Mangrove Microflora As Potential Source Of Hydrolytic .
Indian Journal of Geo Marine SciencesVol. 48 (05), May 2019, pp. 678-684Mangrove microflora as potential source of hydrolytic enzymes forcommercial applicationsBhavya Kachiprath, Solly Solomon, G. Jayanath, & Rosamma Philip*Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences,Cochin University of Science and Technology, Kochi, Kerala, India.[E-mail: rosammap@gmail.com]Received 14 November 2017; revised 23 April 2018The purpose of this study was to isolate and characterize mangrove microflora based on their hydrolytic enzymeproduction. A collection of 100 microorganisms including bacteria, actinomycetes, fungi and yeasts was isolated. The abilityof microbial isolates to degrade hydrolytic enzymes such as amylase, cellulase, chitinase, glutaminase, laccase, ligninase,lipase, protease and tyrosinase were tested and the potent strains were identified based on 16S rRNA and ITS sequencing.More than 90% of the isolates exhibited amylolytic and proteolytic activity. Potent isolates were identified as: Bacillussubtilis (MB1), Bacillus amyloliquefaciens (MB11), Bacillus megaterium (MB23), Bacillus mojavensis (MB28),Streptomyces galbus (MA7), Streptomyces sp. (MA3), Candida parapsilopsis (MY6), Candida etchellsi (MY1), Penicilliumcitrinum (MF5), Aspergillus stellifer (MF12) and Emericella sp. (MF18). These microbes as well as the enzymes are ofpotential importance for commercial applications as bioremediators, detergent additives and nutritional supplements.[Keywords: Mangrove; Microbial enzymes; Yeast; Actinomycetes; Fungus]IntroductionMangrove ecosystem is a reservoir of diversemicrobial communities (bacteria, fungi, algae, planktonand viruses) which play a crucial role inbiogeochemical processes1. In view of the fact thatmicrobial populations are exceptionally diverse interms of their genetic and biochemical properties, theyare considered as a promising source of enzymes withprospective technological applications2,3. In starchprocess industry, chemical hydrolysis of starch hasbeen completely replaced by microbial enzymes4,5.Microbial enzymes are used for various industrialapplications viz., (i) Lipases for the production ofcosmetics, detergents and as components of chemicaland pharmaceutical reagents6,7; (ii) Proteases for thedevelopment of leather, detergents, digestive and antiinflammatory drugs and additives in bioremediationprocesses8,9; (iii) Ligninases in feed, fuel, food,agricultural, paper, textile and cosmetics industries10,11and (iv) Amylases in food, fermentation, textile andpaper industries12,13.Unlike other expensive chemical methods, microbialremediation enables the degradation of toxiccontaminants into simpler compounds without causingany harm to the natural environment. Bioremediationacts as a low-cost, in situ alternative method, which isnon-carcinogenic, non-combustible, widespread andeco-friendly in nature as compared to otherconservative methods. Fungi, as a part ofmycoremediation, also play a significant role in naturalremediation of metal and aromatic pollutants byremoving or degrading toxicants from naturalenvironment.In our study, we isolated and explored bacteria,fungi, actinomycetes and yeasts from mangroveecosystem which showed significant potential forproduction of hydrolytic enzymes that can be used aspotential candidates for bioremediation.Materials and MethodsSample collectionSediment samples were collected from a mangroveecosystem in Malippuram (Lat. 9 59’44.9”N; Long.76 14’12.6”E), Ernakulam, Kerala, using Van-veengrab. Samples labelled, kept in sterile containers andtransported to the laboratory in icebox for furtherprocessing.Isolation of microorganismsApproximately 10 g sediment mixed with 90 mldistilled water and 10-1 to 10-5 dilutions were preparedfor bacterial isolation. ZoBell’s marine agar (peptone– 5 g, yeast extract – 1 g, ferric phosphate – 0.02 g,agar – 20 g, sea water - 1000 ml; pH – 7 to 7.4) was
