Speleothems from Mawsmai and Krem Phyllut caves, Meghalaya, India: some evidences on biogenic activities

ORIGINAL ARTICLE

Speleothems from Mawsmai and Krem Phyllut caves, Meghalaya,India: some evidences on biogenic activities

Sushmitha Baskar Æ R. Baskar Æ Natuschka Lee ÆP. K. Theophilus

Received: 22 February 2008 / Accepted: 27 May 2008 / Published online: 19 June 2008

� Springer-Verlag 2008

Abstract The Mawsmai cave and Krem Phyllut caves,

East Khasi hills, Meghalaya, India has so far not yet

attracted the attention of geomicrobiologists. Observations

and hypotheses on the possible influence of identified

microorganisms for speleothem formations in Meghalaya

are reported for the first time. XRD studies identified cal-

cite in speleothems and gypsum in cave wall deposits as the

dominant minerals. SEM-EDAX showed interesting mi-

crofabric features showing strong resemblance with

fossilised bacteria, calcified filaments, needle calcite and

numerous nano scale calcite crystals, highly weathered and

disintegrated crystals of calcite, that point towards a sig-

nificant microbial influence in its genesis. Thin section

petrography showed laminated stromatolitic features. The

microorganisms identified by conventional isolation and

further evaluation of isolates by molecular techniques

include Bacillus cereus, Bacillus mycoides, Bacillus

licheniformis, Micrococcus luteus, and Actinomycetes.

Microscopic observations also showed unidentifiable cocci

and four unidentifiable strains of CaSO4 (gypsum) precip-

itating bacteria. Experimental studies confirmed that these

bacteria are able to precipitate calcium minerals (calcite,

gypsum, minor amounts of dolomite) in the laboratory.

These results allow us to postulate that species like these

may contribute to active biogenic influence in the cave

formations at Meghalaya.

Keywords Geomicrobiology � Caves �Carbonate speleothems � Calcium minerals �Biogeochemistry � Bacteria

Introduction

Caves have always haunted the imagination of mankind—

already from the start of human history, these natural for-

mations provided shelter to early man and were sought

after earnestly, to provide a ‘safe home’. Caves are also

natural habitats for a wide spectrum of fascinating life

forms. Caves offer natural, experimental study systems of

the subsurface for fundamental geomicrobiological studies,

because they are relatively easy to access (Culver 1982;

Frey 1963). Calcium carbonate speleothems dominate in

most known caves of the world and a number of geomi-

crobiological studies have been reported in literature on

stalactites, stalagmites, helictites, moonmilk, pool fingers

and cave pearls. Microorganisms impact significantly their

environments and in turn microbial communities are

affected and shaped by the geochemistry of their environ-

ment. Recent studies have clearly identified some of the

factors that control the contribution of microbes to CaCO3

precipitation (Castanier et al. 2000; Engel et al. 2003,

2004; Baskar et al. 2005, 2006, 2007). Laboratory experi-

ments support that the microbial species isolated from

these minerals can produce similar crystals from organic

calcium salts in the lab (Rivadeneyra et al. 1993; Warth-

mann et al. 2000; Baskar et al. 2006).

Caves are generally considered as extreme environments

for life. They are mostly resource limited due to the

S. Baskar � R. Baskar (&) � P. K. Theophilus

Department of Environmental Science and Engineering,

Guru Jambheshwar University of Science and Technology,

Hisar 125001, Haryana, India

e-mail: [email protected]

N. Lee

Division Microbial Systems Ecology (MSE),

Department of Microbiology,

Technical University of Munich (TUM),

Am Hochanger 4, 85354 Freising, Germany

123

Environ Geol (2009) 57:1169–1186

DOI 10.1007/s00254-008-1413-y

prevailing aphotic conditions and subsequently there is no

photosynthesis; thus, this drives most cave ecosystems to

depend on allochthonous organic materials for energy

(Poulson and Lavoie 2000; Simon et al. 2003). The entry of

dripping, seeping and flowing water brings energy into the

caves. Groundwater discharges as springs into the passages

of some caves (Sarbu et al. 1996; Egemeier 1981; Angert

et al. 1998; Hose et al. 2000) also serve as high energy-

yielding substrates for some microorganisms. Microorgan-

isms control biogeochemical cycles and their diverse

metabolic pathways convert minerals into chemical forms

that are readily usable by other organisms (Elia 2002).

In the past decade, cave microbiology has emerged as

one of the frontier areas of geobiology research involving

the co-ordinated efforts of microbiologists, chemists and

geologists to address challenging questions regarding

microbial metabolism, biogeochemistry and their role in

mineral precipitation/dissolution. Bacterial communities in

caves are known to acquire energy by, e.g. transforming

aromatic compounds, fixing gases, and oxidising reduced

metals within rocks. By their interaction with minerals,

microbial species play an important role in reshaping the

mineral environment of caves and helps form features such

as stalactites, stalagmites and various cave wall deposits.

