Preliminary Screening and Compositional Analysis of Bacterial Biofilm from Hypogean Environments of Meghalaya, India

· Articles
Authors

Subhro Banerjee and SR Joshi*

Microbiology Laboratory

Department of Biotechnology & Bioinformatics

North-Eastern Hill University

Shillong – 793022, India

*email: srjoshi@nehu.ac.in

Abstract

Hypogean environments comprising of caves and subterranean habitats have long been recognized as a nutrient-deficient environment. To overcome limitation, selfish competition for resources is replaced by cooperative and mutualistic microbial associations of which biofilm formation is a phenomenon. The hypogean habitats in the form of caves in Meghalaya are diverse and some are among the largest in Asia. These caves have so far not yet attracted much attention of geomicrobiologists. On a preliminary scale, two different caves were selected for study of bacterial biofilm at different distances from the cave entrance. A total of 18 different morphotypes of bacteria were obtained from Mawsmai cave and 8 from Mawmluh cave. Moreover, as extracellular substrates can be converted into exopolysachharide of   biofilm bacteria by secreted enzymes, e.g., glucansucrase (which have applications in various industries like pharmaceutical, food, cosmetic, agricultural, photography and mining), we also attempted to screen the most potential glucansucrase producing bacterium from the isolates.

Keywords Bacteria, biofilm, cave, glucansucrase.

Introduction

Microorganisms are able to develop in practically every habitat on earth (Madigan et al., 2003). Their huge diversity and great metabolic capabilities allow these microrganisms to adapt to variety of environmental conditions resulting in an almost high ability to colonize new environments (Nee, 2004; Portillo and Gonzalez, 2008). In spite of these incredible capabilities, microbial activity on natural substrates has been scarcely analyzed and the effects of bacterial growth in nature are barely understood (Taylor et al., 2002; Whitman et al., 2008). Cave microbiology has recently been established as a new interdisciplinary field of microbiology, geology and chemistry dealing with microscopic life that resides in caves and influences natural cave processes. Perpetual darkness, high humidity, almost constant temperature, low airflow and higher CO2 concentrations altogether make the cave (hypogean) ecosystem a unique niche (Rooney et al., 2010). In the last decades the recognition of microorganisms in geologi­cal processes in caves altered our perception of cave eco­systems (Barton and Jurado, 2007). A potential to find specialized higher organisms in such environments is very high (Pipan and Culver, 2007) and can also be expected that a huge variety of different groups of cave-adapted microorganisms will be found.

Barton (2006) defined several features within caves which can be identified as evidence of microbial activity: dots on surfaces, unusual coloration of speleothems, precipitates, corrosion residues, structural changes and biofilms. A biofilm is an assemblage of microbial cells that is irreversibly associated and not removable by gentle rinsing and enclosed in a matrix of primarily polysaccharide material. Moreover, it is becoming clear that these natural assemblages of bacteria within the biofilm matrix function as a cooperative consortium, in a relatively complex and coordinated manner (Caldwell, 1995).

Meghalaya has huge deposits of limestone and abundant rainfall, which is the main reason for the Karst cave formations. All the three hills, namely Khasi, Jaintia and Garo contain limestone of variable quantity and quality. Bacterial community are reported to precipitate calcium minerals (calcite, gypsum, minor amounts of dolomite) postulating that they may contribute to active biogenic influence in the cave formations (Baskar et al., 2008). Reports on characterization of bacteria and fungi are available for some caves of Meghalaya (Baskar et al., 2008; Joshi et al., 2009).