KACHIPRATH et al.: MANGROVE MICROFLORA AS POTENTIAL SOURCE OF HYDROLYTICENZYMES FOR COMMERCIALused for bacterial isolation. Medium used for theisolation of actinomycetes was ISP4 medium (solublestarch – 10 g, K2HPO4- 1 g, MgSO4.7H2O – 1 g, NaCl– 1 g, (NH4)2SO4 – 2 g, CaCO3 – 2 g, agar- 20 g, seawater- 1000 ml; pH – 7 to 7.4) with Bavistin asantifungal agent. Fungi were isolated using l (100 mg/l) as antibacterial agent.Wickerham’s medium (MYGP medium – yeastextract – 3 g, malt extract – 3 g, peptone – 5 g,glucose – 10 g, agar - 20 g, sea water - 1000 ml, pH –7 to 7.4) was used for the isolation of yeasts withchloramphenicol (200 mg/litre) as antibacterial agent.679Pour plate technique was employed and incubationwas done at 28 C for 5 days. Single colonies wereisolated, purified and stored in nutrient agar slants(bacteria and actinomycetes) and malt extract agar(fungi and yeasts) slants for further screening,(Figure 1).Screening for extracellular enzymesIsolates were screened for hydrolytic enzymes viz.,amylase14, cellulase15, chitinase16, glutaminase,laccase, ligninase17, lipase18, protease19 andtyrosinase. Table 1 showed substrates and reagentsused for enzyme assays. Based on the enzymeFig.1 — Enzyme production by mangrove microflora (A - Bacteria, B - Actinomycetes, C - Fungi, D - Yeasts)Table 1 — List of substrates and reagents used for enzyme assaysEnzymeSubstrateReagents used for latin -1%Tributyrin-1%Starch-0.2%Colloidal .02%Tyrosine-0.5%α α - Naphthol-0.005%Mercuric chlorideGram’s iodinePhenol red-Clear ZoneClear zoneClear zoneClear zoneClear zonePink colorationWhite zoneClear zonePurple colour zone
INDIAN J. MAR. SCI., VOL. 48, NO. 05, MAY 2019680production, potent strains were segregated andsubjected to molecular identification.Molecular identification of selected strainsBacteria/actinomycetes were inoculated into nutrientbroth and fungi/yeasts into malt extract broth andincubated at 28 0C at 120 rpm overnight. The cells werepelletized by centrifugation at 12,000 rpm for 15 min.DNA from this pellet was isolated using salting outmethod in which the pellet was thoroughly mixed with500 μl solution I (20 mM EDTA (pH - 8), 50 mM TrisHCl (pH - 8), 20% SDS). Then 5 μl proteinase K (20mg/ml) added to it, mixed well and incubated at 55 Cfor 2 hrs. After that the samples were kept in ice for 10min. 200 μl of solution II (6M NaCl) was added,incubated in ice for 5 min and centrifuged at 8000 rpmfor 15 min. Supernatant was collected, equal volume ofisopropanol was added to it and the samples wereincubated at 4 C overnight. Samples were centrifugedat 11,000 rpm for 20 min. The supernatant wasdiscarded and the pellet was washed in 70% ethanolthrice followed by 100% ethanol. Pellet was air-driedand dissolved in 30 μl TE buffer. Purity of the isolatedDNA was checked using spectrophotometric methodsby measuring 260/280 ratio. DNA concentration andyield were also determined. DNA was visualised on0.8% agarose gel and was shown in Figure 2.Primers used for the amplification of 16S rRNAgene of bacterial and actinomycetes and ITS region offungi and yeasts were given in Table 2. PCRconditions for 16S gene amplification included anPrimers Sequences (5’-3’)27F 5’-AGAGTTTGATCMTGGCTCAG-3’1492R 5'-TACGGYTACCTTGTTACGACTT-3'ITS1 