Biotechnological Applications of Turbinaria ornata and its Endophytes

 

Michelle C. do Rosario, Prativa Poddar, Swarnkumar Reddy, W. Jabez Osborne*

School of Biosciences and Technology, Vellore Institute of Technology, Vellore - 632014, Tamil Nadu, India

 

ABSTRACT:

Endophytes are group of microbes that colonize in plants and other organisms which can be isolated readily. Seaweeds and its endophytes are reservoirs of several bioactive metabolites likes biosurfactants, antimicrobial compounds and several compounds with biological interests. The present study on investigation of biotechnological potential of endophytes from Turbinaria ornata. The presence of endophytes was initially confirmed by SEM analysis and further 7 bacterial strains were isolated. These strains were further screened for their ability in production of biosurfactants and bioactive compounds. The endophytes were tested biofilm formation. The endophytes were tested for heavy metal tolerance in which all the isolates showed resistance to heavy metals and VITJMP7 the most effective isolate which showed most desirable in antibacterial activity and biosurfactant production, exhibited high resistance to heavy metals up to 4000ppm. The isolate was further examined for the uptake of heavy metals by bacterial biofilm by column study. In which the Ca-Ag beads were used as biocarrier matrix for biofilm formation. The Ca-Ag with the developed biofilm showed significantly increased absorption of up to 20% compared to the beads without bioflim.  The effective isolate (VITJMP7) was further molecularly characterised by 16S rRNA gene sequencing and was identified to be the closest neighbour of Cobetia amphilecti (99.59% similarity). In addition, Turbinaria ornata extracts were also evaluated for their antibacterial properties against three pathogens, the methanolic extract showed better activity on all pathogens.

 

 

INTRODUCTION:

Seaweeds are plant-like organisms contributing to about 50% of the global photosynthesis and are thus considered as one of the most important components of the marine ecosystem. They are normally found attached to substrate like rock along the coastal area. Depending on several structural and biochemical features they are classified into 3 major groups^[1] i.e. brown algae (Ochrophyta), red algae (Rhodophyta) and green algae (Chlorophyta).

 

Seaweeds are known to have enormous applications specially as food and medicines and are thus widely used by humans. They are also vital in the pharmaceutical industries due to their ability to produce distinct bioactive compounds^[2].

 

Seaweeds are also known to harbour endophytic microbes like Enterobacter sp., Colletotrichum sp., Phomopsis sp., Phyllosticta sp., Cladosporium sp., etc. The type of endophytes found depends on factors like climatic conditions and location^[2]. Endophytic metabolites are an increased benefit to the seaweeds as they not only serve as antibiotics, drugs or medicines, but are also compounds of high relevance in research as they play important role in nutrient cycling, bioremediation and biodegradation^[3].

 

Recently, with the rapid increase in industrialization and anthropogenic activities, the levels of heavy metals in the environment have also made their presence evident. Heavy metals like chromium, zinc, cadmium etc. are the primary inorganic contaminants in the environment which even at trace concentrations are extremely toxic due to their non-biodegradability and persistent nature ^[4]. They pose a serious threat to the environment and life as they contaminate the water bodies; alter macro and microbiological communities. Heavy metals cause adverse health problems and diseases and thus research work is carried out to identify a suitable remedy. Conventional methods such as chemical precipitation, flotation and adsorption are widely used^[5] however newer cheaper and more effective biological methods are the need of the hour.

 

Based on previous research, it has been found that microbes have developed several resistance mechanisms to counteract the heavy metal stress in their niches^[6]. The present study was conducted to isolate endophytic bacteria from Turbinaria ornata (seaweed) for biosorption, biofilms formation and biosurfactant production.

 

MATERIALS AND METHODS:

Seaweed sampling and study area:

Seaweed samples were collected from costal region of Rameswaram (Latitude: 9.2876254-Longitude: 79.3129291), Tamil Nadu, India. Seaweeds were collected in and placed in sterile polyethylene bags and transferred to laboratory (4°C). The seaweeds collected were identified to be Turbinaria ornate by herbarium at Madurai Kamaraj field station, Mandapam Rameswaram. 

