Different experimental approaches to utilize rhamnolipids in diesel fuel biodegradation

Rhamnolipids are glycolipids containing rhamnose sugar moiety linked to β- hydroxydecanoic acid. Naturally, rhamnolipids and their derivatives are produced by Gram negative bacteria, especially the strains of Pseudomonas. Rhamnolipid production was in fact first time discovered in P. aeruginosa cultures. The most important property of rhamnolipids is that they are surface active compounds (surfactants). On the basis of their surfactant nature, rhamnolipids have many potential commercial applications such as in enhanced oil recovery; clean up of oil spills, as antimicrobial agent for use against phytopathogens and human pathogenic microorganisms and in cosmetic preparations. Other properties of rhamnolipids like their low molecular weight, biodegradability, their production using agroindustrial substrates and low toxicity have conferred them an ability to be used in environmental applications. Diesel is middle distillate of crude oil distillation and contains extremely recalcitrant long chain alkanes as principle constituents. Diesel spills and its extensive use as fuel purpose have been major reasons of environmental pollution. Its hazardous effects are evident in flora and fauna of marine, aquatic as well as terrestrial ecosystems. In the present article, three experimental approaches have been discussed to highlight the potential role of rhamnolipids in the degradation of diesel fuel.

Approach: I - Targeted degradation

Experimental protocol: First activity would be sampling of soil from diesel contaminated site containing indigenous microflora; followed by isolation, characterization and enrichment of selected microbial cultures. The cultures are checked for emulsification activity, IFT (inter facial tension), ST (surface tension), CMC (critical micellar concentration) and Cell surface hydrophobicity during their growth on mineral salt medium added with varying concentrations of diesel. On larger scale, fresh mineral salt medium with diesel is inoculated by such 'activated' culture from previous growth medium. Rhamnolipid (commercial grade) is added at the point of decreased cell surface hydrophobicity. The rate and extent of degradation can be analysed during definite time intervals by growing culture.

As stated before, diesel is principally composed of long chain alkanes but also contains mixture of olefins, polyaromatics and sulfur/nitrogen containing additives. These varying constituents of diesel can be degraded sequentially using the given protocol. Fresh addition of active culture would achieve degradation of maximum components of diesel; there are even chances of degradation of highly recalcitrant components like paraffins which otherwise would remain as leftovers. Rhamnolipid should be added at the point of decrease of cell surface hydrophobicity as at this point most of the biosurfactant produced by microbial cell is released into the medium. This reaction actually makes the biosurfactant useless to the producing cell hindering uptake of chemicals especially n-alkanes. Rhamnolipid addition at this certain point prevents release of biosurfactant in the medium and allows continuous utilization of constituents of diesel. Continuous degradation favors breakdown of those hydrocarbons which do not even support the microbial growth. Thus rhamnolipid not only enhances rate of degradation but also makes the process very efficient. In this case, it is advised to add rhamnolipid externally and biosurfactant produced by culture is not necessarily a rhamnolipid. In fact, biosurfactant produced by culture would facilitate its growth on diesel contaminated medium while as rhamnolipid addition increase hydrocarbon uptake and its subsequent utilization by culture. The growing culture utilizes hydrocarbons (which are diesel constituent/s) as carbon and energy source by degrading them to simpler compounds.

Approach: II - Rhizoremediation

Experimental protocol: Rhizoremediation is natural process of pollutant remediation via green plants. For experimental purpose, crop or forest tree species are suitable. I have tried with soybean, groundnut and eucalyptus saplings. It is better to rear plant under investigation on nursery beds. Plants saplings of desired quantity are then planted directly in diesel contaminated soil which has been filled in garden pots/containers or prepared beds. It is assumed that contaminated soil contains indigenous microflora with hydrocarbonoclastic potential. After establishment of newly planted saplings, commercial grade rhamnolipid at variable concentrations is added by drip irrigation at defined time intervals. Microbial count, viability and degradative activity are assessed weekly by suitable microbiological methods, simultaneously monitoring growth and health of saplings. Diesel fractions left during the course of rhizoremediation in plant sap and soil are determined by sensitive chromatography analyses.

In this case, rhamnolipid induces but the growing plant actually manages the process of biodegradation. It is also possible to avoid poor adaptation or reduced survival abilities of microbes followed by direct addition of purified rhamnolipid (as in 1st approach). Plants provide essential nutrients in the form of root exudates as well as microhabitat conducive to facilitate microbial proliferation, growth and also the subsequent degradation. Diesel would be a contaminant of choice as it contains measurable nonvolatile chemical constituents while other petroleum oils have volatile fractions which are difficult to estimate during such experimental set up.

Approach: III - Induction of nano-emulsification

Size reduction of diesel droplets facilitates their uptake by microbial cells and hence enhances rate of biodegradation. Both the Gram negative, Gram positive or mixed cultures can be used. Total biodegradation time is also reduced as determination of HLB (hydrophile-lipophile balance) value will denote the best time to add rhamnolipid at which maximum dispersion of diesel droplets will occur in aqueous culture medium.

Experimental protocol

• Growth of mixed culture in presence of diesel and rhamnolipid
• Determination of CMC, IFT, ST after definite periods of incubation
• Disappearance of diesel from medium indicates its emulsification
• Emulsion contains micelles
• Determination of emulsion stability and HLB
• Dispersing ability of emulsion will be determined from HLB value
• Re-addition of rhamnolipid and diesel to this emulsion solution
• Diesel molecules will be converted to nanoscale micelles
• Re-inoculation of culture
• Nanomicelles/nanoemulsion will be easily and rapidly utilized by culture

This experimental protocol suggests that growth of microbial culture in diesel and rhamnolipid containing medium is followed by diesel (oil) emulsification. This means that oil and aqueous phase of growth medium which could be detectable as two distinct layers are no more separate but instead diesel has mixed up in aqueous phase. In microscopical observation, such solution shows the presence of oil droplets surrounded by water drops. These oil-water assemblies are referred as micelles and such micellar solution is known as emulsion. In this protocol, further breakdown of emulsion into more oil-water micelles has been proposed so that oil drops would be disintegrated further to very smaller size leading to the formation of Nanomicelles. The resulting nanoemulsion containing nano sized diesel components would be rapidly assimilated by growing microbes in the culture. However, in this experiment concentration of rhamnolipid to be added would require prior determination otherwise it would be critically rate limiting in subsequent diesel remediation.

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