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BIOSURFACTANT ‘SIEVE’: AN ECOFRIENDLY OPTION FOR ENHANCING BIOREMEDIATION IN METALLURICAL FACTORIES

PROJECT SUMMARY
The rhl genes that regulates the synthesis of rhamnolipids in Burkhoderia were engineered into Pseudomonas putida that has a strong biofilm formation property. SPL was used to regulate its expression and a reporter gene, GFP was a tool for quantifying the level of expression in the heterologous host. This would be tested in a flow chamber by observing the remediating effects of this on effluent containing heavy metals using Heavy metal analysis and UHPLC.

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PROJECT DESCRIPTION

The main goal of this research is to combine biosurfactant production and biofilm formation of microorganisms to enhance effective removal of heavy metal from effluent before it gets to soil or water.
Objective 1: To source for high biosurfactant-producing microorganisms from ATCC and do confirmatory tests for their biosurfactant activities using emulsification index

Objective 2: To use Synthetic promoter library (SPL) in engineered genes cloned from the Rhamnolipids producer into Pseudomonas putida for enhanced heterologous production of biosurfactant.

Objective 3: To combine biofilm structure and biosurfactant production in a flow chamber (bioreactor) as a ‘Bio-sieve’ for metallurgical factories where waste effluents are passed before final disposal.

BACKGROUND

In recent years, the need for bioremediation options that are less synthetic, cost effective and ecofriendly has been imperative. This has made environmental scientists go into various research to address the situation. One of the leading causes of pollution globally is the release of xenobiotic materials like plastics, petroleum products, chemicals, hydrocarbons and heavy metals. Most of these are because of anthropogenic activities either intentionally or by mistakes. Overtime, they constitute waste in the environment which degrade slowly or remain undegraded. The roles of microorganisms in remediating the polluted environment has thrown bioremediation to the limelight especially in clean-ups of prominent oil spill that occurred decades ago. Microorganisms degrade using various methods like breaking down of carbon source, conversion of metals to non toxic compounds. Moreover, microbial cell walls consist of polysaccharides, lipids and proteins which have binding abilities with metal (Ahalya et al., 2003). Biosurfactant is another protein product produced by microorganisms which helps the biodegradation of compounds easier. They are surface active substances that are amphiphilic. This is because they have two parts, the hydrophilic or polar side and the hydrophobic or non-polar side. It has been studied that they have higher potential in breaking down heavy metals at sites of pollution than synthetic means (Sriram et al., 2011). Properties such as metal ion affinity, ability to withstand varying pH and temperature helps them to work. (Makkar et al., 2011). They are cost effective ways to treat liquid waste but do not function as effective if they are on soils due to their size that could be as low as 1500 Da ((Banat et al., 2010; Miller, 1995). In comparison, they are less toxic and easily degraded than surfactants of chemical composition (Juwarkar et al., 2008). According to Miller (1995) their mechanism of action includes forming complexes with free, nonionic forms of metals in solution and accumulation at the solid-surface interface through ion exchange, precipitation dissolution, counter-ion association, and electrostatic interaction (Rufino et al., 2012). The high pathogenicity of Pseudomonas aeruginosa which are well known producers of Rhamnolipids is the main reason why attention is being shifted away from them for industrial use. In recent times, the non-pathogenic Burkholderia thailandensis has been shown to share the ortholog genes involved in Rhamnoloipids biosynthesis with Pseudomonas aeruginosa. Genetic engineering has made a way to combine these traits with other useful traits from other organisms like Pseudomonas putida useful in bioremediation (Toribio et al., 2010).