 

Isolation of Endophytes from the Seaweed Sample:

Detection and Isolation of endophytes from the seaweed - Impregnation Technique and enrichment:

Seaweed samples subjected to SEM analysis to confirm the presence of endophytes following the protocol previously reported by pani et al., 2017. Seaweeds were washed in running tap water and surface by sterilised by subsequent immersion in sterile water for 1min, sodium hypochlorite (2.5%) for 4min and finally washed in sterile distilled water for 3 times. Sterilized seaweed was cut using sterile surgical blade Then seaweed samples were dissected into small transverse sections using a sterile surgical blade into 5mm segments and the leaf segments was impregnated onto the surface of Zobells Marine (ZM) Agar. The plates were incubated for 24h at 28±2°C and morphologically distinct colonies were purified and maintained in glycerol stock^[7].

 

For the enrichment technique the seaweed sections were homogenized and transferred to Zobells Marine (ZM) broth and were incubated for 72h at shaking condition. The broth was serially diluted and plating was performed from 10^-4 – 10^-6 upon serial dilution and incubated at 28±2°C. Plates were observed regularly from the first day and the colonies were purified, transferred to fresh culture slants and also maintained in glycerol stock^[8].

 

Screening for the Effective Bacterial Strain:

Seed Culture Preparation:

Seed cultures of all the purified isolates and pathogens were prepared in ZM broth. The isolate was inoculated and the broth was kept in shaking condition till the absorbance of 0.5 OD (10⁸ CFU/ml) was obtained.

 

Antibacterial activity of endophytes:

Cross streak method:

Three pathogenic bacteria [E.coli (MTCC 9721), Salmonella enteric (MTCC 8587) and S. aureus (MTCC 3160)] were tested for antibacterial activity against isolated endophytes. The anti-bacterial was carried out using cross streak method in which the isolated endophytes were cross streaked against the pathogens. The plates were incubated for 24 h at 28±2°C and observed for zone of inhibition^[9].

 

Agar diffusion method:

Antibacterial activity was performed against the secondary metabolites from the isolated endophytes. Secondary metabolites were extracted from the endophytes by mass multiplying the endophytes and further the metabolites were extracted by liquid-liquid extraction by using three solvents: acetone, chloroform and methanol.

 

Antibacterial activity was carried out against E.coli (MTCC 9721), Salmonella enteric (MTCC 8587) and S. aureus (MTCC 3160). Pathogens were swabbed on to nutrient agar and 20µl bacterial extracts was added. Plates were incubated at 28±2°C and antibacterial activity was measure as zone of inhibition around the well.

 

Screening for Biosurfactant Producing endophyte:

Fabric assay:

Biosurfactant production was carried with the supernatant of the effective bacterial isolates cultured in MS media supplemented with 1% glycerol as carbon source^[10]. The bacterial cultures were centrifuged at 7000rpm for 20 min and the supernatants were collected. Fabric assay was performed in clean cotton fabric of about 1cm×1cm pieces. Fabric assay was

 

Haemolytic Activity and oil displacement assay:

Haemolytic activity of the isolates was determined using blood agar technique in which the blood agar and oil displacement assay is a rapid test for biosurfactant detection using crude oil following the protocol reported by Sriram et al., 2011 and were observed for the zone of clearance around the colonies.

 

Drop collapsing test:

The test was carried out in 96 well microtiter plate by placing 2µl of mineral oil which was allowed for equilibrate for 30 min at room temperature. Further 5µl of cell free supernatant was added to the wells on to the oil layer and the shape of the oil drop was observed after 1 min^[11]. Presence of biosurfactant was characterized by flattened drops, Milli-Q water was used as negative control.

