TO EXPLORE ANTIMICROBIAL AND METAL MEDIATION PROPERTIES OF RHAMNOLIPID EXTRACTED FROM PSEUDOMONAS SPP. FROM METAL CONTAMINATED SOIL Mahulkar

Mahulkar Ankita Vidyadhar Vaijayanti Ramnarain Ruia College, Matunga L. Nappo Road, Matunga, Dadar East, Mumbai, Maharashtra 400019. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 01 December 2019 Final Accepted: 03 January 2020 Published: February 2020

The heavy metal contamination and disposal of waste frying oil, as well as resistance, occurs due to the hydrophobic nature of antibiotics is a universal issue. The challenge is to develop a method in which biological molecules produced by the low-cost substrate used to reduce metal-associated toxicity in metal-contaminated waste. Along with that, such biological molecules can use alone or synergistically to makes the microbial cell surface more susceptible to accumulate hydrophobic substance (antibiotic) into the cell for restoring resistance. For this purpose, biosurfactant producing bacteria were isolated from a metalcontaminated soil sample and identified as Pseudomonas spp..The result showed that extracellular glycolipid produced by metal isolate was Rhamnolipid. Maximum rhamnolipid production observed when the culture was incubated for 6-8 days in medium containing different amount of mannitol and peptone. Fried oil also acts as the best carbon source giving excellent rhamnolipid production. Moreover, extracted rhamnolipid found to be antimicrobial against gram-positive, gramnegative organisms and fungi as well. It also increases its microbicidal activity of ampicillin antibiotic when used synergistically with rhamnolipid. Different concentrations (60, 80µg/ml) of rhamnolipid were evaluated and compared for the efficiency of heavy metal [Cr (VI)] removal. The result showed that superior adsorbent having 80 µg/ml concentration of rhamnolipid reduced 46%, 39%, 32%, 19% of 10ppm, 20ppm, 60ppm, 80ppm of hexavalent chromium respectively. 75 dirhamnolipid (rhamnosyl-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate) respectively (Reis, R. S., Pereira, A. G., Neves, B. C., & Freire, D. M. G. , 2011).A heavy metal such as hexavalent chromium is soluble in polluted water and carcinogenic, too (Kim and Om, 2013). The interaction between rhamnolipid and heavy metals leads to the separation of metal from the polluted stream through binding of metal either to the external layer of the microbial cell or within the cell wall barrier that promotes the entry of metal into the cell (Abdurrahim A Elouzi et al., 2012). Extracellular polymers or microbial produced anions facilitate the volatilization of the metal either by the metal precipitation or by biotransformation into the cells (Abdurrahim A Elouzi et al., 2012). Rhamnolipids are known as a source for rhamnose, used for the manufacture of high-quality flavor compounds as well as in cosmetic and healthcare industries, biodegradation and bioremediation of xenobiotics, biocontrol (Vanavil, Perumalsamy and Rao, 2014). Pharmaceutically, antibiotic resistance is a global issue, such as hydrophobic antibiotics are those who do not support entry inside the cells efficiently (K.,A.,L.,T., 2014). Rhamnolipid biosurfactant is known for the uptaking of hydrophobic antibiotics to improve their activity. It also demonstrates antimicrobial activity against various microorganisms by interaction with biological membrane systems, especially phosphatidylethanolamine moiety, which disrupts microbial membranes (Das, Yang and Ma, 2014). This study aimed to check the ability of Rhamnolipids to bind to the heavy metals that usually contaminate water as well as its antimicrobial activity along with comparing the penetration of single antibiotic (ampicillin) and combination with biosurfactant on the target site.

Material and Methods:-
Rhamnolipid production, extraction, and purification: Metal contaminated soil sample was collected from western railway workshop (Parel) and checked for the presence of Pseudomonas spp. as described in Bergey's manual. Biosurfactant producers Pseudomonas spp. screened by oil displacement test, drop collapse test, blood agar plate method, and rhamnolipid production confirmed by blue agar plate methodas previously described in Santhini and Parthasarathi, 2014. Rhamnolipid produced (g/L) as described in Hayder, Nadhem,2012  ) with 2 % olive oil was extracted, purified, and quantified by using orcinol assay (K.,A.,L.,T., 2014). The thin-layer chromatography method performed to characterize the rhamnolipid (Das, Yang and Ma, 2014).

