–methanol extracts, with Rf values identical to cardiolipin, phosphatidylethanolamine and phosphatidylserine standards. From TLC results, it is apparent that phospholipids were present in R. ruber surfactant in small amounts.

Interestingly, the composition of phospholipids found in chloroform–methanol extracts was similar to that of R. ruber cell phospholipids during growth of bacteria in medium with n-hexadecane ŽKuyukina et al., 2000.. This suggests that chloroform–methanol was able to extract structural phospholipid components of the rhodococcal cell envelope. The results of GC analysis shown in Table 3 indicate that fatty acid profiles of crude extracts examined were generally similar, and differed only in the proportion of individual fatty acids. Thus, characteristic for all surfactant extracts was the presence of hexadecanoic acid as the predominant saturated fatty acid, ranging from 35% to 65% of the total. Another major saturated acid found in all extracts was tetradecanoic acid Ž5–11% of the total.. The MTBE and chloroform–methanol extracts contained a significant amount of saturated fatty acids Ž80% and 72–73% of the total, respectively., whereas the dichloromethane extract was distinguished by a lower concentration Ž44%. of these compounds. Characteristic rhodococcal cell lipids Žbranched-chain Ž10-methyl. fatty acids. were found only in chloroform–methanol extracts. Individual fatty acids were present in crude extracts in various proportions, e.g. the MTBE extract contained, apart from the two predominant acids mentioned above, considerable

154

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Fig. 1. TLC plate of glycolipids from R. ruber biosurfactant isolated by different solvent systems. Running solvent: chloroform– methanol–water Ž85:15:2.. Visualisation: sprayed with phenol–sulphuric acid and heated to 1058C. Lanes 1 and 2—MTBE extract; lanes 3 X and 4—dichloromethane extract; lanes 5 and 6—chloroform–methanol Ž1:2. extract; lane 7—trehalose 6,6 -dicorynomycolate.

amounts of oleic Ž13.4%. and eicosenoic Ž4.1%. acids. Fatty acid composition of dichloromethane extract differed from that of MTBE extract by increased concentrations of some unsaturated Žhexadecenoic, oleic and eicosenoic. acids, and correspondingly lower content of saturated Žtetradecanoic and hexadecanoic. acids. Moreover, alternatively to other extracts tested, dichloromethane extract contained 4.8% of long-chain unsaturated heneicosadienoic Ž21:2. acid. Two tested chloroform–methanol extracts were similar, and contained higher concentrations of some unsaturated Žpentadecenoic and tetradecenoic. and saturated Žstearic and heneicosanoic. acids in comparison to MTBE and dichlo-

romethane extracts. Also, the chloroform–methanol Ž2:1. extract was characterised by considerable amount of minor fatty acids. MTBE has been shown to be a good solvent for extraction of biosurfactant from Rhodococcus culture with high product recovery Ž10 grl.. Crude biosurfactant isolated using MTBE had efficiency ŽCMC, 130–170 mgrl. and functional characteristics Žsurface and interfacial tensions, 29 and 0.9 mNrm. comparable to those of extracts obtained by other solvent systems. Isolated surfactant complex contained 10% polar lipids, mostly glycolipids, which are characterised by maximal surface activity ŽKretschmer et al., 1982.. Ultrasonic treatment of the

M.S. Kuyukina et al.r Journal of Microbiological Methods 46 (2001) 149–156 Table 3 Fatty acid composition of the crude surfactants extracted using different solvent systems Žpercent of the total amount of fatty acids. Fatty acid

MTBE CH 2 Cl 2

CHCl 3 r CHCl 3 r CH 3 OH Ž1:2. CH 3 OH Ž2:1.

11:1 12:0 12:1 13:0 13:1 14:0 14:1 15:0 15:1 16:0 16:1 10Me16:0 17:0 17:1 18:0 18:1 18:2 10Me18:0 20:0 20:1 21:0 21:2 Saturated fatty acids Unsaturated fatty acids

– 2.0 – 0.1 – 11.0 – 0.5 0.2 65.0 0.9 – – 1.7 1.6 13.4 – – – 4.1 – – 80.2

– 1.2 – 0.4 1.1 5.0 – 1.4 1.7 34.5 9.6 – 1.3 0.5 – 27.4 0.3 – – 11.7 – 4.8 43.8

– 0.5 – 0.2 – 6.5 0.6 0.8 9.7 55.7 13.0 0.2 – 0.9 5.1 2.8 – 0.2 0.9 – 2.9 – 73.0

0.3 0.8 0.1 0.1 1.1 6.0 0.3 0.2 9.6 57.5 12.5 0.2 – 0.7 2.8 – 3.1 – 0.6 1.1 4.0 – 72.2

20.3

57.1

27.0

28.8

extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. There are several advantages of using MTBE as a solvent in the bioprocess industry, e.g. biosurfactant production. MTBE has lower toxicity and is more easily biodegraded than many solvents; therefore, it meets the criteria of the Hazardous Solvent Replacement Program aiming to substitute chlorinated solvents Že.g. chloroform, dichloromethane., traditionally used in industrial processing, with more environmentally friendly products ŽSteffan et al., 1997; Sherman et al., 1998.. MTBE is a relatively inexpensive and highly available compound. It is in the top 50 production chemicals in the USA due to its use as a fuel additive Žoctane enhancer, fuel oxygenate.. MTBE, due to its relatively low heat of vaporization Ž6.78 kcal moly1 . compared to other

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solvents Žvalues for methanol and chloroform are 8.95 and 7.97 kcal moly1 ., could be easily recovered and reused in downstream processing bioreactors ŽFiechter, 1992; Schmid et al., 1998; National Center for Manufacturing Sciences, 2000.. Additional characteristics of MTBE, such as low flammability and explosion safety, are important for the use of this solvent as extraction agent in industrial scale biosurfactant production. Biosurfactant extraction is one of the several pressing issues concerning their use as replacements for synthetic surfactants. One of the least well known aspects of microbial biosurfactant production is regulation of production, which is hindered by lack of knowledge of the molecular genetics. Whereas progress in this respect has been made with rhamnolipid and surfactin production ŽSullivan, 1998., very little is known about genetic regulation of biosurfactant production in the rhodococci. Given this lack of progress, studies which optimise extraction and purification of rhodococcal biosurfactants are means of improving recovery without genetic intervention.

Acknowledgements This work was supported by the Royal Society grant Ž638072 P750rLJH. and the grant from the Ministry for Science and Technology of the Russian Federation ŽNo. 991F..

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