 

Biofilm Production:

The isolates were screened for their ability to form biofilms by tube tube assay. LB broth was inoculated with 2% of the seed culture and was incubated for 72 h at 28±2°C. Further the broth was discarded and tubes were washed with Phosphate Buffer Saline (PBS) (pH 7.3) and dried. Staining of dried tubes was carried out with 0.1% (w/v) crystal violet. Excess stain was removed by washing the tubes with PBS. Formation of biofilm was confirmed visually by the presence a visible film on the wall of the tube^[12].

 

Quorum sensing bioassay (T-streak assay):

The isolates were tested for their ability to produce N-Acyl Homoserine Lactone(N-AHL) was detected by C.violaceum CV206 mutant strain (CV026), which produces violacein in response to N-AHL. The mutant strain C.violaceum CV206 was streaked as a horizontal line on LB media and the isolates were streaked perpendicularly. The plates were incubated for 24 h and observed for violacein production^[13].

 

Heavy metal resistance and Minimal Inhibitory Concentration:

Isolates were screened for resistance against three metals: cadmium, lead and chromium. Heavy metal resistance was tested with 100ppm concentration for each metal individually. Nutrient Broth (NB) was aliquoted in test tubes and sterilized. Then heavy metal solutions were added to the NB to get final concentration of 100ppm. Further tubes were inoculated with 2% inoculum and incubated at 28±2°C for 48h. The heavy metal tolerance was quantitatively measured in terms of turbidity. The test tubes with growth was considered as resistant and the tubes with lack of turbidity was considered as sensitive.

 

 Minimal inhibitory concentration of endophytes was performed using NB supplemented with various concentrations of heavy metal solutions ranging from 500 to 5000ppm. Upon incubation the turbidity in the test tubes indicated the growth of the endophytes. Further, to confirm the presence of the microorganism in the broth, drop plate assay was carried out^[14].

 

Morphological, biochemical and molecular characterization of the effective isolate (VITMPJ7):

The bacterial isolates were characterized for their morphologically by gram staining and hanging drop. Further the biochemical profile of the isolates was characterized by and biochemical properties by IMVIC, oxidase, catalase test^[15]. 

 

Based on the screening assays it was found that the isolate VITMPJ7 to be the effective strain  and was further it was subjected to molecular characterization by 16S ribosomal DNA (rDNA) sequence of the effective bacterium was amplified by PCR using universal primers of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

 

Biofilm Based Bio removal of Cadmium in Packed Column:

Calcium Alginate biocarrier matrix was prepared by dissolving the sodium salt of alginic acid (4% v/w) in distilled water followed by adding it into 0.2 M calcium chloride and incubated in 4 to 8°C to facilitate polymerization^[16].

 

Bio removal experiments were carried out in glass column of 30cm height, 3cm diameter. Column was packed with Ca-Ag beads and column was eluted with distilled water before biofilm formation to attain even bed height and void volume. For the formation of biofilm on Ca-Ag matrix the effective bacteria VITMPJ7 was harvested from fresh broth by centrifugation was suspended in PBS and passed through the column for effective formation of biofilm. The biofilm formation was further confirmed by spread the homogenized bead onto the nutrient agar plate. Bead without biofilm severs as negative control.

 

The ability of the isolate to adsorb the heavy metals the column was eluted with heavy metal solution (100 ppm). The sorbate was collected at an interval of 10 min for 1h. The sorbate was analysed for the elution of heavy metal by Atomic Adsorption Spectroscopy^[17].

 

Detection of Bioactive Compounds Present in the Seaweed:

Extraction of bio actives and anti-bacterial activity from Turbinaria ornata:

Leaves of Turbinaria ornata were washed and blotted to dry. Dried leaves were homogenised with various solvents: methanol, chloroform and acetone; extractions with different solvents were carried out separately. Extractions were concentrated using rotary vacuum evaporator, concentrates were dissolved in DMSO to prepare the desired stock concentration. The extracts of turbinaria ornata with all three solvent were analysed for the bioactive compounds by gas chromatography-mass spectrometer (GC-MS)^[18].

