Introduction

East Kolkata  Wetland (EKW), earlier known as East Calcutta  Wetland (22° 27’ N 88° 27’ E) is also rich in  biodiversity as it contains about 104 plant species,  about 20 important mammalian species, various  threatened reptiles, more than 40 bird species including  both local and migratory types, 52 endemic varieties of  fishes of which 34 are threatened and a huge number of  diverse microbial populations including those, playing  active roles in the wastewater treatment.

This vast area (EKW) includes intertidal marshes,  salt meadows with significant waste water treatment  areas like settling ponds (locally known as Jheels and  Bheries), Sewage Canals (RSC) and farms, oxidation  basins, dumping ground, green zone and cultivable  lands (Ghosh, 1998). EKW was designated as ‘wetland  of international importance’ under ‘Ramsar convention’  on 19th  August, 2002 and as ‘Ramsar site’ in November,  2002 (Ghosh, 1998; Furedy and Ghosh, 1984). Including ECW, total 19 wetlands in India were designated as Ramsar till 2002 .

East Kolkata Wetland shows an huge  diversity of flora and fauna both at the macro and micro level. Microbial Diversity is an integral part of biodiversity which includes bacteria, archaea, fungi, algae, protozoa and protists. Microbial diversity  of this region is still a hidden  asset that needs to be explored and conserved.  Soil samples collected from ECW shows the presence of various new strains of microbes which are not only ecologically important but also have commercial value. They are capable of degradation of toxic chemicals like nitrophenol, nitroaromatic compounds,pesticides etc. , bioremediation of heavy metals, oil contaminated soil and toxic compounds , degradation and recycling of woody tissues of plants, and nitrogen fixation along with the cyanobacters; other bacteria playing important roles in metal accumulation, oil degradation, antimicrobial compound production, enzyme production etc.

Fungi include hundreds of species which are of tremendous economic importance to man. In fact our lives are intimately linked with those of fungi. Hardly a day passes when we are not benefited or harmed directly or indirectly by these organisms. They play an important role in medicine yielding antibiotics, in agriculture by maintaining the fertility of the soil and causing crop and fruit diseases, forming basis of many industries and as important means of food. Some of the fungi are important research tools in the study of fundamental biological processes.

Some of the fungi particularly molds and yeasts play a negative role by causing spoilage of stored goods such as foodstuffs, textiles, leather, rubber, plastic, timber and even glass. but this richness of diversity amongst bacterial, fungal and virus species has yet to be catalogued particularly in East Kolkata Wetlands , West Bengal. Isolating, culturing and cataloguing of fungi are a challenging  task and began  recently with the development of new technology. But microbial diversity including fungi is one of the challenging  areas of biodiversity research.   The biological diversity of the Indian subcontinent is one of the richest in the world. India is recognized as one of the 12 mega diversity regions of the world. Nearly 72% of India’s bio-wealth is constituted by fungi (~18%), insects (~40%) and angiosperms (~13%). Thus, India’s contribution to the global diversity is around 8%. The most important mega-diversity centers are Western Ghats, Northeastern hill regions, Andaman Nicobar islands, mangrove forests of Sunderban area, silent valley of Kerala, Chilka lake of Orissa, Sonar Lake of Maharashtra , the Himalayan region, East Kolkata Wetland etc. India’s rich microbial diversity (14,500 species of fungi, 2000 lichens, 17,000 flowing plants are currently known), has not been adequately enumerated and catalogued. Apart from this there is no such recognized data bank of microbial resources specially on bacteria and fungi. Very few works has been done in the field microbial biodiversity in West Bengal. So it might be an important work for future to work in this field.

A Brief Introduction to Wetlands

A patch of land that develops pools of water after a rain storm would not be considered a “wetland” even though the land id wet. Wetlands have unique characteristics: they are generally distinguished from other water bodies or landforms based on their water level and on the types of lands that live with in them. Specifically, Wetlands are characterized as having a water table that stands at or near the land surface for a long enough period each year to support aquatic plants. Wetlands have also been described as ecotones. The largest Wetlands in the world include the swamp forest of the Amazon and the Peatlands of Siberia. Wetlands play an important role in their ecology. Such wetlands are important breeding grounds for waterfowl and wildlife in general.