India has a large number of unexplored caves of which

the Meghalaya caves are the largest, which have so far not

yet attracted scientific attention. This study reports the first

geomicrobiological investigations, general geochemistry,

in vitro precipitation experiments, and provides further

indications on the significant role of bacteria as geochem-

ical agents in the genesis of the speleothems from two

caves in East Khasi hills, Meghalaya (India).

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 sub-

continent) and the caves in the East Khasi Hills are Krem

Phyllut, Mawsmai, Krem Mawmluh, Krem Soh Shympi

and Krem Dam.

The Khasi Hills (Meghalaya Plateau) is an uplifted

Precambrian crystalline complex and forms the northeast-

ern 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 (25�0700; 25�4100 North

Latitudes and 91�2100; 92�0900 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

(Fig. 1a–c). The Khasi group consists of sandstone and

conglomerate of Jadukata formation overlying the feld-

spathic 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. A number of prominent linea-

ments trending NE–SW and E–W are present in the

plateau; the most prominent structural feature is the E–W

trending Dauki fault, which marks the southern border of

the plateau. The granite plutons occur in greater number in

the eastern part of the plateau compared to the western part.

Meghalaya has huge deposits of limestone and abundant

rainfall, which is the main reason for the Karst cave for-

mations. All the three hills, namely, Khasi, Jaintia and

Garo contain limestone of variable quantity and quality.

The important limestone deposits in the East Khasi Hills

are in Cherrapunji. This deposit is located in the

Mawmluh-Mawsmai Hills (study area) south of lower

Cherrapunji. The deposit is about 1.40 km in area. The

deposit is of a composite nature made up of limestone in

the upper part and dolomite in the lower part. These caves

are not so easily accessible for the public since they are

situated in a hilly, uninhabited area and are thus scarcely

visited. Here, we report our initial geomicrobiological

studies of two of the Meghalaya caves, the Mawsmai and

the Krem Phyllut.

Sites of study

Mawsmai cave

These caves are located amidst a thickly forested zone and

are 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

only cave fauna observed were spiders and rodents in

rather sporadic amounts. The cave was moist and only

dripping water could be observed. The average annual

temperature of the inner cave is around 15–19�C (Table 1).

The length and diameter of the stalactites ranged from

7–10 cm length, 8–15 cm diameter (small) to 50–1.5 m

length, 50–100 cm diameter (large).

1170 Environ Geol (2009) 57:1169–1186

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Krem Phyllut cave

This cave is also situated in Mawsmai village, south of

Cherrapunjee. This cave has a large section of fossil pas-

sage, two stream ways that join inside the cave and then

leave out of the cave. This cave was relatively long (total

length of 1,003 m, width 4.5 m, height 15 m) and had three

entrances (approximately of 2 m height, 2.5 m width).

Inside the cave the observed zones include a photic zone,

twilight zone with limited light penetration and a deeper

zone, which was totally aphotic. The average annual tem-

perature of the deep aphotic inner cave wall was

approximately 15–17�C and discharged two springs that

run approximately 500 m through the length of the cave.

The approximate length and diameter of the stalactites

ranged from 6–7 cm in length, 25–30 cm in diameter

(small) to 30–40 cm in length, 50 cm in diameter (large)

and the columns were 40–45 cm long and diameter of the

upper end was 40 cm and that of the lower end was 60 cm

(Table 1). The speleothems at the entrance were large and

towards the interior they were smaller in size. Interestingly,

some of the stalactites observed showed nodular and pop-

corn-type morphologies (Fig. 2a–c). The main cavern

extends to about 50–60 m and narrows to a much smaller

tunnel. Gypsum deposits on the cave walls were found

here. The characteristic fauna here consisted of sporadic

amounts of prawns and rats.

Methodology

Sampling

Samples for the studies were taken from minimal con-

taminated (not disturbed by human/anthropogenic

activities) areas of the cave and were collected with sterile

disposable gloves, sterile forceps, sterile ziplock sachets

and sterile autoclaved bottles. Ten aliquots of spring

waters (2 l each) and ten speleothem samples (0.5 kg

each) (including stalactites, stalagmites, cave wall

Fig. 1 a Location map of the study area. Inset shows its position

in India. b Geological map of Meghalaya (Ghosh et al. 2005).

c Geological map of the study cave area (Ghosh 1940; Garg and Jain

1995). 1 Alluvium, 2 Kopili formation (shale/sandstone), 3 Sylhet

limestone, 4 Therria formation (calcareous bands, sandstone, shale),

5 Langpar formation and Um Sohryngkew formation (calcareous

shale, marl and mudstone), 6 Sylhet trap, 7 Archaen, 8 K/T Sect., 9Um Sohryngkew river

Environ Geol (2009) 57:1169–1186 1171

123

deposits and pool sediments), three each from different

areas of the cave (photic, aphotic and twilight areas) were

collected in sterilised bottles, sterile ziplock sachets and

stored at 4�C for up to 30 days before different geo-

chemical and microscopic analysis. The microbial

community structure was characterised and identified

using a combination of conventional isolation efforts and

molecular techniques of isolated specimens, examination

of morphology by SEM, and subsequent quantitative

analyses by EDX.