 

The anti-bacterial activity of the seaweed extract was determined against three pathogens (E. coli, Salmonella and S. aureus). All extractions were dissolved in respective solvents to obtain a final concentration of 10 mg/ml and anti-bacterial activity was examined by protocol described by Sriram et al., 2011.

 

Figure1: A- Surface sterilised of Leaf (non-sectioned), B- Leaf Section, C-Surface sterilised Stem (non-sectioned), D- Stem section.

 

RESULT AND DISCUSSION:

Isolation of Endophytes from the Seaweed Sample:

Detection of endophytes by SEM:

The presence of endophytes in the seaweed was very much evidenced with SEM analysis. (Fig. 1). The electron micrographs showed distinct morphology of the endophytes in leaf and stem sections (Fig.4, b and d). whereas the surface of leaves and stem did not show any microbial colonization (Fig.1, a and c) making clear that the potential seaweed has endophytes.

 

Isolation and identification of effective endophytes:

From the marine algae collected a total of 7 (VITJMP1, VITJMP2, VITJMP3, VITJMP4, VITJMP5, VITJMP6 and VITJMP7) morphologically distinct endophytic bacteria were purified and considered for the further study, which were then purified and maintained in glycerol stock.

 

Morphological identification showed, 5 endophytes belongs to gram positive and 2 belongs to gram negative endophytes. The biochemical profile of the isolates was given in table 1.

Table 1: Biochemical test of endophytes

 

Table 2: Antimicrobial activities of bacterial metabolites

Pathogens	Kanamycin	Endophytes	Acetone	Chloroform	Methanol
E. coli (MTCC 9721)	22	VITJMP1	11	10	13
		VITJMP2	13	8	6
		VITJMP3	11	9	11
		VITJMP4	9	5	8
		VITJMP5	9	6	6
		VITJMP6	12	13	15
		VITJMP7	16	14	12
Salmonella enteric (MTCC 8587)	25	VITJMP1	13	11	9
		VITJMP2	11	12	7
		VITJMP3	8	9	8
		VITJMP4	-	5	-
		VITJMP5	7	8	10
		VITJMP6	8	9	8
		VITJMP7	18	16	17
S. aureus (MTCC 3160)	22	VITJMP1	12	10	11
		VITJMP2	11	14	12
		VITJMP3	14	13	10
		VITJMP4	7	-	-
		VITJMP5	9	8	11
		VITJMP6	9	6	8
		VITJMP7	14	19	16

 

Biochemical test showed all the isolates showed no formation of tryptophanase enzyme which was evidenced by indole test. Among all isolates VITJMP3 showed positive for methyl-red test which was confirmed by its ability to ferment glucose to give stable acidic end product^[19]. All isolates except VITJMP6 and VITJMP7 showed negative for catalase production. Where VITJMP1 and VITJMP5 showed the production of oxidase where other isolates showed lack of oxidase production^[20].

 

SCREENING FOR THE EFFECTIVE BACTERIAL STRAIN:

Screening for endophytic isolates capable of producing antimicrobial activity:

The antibacterial activity of isolated endophytes with various organic extracts (acetone, chloroform and methanol) against three bacterial pathogens.

 

The antibacterial activity by cross streak showed that the isolated endophytic bacteria showed notable antibacterial activity by formation of clear zone around the cross streaked endophyte. Which was further quantified by the agar diffusion method where the bacterial metabolites exhibited significant antibacterial activity against pathogens. All seven isolates exhibited antibacterial activity against E. coli. Where in case of S. enteric VITJMP4 showed no zone of inhibition for acetone and methanolic extracts and it showed no zone against S. aureus. The data in Table.2 showed that the more susceptible bacteria against all pathogens was VITJMP7 which showed highest zone of inhibition of about 18 mm with acetone extract against S. enteric.    

