Rhamnolipid production by indigenous Pseudomonas aeruginosa J4 originating from petrochemical wastewater

Abstract

Pseudomonas aeruginosa J4, isolated from wastewater of a petrochemical factory located in southern Taiwan, was used to produce rhamnolipid from a variety of carbon substrates, including hydrophilic substrates, vegetable oils, and mineral oils. The P. aeruginosa J4 strain was able to assimilate the seven carbon substrates examined (namely, glucose, glycerol, olive oil, sunflower oil, grape seed oil, diesel, and kerosene), whereas it grew less efficiently in mineral oils (esp., kerosene). Rhamnolipid production from the J4 strain was affected by temperature and agitation rate, as 30 °C and 200 rpm agitation were favorable for rhamnolipid production. The rhamnolipid concentration (C_RL) and production rate ($v_{\text{RL}}$) was also influenced by the carbon sources used to grow the J4 strain. Similar $v_{\text{RL}}$ (10–12 mg/h/L) and C_RL (1400–2100 mg/L) were obtained from using glycerol, glucose, grape seed oil, and sunflower oil as the sole carbon substrate, while using olive oil delivered the best rhamnolipid production. Maximum C_RL (3600 mg/L) and $v_{\text{RL}}$ (26 mg/h/L) were attained at 10% olive oil. P. aeruginosa J4 also utilized diesel and kerosene for rhamnolipid production but with much lower C_RL and $v_{\text{RL}}$ values Rhamnolipid was purified (nearly 90% pure) from the culture broth. Mass spectrometry and NMR analysis indicate that the purified product contained two types of commonly found rhamnolipids: l-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL1) and l-rhamnosyl l-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL2). The rhamnolipid product can reduce the surface tension of water to 31 mN/m with a critical micelle concentration of nearly 50 mg/L. The biosurfactant also achieved a maximum emulsion index of 70 and 78%, for diesel and kerosene, respectively, at a low concentration of about 300 mg/L.

Introduction

There are over 3000 gas stations in Taiwan, containing more than 10,000 oil storage tanks underneath. About half of the gas stations have been operated for over 10 years, and a recent investigation by Taiwan's EPA showed that leaking problems began to occur at a number of aged oil storage tanks, causing oil contamination in nearby soils and groundwater. It is, therefore, extremely critical to clean up and soundly control the oil-contaminated sites before the situation further deteriorates. The efficiency of biodegradation of oil compounds has been limited by poor mass transfer due to high hydrophobicity of the oil compounds, leading to low aqueous solubility [1], [2], [3], [4], [5], [6]. One of the approaches to enhance biodegradation of oils was to use surfactants, which played a crucial role in facilitating bioremediation of crude oil during the Exxon Valdez spill incident in Alaska [7]. Surfactants could increase solubility of oils in water to enhance the bioavailability of the hydrophobic substrates, leading to higher oil degradation rates. However, synthetic surfactants are unsuitable for bioremediation applications since they may cause toxic effects to the environment or result in secondary pollution. Thus, biosurfactants, natural products of a variety of microorganisms [8], [9], appear preferable for the environmental applications. In addition to bioremediation, biosurfactants also find their applications in medical, cosmetic, and food industries, etc. [5]. Biosurfactants have recently received much more attention due to its potential to become an environment-friendly alternative to conventional chemically synthetic surfactants. However, the factors restricting commercial application of biosurfactants have been the low yield and high production cost. Therefore, there is an urgent need to develop an efficient and cost-effective bioprocess for the production of biosurfactants.

Rhamnolipid, a glycolipid-type biosurfactant primarily produced by Pseudomonas aeruginosa [10], [11], is among the most effective biosurfactants and has been applied in various industries and bioremediation [5], [8], [12], [13]. Rhamnolipid surfactants were found to be able to release three times as much oil as water alone from the oil-contaminated beaches in Alaska [7]. Previous work showed that P. aeruginosa is able to produce six types of rhamnolipids, which possess similar chemical structure and surface activity and have an average molecular weight of 577 [14]. Rhamnolipid can reduce surface tension of water from 72 to 30 mN/m [15] with a critical micelle concentration of 27–54 mg/L [16]. Although rhamnolipid is not the strongest biosurfactant available, it is well suited for applications in bioremediation of oil pollutants due to having high emulsification activity and minor antibiotic effects [17].

In this study, an indigenous bacterial strain capable of producing biosurfactant was isolated from wasterwater of a local petrochemical factory. The strain, identified as P. aeruginosa J4, was found to be able to produce rhamnolipid and was thus evaluated for its potential for commercial-scale production of the biosurfactant. The isolated strain was cultivated with different media and conditions to determine the optimal culture strategy for rhamnolipid production. Different carbon sources, especially oils (e.g., diesel, kerosene, olive oil, sunflower oil, grape seed oil, etc.), were adopted to explore their effects on the yield of rhamnolipid. The rhamnolipid present in the supernatant of P. aeruginosa J4 culture was purified and identified by NMR and mass spectrometry. Surface activity of the rhamnolipid produced from P. aeruginosa J4 was analyzed in terms of surface tension reduction and emulsification activity for oil substrates. The objective of this work was to assess the potential and commercialized feasibility of using the indigenous bacterium for the production of rhamnolipid.