The macro and micro nutrients supplemented to the growth medium are helpful in the metabolism and effective growth of microbes. The media that were used for microbial growth previously consisted of meat extract, urine, soya flour, potato pieces, and sprouted barley. Recently, commercially available culture media consisted of yeast extract, casein and beef extract. These media are prepared by laborious methods such as maintenance, sterilization and supplementation to be useful for the effective growth of microbes.
The coconut water or liquid endosperm in the cavity of the coconut fruit comprises of nutrients like 95.5 percent of water, 0.1 percent of fat, 4 percent of sugars, 0.02 percent of calcium, 0.5 percent of iron and 0.01 percent of phosphorus, amino-acids, vitamin B complex, mineral salts, cytokines and vitamin C. Coconut water is considered to be a rich nutrient in the medium for the growth of microbes, fungus and in plant tissue culture. Studies have reported that coconut water was added to supplement carbon and nitrogen to MRS media to produce Exo-polysaccharide by Lactobacillus confuses. Coconut water was also used in the growth medium previously in the release of delta-endotoxin from Bacillus thuringiensis according to another research study.
Recombinant protein production has become very important recently due to their applications in medicine. Production of recombinant proteins has been a big project for most of the industries. So, there is a necessity for the medium to be effective without toxins for supporting expression. Tender coconut water is a safe medium to be used for therapeutic purpose as it is devoid of endotoxins and is derived from plants.
Strains of E.coli
The E.coli strains used for protein expression investigations were BL21 (DE3) and BL21 (DE3) pLysS. Those used for growth studies were P.pastoris GS115 and E.coli C41 (DE3).
The coconut water was extracted from the tender coconut fruit after removing the mesocarp and endocarp. The coconut water is centrifuged for twenty minutes at 4000 rpm and then filtered, followed by sterilization using 0.22 um filter. The TCW medium is prepared by mixing 25 ml of filtered and sterilized TCW with 25 ml of 4 percent agar. This mixture is microwaved for about 60 seconds.
The components like ammonium, magnesium and sodium sulphates, potassium phosphate, glycerol, lactose, glucose and amino acid are supplemented to the TCW medium. Estimation of metabolites and amino acid was carried out by Liquid chromatography-Mass spectrometry/Selected Reaction monitoring method. The total nitrogen was evaluated by the Kjeldahl conventional method while the total carbohydrate was evaluated by a phenol sulfuric acid method.
E.coli C41 (DE3) culture was streaked on the LB agar plates and the culture of P.pastoris GS115 was streaked on yeast-peptone-dextrose agar plate and incubated. The culture is then inoculated in higher quantity medium and incubated. The growth of bacteria is measured by determining the optical density of the extract at 600 nm. The protein expression of the bacterial culture was studied by transforming BL21 strain with MBP and MBP-TEV fusion. The fragment mEos2 was transformed into BL21 (DE3) pLysS. Overnight incubated E.coli cultures are induced with the fused constructs and grown in the medium. Their optical density was measured and are harvested to check their protein expression levels by SDS-PAGE.
Results of the study
E.coli grown in TCW showed the maximum optical density of 1.55 at 600 nm after 12 hours of incubation. The growth in TCW was compared with that of in M9 medium where the optical density was 1.70 after 12 hours of incubation. Though the pH required for the growth of E.coli is 6.5 to 7, the pH of TCW (4.7+/-0.2) was not disturbing the growth of bacteria. P.pastoris was also grown in TCW which showed the OD at 600 nm as 8.7 after 64 hours of incubation.
As the chemical composition of coconut varies from one fruit to another, the biomass of coconut that influences the growth of bacteria was studied by investigating the ODs of the samples at 600 nm after 12 hours incubation. The ODs were in between 0.15 and 2.5. The chemical profile of the coconut water is different based on the location, maturity and variety of coconut fruit. Some studies have reported that the ratio of fructose, glucose and sucrose was found to differ based on the maturity of the coconut fruit. The composition of potassium, chloride, sulphur and iron vary in TCW medium based on the extent of its maturation.
Addition of glucose, glycerol and lactose to TCW medium did not show any improvement in the growth of E.coli while little growth was observed in the case of P.pastoris. This shows that carbon source is not a limiting component in TCW medium for E.coli.
E.coli biomass increased by 4 times when TCW was supplemented with Ammonium sulphate, by 3 times when supplemented with amino acid, and by 2.7 times when supplemented with magnesium sulphate. Growth improvement was negligible even in the case of sodium sulphate supplementation. E.coli growth enhanced by 6 times when TCW was supplemented with 25 mM ammonium sulphate while the increase was only by 4.5 times when 37mM and 50mM of the same chemical was added. So, nitrogen was found to be a limiting factor for bacterial growth.
The biomass of P.pastoris increased by 1.5 times when TCW was supplemented with ammonium sulphate, by 1.4 times when amino acid cocktail was added. The biomass decreased due to potassium phosphate supplementation.
Therefore, 25 mM ammonium sulphate was concluded to be beneficial to supplement TCW for E.coli growth in large scale applications.
The E.coli grown with transformed constructs (MBP, MBP-TEV protease and mEoS2) showed higher expression of recombinant protein in TCW supplemented with 25mM ammonium sulphate. The carbon sources in TCW are found to be hindering the growth of bacteria and YPD media were found to be beneficial to the bacterial growth.
Narendrakumar Sekar, Soumya Kariyadan Veetil and Muniasamy Neerathilingam. Tender coconut water an economical growth medium or the production of recombinant proteins in Escherichia coli. BMC Biotechnology 2013, 13:70.
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