Optimization of conditions for biosurfactant production conditions: Parameter 1: Different time interval:
For this, metal isolate grown in 100 ml of Nutrient broth (Himedia) containing 2 % olive oil for 8-10 days (Patil, Pendse and Aruna, 2014) and production of biosurfactant (rhamnolipid) was determined each day by performing oil displacement test.

Parameter3:
Varying concentration of carbon and nitrogen sources: 3ml of the inoculum (0.1 OD of 18 hours old actively growing culture) inoculated into each of the production medium containing different carbon and nitrogen source (2 % mannitol + 1% peptone and 1 % mannitol + 2 % peptone) as described in Subasioglu,Cansunar(2008) and Rikalovic et al., (2012). The flasks had incubated at 37 0 C for 6-8 days under static and shaker conditions and checked for optimum rhamnolipid production by performing orcinol assay, which states rhamnolipid concentration.

Applications:
Heavy metal removal: For standard chromium sample: Weighed 141.4 mg of potassium dichromate in 1000 ml of distilled water, which has chromium concentration 50µg/ml (50 ppm). So, to prepare a standard stock [80 µg/ml (80 ppm)] solution, dissolved 226.2 mg of K 2 CR 2 O 7 in 76 1000 ml round bottom flask and diluted it with distilled water till the mark. Pipette measured volumes from standard chromium solution to give a concentration of 10 ppm, 20 ppm, 60 ppm, 80 ppm. To this, a drop of conc.H 3 PO 4 solution and 1 ml of diphenyl carbazide solution added and kept for 5 minutes in the dark to form red-violet coloration and the absorbance measured using a visible spectrophotometer (WPA Biowave II) at 540 nm as described in Kim and Om (2013) and https://www.epa.gov/sites/production/files/2015-12/documents/7196a.pdf.
To determine the highest concentration of rhamnolipid required to remove chromium: 100 ml of chromium (50 ppm or 50g/ml) solution incubated with 1 ml rhamnolipid having a concentration of 60 g/ml and 80 g/ml along with a drop of conc. H 3 PO 4 solution, Incubated for 24 hours, then added 1 ml of diphenylcarbazide solution and allowed to stand for 5 minutes in the dark to form red-violet coloration.Absorbance measured using a visible spectrophotometer (WPA Biowave II) at 540 nm.
To determine the removal of chromium (10 ppm, 20 ppm, 60 ppm, 80 ppm) by rhamnolipid: Antimicrobial activity of extracted rhamnolipid: 1 ml culture suspension of pathogenic organisms such as Candida albicans, Aspergillus niger, Salmonella typhi, Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus inoculated in sterile nutrient agar (Himedia) for bacteria (Govindammal M and Parthasarathi R. 2013) and potato dextrose agar (Himedia) for yeast and fungi (Hayder, Nadhem,2012), and poured in the sterile Petri plate and wells made by using sterile cork borer and filled with extracted crude rhamnolipid as described in Onbasli and Aslim (2008). Then plates incubated at respective temperatures for 24-48 hours and checked for the zone of inhibition.

Combine activity of extracted rhamnolipid with antibiotic ampicillin against Klebsiella pneumonia:
To study the combined effect of extracted rhamnolipid and ampicillin against Klebsiella pneumoniae combination of the procedure follows as described in Mizoguchi, Suginaka, and Kotani (1979), White et al., (1996) and Ashayeri-Panah, Feizabadi and Eftekhar, (2014). 1 ml culture suspension of Klebsiella pneumoniae of 0.1 OD was mixed with 20 ml of sterile nutrient agar (Himedia) and poured in the sterile petriplate. The sterile filter paper (10 mm) soaked in a crude rhamnolipid solution and other in ampicillin antibiotic solution (1:5 diluted ampicillin injectable 25mg/ml, Sandoz) and placed perpendicular on media to analyze its synergistic action.

