Investigation Of Eco-Friendly Nontoxic Novel Biosurfactant From Marine Bacteria Alcanivorax Borkumensis A Prashanth Rajan, 2nd Year, B.Tech Biotechnology Department of Biotechnology, Karunya Institute of Technology and Sciences, Coimbatore, TN. Mobile :8056606621, Email:prashanth07052001@gmail.com
ABSTRACT Biosurfactants (BS) are exceedingly superior and exceptionally thermostable then chemical surfactants. This may be due to protracted extreme environment. BS has invaded all anthropogenic commercial products to name a few, pharmaceutical, agricultural, petroleum, confectionary, bioremediation and cosmetic and nutraceutical products. The exploration of Indian Marine bacteria for their biosurfactants production remain virgin. This lacuna lead to the present study. Bacteria from water and sediment samples from Pulicat Lake which is known as paradise for marine microbiologist, were enriched in Marine broth solution (BD Difco 2216). After 7 days of controlled bacterial growth in presence of crude oil, cell free supernatant were extracted and subjected to BS assays. The result of emulsification assay, oil spreading test and drop collapse test confirmed the presence of BS producing bacteria. The results inveterate to species Alcanivorax sp. or Alcanivorax borkumensis
Global market survey of Bio surfactant companies
1.Introduction Versatility of Biosurfactant (BS) has made it inevitable in commercial laundry, food processing industry, petroleum industry, fuel formulation, cosmetic industry, demulsifying agents and anti-corrosive agents. BS has also anchored itself as active medical compound having antimicrobial, anticancer, antiadhesive agent, immunological adjuvants, and antiviral effects. It has acclaimed importance in agro-base industry in agrochemical formulations, biopesticide, biocides etc.
Globally billions of dollars are poured in establishment of biosurfactant companies. The chief companies producing BS are BASF Cognis (Germany), Boruta-Zachem (Poland), Surfactin, and Ecover (Belgium), EcoChem Organics Company (Canada ), Croda International PLC (Snaith, England),
1.1 Biosurfactant Vs Chemical surfactant 1. 2. 3.
Sun Products Corporation (Marine bacteria were successfully seen growing in interface of USA), AkzoNobel (Netherlands), Mitsubishi Chemical Corporation (Japan), Jeneil Biotech ( Wisconsin, USA), Sigma-Aldrich Co (Germany)., AGAE Technologies (Corvallis, USA ), oil/water. Connecticut
Biosurfactant proved to be more biocompatible and biodegradable than chemically-derived surfactants (Marchant and Banat 2012) BS are comparatively, extremely thermostable and has wide spectrum pH activity (Constantina et al.,2020 ) BS Rhamnolipids coated on face masks prevents and reduces the spread of respiratory viruses, COVID -19 SARS-CoV2 and enveloped viruses (Ling et al., 2020)
Evonik Industries AG (Germany), Soliance (France), Logos Technologies (Virginia USA),
1.2 Marine Bacteria
TensioGreen Technology Corp (USA),
The marine environment is a treasure chest of bio-prospective compound yielding microbes. Metagenomics unwrap that, only 1% percent of marine microbes are culturable in Laboratory.
Biotensidon (Baden-Württemberg Germany), and Saraya Co. (Japan). Recently China has also established biosurfactant company -Urumqi Unite.
Numerous bacteria are reported to be active at oil/water interphase such as Pseudoalteromonas,Oleiphilus,Cycloclasticus,Halomonas,Marinomonas,Alcanivorax , Planococcus,Marinob acter,Neptunomonas,Oleispira,Marinobacter,Neptunomonas,Vibrio,Staphylococcus,Geobacillus, Sphingomona and Micrococcus Present extensive literature search on BS yielding marine bacteria revealed a scientific void in this arena.
RESULTS
India needs to develop neoteric biosurfatant companies to compete with international players before it is too late.
Enrichment and isolation
S. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Bacteria
Biosurfactants
Acinetobacter Serratia marcescens Rhodococcus erythropolis Rhodococcus glutinis Bacillus sp Mycobacterium spp. Nocardia erythopolis Pseudomonas spp. Pseudomonas Pseudomonas fluorescens Agrobacterium spp. Pseudomonas aeruginosa Rhodococcus sp P. flurescens Serratia rubidea Lactobacillus fermentum P. aeruginosa Kocuria marina BS-15
References
Emulsan and alas an Serrawettin Trehalose lipids
Fakruddin et al. (2012) Lia et al. (2009) Muthusamy et al. (2008)
Polyol lipids Surfactin,rhamnolipid, lichenysin Trehalose lipids Trehalose lipids Ornithine lipids Rhamnolipid, glycolipids Viscosin
Amaral et al. (2006) Fakruddin et al. (2012)
Ornithine lipids Rhamnolipids
Desai and Banat. (1997) Jadhav et al. (2011)
Viscosin &trehaloselipids
Carbohydrate-lipid Rhamnolipids Diglycosyl diglycerides
Fakruddin et al. (2012) Nerurkar et al. (2009) Jadhav et al (2011) Mulligan et al.(2001)
Protein PA lipopetide
Hisatsuka et al. (1971) Sarafin, et al. (2014)
Muthusamy et al. (2008 Muthusamy et al. (2008 Desai and Banat (1997) Fakruddin et al. (2012) Banat et al. (2010)
Current investigation was carried out in order to bridge the gap, in exploitations of marine bacteria in terms of its efficiency to produce industrially potential biosurfactant from Indian marine water.