East Calcutta Wetlands

The East Calcutta Wetlands, also known as the East Kolkata Wetlands (22° 27’ N 88° 27’ E), are a complex of natural and human-made wetlands lying east of the city of Calcutta (Kolkata), West Bengal in India. The wetlands to the east of Kolkata comprises a large number of water bodies distributed across the districts of South and North 24 Parganas. The multifunctional wetland ecosystem is spread over 12,500 hectares

The East Calcutta Wetland has, along with the wetlands, 254 sewage-fed fisheries, agricultural and solid waste farms and some built up areas. The resource recovery system developed by the local people over many years using waste water from the city is the largest and the only one of its kind in the world. The name East Calcutta Wetlands was coined by Dr. Dhrubajyoti Ghosh, who reached this incredible but neglected part of the city. Dhrubajyoti Ghosh, a civil engineer, prepared the first report for the Department of Fisheries in 1983. He became the first director of the newly established Institute of Wetland Management and Ecological Design (IWMED) in 1986. EKW was designated as ‘wetland of international importance’ under ‘Ramsar convention’ on 19th August, 2002 and as ‘Ramsar site’ in November, 2002

Figure 1: Map of East Kolkata Wetlands Click here to View figure

Review of Literature 

There are some reports on microbial biodiversity at national and international level. The biota of marine microorganisms has developed unique metabolic and physiological functions that not only ensure survival in extreme habitats, but also offer a potential for the production of novel enzymes for potential exploitation. Mangrove ecosystem is nutritionally very rich and widely diverse group of organisms can survive in this extreme habitat. Out of the large number of species examined, only a fraction of marine bacteria have been isolated and cultured. Among them, alkaliphilic Bacillus strains are of considerable importance in biotechnological applications (Fritze et al., 1990, Kumar. and Takagi, 1999, Kumar et al. 2004). A novel B. lehensis (MLB2 (T)) was reported recent from Leh region, Jammu and Kashmir and B. licheniformis SPT27, a producer of extracellular alpha amylase was isolated from the alkaline soil of the eastern coastal region of Bombay, Gujarat. However, in an extensive survey of microbial diversity at marine salterns near Bhavnagar, Gujarat, no Bacillus sp. was documented (Ghosh, et al. 2007, Aiyer, 2004, Deve, and Desai, 2006).

Pushpangadan and Narayanan (2001) made attempt to organize systematics and biodiversity research in India. Diversity of microflora in the gut and casts of tropical composting earthworms reared on different substrates was studied by Parthasarathi et al. (2007). They isolated and identified the strains able to fix nitrogen, produce extracellular enzymes like protease, cellulase, xylanase, and amylase, and solubilize inorganic phosphates. Molecular analysis of microbial diversity of Lonar soda lake (Indian Soda Lake) was analysed by Wani et.al. (2006). Bacterial diversity of East Calcutta wetland and their possible identification was done by Ghosh et.al. (2007).

Surajit Das et al. (2006) reported about the marine microbial diversity & its importance. Halobacteria (Halococcus) isolated from mangrove sediments produce L-asparaginase etc. Joshi et al. (2008) reported the cultivable bacterial diversity alkaline Lonar Lake, India. Most of their isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.

East Calcutta Wetland is low lying area of about 12500 hectares on the eastern region of Calcutta. It is acting as a natural sewage treatment plant to the city and side by side generates product like paddy, vegetables and fish utilizing the sewage. It receives effluents from domestic activities, industries, tanneries, battery manufacturing units as well as health sectors. The purification of the waste products is mainly based on microbial activity. The hot and humid climate all throughout the year favors this site to act as an incubator for diverse group of microbes. Thus the site was selected to explore wide variety of microbes which can be applicable in biotechnology and bioremediation. Bacterial diversity of East Calcutta wetland and their possible identification was done by Ghosh et.al. (2007).

Aim of the present Work 

The central aim  of the present work  to isolate the biologically diverse group of fungi from locality of selected natural spot of East Kolkata wetland of South 24-parganas including Gosaba Island . The purification and indexing of the isolates will be done thereafter. The microbes will be preserved for future use. The identification and cataloguing of the isolated microbes will be done. As there is no such report on the micro fungi of above said area, this study will help to prepare a catalogue/data bank of total fungi including micro fungi of the selected area.

Materials and methods 

Soils were collected from four different agro-ecosystems of Dhapa landfill site at the East Kolkata Wetlands .  Slide were prepared by staining the mycelium by cotton blue dye and observed under oil immersion microscope. Identification of the microbes have done morphologically by light microscopy, Phase contrast Microscopy and 3-D Transmission electronic microscope (model no-TM1500) and picture available in Pictorial Atlas Of Soil and Seed Fungi (Author- Tsugeo watanabe )

Isolation of Soil Sample

Saline  Soil samples were collected from different corner of East Kolkata Wetlands  . The sample were taken  at least 50 inches distance from shore and  from 7 inches depth and mixed well and put in sterile plastic containers . These were then shifted to the laboratory for further analysis . The laboratory which were used for conducting the said work was  , Department of  Microbiology , Bidhannagar College , Salt Lake , Govt. of West Bengal .