Geochemical analysis

The major and trace element rock geochemistry of the

speleothems (Na2O, MgO, SiO2, K2O, CaO, P2O5, Al2O3,

TiO2, MnO, Fe2O3, Sr, Ni, Cu, Zn, Ga, Pb, Th, Rb, U, Y,

Zr, Nb) were analysed using an automated sequential X-ray

fluorescence spectrometer WDXRF (Siemens SRS 3000,

Wadia institute of Himalayan Geology, Dehradun). The

geochemistry of the spring, pool and dripping water sam-

ples (Mn, Cu, Zn, Cd, Ba, Sr, Cr, Ag, Mg, Si, V) were

analysed using ICP-MS (Wadia Institute of Himalayan

Geology, Dehradun). The acidity, alkalinity, hardness,

conductivity and pH of spring waters and total organic

carbon content of speleothems was determined by using

standard procedures at the Environmental Geology Labo-

ratory, Guru Jambheshwar University of Science and

Technology, Hisar, India.

Electron microscopy and mineralogy

Scanning electron microscopic (SEM ZEISS EVO 40

EP, resolution, 3.0 nm SE and HV; magnification,

7–1,000,0009; accelerating voltage, 0.2–30 kV; WIHG

Dehradun) studies were performed on the speleothems and

bacterially precipitated (in vitro) mineral samples. EDX

(EDX microanalyser Bruker LN2 Free X Flash 4010SDD

detector, resolution, 129 eV at Mn ka9 (5.98 keV) and XRD

(X0Pert Pan X-ray diffractometer, WIHG, Dehradun) was

used for quantitative estimation of the chemical composi-

tion of the minerals. The speleothem samples and samples

postulated to contain biomass were fixed onto aluminum

stubs with two-way adherent tabs with conductive paint,

and allowed to dry (Leveille et al. 2000). They were then

gold-coated by sputtering for approximately 2–3 min.

Some of the speleothem samples were acid-etched (3 and

10% HCl) before SEM examination (Melim et al. 2001).

For EDX the samples were coated with carbon (CC7650

carbon evaporation coater) and then transferred to the

sample chamber of the instrument prior to imaging. The

SEM was operated with a working distance of 15 mm for

optimum imaging and to minimize charging and sample

damage. For X-ray analysis, an accelerating voltage of

Table 1 General characteristics of speleothems from East Khasi Hill caves, Meghalaya

S.no. Sample no. Type Mineral Length (cm) Diameter

(cm)

Temperature

(�C)

Zone Distance from

cave entrance (m)

Mawsmai cave (MC1R)

1 MC1R-12 Stalagmite Calcite 7–50 8–100 17 Aphotic 90

2 MC1R-18 Stalactite Calcite 15–35 10–90 16 Aphotic 100

Krem Phyllut cave (MC2R)

3 MC2R-02 Stalactite Calcite 30–40 35–50 15–17 Aphotic 90–100

4 MC2R-08 Cave wall deposit Gypsum 12–15 15 16 Aphotic 260–270

5 MC2R-11 Column Calcite, aragonite 40–45 40 Upper,

50 lower end

16 Aphotic 330

6 MC2R-13 Pool water sediment SiO 20–25 10–20 17 Aphotic 360

7 MC2R-15 Stalagmite Gypsum 15–20 25–30 17 Aphotic 400

8 MC2R-23 Cave wall deposit Calcite 7–50 30 16 Aphotic 70–80

9 MC2R-25 Stalagmite 7–40 6–30 17 Twilight 20

Fig. 2 Speleothems, pool waters from Meghalaya caves. b Nodular

and popcorn crusts (encircled)

1172 Environ Geol (2009) 57:1169–1186

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15 kV was used to obtain sufficient X-ray counts. Samples

for XRD were dried, powdered and scanned between 4�and 64� 2h at 1� 2h min-1. The minerals were identified

using the JCPDS–ICDD, XRPD database (2000).

Microbiological analysis

Attempts to isolate bacteria from the speleothems and cave

wall deposits from both of the caves were made for identi-

fying the culturable, aerobic, heterotrophic fraction of the

total microbial community. Stalactite, stalagmite and cave

wall deposit samples (1 g) were powdered in a sterilised set

of mortar and pestle, suspended in sterile 0.9% saline solu-

tion and briefly vortexed. Thiosulphate and sulphite agar

(tryptone 10 g l-1, sodium sulphite 1 g l-1, agar 20 g l-1)

based media were used to isolate bacterial isolates from the

cave wall deposits at pH values of 4–6. Standard nutrient-

rich media such as nutrient agar and B-4 agar (Boquet et al.