Study area and geology

There are more than 1,000 caves in Meghalaya (a few of them forming one of the longest caves on the Indian subcontinent) and the caves in the East Khasi Hills are Krem Phyllut, Krem Mawsmai, Krem Mawmluh, Krem Soh Shympi, Krem Mawjyngbuin and Krem Dam. The Khasi Hills (Meghalaya Plateau) is an uplifted Precambrian crystalline complex and forms the northeastern extension of the Indian Peninsular Shield. It is an E–W trending oblong horst block elevated about 600–1,800 m above the Bangladesh plains in the south and separated from Peninsular India by the Rajmahal-Garo gap (Ghosh et al., 2005). The caves investigated in this study are located on the East Khasi Hills (25007”; 25041” North Latitudes and 91021”; 92009” East Longitudes), bounded by Ri-Bhoi District on the north, Karbi Anglong District on the north east, Jaintia Hills district on the east, Bangladesh on the south and West Khasi Hills district on the west . The Khasi group consists of sandstone and conglomerate of Jadukata formation overlying the feldspathic sandstone of Mahadek formation. Isolated patches of older Alluvium overlie the Tertiary rocks along the southern fringes of Khasi Hills and recent alluvium is found in the river valleys in the northern foothills region. The Proterozoic meta sedimentary Shillong group and the basement gneissic complex make up most of the Meghalaya plateau (Ghosh et al., 2005). The southern part of the plateau is covered by cretaceous Sylhet basalt and tertiary shelf sediments.

In the present study, we report the process of isolation and cultural characteristics of biofilm bacteria from two of the Meghalaya caves, the Mawsmai and the Mawmluh.

Mawsmai cave

The cave is located south of lower Cherrapunji amidst a thickly forested zone and is quite small (160 m long, 15 m high, width 4–10 m), but the inner parts are large enough to facilitate easy movement within them. It is an important site for tourism, but the cave speleothems are not allowed to be touched by the public due to strict rules set by the state government cave authorities. The main entry to these caves is located close to the Mawsmai village and the entry is a fairly narrow (1.8 m) vertical opening. The cave was totally aphotic and has myriads of stalagmites and stalactites. The cave was moist and only dripping water could be observed.

Mawmluh cave

The cave, also known as Mawkhyrdop cave, is located at a distance of approximately 1 km west of Sohra (Cherrapunji), adjacent to the small hamlet of Mawmluh and at a distance of 10km from Mawsmai cave. Oldham (1859) first reported the existence of Krem Mawmluh. The cave is about 7.1 km long (Brooks and Brown, 2007) and the main entrance lies at the bottom of the western flank of the Lum Lawbah river. The main entrance to the cave is partly blocked from a cave-in, but remains accessible. Unscientific quarrying/mining activities of limestone in the karst area directly or indirectly led to partial collapse of a portion of this cave despite early warning calls. This part of the cave gets flooded by rain water during monsoons. As the sampling time was during the monsoons in the month of July, we could collect biofilm samples only from the cave entrance rather than venturing inside. Several narrow water streams ensuing from the bottom level of the cave entrance took the shape of a big river stream about 10–12 m wide, inside the cave.

Materials and Methods

Biofilm Sampling

Using aseptic techniques, stalactite, stalagmite and column samples were scrapped  in situ with sterilized disposable gloves and scalpel from minimal contaminated (not disturbed by human/anthropogenic activities) areas and then placed into sterile sample containers. Samples were taken from different distances from the cave entrance (0, 10, 20, 30m). Biofilm samples from four different sites were taken from Mawsmai Cave (MM1, MM2, MM3 and MM4) and from one site from Mawmluh Cave (ML5). The ambient temperature and relative humidity at the sites were measured by using a hygro-thermometer. The geographical location and altitude of the site were recorded by using digital GPS (Garmin 7.6). The samples were transported to the laboratory where it was stored at 40C until processing.

Isolation of bacteria

The culturable and aerobic heterotrophic bacterial communities were isolated from the collected samples. The samples (1g) were aseptically transferred into 9 ml sterile Ringer salt solution [SRL, India] (1/4 strength) and vortexed briefly for 2 minutes. Sample dilutions ranging from 10-1 to 10-5 were plated in triplicates onto 4 different media, viz.  Nutrient Agar, R2A Agar, Thiosulphate Agar [HiMedia, India] and B4 Agar [2.5 gl-1 calcium acetate, 4 gl-1 yeast extract, 10 gl-1 glucose and 18 g l-1 agar] (Baskar et al., 2006) and were spread uniformly with a sterile spreader. The plates were incubated at 25oC (to mimic cave temperatures) in an inverted position for 3 days and the CFU were recorded. Nutrient Agar was used as the standard nutrient rich media in the laboratory; R2A Agar was used as the minimal media to enumerate the aerobic heterotrophic count; isolation of microbes on B-4 Agar and Thiosulphate Agar were used to investigate the growth of microbes on a calcite and sulphur-rich substrate respectively. Controls consisting of autoclaved distilled water and 0.9% saline solution were also incubated.  Individual colonies were selected (based on color, colony morphotypes) and purified by repeated streaking. For short-term preservation, the isolates were streaked on respective agar slants and stored at 40C for 30 days before renewed inoculation. The isolates were also stored at -70°C in Tryptone Soya Broth containing 12.5% glycerol for future analysis (Stepanovic et al., 2004). Each isolate was subjected to Gram staining and was examined for cellular morphology and arrangement. In addition, the strains were streaked on nutrient agar with 50 ?g/ml crystal violet (Holding, 1960).

