Applied Biochemistry and Microbiology, Vol. 37, No. 1, 2001, pp. 7175.

Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 37, No. 1, 2001, pp. 8085. Original Russian Text Copyright 2001 by Turkovskaya, Dmitrieva, Muratova.

A Biosurfactant-Producing Pseudomonas aeruginosa Strain

O. V. Turkovskaya, T. V. Dmitrieva, and A. Yu. Muratova
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entuziastov 13, Saratov, 410015 Russia
Received October 27, 1999

AbstractA Pseudomonas aeruginosa strain producing an extracellular surfactant (biosurfactant) was isolated. The growth of this strain, referred to as 50.3, on a mineral glycerol-containing medium produces an emulsifying activity (60%) and decreases the surface tension of the culture liquid by a factor of 2.8 (to 25 mN/m). The optimum conditions for its growth and production of biosurfactants are intense aeration, pH 7.08.0, and the presence of Mg2+. The optimum biosurfactant properties were achieved when glucose was used as the only source of carbon and energy and NH4Cl was used as a source of nitrogen. The biosurfactant was isolated from the culture liquid by extraction and precipitation.

Synthetic surfactants appeared relatively recently, and their unique properties revolutionized both housekeeping and industry. The replacement of synthetic surfactants by biological ones may prove to be no less revolutionary. Biological surfactants are less toxic, less allergenic, biodegradable, and therefore less detrimental to the environment. They can be produced from various substrates, including industrial waste [1, 2]. They are as diverse in chemical structure and properties as the producer microorganisms [35]. Synthetic surfactants are environmentally hazardous. Another reason for the search for biological surfactants is the depletion of oil resources required for the production of synthetic surfactants. This makes microbial surfactants even more promising. The oil industry (oil production, processing, and transportation, as well as recovery of oil-polluted objects) is a rapidly developing eld of application for biosurfactants [68]. Uses of biosurfactants in other industries (food production, pharmacology, microbiology, cosmetics, research, etc.) is less investigated. However, biosurfactants and bioemulsiers can be cost-effective exactly in these elds owing to the increasing demand for natural products [9]. This is a preliminary study, and its goal is to select microorganisms producing biosurfactants and emulsiers with desired properties. Information on the producer strains and their products is of great importance. The objectives of the present study were isolation of a biosurfactant-producing microbial strain, investigation of factors affecting the biosynthesis of the biosurfactant, and general characterization of the product. MATERIALS AND METHODS Cultures producing biosurfactants were isolated from petroleum-polluted soils and microbial associations formed in pilot devices modeling the purication

of waste waters containing mineral oils. Enrichment cultures were prepared from the samples on M9 mineral medium [10] supplemented with kerosene (0.5 g/l). Glycerol and kerosene (1 : 1) were added in the rst month of cultivation to accelerate the microbial growth. Isolation of microorganisms from the enrichment cultures and associations obtained from purication devices was performed by sowing dissolved samples on solid media. The surfactant production capacity was determined on medium 5 containing K2HPO4 (7.0 g/l), KH2PO4 (3.0 g/l), MgSO4 7H2O (0.1 g/l), (NH4)2SO4 (1.0 g/l), FeSO4 7H2O (0.0025 g/l), ZnSO4 7H2O (0.0025 g/l), MnSO4 (0.0025 g/l), and CaCl2 (1 ml of 1% aqueous solution) [11]. The following carbon and energy sources were used: glycerol (1.2%), glucose, sucrose, sorghum syrup, vegetable oil (1%), and ethanol (2%). The media were adjusted to pH 7.2. Cultivation was performed at 2830C in 250-ml Erlenmeyer asks (liquid volume 50 ml) by shaking at 160 rpm for ve days on a UVMT-12-250 shaker. The biomass growth was monitored by measuring A440 on a KFK-2 photocolorimeter (USSR). Surface tension of the culture liquids () was measured stalagmometrically [12] after removing cells by centrifugation at 8000g for 20 min. The method is based on measuring the weight of a drop of the supernatant that slowly (within 2060 s) accumulates on the end of a capillary tube. At the break-away point, the weight of the drop is equal to the surface tension. The emulsifying activity was determined according to Cooper [13]. After cell removal by 20 min of centrifugation at 8000g, the culture liquid was shaken with kerosene (3 : 2 v/v) for 20 min and left for 48 h at room temperature. The emulsifying activity (E48) was determined as the emulsion/total liquid volume ratio multiplied by 100. To determine the thermal stability of the biosurfactant, asks with cell-free culture liquid were incubated

0003-6838/01/3701-0071$25.00 2001 MAIK Nauka /Interperiodica