Diatoms play a major component and dominant life form of the phytoplankton community. Diatoms are made up of complex silica cell wall and microscopically single-celled autotrophic eukaryotic algae belonging to the class Bacillariophyceae and kingdom Protista1. The tiny algae diatom range from 2 and 500µm in length or diameter and their habitation vary from ocean to freshwater systems exhibiting seasonal variations during monsoon seasons2,3. They are usually yellowish to brown colour in nature and found among the surface of plants, moist soils and in the oceans4. They primarily form a salient link between autotrophic and heterotrophic productions which constitutes a major component of aquatic ecosystem. They constitute an important role in the food chain and are useful as valiant bio indicators of the system where their presence is enhanced in unpolluted waters5. Diatoms generate greater organic carbon in the sea which is equivalent to all the terrestrial rainforests together thus contributing major portion of the photosynthesis in the earth’s atmosphere6,7. Diatoms are considered as the key vectors for biological carbon in the oceans8-10 and the various recent studies in the wide distribution of diatom morphology, elemental composition, and community structure play major factors in understanding the carbon export efficiency11-13. The importance of diatom research has been extensively studied in the areas of taxonomy, ecology, biomonitoring and algal-biotechnology over the last few decades14. The diatom taxonomy was initially started with microscopic observations15 and its identification was based on two parameters either with ribosomal sequences16 or with the morphology, shape of frustules and the external silica cell wall17. The Phylogeny, morphometric18 and teratological structures in diatoms19,20 forms the basis for culturing the diatoms. Diatom culturing for obtaining axenic cultures can be done using the manual isolation technique or automated isolation technique as well as the combination of both21-24. In the present study, the isolated diatoms were cultured using both manual and automatic isolation procedures and the identification was based on the morphological, shape of frustules and the silica cell wall of the diatoms using a Trinocular microscope attached to the monitor.
MATERIAL AND METHODS
The pelagic regions of the coast at Marakkanam, Villupuram district, Tamil Nadu, India [Figure No.1] were selected for the collection of the samples. The sampling was done during June to September 2019 at the latitude of 12.2°N and longitude of 79.95°E.
The water samples were collected manually using plastic water bottles volume 2 Litres capacity by using a plankton net mesh size 25μM. About 50 liters of seawater was collected from four different locations which were filtered through plankton net and transferred to 100mL glass bottles. Collected samples were enriched with nutrients (Sodium Nitrate, Sodium Dihydrogen Orthophosphate, Sodium Silicate, Ferric chloride, EDTA, Trace Metals and Vitamins based on f/2 medium stock solution25,26 Table No.1. The enriched samples were incubated in the laboratory with ambient illumination using 20-watt white fluorescent tube lights and the temperature was maintained between 26°C to 28°C for 12:12 hour light and dark cycles. The sample from the plankton net was prepared directly for the isolation and identification process.
Culturing and isolation
The culturing procedures were done using the samples from the plankton net which were subjected to general isolation methods. Techniques like centrifugation, rarefaction and filtering methods were employed to enhance the cell numbers and in order to separate the mixed samples they were primarily set for centrifugation process at 500rpm for about 15 to 20 minutes27. The centrifugation process results in the precipitation of the contaminants and zooplankton and also eliminates the tiny organisms such as bacteria tom the uppermost layer of the aqueous solution28. The bottom water column was carefully withdrawn using a micropipette and subjected to a serial dilution process29,30. The serial dilution was performed using 10mL test tubes enriched with F/2 medium and the dilutions were done from 10-1 to 10-10 and were incubated at 26°C to 28°C for 24 hrs under illumination. The serially diluted samples containing the diatoms were then plated using agar plating technique31 via micro capillaries for the isolation process. The agar medium was prepared by using 2% agar (Hi-media) and then autoclaved at 120°C for 15 min at 15 lbs. After autoclaving, the semi cooled agar medium was enriched with F2 medium and poured into Petri dishes of 20ml capacity per plate. The samples from serial dilution (10-1 to 10-10) of 0.1mL were spotted in the middle of the agar plates and spread evenly on the surface using a glass spreader. The plates were sealed with parafilm to avoid drying out and incubated in the culture shelve under proper illumination at an ambient temperature ranging between 26°C to 28°C. The emerged colonies on the plates were and restreaked onto fresh agar medium32. Pure unialgal colonies were obtained after repeated streaking on fresh agar media and confirmed by microscopic observation. The pure colonies were then transferred into the liquid medium by inoculating a single colony into each well of the 24 and 96-wells micro titer plate containing 1mL and 200μL of f/2 medium, respectively.
The cleaned diatoms were preserved in 4% formalin added with glycerin.