Microscopic examination (Gram staining)

Gram staining was performed with Gram Stains-Kit [HiMedia, India]. The slide was blotted dry, covered with cover-slip and mounted on a microscope (Leica DM 5500) with immersion oil under 100X magnification.

Physiological characteristic (growth in liquid media)

Bacterial cultures were transferred with sterile inoculating loop into the sterile Tryptone Soya Broth [HiMedia, India] tubes. The inoculated tubes along with the control was incubated at 30oC for 24 hr and observed.

Screening methods for detection of biofilm formation using different methods

Congo Red Agar method

It is a method of screening biofilm formation, which requires the use of a specially prepared solid medium -brain heart infusion broth (BHI) supplemented with 5% sucrose and Congo red. Congo red was prepared as concentrated aqueous solution and autoclaved at 121°C for 15 minutes, separately from other medium constituents and was then added when the agar had cooled to 55°C. Plates were inoculated and incubated aerobically for 24 to 48 hours at 37°C. Black colonies with a dry crystalline consistency were indicative of positive result. Weak slime producers usually remained pink, though occasional darkening at the centers of colonies was observed. Darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an indeterminate result.

Modified Congo Red Agar method

Modified Congo Red Agar used as new alternative method for detecting slime production is reliable but this media has a shortcoming of variations in black pigment formation. However, modification on the agar constituent is hypothesized to improve the outcome on biofilm identity determination.

Screening for glucansucrase activity

The isolates were screened for glucan producing strains by inoculating in medium containing: (gl –1): Sucrose, 50.0; Tryptone, 10.0; Yeast extract, 1.0; K2HPO4, 2.5; the pH was adjusted at 7.0 and autoclaved at 121oC for 15 minutes. After autoclaving, 0.005 % sodium azide was added aseptically. Bacterial isolates showing highly viscous slimy growth on sucrose agar plate were selected (Qader, 2001).

Genomic DNA isolation, 16S rRNA gene amplification and sequencing

Genomic DNA isolation was done by HipurTM  Bacterial Genomic DNA Purification Spin Kit (HiMedia, India) and the DNA bands were visualized in 0.8% agarose gel stained with ethidium bromide. Genomic DNA content and purity were estimated using a NanoVue Plus Spectrophotometer (GE Healthcare, Sweden). Bacterial 16S rDNA was amplified by using the universal bacterial 16S rDNA primers, 27F [5’- AGA GTT TGA TCC TGG CTC AG – 3]’ and 1541R [5’- AAG GAG GTG ATC CAG CCG CA – 3’] (Cao et al., 2003) under the following conditions in Gene AMP PCR system 9700 (Applied Biosystems, USA): initial denaturation for 5 min at 94ºC, followed by 35 cycles consisting of denaturation at 94ºC for 1 min, annealing at 55ºC for 1 min, elongation at 72ºC  for 2 min and then cycling was completed by a final elongation step for 5 min at 72ºC. A control tube containing sterile water instead of DNA solution was used as a negative control. PCR products were analyzed by electrophoresis in 1.5% (w/v) agarose gel in 1xTAE buffer with ethidium bromide (0.5µg/ml). PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Germany). DNA content of the PCR products was estimated as above. Sequencing reactions of the 16S rDNA fragments were performed with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA).

Phylogenetic Analysis

The 16S rRNA gene sequence of the isolates and their closest match were retrieved from EzTaxon server [http://www.eztaxon.org/] (Chun et al., 2007) and aligned using Clustal W with MEGA software version 4.1 (Tamura et al., 2007) with Arcanobacterium pyogenes as the outgroup organism. Neighbor-joining method (Felsenstein, 1985) was employed to construct the phylogenetic tree with 1000 bootstrap replications to assess nodal support in the tree.