5 -CCGTAGGTGAACCTGCGG-3’ITS4 5’-TCCTCCGCTTATTGATATGC-3’Characteristics of the selected culturesHydrolytic enzyme production potential of selectedcultures was shown in Table 3. Bacterial oliquefaciens (MB11), Bacillus megaterium(MB23) and Bacillus mojavensis (MB28) exhibitedsignificant protease production; moderate productionTable 2 — Primer sets used for the identification tsResultsScreening for extracellular enzymesWe have tested the ability of 32 bacteria, 20actinomycetes, 4 yeasts and 41 fungi for enzymeproduction. In the case of bacteria, 90% exhibitedprotease production and 70% cellulase production.Among actinomycetes, 95% showed protease activity,90% amylase, 58% lipase, 50% cellulase, 50%ligninase, and 40% glutaminase production. Morethan 50% of the fungal isolates showed amylase,protease and lipase activity. Among yeasts, 80%showed lipase production, 40% showed cellulase andprotease production and 20% strains exhibitedglutaminase and ligninase production (Fig. 1).Molecular identification of selected strainsThe nucleotide sequences first edited with BioEditsoftware and compared with NCBI database throughBLAST searches and the isolates were identifiedbased on similarity. The sequences were aligned withmultiple alignment and phylogenetic trees constructedby MEGA 5.2. Potent bacterial isolates were Bacillussubtilis (MB1), Bacillus amyloliquefaciens (MB11),Bacillus megaterium (MB23), and Bacillusmojavensis (MB28). Potent actinomycetes includedStreptomyces galbus (MA7) and Streptomyces sp.(MA3). Yeasts were Candida parapsilopsis (MY6)and Candida etchellsi (MY1). Potent fungal strainsincluded Penicillium citrinum (MF5), Aspergillusstellifer (MF12), and Emericella sp. (MF18).Fig. 2 — Gel image of isolated DNA from potent isolates usingsalting out methodSl. No Microorganismsinitial denaturation at 94 C for 5 min followed by 35cycles of denaturation at 94 C for 45 s, annealing at58 C for 45 s, extension at 72 C for 1 min and a finalextension at 72 C for 10 min. PCR conditions for ITSamplification included an initial denaturation at 94 Cfor 5 min followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 56 C for 45 s, extension at72 C for 1 min and a final extension at 72 C for 10min. Amplicons were visualised on 1% agarose gelusing a gel documentation system (Syngene) and areshown in Figure 3 (A, B, C & D). PCR products weresequenced at SciGenom, Kochi, India.
KACHIPRATH et al.: MANGROVE MICROFLORA AS POTENTIAL SOURCE OF HYDROLYTICENZYMES FOR COMMERCIAL681Table 3 — Hydrolytic enzyme production by selected isolatesSl. No MicroorganismsHydrolytic EnzymesCellulase Tyrosinase Laccase Glutaminase Chitinase Amylase LigninaseProtease LipaseBacteria1234Bacillus subtilis (MB1)Bacillus amyloliquefaciens(MB11)Bacillus megaterium (MB23)Bacillus mojavensis (MB28)* -- - -- - -- - - -- - - - -- --- - Actinomycetes567891011Streptomyces galbus (MA7)Streptomyces sp. (MA3)FungiAspergillus stellifer (MF12)Penicillium citrinum (MF5)Emericella sp. (MF18)YeastsCandida parapsilopsis (MY6)Candida etchellsi (MY1)*Clear zone is indicated by: ( ) 0.5 -1cm; ( ) 1-2cm; ( ) above 2cm; and (-) no activityof amylase, ligninase and lipase and weak productionof cellulase and glutaminase. Streptomyces galbus(MA7) and Streptomyces sp. (MA3) showed moderateproduction of all the tested enzymes excepttyrosinase, laccase and chitinase. Penicillium citrinum(MF5), Aspergillus stellifer (MF12) and Emericellasp. (MF18) showed very high glutaminase production.Only Penicillium citrinum (MF5) exhibited chitinaseactivity. Yeasts, Candida parapsilopsis (MY6) andCandida etchellsi (MY1) exhibited