Microorganism and growth:
The Pseudomonas spp. obtained from the metal contaminated soil sample. Identification and characterization of the isolate were carried out by biochemical tests, as mentioned in Bergey's manual of systematic bacteriology. The tested isolate showed a positive result for the oil displacement, drop collapse test, and blood agar plateindicates the presence of biosurfactant. The tested strain gives a positive CTAB (Cetyl trimethylammonium bromide) test, which confirms the presence of extracellular anionic glycolipids (rhamnolipids). The result showed that rhamnolipid production was taking place during the stationary phase, where the limitation of a nutrient in the microbial culture promotes rhamnolipid production (Vanavil,

Optimization for rhamnolipid production: Parameter 1: Day interval:
The metal isolate showed a maximum production of rhamnolipid in 6-8 days (Graph 1a). It is the time taken by Pseudomonas spp. to reach the stationary phase having low nutrient conditions that promote rhamnolipid production (Zhi, L. U. O., Xing, Y., Hua, Z., Guang, Z., Zhi, L. I. U., Ya, Z. H. U., 2013). After the 10 th day, there is a decrease in rhamnolipid concentration due to cell lysis caused by its metabolic toxic product formation as well as nutrient depletion, which affectsrhamnolipids production. So, for the optimization, the metal isolate was incubated at 37 0 C for 6-8days.

Parameter 2: Presence of substrate:
These results (Graph 1b) suggested that higher rhamnolipid production observed under shaker conditions than static conditionsindicate that rhamnolipid production and bacterium growth activity have a close relationship (Zhi, L. U. O., Xing, Y., Hua, Z., Guang, Z., Zhi, L. I. U., Ya, Z. H. U., 2013). The result suggests that culture maintained in Luria Bertani broth also showed rhamnolipid production due to the onset of the stationary phase, and the Limitation of a nutrient in the microbial culture promotes rhamnolipid production. Though this medium rich in nitrogen compounds, supplemented olive oil acts as the best carbon substrate (Abouseoud, Maachi, and Amrane, 2007), which allows for better biosurfactant production; insoluble substrates lead to better performance of rhamnolipids. Pseudomonas spp. is lipase positive, which facilitates fatty acid assimilation, which presents in olive oil (Abouseoud, Maachi and Amrane, 2007). Also, less production observed in the absence of olive oil indicates that Pseudomonas spp. also produce rhamnolipids when hydrophobic carbon sources were not there. As secondary metabolite, rhamnolipid was usually synthesized under nutrient-limited conditions and act as biosurfactant, which will reduce surface tension between waste frying oil and culture broth, which help microbes to assimilate fried oil containing fatty acids and used as carbon source (Xiangsheng, Z. et al. 2012) leads to limit nitrogen content thus promote rhamnolipid production.  Chromium assay (Method 7196) is used to calculate the concentration of dissolved chromium (VI). Therefore, to determine total chromium, all chromium of potassium permanganate was converted to the hexavalent state by oxidation, forms red-violet coloration when reacted with diphenylecarbazide under acidic conditions. Absorbance measured using a visible spectrophotometer at 540nm (Website -https://www.epa.gov/sites/production/files/2015-12/documents/7196a.pdf.).
To determine the highest concentration of rhamnolipid required to remove hexavalent chromium: Chromium assay performed to estimate hexavalent chromium concentration in aqueous solution ( Table 1). The result showed that 60 µg/ml concentration of biosurfactant removes 31% of chromium (VI), and 80 µg/ml concentration of biosurfactant removes 48% of chromium (VI) and act as a superior adsorbent (Graph 2a). So, 80 µg/ml concentration of biosurfactant used further to check the removal of different concentrations of chromium. Table 1:-Effect of rhamnolipid on 50 ppm concentration of chromium (VI).
Graph 2a: Efficiency of rhamnolipid on hexavalent chromium removal.