Discussion

Biotechnological and bioremediation potential of cave inhabiting microorganisms is still scarce and have not been explored from cave environments. Many microbes have the potential to harbor different important substances which can be effective under low (cave) temperature and thus interesting for industry such as antibiotics and tumor suppression potentials. For example, in Carlsbad Cavern novel spe­cies of microorganisms that can degrade complex haz­ardous aromatic compounds, such as benzothiazola and benezenesulfonic, were isolated (Mulec, 2008). Microbes can use these compounds involved in the manufacture of plastics as an energy source for their growth (Barton, 2006). Cave microbiology offers immense poten­tial to study evolutionary relationships and use of alternative sources of energy developed by microbes for scavenging of scarce nutrients in oligotrophic environments. These microbes can also provide to unanswered queries of evolution and help us to identify the geochemical signatures of life. Also, understanding the genesis of biofilm has potential applications as a palaeoclimatic indicator, since it appears to form in certain restricted environmental conditions (Borsato et al., 2000).

The results of this study revealed considerable spatial bacterial diversity associated with biofilm in the hypogean environments studied suggesting that the microbial community associated with biofilm may be more diverse than previously thought. A subject to be investigated is the possibility that different colored biofilms might preferentially develop on areas with slightly different organic nutrient supplies or environmental conditions. Stomeo et al., 2009 have shown that different organic and inorganic compounds can limit growth of bacterial biofilms on cave substrate. These results further suggest that microbes may have important functional roles in subterranean environments, possibly exploiting ‘microniche’ habitats, as suggested by previous studies (Benzerara et al., 2004).

The enzyme glucansucrase screened in this study have broad applications in the biotechnology industries. They have made a remarkable impact in the world of biotechnology because of their applications in the food, cosmetic, agricultural, photography and fermentation industries. The most promising application of glucansucrase and glucan is their use as protective colloid in blood plasma volume expander, flocculation, stabilization, lyopholization and cosmetic ingredient formulation (Leathere et al., 1995). Other applications include its use as gel permeation matrices in research and various industries for the separation purposes of various products. Hence, the enzyme is significant for industrial perspective and therefore the isolates showing maximum activity can be used for the elaboration of glucansucrase and glucan on a large scale.

Acknowledgement

The authors acknowledge the research grant received from Department of Information Technology (Ministry of Communication & Information Technology), Govt. of India to undertake the present study. SB would also like to thank Mr.Vishal Joshi for his assistance in collecting the samples.

 

 

Table 1: Physical parameters of the caves

Cave Ambient Temperature (oC) Relative Humidity (%) Latitude Longitude Elevation (metres)
Mawsmai

24.6

89

N 25o14.68′

E091o43.48′

1221

Mawmluh

30

93

N 25o15.548′ E091o42.749′

1274

Table 2: Source of sampling for biofilm from the caves

Sample No.

Distance from cave entrance (metres)