 

Screening for endophytic isolates capable of Biosurfactant Production:

Fabric assay: Fabric assay showed the ability of the endophytes to produce biosurfactant on washing the cloth pieces stained with coffee, mud and chocolate with cell free broth, it was observed that VITJMP4, VITJMP6 and VITJMP7 were considerably effective in removing the stains from the cloth when compared to the detergent solution, used as positive control and distilled water, used as negative control (Table.3).

 

Table 3: Fabric assay

 

Haemolytic Assay:

From seven endophytes isolated four showed haemolysis, where VITJMP7 a maximum haemolytic activity of 1.8cm and VITJMP4 showed a least activity of 0.4cm (Table 4). Screening of biosurfactant production through haemolytic activity is the widely used method for the effective screening of biosurfactant production. Carrillo et al., in their work reported that surfactant produced by B. subtilis lysed red blood cells and they have also reported that blood agar method is a crucial primary method for the production of biosurfactant.

 

Oil Displacement assay:

The cell free supernatant was tested for oil displacement which is the simple and most effective method for the biosurfactant detection^[21]. Among the 7 endophytes tested 4 showed positive for oil displacement. On addition of the cell free broths of the isolates VITJMP1, VITJMP3, VITJMP6, VITJMP7 showed notable clear zone in oil layer, where the formation of a hollow zone indicating the biosurfactant production by the endophytes (Table 4).

 

Drop collapsing assay:

Drop collapse assay resulted in corroboration with oil displacement results, where the isolates which exhibited positive for oil displacement showed positive for drop collapsing (Table 4). From these results the biosurfactant production by the isolated endophytes were significantly proved. Further with these results it can be stated that the displacement of oil in oil displacement assay and the time in drop collapse assay directly signifies the amount of biosurfactant. In which the diameter of the oil displaced is directly proportional to the concentration of biosurfactant and time taken for oil collapse is inversely proportional to the concentration of biosurfactant.

 

 

Table 4: Screeing for effective endophytes

 

 

Biofilm formation by endophytes:

Test Tube Assay:

The Test tube assay was performed to check for biofilm production by the isolates. The crystal violet rings formed around the tubes containing  the cultures VITJMP7 and VITJMP1 indicated that the 2 cultures showed biofilm production. The intensity of the violet colour showed that VITJMP7 showed more biofilm production compared to VITJMP1 (Table 4). All endophytes showed negative for AHL production.

 

Heavy metal resistance and Minimal Inhibitory Concentration:

Bioremediation, notable eco-friendly approach for the removal of heavy metals from contaminated sites. In present study all the isolated endophytes where able to resist heavy metals at minimal concentration of 100 ppm. After incubation period all the isolates showed growth in MS agar with supplemented heavy metals. Further the endophytes where tested for minimal inhibitory concentration of heavy metals with increasing concentrations.

 

The isolates screened for metal resistance in MS broth with various concentrations of heavy metals. All seven endophytes showed resistance to all tested metals, while VITJMP7 showed highest MIC of around 4000 ppm to all metals. Where VITJMP4 showed less tolerance to heavy metals (Fig 2). Further MIC was confirmed by drop plate technique.  

Characterization of the effective isolate (VITMPJ7):

The effective isolate VITJMP7 was further molecularly characterised using 16s rRNA gene sequencing and identified to be the closest neighbour of Cobetia amphilecti- KMM 1561-AB646236 (99.59% similarity).

 

Biofilm based bio removal of heavy metals in packed column:

To investigate the ability of biofilm on removal of heavy metal from the aqueous solution, the column was packed with Ca-Ag bio carrier matrix. Further the metal adsorption ability of the effective endophyte VITJMP7 the metal solution was passed through the Ca-Ag beads with biofilm. The absorption ability of the isolate was assed by the concentration of heavy metal in the eluent and was compared with absorption ability of the beads without biofilm. Where the effective endophyte VITJMP7 showed increased absorption of about 50% for all metal solution where the alginate beads without formed biofilm showed 35% absorption.