JUSTIFICATION AND RATIONALE

Synthetic Promoter Library (SPL) is an engineered promoter sequences that could be used to regulate the expression of Rhamnolipid biosynthesis genes. Whole genome sequencing has shown that Burkhoderia thailandensis has similar orthologs of genes with Pseudomonas putida with the former having a better arrangement of the rhlA, rhlB rhlC operons as a gene cluster whereas Pseudomonas putida rhlA and B are situated in the same operon, while the rhl C is in a different operon. This makes Burkhoderia thailandensis a preferred choice in the genetic engineering of these cells for a higher production of rhamnolipids using Synthetic promoter library (Dubeau et al., 2009). Also, it has been shown that production of rhamnolipids increased the formation of biofilm and this could be applied industrially to remove metal ions from metallurgical effluent in a treatment plant (Wigneswaran et al., 2016). The ripple effect of finding more efficient ways to reduce soil or water heavy metal pollution from industries include the alleviation of global warming, assuaging rate of soil or water pollution from effluents improving the environment at large.

METHODS
Objective 1: To source for high biosurfactant-producing microorganisms from ATCC and do confirmatory tests for their biosurfactant activities using emulsification index

Pure cultures of Burkhoderia thailandensis, P. putida KT2440 and Escherichia coli cells will be purchased in ATCC and maintained in Lysogeny broth medium. To confirm the efficiency of the biosurfactant to be produced, emulsification tests will be done. Colonies of the pure Burkhoderia thailandensis culture would be suspended in a 2 ml test tube that has mineral salt medium and incubated for 48 hours. After incubation, an equal volume of hydrocarbon would be added to the tube and vortexed at maximum speed for 1 minutes, this is left to stand for 24 hours. The layers formed between the cell culture and hydrocarbon column would be measured and emulsification index is calculated as a percentage (Bodour et al., 2004). The emulsion layer height is measured and divided by the entire height of the mixture and multiplied by 100. Emulsion stability of E24 >50 would be expected for a good strain (Sarrubo et al., 2007).

Objective 2: To use Synthetic promoter library (SPL) to regulate expression of engineered genes cloned from the Rhamnolipids producers into Pseudomonas putida for enhanced heterologous production of biosurfactant. The SPL will be made by combining the promoters that are from genome sequence from Burkhoderia and P. putida (Solem et al., 2002)

The DNA from Burkhoderia will be extracted and PCR amplification of the rhlABC operon will be done using Taq Polymerase and specific primers. The vector and PCR product will be digested and ligated. Furthermore, cloning of the vector will be done and the transformed cells of P. putida will be obtained.
SPL will be designed according to Wigneseram (2007) by removing the 16SrRNA promoter sequences from B. thailandensis and P. putida to make an engineered promoter. A strain of E. coli will be used for molecular procedures for DNA. Restriction enzymes will be used to cut at specific sites of the B. thailandensis and P. putida of (accession number). PCR will be done to amplify the promoter fragment containing the start codon gene from each strain. The PCR product and vector will be digested with Restriction enzymes at specific sites and ligation with T4 ligase will be done. Consequently, transformation of E. coli cells by cloning the vector into them will follow. The cells will be inoculated into LB agar medium with a selectable marker and grown overnight. The cells will be harvested, and plasmid extracted. The plasmids will be cloned into transformed P. putida (Rud et al., 2006)
The whole plasmid will be introduced into P. putida by electroporation method. The construct with the SPL will be placed in front of the rhlABC operon and a gfp reporter would be placed downstream in the vector to measure the expression of the heterologous genes in a flow cytometer.
The transformed P. putida that has the SPL will be cultured in LB medium with Tetracycline in a microtiter plate overnight. The cultures will be retrieved for dilution and incubated for additional time. This will be passed through a flow cytometer which will be used to measure the gfp expression (Wigneseram et al., 2016).

Objective 3: To combine biofilm structure and biosurfactant production in a bioreactor as a ‘Bio-sieve’ for metallurgical factories where waste effluents are passed before final disposal.

Application of biosurfactant production and biofilm formation would be tested in a flow chamber. To create the ‘biosieve’ effect, biofilm will be made in a chamber that contains a FAB medium that contains 10mM sodium citrate as a suitable carbon source. Transformed Pseudomonas putida cells are inoculated and the effluent which serves as substratum would be passed through the flow cell and collected at the outlet for analysis (Sternberg et al., 1999). Furthermore, tests such as COD, BOD, and heavy metal would be carried out to determine the extent of transformation. 50 ml of the sample will be centrifuged at 10000rpm for 10 minutes and the supernatant will be analyzed for reduction in the metal (Singh and Vaishya, 2017). Rhamnolipids would be quantified using UHPLC.