Zone

Speleothem type

Biofilm morphology

MM1

20

Aphotic

Stalactite

Deep green

MM2

30

Aphotic

Stalactite

Blackish green

MM3

20

Aphotic

Stalactite

White

MM4

10

Twilight

Stalactite

White

ML5

0

Photic

Cave Wall Deposit

Deep green

Table 3: Colony characteristics of the isolated bacteria

Isolate

Colour

Shape

Size

Opacity

Elevation

Surface

Edge

Consistency

MM1-1

Orangish

Irregular

Variable

Opaque

Raised

Smooth

Undulate

Highly butyrous

MM1-2

Creamish

Irregular

Punctiform

Opaque

Convex

Smooth

Entire

Butyrous

MM1-3

Dull yellow

Circular

Punctiform

Transluscent

Convex

Smooth

Entire

Butyrous

MM1-4

White

Circular

4mm

Transparent

Convex

Dull

Entire

Viscid

MM1-5

Pale cream

Circular

3mm

Transluscent

Convex

Smooth

Entire

Butyrous

MM1-6

Off white

Irregular

5mm

Transluscent

Convex

Glistening

Undulate

Viscid

MM1-7

Creamish

Circular

3mm

Opaque

Convex

Smooth

Entire

Butyrous

MM1-8

Creamish

Irregular

Punctiform

Transluscent

Raised

Smooth

Entire

Granular

MM1-9

Bright yellow

Circular

Punctiform

Transluscent

Convex

Smooth

Entire

Butyrous

MM2-1

Pale cream

Circular

Punctiform

Transparent

Convex

Smooth

Entire

Butyrous

MM2-2

Pale white

Circular

2mm

Opaque

Convex

Dull

Entire

Hard

MM2-4

White

Circular

Punctiform

Transparent

Convex

Smooth

Entire

Butyrous

MM2-5

Off white

Circular

Punctiform

Transluscent

Convex

Dull

Entire

Lightly butyrous

MM2-6

Pale white

Circular

Punctiform

Transparent

Convex

Dull

Entire

Butyrous

MM2-7

Creamish

Circular

3mm

Transluscent

Convex

Glistening

Entire

Viscid

MM2-8

Creamish

Circular

2mm

Opaque

Convex

Smooth

Entire

Butyrous

MM2-9

Off white

Irregular

2.5mm

Opaque

Umbonate

Rough

Entire

Viscid

MM4-1

White

Circular

2mm

Opaque

Convex

Smooth

Entire

Granular

ML5-1

Yellow

Rhizoid

-

Transparent

Flat

Smooth

Rhizoid

Butyrous

ML5-2

Creamish

Circular

4mm

Transluscent

Convex

Glistening

Entire

Butyrous

ML5-3

Off white

Irregular

4mm

Transparent

Umbonate

Rough

Dentate

Viscid

ML5-4

Light yellow

Circular

Punctiform

Transluscent

Convex

Smooth

Entire

Butyrous

ML5-5

Pale white

Circular

2mm

Transparent

Convex

Smooth

Entire

Butyrous

ML5-6

Pale yellow

Circular

Punctiform

Transparent

Convex

Smooth

Entire

Butyrous

ML5-8

Creamish

Circular

1mm

Transparent

Convex

Smooth

Entire

Butyrous

ML5-10

Deep cream

Circular

2mm

Opaque

Convex

Smooth

Entire

Butyrous

Table 4: Growth pattern of the isolates in broth cultures

Isolate

Amount of growth

Surface growth

Turbidity

Deposit

MM1-1

Moderate

Absent

Absent

Granular

MM1-2

Moderate

Absent

Uniform

Disintegrates on shaking

MM1-3

Profuse

Absent

Uniform

Disintegrates on shaking

MM1-4

Profuse

Absent

Flocculent

Viscid

MM1-5

Profuse

Absent

Uniform

Absent

MM1-6

Moderate

Absent

Uniform

Disintegrates on shaking

MM1-7

Profuse

Pellicle that disintegrates on shaking

yellow

Flocculent

MM1-8

Scanty

Pellicle that does not disintegrates on shaking

Absent

Absent

MM1-9

Moderate

Absent

Uniform

Disintegrates on shaking

MM2-1

Profuse

Absent

Uniform

Viscid

MM2-2

Moderate

Absent

Absent

Flocculent

MM2-4

Profuse

Absent

Uniform

Disintegrates on shaking

MM2-5

Profuse

Absent

Absent

Flocculent

MM2-6

Moderate

Formation of ring

Uniform

Absent

MM2-7

Profuse

Absent

Uniform

Disintegrates on shaking

MM2-8

Profuse

Absent

Uniform

Disintegrates on shaking

MM2-9

Profuse

Absent

Flocculent

Flocculent

MM4-1

Scanty

Absent

Uniform

Granular

ML5-1

Moderate

Absent

Uniform

Disintegrates on shaking

ML5-2

Profuse

Absent

Uniform

Disintegrates on shaking

ML5-3

Profuse

Pellicle that disintegrates on shaking

Uniform

Flocculent

ML5-4

Profuse

Absent

Uniform

Disintegrates on shaking

ML5-5

Profuse

Absent

Uniform

Disintegrates on shaking

ML5-6

Scanty

Absent

Uniform

Flocculent

ML5-8

Profuse

Absent

Uniform

Disintegrates on shaking

ML5-10

Profuse

Absent

Uniform

Disintegrates on shaking

Table 5: Comparative degree of  biofilm formation by the bacteria on two different media and their glucansucrase activity