protease, lipase,cellulase, glutaminase and ligninase production.DiscussionMangrove ecosystem, located as a transition zonebetween land and sea, is one of the most dynamic andvariable ecosystems due to its constantly fluctuatingpatterns of tidal flooding, salinity, oxygen, sea surfacetemperature, pH, muddy and anaerobic soil, windpattern and nutrient availability. To exist in such afluctuating environment, the mangrove microbialcommunity possesses unique and specific adaptationslike salt tolerance, biosynthetic potential and heavymetal resistance compared with their terrestrial andmarine counterparts20. Many studies reported thatmicrobial consortia with almost all enzymatic abilitiesare essential to degrade complex hydrocarbon mixturessuch as crude oil from industrial waste water, freshwater and marine environments21-23. Bacteria are themost dynamic agents in petroleum degradation and theyperform as primary degraders of spilled oil in variousenvironments24. Several studies have proved thecapability of microbial communities like uorescens , Pseudomonas desmolyticum27 andBacillus sp.28 on decolourization of pulp and paperindustrial effluents and normalization of otherphysicochemical parameters, such as odour,temperature, pH, BOD, COD, calcium, magnesium,chlorine, total solids, total dissolved solids, totalsuspended solids and total hardness of textile dyeeffluents. They naturally biodegrade pollutants, severaltypes of toxic, carcinogenic and mutagenic chemicalssuch as dispersants, levelling agents, acids, alkalies andvarious dyes and thereby remove them from theenvironment. In our study, 60-95% of isolated bacterialstrains including Bacillus subtilis (MB1), Bacillusamyloliquefaciens (MB11), Bacillus megaterium(MB23) and Bacillus mojavensis (MB28) showedcellulase, amylase, ligninase and protease productionand 20-40% showed lipase and glutaminase production.Bacillus subtilis is a biosurfactant producingmicroorganism whose decolourization ability wasreported as 92-97% and has the ability to producelipopeptide antibiotics29. Studies proved that Bacillussubtilis utilize the crude oil components as carbon andenergy sources, degrade heavy metals like mercury,chromium, etc. and is a significant candidate forbioremediation in coastal and marine environment30,20.
682INDIAN J. MAR. SCI., VOL. 48, NO. 05, MAY 2019Out of more than 10,000 known antibiotics,50–55% are reported to be produced bystreptomycetes and are considered economically themost significant among the actinomycetes31. In thepresent study, we could get potent actinomycetes,Streptomyces galbus (MA7) and Streptomyces sp.(MA3). Actinomycete isolates were highly potent interms of cellulase, glutaminase, amylase, ligninase,protease and lipase production. Streptomyces arereported to be involved in the breakdown andrecycling of organic compounds and degradation ofpetroleum hydrocarbons32.Fungal strains, classified under basidiomycetes arereported as the best producers of ligninolyticenzymes. They can be considered as the ideal sourcein studies related to environmental and industrialapplications including the biological treatment oflignocellulosic substrate for biofuel production33.Ligninolytic enzymes are able to degrade a broadrange of compounds via free radical-mediatedoxidizing reactions34. In the present study, we got twopotent fungal strains viz., Aspergillus stellifer (MF12),Penicillium citrinum (MF5) and Emericella ng, oil and gas production alwaysgenerated hyper saline wastes containing highconcentration of salts, oil, organic acids, heavy metalsand radionuclides35. Penicillium sp. can tolerate