Marine bacteria were successfully seen growing in interface of oil/water in marine broth solution (BD Difco 2216) was mixed into deionized water at a concentration of 37.4 g/L, along with 2 g/L supplemented KNO3. The interface of the octane and aqueous phase of the pour plate promotes the growth of oil-degrading, octane consuming microbes due to the lack of nitrogen sources in the media.
Bacterial isolation Consortia of marine bacteria were seen growing at the interface and consumption of octane visually confirmed the presence of marine bacteria. The isolate was named as KITPR-1.
Bacterial Morphology studies Bacteria were found to be rod-shaped, gram-negative bacillus. Bacteria were non flagellated.
Biosurfactant producing isolates Marine bacteria that were capable of consuming crude oil and degrading octane were successfully cultured for 7 days at 30o C in an orbital shaker.
Preparation of cell-free culture media The extracted supernatant was transparent and was stored at 4o C. The biosurfactant assays were performed at the room temperature
Conclusion Emulsification assay The emulsification assay showed positive results as it showed absorbance at 400 nm.
Future work Oil spreading test The negative control with double distilled water did not give any clearing zone. Polyethylene glycol tert-octyl phenyl ether which was a positive control showed clearing zone similar to the cell-free supernatant which confirms that isolate was capable of producing biosurfactant
Objectives 1. 2.
To isolate Marine Bacteira using selective media To elucidate bio surfactant producing capacity of isolate.
2.1 Sample site Pulicat Lagoon is the second largest brackish water lagoon in India, measuring 759 square kilometers. Pulicat lake is selected for the isolation of marine bacteria producing BS as it is one of the richest hotspots for marine microbial isolation. This lake brims with winged migratory birds, enriching the lake with marine bacteria of the whole world in a small marine habitat. The soil and water samples from the lake were collected in sterile brown bottles and was transported at 4oC (APHA et al., 2012protocol).
Figure 1 shows the site map and migratory birds Flamingos at Pulicat Lake.
The molecular studies, 16Sr RNA sequences may reveal the phylogenetic taxonomic classification of KITP-1. Submission of the sequence in NCBI. Biosurfactant can be purified and subjected to FTIR, GC-Mass Spectroscopy for chemical structure elucidation. Industrially important BS can be used in shampoos, tooth paste and other commercial products. REFERENCES
Drop collapse test The cell-free supernatant showed flattening inside the microwell plate when absorbed through a magnifying glass. Double distilled water was added in the positive control well to visualize the rounding of water droplets
DISCUSSION 2. Material and Methods
Marine bacteria capable of producing Biosurfactant was successfully isolated and BS assays confirmed the production of biosurfactant by KITP-1. The data from previous researchers confirmed the bacteria to be Alcanivorax borkumensis.
Marine bacteria that are capable of oil-degrading belong to several families like Shewanellaceae, Rhodospirillaceae, Rhodobacteraceae, Oceanospirillaceae, Halomonadaceae, and Pseudomonadaceae. KITPR-1 may not be Shewanellaceae as these are gammaproteobacterium that is found in a cold marine environment with high pressure. KITPR-1 was aerobic bacteria so it does not belong to Rhodospirillaceae (alphaproteobacteria) as they grow in an anaerobic marine environment. KITPR-1 was cultured easily in the laboratory whereas Rhodobacteraceae belong to alphaproteobacterial which comprises of unculturable bacteria. Oceanospirillaceae belongs to gamma proteobacteria similar to Halomonadaceae. Pseudomonadaceae has polar flagella whereas KITPR-1 was a non-flagellated bacterium. According to Marion et al., (2019) Shewanellaceae, Rhodospirillaceae, Rhodobacteraceae, Oceanospirillaceae, Halomonadaceae, and Pseudomonadaceae were reported to be oildegrading bacteria found in the oil/marine water interface but they fail to produce biosurfactant. Therefore, we conclude KITPR-1 does not belong to any of these families. The results of KITPR-1 coincide with only one bacterium, namely Alcanivorax borkumensis. KITPR-1 was a non-flagellated marine rod-shaped gram bacterium similar to Alcanivorax borkumensis. Explicit evidence that A. borkumenis produces biosurfactant (Marion et al., 2019) among all the marine bacteria uniquely showing a positive result for drop collapse test, analogous to KITPR-1. Alcanivorax borkumensis was discovered in 1998 (Yakimov et al., 1998) is used as oil degrading. The Alcanivorax borkumensis is a Gram-negative γ-proteobacteria that is omnipresent in the marine environment. Two strains of Alcanivorax borkumensis is used for bioremediation of oil spills, hydrocarbons ATCC 700651 and SK2.
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Acknowledgments: Author wants to thank Karunya Institute of Technology and Sciences, Coimbatore for facilitating research environment for us to do research. Author extend heartfelt thanks to Biotecnika KITS, ABLE, IISc, IBAB, Sea6 Energy, Skanray, Renalyx, CosmosID, Stempeutics for giving this opportunity to showcase research potential.
Poster presentation in event "Online Digital Presentation In The Emerging Area Of Biotechnology” Final Live Presentation: 10th September 2020