Isolate

Congo Red Agar

Modified Congo Red Agar

Glucansucrase activity

MM1-1

Pink

Black with dry consistency

-

MM1-2

Black with no dry consistency

Pink with occassional dark centres

-

MM1-3

Pink

Pink

-

MM1-4

Pink

Pink with occassional dark centres

-

MM1-5

Pink

Pink

-

MM1-6

Pink

Pink

-

MM1-7

Pink

Pink

++

MM1-8

Pink

Pink

-

MM1-9

Pink with occassional dark centres

Pink

-

MM2-1

Pink

Pink

-

MM2-2

Pink

Pink

-

MM2-4

Pink

Pink

-

MM2-5

Black with dry consistency

Black with dry consistency

-

MM2-6

Black with no dry consistency

Pink

++

MM2-7

Pink

Pink

+

MM2-8

Pink

Pink

-

MM2-9

Black with no dry consistency

Pink

-

MM4-1

Pink

Pink

-

ML5-1

Pink

Pink

-

ML5-2

Pink

Pink

+/-

ML5-3

Black with dry consistency

Pink

+++

ML5-4

Pink

Pink

++

ML5-5

Pink

Pink

+++

ML5-6

Pink

Pink

-

ML5-8

Pink

Pink with occassional dark centres

+

ML5-10

Pink

Pink

-

[‘-’ = no activity; ‘+’ = less activity; ‘++’ = intermediate activity; ‘+++’ = high activity; ‘+/-’ = variable activity]

Table 6: Biochemical, morphological and microscopic parameters of the isolates

Isolate

Gram Reaction

Cell Shape

Cell Length (µm)

Arrangement of Cells

ML5-1

positive

rod

1.8

isolated

ML5-2

positive

rod

0.8

isolated

ML5-3

negative

rod

1.72

cluster

MM1-1

positive

coccus

1.05

tetrad

MM1-3

positive

coccus

1.05

isolated

MM1-6

negative

coccus

0.9

isolated

MM1-7

negative

rod

0.82

isolated

MM1-8

negative

rod

0.67

isolated and also in groups

MM2-9

positive

spiral

1.8

short chains

Table 7: Colony Forming Units (CFU) of biofilm bacteria from various sites in respective media and the isolates selected for the study

MM1

MM2

MM3

MM4

ML5

CFU × 103

Isolates

CFU × 103

Isolates

CFU × 103

Isolates

CFU × 103

Isolates

CFU × 103

Isolates

B4 Agar

2.5

MM1-1

MM1-2

MM1-3

MM1-4

1.3

MM2-1

MM2-2

MM2-9

4.9

-

-

-

7.2

ML5-10

Thiosulphate Agar

28.4

-

51

MM2-5

9.2

-

-

20

-

Nutrient Agar

1080

MM1-8

MM1-9

11000

MM2-7

MM2-8

2600

-

-

-

23.2

ML5-1

ML5-2

ML5-3

ML5-8

R2A Agar

76

MM1-5

MM1-6

MM1-7

237

MM2-4

MM2-6

240

-

30

MM4-1

130

ML5-4

ML5-5

ML5-6

 

 

Fig.1:(a) Pure Culture showing well defined isolated colonies on Nutrient Agar plate (isolate ML5-2)

Fig. 1 (b) Gram Staining revealing the organism as a Gram-positive with short rods.

Fig. 2: The phylogenetic tree contructed for the isolate using Neighbour-Joining method with 1000 bootstrap nodal support. (representative isolate ML5-2)