Effect of rhamnolipid on various chromium concentrations:
After the addition of biosurfactant (Rhamnolipid), a remarkable reduction in the chromium(VI) concentration was observed ( Table 2). The result showed that 80 µg/ml concentration of biosurfactant removes 46%, 39%, 32%, 19% of 10ppm, 20ppm, 60ppm, 80ppm of hexavalent chromium respectively (Graph 2b). The anionic rhamnolipid carry a negative charge, and thus, when the molecule finds a cationic metal having a positive charge, it forms an ionic bond that is stronger than the bond between the chromium metal and the water, which enables chromium to precipitate (Pacwa-Plociniczak, Plaza, Piotrowska-Seget, & Cameotra, 2011). A higher concentration of metal may affect Rhamnolipid, which ultimately affects metal removal efficiency. The incorporation of biosurfactant (Rhamnolipid) known to bound to the free form of heavy metals from polluted water, which lowers the solution-phase activity of the heavy metal, therefore, promotes desorption (Elouzi, Akasha, Elgerbi, & El Gammudi, 2012).

Antimicrobial activity of extracted rhamnolipid:
The result (Graph 3) showed that the antimicrobial activity of extracted rhamnolipid against Gram-positive bacteria were more effective than Gram-negative microorganism. It was achievedby disturbing cell membrane structure by interaction with phospholipids and membranous proteins (Samadiet al.,2012). Also, the lipopolysaccharide content of Gram-negative microorganism membranes limits the entry of rhamnolipid because LPS increases the negative charge of the cell membrane causes repulsive interaction between its lipid portion and anionic rhamnolipid (Samadi et al., 2012). Less zone of inhibition observed in fungi, but yeast has not inhibited by rhamnolipid, because the insufficient concentration of rhamnolipid fails to disturb the cell membrane leads to possess tolerance against rhamnolipid. Metal soil isolates contained only monorhamnolipid, which is known to have a high emulsification index (Das, Yang, & Ma, 2014) that enhances its penetration in the gram-negative cell wall. Adherence of rhamnolipid to the microbial cell wall leads to a breakdown of cell membrane integrity as well as alters its metabolism, which signifies an antimicrobial mode of action (Govindammal M and Parthasarathi R, 2013).

Combine action of extracted rhamnolipid and antibiotic ampicillin against Klebsiella pneumoniae:
Klebsiella pneumoniae had grown on sterile nutrient agar, on which strips soaked in rhamnolipid and ampicillin solution kept perpendicularly. The unite activity of A & B is larger than that of A alone or B alone (Fig.1)  that inhibits transpeptidase and D-alanine carboxypeptidase activity, which stops cell wall synthesis leads to cell damage (Mizoguchi, Suginaka, & Kotani, 1979). Klebsiella pneumoniae has extended-spectrum β-lactamase (ESBL) encoding genes that are mainly present on plasmids that carry antibiotic resistance determinants leads to infection (Ashayeri Panah, Feizabadi, & Eftekhar, 2014). β-lactamase present inside the periplasmic space of the bacterial cell, which hydrolyzes β -lactam ring of ampicillin, thus forms resistance against ampicillin (Mizoguchi, Suginaka, & Kotani, 1979). The cytoplasmic membrane has considered the primary site of cellular damage (Samadi et al., 2012). Ampicillin entry into the cell membrane of β-lactamase producing strains was achieved by rhamnolipid by causing the poresthat increase the cell membrane permeability facilitating the penetration of ampicillin through periplasmic space (Lotfabad, Shahcheraghi, & Shooraj, 2013) results in cell lysis. Therefore, it concluded that the enhancement of susceptibility of Klebsiella pneumoniae to ampicillin was probably due to cytoplasmic membrane disruptionand attributed to a significant increase in cell membrane permeability.
A +B = Sum of both effects A and B = Additive Effect. A + B > Sum of both effects A and B = Synergistic Effect. A + B < Sum of both effects A and B =Antagonistic Effect.

Conclusion:-
Our finding has demonstrated that the isolated Pseudomonas strain of metal contaminated soil in the western railway workshop, Parel, was found to be the potent producer of biosurfactant (Rhamnolipid). It produces a higher amount of rhamnolipid when waste fried oil supplemented as a substrate. It also possesses antimicrobial activity against pathogenic strain; thus, it may use as an antimicrobial agent after clinical trials. As well as it has the potential to chelate chromium thus can be feasibly utilized towards in situ bioremediation of industrial waste.