So all the soil analysis was carried out there . Soil sample were collected from the localities like  Tree Garden Land ( Bally -1 & 2 ) , Beside Tree ( Jharkhali ) , Beside Pond (Hirenmaypur), Rice Land (Hirenmaypur, Gosaba Bally – 1 & 2), Trees Land (Hirenmaypur), Rice Land (Main Gosaba, 9 No. Bali), Garden Land (9 No. Bali, Malopasa, Rajabazar), Under Trees Land (Malopasa), Bantala tannery, Dhapa, Bherry

Isolation of Fungi 

Fungus were isolated by 10 gms dry soil in 90 ml sterile distilled water, shake well for 15 minutes and kept for one hour for settling. After that serial dilution were made in 9 ml sterile distilled water. 0.1 ml sample were poured in agar plates and spread well by glass spreader and kept in incubator at 320 C.  Optimization of the growth media were done by different media like Potato Dextrose Media, Molt Agar Media and Czapek-Dox agar media and Czapek-Dox agar media was found to be most suitable. Cultivations were made on both still culture or shaking culture methods. Purifications were done by tube dilution methods.

Composition of Potato Dextrose Ager

Potato – 200 gm

Dextrose – 20 gm

Ager – 20 gm

Distilled Water – 1000 ml

pH – 5.6

Procedure

Fungus were isolated by 10 gms  soil in 90 ml distil water, shake well and kept for settling. pH was measured by pH- meter. After that serial dilution were made in 9 ml sterile distilled water. 0.1 ml sample were poured in agar plates and spread well by glass spreader and kept in incubator at 37⁰

Purifications were done by tube dilution methods. Preservation was done in Liquid paraffin .

Result and Discussion

Isolation of soil samples

Soil sample were collected from different corners such as Trees Garden Land (Bally – 1 & 2), Beside Tree (Jarkhali), Beside Pond (Hirenmaypur), Rice Land (Hirenmaypur, Gosaba Bally – 1 & 2), Trees Land (Hirenmaypur), Rice Land (Main Gosaba, 9 No. Bali), Garden Land (9 No. Bali, Malopasa, Rajabazar), Under Trees Land (Malopasa), Bantala tannery, Dhapa, Bherry .

Figure 2: Date of Collection 07.07.2016 &  08.07.2016 (Near Gosaba) Click here to View figure

Figure 3: date of Collection 17.07.2016  (Naktala Road, Bantala tannery,Bantala sewage desposal cannel, Dhapa) Click here to View figure

Plate Cultures of the Isolated Organism

After inoculation of soil sample into Potato Dextrose Agar plate various type of fungus colony were appeared.

Figure 4: Isolated organism from Soil, Collection Date:- 07/07/2016 Click here to View figure

Figure 5: Isolated organism from Soil, Collection Date :-08/07/2016 Click here to View figure

Figure 6: Isolated organism from soil, Collection Date :- 17/07/2016 Click here to View figure

Figure 7: Isolated organism from soil of Dhapa Click here to View figure

Figure 8: Isolated organism from soil of Bhery Click here to View figure

Table 1: Represent the microbial count of the soil sample collected on 07.07.2016

Table 2: Represent the microbial count of the soil sample collected on 08.07.2016

Table 3 : Represent the microbial count of the soil sample collected on 17.07.2016

Table 4: Represent the microbial count of the soil sample collected on 17.07.201

Purification and Preservation

Organisms were Purified by serial dilution method and inoculated in a slant, left for incubation at 37°. After sufficient growth slants were preserved by autoclaved paraffin.

Figure 9: Preservation of Purified Organism Click here to View figure

Figure 10: Preservation of Purified Organism Click here to View figure

Observation  Tables 

Table 5: Date Of Collection 07/07/2016

Table 6: Date of Collection 08/07/2016

Table 7: Date of Collection 17/07/2016

Table 8: Date of Collection 17/07/2016             

Identification 

Based on microscopically   examination (LEICA DM 1000) and picture available in Pictorial Atlas Of Soil and Seed Fungi (Author- Tsugeo watanabe ) some fungi are identified

Table 9 Click here to View table

 Conclusion

An understanding of Microbial Diversity  is of enormous importance because it will help in understanding  different processes of earth and which may present potent novel microorganism which will play a vital role in screening of  bioactive compound . One of the most unexplored area relating to biodiversity in west Bengal is selected natural spot of  East Kolkata Wetland of  the present study , because there is no such records of available fungi in these areas . Isolation , documentation and conservation of these resources are necessary an important because the study of these diverse conditions may play an important role in future in this field  .

The present work deals with seven micro-organisms , which was identified by microscopic studies after isolating and purifying several fungi from diverse areas of East Kolkata Wetland . It is expected that out of seven isolated organisms one or two new genus of fungi may be obtained . These are several characteristic feature like pesticide degradation , chromium degradation , cellulase degradation capabilities and may producing any important medical molecule etc. Which were not explored due to lack of time may be explore in future .

Acknowledgment

The author  would like to thanks from his inner core of  heart to the Department of  Microbiology , Bidhannagar College , Salt Lake ,  Govt. of  West Bengal  for conducting the above work at said laboratory and department of Environment, Govt. of  West Bengal for financial assistance to main author Dr. Abul  Kalam,  the principal investigator of this project.

References

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