REFERENCES

Ahalya, N., Ramachandra, T. and Kanamadi, R. (2003). Biosorption of heavy metals. Research Journal of Chemistry and Environment. 7: 71-79.
A?çi, Y., Nurba?, M. and Açikel, Y.S. 2007. Sorption of Cd (II) onto kaolin as a soil component and desorption of Cd(II) from kaolin using rhamnolipid biosurfactant. Journal of Hazardous Materials 139: 50–56.
Banat, I.M., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M.G., Fracchia, L., Smyth, T.J. and R. Marchant. 2010. Microbial biosurfactants production, applications and future potential. Applied Microbiology and Biotechnology 87: 427–444.
Dubeau, D., Déziel, E., Woods, D.E. and Lépine, F., 2009. Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids. BMC microbiology, 9(1), p.263.
Juwarkar, A.A., Dubey, K.V., Nair, A., and Singh, S.K. 2008. Bioremediation of multi-metal contaminated soil using biosurfactant—A novel approach. Indian Journal of Microbiology 48: 142–146.
Makkar, R.S., Cameotra, S.S., and Banat, I.M. (2011). Advances in utilization of renewable substrates for biosurfactant production. Applied Microbiology and Biotechnology Express 1: 5. doi: 10.1186/2191-0855-1-5
Miller, R.M. (1995). Biosurfactant-facilitated remediation of metal-contaminated soils. Environmental Health Perspectives 103: 59–62.
Rud, I., Jensen, P.R., Naterstad, K. and Axelsson, L., 2006. A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology, 152(4), pp.1011-1019.
Rufino, R.D., Luna, J.M., Campos-Takaki, G.M., Ferreira, S.R.M., and Sarubbo, L.A. 2012. Application of the biosurfactant produced by Candida lipolytica in the remediation of heavy metals. Chemical Engineering Transactions 27: 61–66.
Sandrin, T.R. and Maier, R.M. (2003). Impact of metals on the biodegradation of organic pollutants. Environmental Health Perspectives 111: 1093-1101.
Sarubbo, L.A., Farias, C.B. and Campos-Takaki, G.M. (2007). Co-utilization of canola oil and glucose on the production of a surfactant by Candida lipolytica. Current Microbiology, 54(1), pp.68-73.
Singh, K.K. and Vaishya, R.C., 2017. Bioremediation of Heavy Metal Using Consortia Developed from Municipal Wastewater Isolates. In Souvenir of International Seminar on Sources of Planet Energy.
Solem, C. and Jensen, P.R., 2002. Modulation of gene expression made easy. Applied and environmental microbiology, 68(5), pp.2397-2403.
Sriram, M.I., Gayathiri, S., Gnanaselvi, U., Jenifer, P.S., Mohan Raj, S., and Gurunathan, S. 2011. Novel lipopeptide biosurfactant produced by hydrocarbon degrading and heavy metal tolerant bacterium Escherichia fergusonii KLU01 as a potential tool for bioremediation. Bioresource Technology 102: 9291–9295.
Sternberg, C., Christensen, B.B., Johansen, T., Nielsen, A.T., Andersen, J.B., Givskov, M. and Molin, S., 1999. Distribution of bacterial growth activity in flow-chamber biofilms. Applied and Environmental Microbiology, 65(9), pp.4108-4117.
Toribio, J., Escalante, A.E. and Soberón?Chávez, G., 2010. Rhamnolipids: production in bacteria other than Pseudomonas aeruginosa. European journal of lipid science and technology, 112(10), pp.1082-1087.
Wigneswaran, V., Nielsen, K.F., Sternberg, C., Jensen, P.R., Folkesson, A. and Jelsbak, L., 2016. Biofilm as a production platform for heterologous production of rhamnolipids by the non-pathogenic strain Pseudomonas putida KT2440. Microbial cell factories, 15(1), p.181.

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