andtransform heavy metals and xenobiotic compoundsinto less mutagenic products and are potentiallyinteresting to remediate pollutants in the presence ofsalt for biological treatment without damage36,38. Theplant polymers, cellulose and lignin which are verysturdy compounds that give plants their structure aredegraded by fungal enzymes39,40. In this study, 10% ofthe isolated fungal isolates showed chitinase andligninase production and more than 50% fungalstrains showed amylase, protease and lipaseproduction.Marine yeasts have been isolated from differentsources such as seawater, hydrothermal vents, marinedeposits, seaweeds, fish, marine mammals, sea birdsand also from hypersaline habitats. Marine yeastsmostly included Candida spp., Cryptococcus spp.,Debaryomyces spp., Rhodotorula spp., Metchnikowiaspp., Kluyveromyces spp., Rhodosporidium spp.,Pichia spp., Hansenula spp., Saccharomyces spp.,Trichosporon spp., and Torulopsis spp.41,42. Severalyeast strains possess filaments or pseudohyphae andproduce hydrolytic enzymes to endorse efficientdisintegration of substrates. Potent yeast strainsobtained from this study included Candidaparapsilopsis (MY6) and Candida etchellsi (MY1).Bioremediation of diary effluents by yeast strainsincluding Candida intermedia and Kluyveromycesmarxianus was already reported43. Candida utilis,Candida albicans, C. tropicals, and other Candidaspp., were reported to have played prominent role inthe control of algal blooms, indicating their potentialuse in the control of aquatic weeds, bio-ethanolproduction and bioremediation of environmentalpollutants such as heavy metals44-47. This studyindicated that the mangrove microflora, especially theactinomycetes are potential source of hydrolyticenzymes and can find varied applications in industries.AcknowledgementThe authors are grateful to the Head, Departmentof Marine Biology, Microbiology and Biochemistryand Director, National Centre for Aquatic AnimalHealth (NCAAH), Cochin University of Science andTechnology, for providing necessary facilities for thisstudy. Thanks are also due to Mrs. Divya T. Babu,Ms. Manomi S., Mrs. Archana K., Mrs. Neema Joband Mrs. Emilda Rosmine from Department ofMarine Biology, Microbiology and Biochemistry andDr. Jayesh Puthumana, National Centre for AquaticAnimal Health (NCAAH) for their scientific support.References1 Thatoi, H., Behera, B.C., Mishra, R.R., Dutta, S.K., Biodiversityand biotechnological potential of microorganisms frommangrove ecosystems: a review, Ann Microbiol., (2012) DOI10.1007/s13213-012-0442-7.2 Thatoi, H., Behera, B.C., Mishra, R.R., Ecological role andbiotechnological potential of mangrove fungi: a review, Mycol.,4(1) (2013) 54-71.3 Anbu, P., Gopinath, S.C.B., Chaulagain, B.P., Tang, T.H.,Citartan, M., Microbial enzymes and their applications inindustries and medicine, BioMed Res Int., (2015) Article ID816419, 3 pages. doi:10.1155/2015/816419.4 Milshteyn, A., Schneider, J.S., Brady, S.F., Mining themetabiome: identifying novel natural products from microbialcommunities, Chem Biol., 21 (9) (2014) 1211–1223.5 Pandey, R.K., Tewari, L., Microbial enzymes involved in starchprocessing industries, Scholarly J. Biol. Sci., 4(1) (2015) 1-3.6 Zhang, C., Kim, S.K., Research and application of marinemicrobial enzymes: status and prospects, Mar Drugs., 8 (2010)1920-1934. doi: 10.3390/md8061920.7 Choudhury, P., Bhunia, B., Industrial application of lipase: areview, Biopharm J., (2015) MJ/article/view/11/html5. ISSN2454-1397.8 Sawant, R., Nagendran, S., Protease: an enzyme with multipleindustrial applications, World J Pharm Pharm Sci., 3(6) (2014)568-579.