Morphological Identification and characterization
The cultured species were examined using Trinocular microscopy (Labomed LX 400) for the identification of diatoms in the genus and species level. The identification of the larger diatom isolates was based on the morphological characteristics and phytoplankton identification books33-37. The isolated diatoms were also identified with the help of eminent algologist from India and Belgium.
RESULTS AND DISCUSSION
The diatoms from the pelagic seacoast of Marakkanam were isolated and identified belonging to 12 genera [Figure No.3D] namely Trieres, Chaetoceros, Eupyxidicula, Odontella, Coscinodiscus, Guinardia, Lauderia, Skeletonema, Conticribra, Asterionellopsis, Nitzschia, Craticula, 9 orders [Figure No.3C] namely Eupodiscales, Chaetocerotales, Stephanopyxales, Coscinodiscales, Rhizosoleniales, Thallasiosirales, Rhaphoneidales, Bacillariales, Naviculales, 6 subclass [Figure No.3B] namely Thallasiosirophycidae, Chaetocerotophycidae, Archaegladiopsophycidae, Coscinodiscophycidae, Urneidophycidae, Bacillariophycidae, 3 class [Figure No.3A] namely Mediophyceae, Coscinodiscophyceae, Bacillariophyceae and 14 species were identified as Trieres mobiliensis, Trieres chinensis, Chaetoceros calcitrans, Eupyxidicula turris, Odontella aurita, Coscinodiscus radiatus, Guinardia striata, Lauderia annulata, Skeletonema marinoi, Skeletonema costatum, Conticribra weissflogii, Asterionellopsis glacialis, Nitzschia acicularis and Craticula cuspidata.
Valves elliptical or lanceolate (bipolar). An elevation with an ocellus at each pole. Cells in straight (united by both elevation). Two or more labiate processes per valve, usually with long external tubes. Numerous small chloroplasts observed lying against valve wall with prominent elevations and valve face flat or concave or bulging in the middle. The middle part of valve face was flat or slightly concave, external tubes of processes and elevations diverging38,39 [Figure No.2A and Table No.1].
Valves elliptical or lanceolate (bipolar). An elevation with an ocellus at each pole. Two or more labiate processes per valve, usually with long external tubes. Numerous small chloroplasts lying against valve wall. Cell wall weakly silicified, middle part of valve face shaped in various ways, Processes close to slender elevations, valve face between processes flat or concave39, 40 [Figure No.2B and Table No.1].
Cellulae solitariae, cylindricae. Valvae ellipticae, planae vel convexae a centro, 5-16μ longae. Zona connectivales fere major quam 1/3 alti frustuli. Setae tenues, rectae, 4-5 tanto longior prae axe longo valvae, a margine valvae at ad zonam connectivaleum frustulorum diagonaliter directae41 [Figure No.2C and Table No.1].
Cylindrical, sometimes nearly spherical, capsuleshaped cells. Valves are domed with large hexagonal areolae. Cells have numerous discoid or lobed chloroplasts. Some species form resting spores42, 43 [Figure No.2D and Table No.1].
Heavily silicified cells form curving or spiraling chains, joined by mucous pads on ends of elevations (horns). Numerous small chloroplasts. Cells can be solitary44 [Figure No.2E and Table No.1].
Frustule discoid. Cingulum with three bands, valvocopula more than double the width of one of the two other bands. All bands with hyaline broad marginal borders, slightly rounded ends, areolae in regular rows, 5-6 in μm, broad ligulae with more irregular structure. The band in abvalvar direction with a horizontal marginal flange. Valve flat with rounded margins. Mantle shallow. Valve diameter 90-40μm. Central hyaline area with varying number of branches and a small circular hole externally. Locular areoloe 4-6 in 10μm on valve face, 6-8 in 10μm on valve mantle in radial rows45 [Figure No.2F and Table No.1].
Cylindrical cells that form straight, curving and sometimes spiraling chains. Valves flat but with rounded edges. External process is marginal, and fits into a shallow depression in adjoining valve. Girdle bands appear as collars but hard to see. Small numerous chloroplasts was observed46 [Figure No.2G and Table No.1].
In valve centre an area set off from rest of valve by a silicified ring. Irregularly shaped areolae, more than 30 in 10μm, in marginal zone. Radial, dichotomously branched costae, more than 30 in 10μm, separated by usually 2 rows of minute pores, present on rest of the valve. Structured tubuli usually more in marginal zone than in central part of the valve with long exernal tubes and short internal tubes. One large labiates process at some distance from valve margin47 [Figure No.2H and Table No.1]
Cells in girdle view are rectangular, with rounded corners, 2.0-7.0μm wide and 2.0-10.0μm high. The colonies are chain like, long, consisting of 15-30 cells, strait or slightly curved. The chloroplasts (one or two) are large, plate like, located near the cell wall. The valves are rounded, slightly convex48 [Figure No.2I and Table No.1].