References

  1. Barton, H.A. & V. Jurad o, 2007: What’s up down there? Microbial diversity in caves. – Microbe (Washington, D.C.), 2, 3, 132-138, Washington.
  2. Pipan, T. & C. Culver, 2007: Regional species richness in an obligate subterranean dwelling fauna – epikarst copepods.- Journal of biogeography, 34, 854-861, Oxford.
  3. Barton, H.A., 2006: Introduction to cave microbiology: a review for the non-specialists. – Journal of cave and karst studies, 68, 2, 43-64, Huntsville.
  4. Rooney., D.C., Hutchens., E., Clipson., N., Baldini., J. and  McDermott, F. (2010). Microbial community diversity of moonmilk deposits at Ballynamintra Cave, Co. Waterford, Ireland. Microbial Ecology. 60:753–761.
  5. Madigan, M., Martinko, J. and Parker, J. (2003). Brock biology of microorganisms. Prentice Hall Incorporation.
  6. Nee, S. (2004). More than meets the eye. Nature. 429:804–805.
  7. Portillo, M.C. and Gonzalez, J.M. (2008). Microbial communities and immigration in volcanic environments of Canary Islands (Spain). Naturwissenschaften . 95:307–315.
  8. Taylor, J.P., Wilson, B., Mills, M.S. and Burns, R.G. (2002). Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques. Soil Biology Biochemistry. 34:387–401.
  9. Whitman, W.B., Coleman, D.C., Wiebe, W.J. (2008). Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences95:6578–6583.
  10. Baskar S, Baskar R, Mauclaire L, McKenzie JA (2006) Microbially induced calcite precipitation in culture experiments: possible origin for stalactites in Sahastradhara caves, Dehadrun, India. Curr Sci 90:58–64.
  11. Baskar, S., Baskar, R., Lee, N. and Theophilus, P.K. (2008). Speleothems from Mawsmai and Krem Phyllut caves, Meghalaya, India: some evidences on biogenic activities. Environmental Geology. 57(5): 1169-1186.
  12. Joshi, S.R., Saikia, P. and Pyngrope, M.H. (2009). Microbial communities associated with cave systems in Meghalaya, India. Online Journal of Biotechnology Research. 1(3): 84-92.
  13. Oldham, T. 1859. On geological structure of a part of the Khasi Hills, Bengal with observations on the meteorology and ethonology of that district. Mem Geol Surv India 1:99–210.
  14. Brooks S, BrownM. 2007. Caving in the abode of the clouds:Meghalaya, India. Speleol Bull Br Cav 9:32–34.
  15. Holding AJ (1960) The properties and classification of the predominant Gram-negative bacteria occurring in soil. J. Appl. Bacteriol., 23: 515-525.
  16. Ghosh S, Fallick AE, Paul DK, Potts PJ (2005) Geochemistry and origin of Neoproterozoic Granitoids of Meghalaya, Northeast India: implications for linkage with amalgamation of Gondwana Supercontinent. Gondwana Res 8(3):421–432.
  17. Stepanovic S, Cirkovic I, Ranin L, Svabic-Vlahovic M (2004) Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett Appl Microbiol 38: 428-432.
  18. S.A. Qader, L. Iqbal, H.A. Rizvi and R. Zuberi, Production of dextran from sucrose by a newly isolated strain of Leuconostoc mesenteroides (PCSIR-3) with reference to Leuconostoc mesenteroides NRRL-B512F, Biotechnol Appl Biochem 34: (2001), pp. 93- 97. (s)
  19. Cao X, Liu X, Dong X (2003) Alkaliphilus crotonatoxidans sp. nov., a strictly anaerobic, crotonate-dismutating bacterium isolated from a methanogenic environment. Int J Syst Evol Microbiol 53: 971–975.
  20. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57: 2259-2261.
  21. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599.
  22. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.
  23. Janez Mulec (2008) Microorganisms in hypogeon: examples from Slovenian Karst Caves. Acta Carsologica 37/1.
  24. Borsato A, Frisia S, Jones B, Van der Borg K. 2000. Calcite moonmilk: Crystal morphology and   environment of formation in caves in the Italian Alps. J Sediment Res 70(5):1179–1190.
  25. Stomeo F, Portillo MC, Gonzalez JM (2009) Assessment of bacterial and fungal growth on natural substrates. Consequences for preserving caves with prehistoric paintings. Curr Microbiol 59:321–325.
  26. Benzerara K, Menguy N, Guyot F, Skouri F, Luca G, Barakat M, Heulin T (2004) Biologically controlled precipitation of calcium phosphate by Ramlibacter tataouiniensis. Earth Planet Sci Lett 228:439–449.
  27. T.D. Leathere, G.T. Hayman and G.L. Cote, Rapid screening of Leuconostoc mesenteroides mutants for elevated proportions of alternan to dextran, Curr Microbiol 31 (1995), pp. 19-22.
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