KACHIPRATH et al.: MANGROVE MICROFLORA AS POTENTIAL SOURCE OF HYDROLYTICENZYMES FOR COMMERCIAL9 Singh, R., Mittal, A., Kumar, M., Mehta, P.K., Microbialproteases in commercial applications, J Pharm Chem BiolSci., 4(3) (2016) 365-374.10 Chen, H.Y., Xue, D.X., Feng, X.Y., Yao, S.J., Screeningand production of ligninolytic enzyme by a marinederived fungal Pestalotiopsis sp. J63, Appl BiochemBiotechnol., 165 (2011) 1754–1769 DOI 10.1007/s12010011-9392-y.11 Bonugli-Santos, R.C., Durrant, L.R., Sette, L.D., Theproduction of ligninolytic enzymes by marine-derivedbasidiomycetes and their biotechnological potential in thebiodegradation of recalcitrant pollutants and the treatmentof textile effluents, Water Air Soil Pollut., 223 (2012)2333–2345. doi: 10.1007/s11270-011-1027-y.12 Sahoo, K., Dhal, N.K., Das, R., Production of amylaseenzyme from mangrove fungal isolates, Afr J Biotechnol.,13(46) (2014) 4338-4346.13 Sundarram, A., Krishna Murthy, T.P., α-amylaseproduction and applications: a review, Appl EnvironMicrobiol., 2(4) (2014) 166-175.14 Hankin, L., Anagnostakis, S.L., The use of solid mediafor detection of enzyme production by fungi, Mycologia.,67(3) (1975) 597-607.15 Teather, R.M., Wood, P.J., Use of congo redpolysaccharide interactions in enumeration andcharacterization of cellulolytic bacteria from the bovinerumen, Appl Environ Microbiol., (
Mangrove microflora as potential source of hydrolytic enzymes for commercial applications Bhavya Kachiprath, Solly Solomon, G. Jayanath, . than 50% of the fungal isolates showed amylase, protease and lipase activity. Among yeasts, 80% showed lipase production, 40% showed cellulase and protease production and 20% strains exhibited glutaminase and ligninase production (Fig. 1). Molecular .
karya tulis dalam bentuk laporan apapun tanpa izin Unismuh Makassar. ABSTRAK . Tumbuhan mangrove merupakan ekosistem hutan tropis yang letaknya berbatasan antara darat dan laut (Zhou et.al, 2006). Mangrove merupakan . ekologi, maupun sumber daya ilmiah dan budaya. Tumbuhan mangrove
design and establishment of a marine plant (specifically mangrove) nursery; outline specific growth characteristics of selected mangrove species; provide details of the impacts of pruning trials on nursery stock; and describe trial restoration methods for tidal fish habitats by selective use of nursery-reared mangrove stock.
perikanan payau dengan kolam jebak ataupun keramba jaring apung. Saat ini pemanfaatan tumbuh-tumbuhan di ekosistem mangrove sebagai bahan pangan dan untuk pengobatan semakin meningkat. Buah dari tumbuhan mangrove dapat diolah menjadi bahan pangan pengganti makanan pokok yang mengandung karbohidrat. Dengan demikian, selain beras,
Penelitian Struktur Komunitas Mangrove di Pulau Keter Tengah Kabupaten Bintan Provinsi Kepulauan Riau bertujuan untuk mengetahui struktur komunitas mangrove dan kondisi lingkungannya saat ini (eksisting). Penelitian ini telah dilakukan pada bulan Februari sampai Mei 2013 dengan metode transek kuadran. Jalur transek terpanjang
pada 6 plot. Struktur Komunitas Analisis struktur komunitas pada area Gisik Pantai Pasirmendit, yang kami amati adalah mangrove sejati. Berdasarkan pengamatan, terdapat empat jenis mangrove sejati yang tumbuh di area Gisik Pantai Pasirmendit yaitu,Avicennia marina, Avecennia ovisinalis, Rhizophora appiculata, dan Sonneratia caseolaris.
on macrobenthic fauna from mangroves (Lee and Dittmann, 2008). Finally, we refer to Aaron Ellison’s preface to this Aquatic Botany Special Issue on ‘Mangrove ecology—applications in forestry and coastal zone management’ for a state-of-the-art of mangrove ecosyste
Mangroves and mangrove ecosystems have been studied extensively but remain poorly understood. With continuing degradation and destruction of mangroves, there is a critical need to understand them better. Aspects of mangrove biology have been treated in several recent reviews. Tomlinson (1986) described the
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