Short and cylindrical cells connected by undulated chains which are long and with spines of marginal rings straight and undulated Convex to flat valve face observed. A dotted ring was observed between adjacent cells with midway length interlocking spines. A central with two chloroplasts was observed. Tubular and semicircular strutted processes in cross section were observed. One labiate process is present near the center of the valve inside the ring of processes47 [Figure No.2J and Table No.1].
Valves are round, flat, with short mantles. The frustules are relatively lightly silicified. Fine areolae were observed and the light microscopic view of the structure was not clearly visible. A small external opening with a ring of marginal fultoportulae was present. The center of the valve face was observed with central fultportulae between three to six numbers. The margin of the valve was observed with a single prominentrimoportula49,50 [Figure No.2K and Table No.1].
It is a Pennate diatom. The cells are connected by star-shaped or spiraling chain valve faces. A single cell with two chloroplasts was observed. The symmetric valves towards apical axis and asymmetric valves towards transapical axis were observed. Linear-lanceolate shaped valves with capitate ends were observed in valve view. Stellate colonies are formed with living cells attached by mucilage pads at the foot poles or basal ends. Living colonies present cells in girdle view shoes the presence of living colonies in the cells where as the processed samples contain single valve with break up the colonies in the valve view. The light microcopy revealed the presence of small spines on the valve margins51 [Figure No.2L and Table No.1].
Valves are lightly-silicified. The valve with parallel sides and sharp tapering to narrow apices was observed at the central part. The evenly spaced fine fibulae which are restricted towards the margin through the central valve was observed with a density 15-20 in 10µm. The light microscopy revealed the absence of central nodule with unclear striae52 [Figure No.2M and Table No.1].
Wide rhombic-lanceolate valves at the center and tapering to narrow apices were observed. The entire valve was observed with parallel and equidistant striae throughout. An orthostichous pattern was observed with thin transapical costae intersecting perpendicular to the longitudinal forming striae. Small, elliptic areolae, approximately 11-15 in 10µm in the transverse direction and 23-25 in 10µm longitudinally were observed in striae. Wide central area with narrow axial area and slightly concave margins were observed. Filiform raphe observed. Expanded proximal raphe ends which are straight, or slightly hooked in the same direction and hooked distal raphe ends which are deflected in the same direction was observed37,51,53. [Figure No.2N and Table No.1].
In the present study, the isolated and identified diatoms from the Marakkanam Sea coast determine the rich nutritional profile for the growth of the organisms and as bio-indicators for the water quality. The diatoms isolated from the coast may serve multiple purposes including as a feed for shrimp larvae, post larvae, oysters and copepods as well as for fuel precursors in liquid. The dead diatoms in the sea bed along with organic matter forms diatomite which is used as a porous material in filtration of sugars and alcohols54-57. The diatoms Chaetoceros calcitrans, Skeletonema costatum and Conticribra weissflogii are widely used in aquaculture as live feed58-60. The diatom Skeletonema marinoi are rich in phenolic compounds and the most available antioxidant substance in photosynthetic organisms making it valiant in the pharmaceutical industry61,62. The diatoms belonging to the genera Trieres, Coscinodiscus, Nitzschia may be used as experimental foods for larvae of grey mullet species63. The biosynthesis of gold nanoparticles using diatom Eupyxidicula turris plays a crucial role in nano-biotechnology applications64. Odontella aurita which is widely used as a food supplement due to its rich EPA content and fucoxanthin may help in the management of obesity especially abdominal fat65. The diatom genera such as Navicula, Lauderia and Asterionellopsis can be tapped for possible biofuel production and as a source for EPA66-69. Thus the isolated diatoms find their applications in aquaculture as a live feed, as a food supplement in the pharmaceutical industry, possible biofuel production, biosynthesis of nanoparticles in the nanotech industry, used as experimental foods and as antioxidant substances as well as for various bioactive substances.
The diversity of diatoms from the northeast pelagic coast of Marakkanam was studied by isolation and identification techniques. The twelve genera with nine orders of diatoms confirm the rich diatom community present in the Marakkanam coast. The isolated diatoms are economically important and find their applications in various fields such as aquaculture, nanotechnology, food industry, pharmaceutical industry, biofuel production and medicinal properties. The culturing techniques employed in the study enable the diatoms to be cultured in mass scale production for the above mentioned applications. The remarkable properties and applications of diatoms can be tapped and studied further for the economic development and new compound identification with useful properties for the aquatic and human ecosystems. Thus the diatoms isolation and identification from the Marakkanam coast portrays the rich diversity and forms the basis for their further advanced research beneficial to mankind and in water quality indicator of the coast.
This article was originally published in Asian Journal of Research in Pharmaceutical Sciences and Biotechnology, 9(1), 2021, 7-18. https://doi.org/10.36673/AJRPSB.2021.v09.i01.A02.