 

Figure 2. Minimal inhibitory concentration of endophytes against heavy metals a). Lead, b). Cadmium, c). Chromium 

 

Figure 3: Phylogenetic tree, showing the closest neighbours of the effective strain VITJMP7

 

Figure 4: Biosorption of heavy metals a). Biosorption without biofilm b). Biosorption with biofilm

 

The isolate exhibited significantly very less absorption compared to the Ca-Ag beads without biofilm formation (Fig 4). The absorption of metals by alginates beads was dependent on several factors like acidic nature of the environment, ionic strength and swelling ability of the beads. The ion exchange also plays a crucial role in metal uptake.

 

DETECTION OF BIOACTIVE COMPOUNDS PRESENT IN THE SEAWEED:

Screening for Antimicrobial activity of Turbinaria ornate extracts (well diffusion assay):

The methanolic extract showed effective antimicrobial activity against all the three pathogens i.e, E. coli, S. aureus and Salmonella enterica. The acetone extract showed antimicrobial activity against 2 pathogens, S.aureus and Salmonella enterica while chloroform extract showed antimicrobial activity only against E. coli (Table. 5).

 

Table 5: Antimicrobial activities of extracts from Turbinaria ornate

 

DISCUSSION:

Endophytes, the less exposed group of microbes with various biotechnological potentials. The present study reports on the isolation of endophytes from Turbinaria ornate producing potential bioactive compounds. Endophytes are treasure of various bio active compounds, which is dependable source of several bioactive compounds and chemically significant compounds^[22]. A total of seven isolates were obtained from the seaweed which were capable of producing biosurfactants, oil spreading assays is one of the reliable techniques used to screen large number of protentional specimens. A Study conducted by Rengathavasi Thavasi et. al., 2011 suggested that crude oil can be used as a hydrophobic substrate to isolate large number of biosurfactant producing bacterial strains as it reduces the time taken in screening^[23]. In the current research, the positive results obtained from the oil spreading, drop collapse and fabric assays, confirmed the presence of bioactive compound in the isolate’s cell free culture broth. The biosurfactant production was confirmed by haemolytic assay which is the most reliable test for biosurfactant production^[24]. Biosurfactants are known to play important role in reduction and inhibition of biofilm production by pathogens.  So, isolates were assessed for the antibacterial activity against most common bacterial pathogens, the cross-streak method confirmed the antibacterial potential of the isolates which was then quantified by the extraction of bioactive secondary metabolites with organic solvents.  The methanolic extract of seaweed, Turbinaria ornate also showed notable antimicrobial activity against three pathogens (E.coli, salmonella spp. And S aureus), thus signifying that the seaweed extracts contain certain bioactive compounds that can prevent the pathogenic invasion. Experiment of Hosam O. Elansary et, al., 2016 using seaweed extracts (Ascophyllum nodosum) on mint and basil plants showed beneficial impacts on the plant growth, increased leaf number, increase in plant height and increases in the essential oil content. Also, plants treated with A. nodosum showed greater antibacterial potential than the control.

 

Further, the effective endophyte showed resistance to heavy metals up to 4000 and thus can be used as an effective tool in the process of bioremediation. Presence of heavy metals in the aquatic environment causes harmful and toxic effects, so it’s removed is very essential^[25]. Wael M.Ibrahim, 2011 studied the biosorption of heavy metal Co(II), Cd(II), Cr(III) and Pb(II) ions from aqueous solution by four species of red seaweeds Corallina mediterranea, Galaxaura oblongata, Jania rubens and Pterocladia capillacea, the final outcome showed the maximum take up of the metal was 105.2mg/g at biomass dosage 10g/L at pH 5 and incubation time of 1h, which emphasize that seaweeds gives a promising, efficient, and cost-effective way of removing or reducing the heavy metal toxicity from the environment. This supports the present outcome, where the isolated endophyte is effective in biosorption of heavy metals and it also showed high resistance. From the present outcome the endophyte isolated from seaweed has effective potential in various aspects like biosurfactant production, antibacterial activity and also in heavy metal tolerance.

 

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Accepted on 25.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4231-4238.