1973) were used to isolate bacteria from the speleothems.

The agar plates were inoculated with sample dilutions

ranging from 101 to 106 and incubated under aerobic con-

ditions between 15 and 25�C (photic, aphotic conditions

separately designed to mimic cave temperatures) for

2 weeks. The initial plate counts were approximately

12 9 102 g l-1 (sulphite agar), 16 9 101 g l-1 (thiosul-

phate agar), 40 9 102 g l-1 (nutrient agar), 32 9 102 g l-1

and contained a large variation of colony-morphotypes, of

five different colours (yellowish, pale cream, creamish,

whitish brown, red). Individual colonies were selected

(based on differences in colour, colony morphotypes) and

purified by repeated streaking. For short-term preservation,

the isolates were streaked on respective agar slants and

stored at 4�C for 30 days before renewed inoculation.

Microbe–mineral precipitation experiments were designed

using temperatures and salt concentrations to mimic the cave

environment. Bacterial isolates capable of precipitating

minerals were streaked for purity and further identified by

conventional and molecular techniques and the mineral type

and chemistry of the precipitated bio-mineral was deter-

mined using XRD and EDX. Altogether 30 purified isolates

were obtained and five of these isolates were deposited at the

strain collection MTCC (Microbial Type Culture Collection

and Gene Bank), Institute of microbial technology, IM-

TECH, Chandigarh, India. Strains 2, 4, and 8 were screened

by conventional and DNA sequencing identification meth-

ods, whereas strains 5 and 13 were characterised by

conventional phenotypical methods. The origin of the strains

and their designations are listed in Table 2.

The isolates were identified using conventional (mor-

phological, biochemical and physiological tests) and DNA

sequencing identification methods, based on PCR amplifi-

cation of 16S rRNA genes (16S rDNA) and partial

sequencing (*800–1,000 bp). The partial 16S rRNA gene

sequences were compared with the large nucleotide data-

base of the National Center for Biotechnology Information

NCBI by submitting the sequences to BLAST (Altschul

et al. 1990, http://www.ncbi.nlm.nih.gov/). All (three in

all) sequences, as well as relevant reference 16S rRNA

gene sequences identified by BLAST search in NCBI were

imported to the phylogenetic software ARB (Ludwig et al.

2004; http://www.arb-home.de) for alignment and sub-

sequent calculation of a phylogenetic tree based on the

neighboring joining hood algorithm, to confirm the iden-

tities suggested by BLAST. The sequences retrieved in this

study have been submitted to NCBI, and their accession

numbers are: EU495983, EU495984 and EU495985.

Results and discussions

The purpose of this study was to investigate the biogenic

influence on the cave formation in two of the Meghalaya

caves in the Indian subcontinent. For this, we combined

geochemical, scanning electron microscopical, microbio-

logical and in vitro precipitation studies. Here, we present

our results from these studies on ‘‘Speleothem carbonates’’

as well as on ‘‘Gypsum and cave wall deposits’’. In ‘‘Bio-

genic influences on speleothem carbonates’’ and ‘‘Biogenic

influence on gypsum and cave wall deposits’’, we present

our hypotheses on biogenic influences on these systems.

Speleothem carbonates

Petrographic observations

It is crucial to determine the conditions under which spe-

leothems form by studying the fabrics preserved in the cave

precipitates/deposits. The criteria for recognising microbes

and microbial activity in speleothems and cave deposits

include documentation and recognition of mineralised

microbes, recognition of stromatolitic structures that are

wavy or laminated structures formed by microbes that trap

and bind detrital grains to a substrate or act as nucleation

sites for mineral precipitation and the identification of

fabrics/textures that are known to be indicative of micro-

bial activity (Jones 2001).

Petrography of our speleothem samples showed alter-

nating white and dark-laminated bands and voids (Fig. 3a,

b), and the presence of lithified structures and internal

fabrics similar to microbialites along with some organic

inclusions. Dark coloured clotted peloidal fabrics (Fig. 3c)

were observed, which may be interpreted as calcified

bacterial aggregates since such formations have quite often

been reported in stromatolites, thrombolites, travertines,

and in reefs (Chafetz and Buczynski 1992; Pedley 1992;

Riding 2000; Baskar et al. 2007).

Environ Geol (2009) 57:1169–1186 1173

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http://www.ncbi.nlm.nih.gov/

http://www.arb-home.de

Geochemical investigations

The spring waters were pH neutral (7.4–7.8). The speleo-

thems had a TOC content of approximately 0.371–1.984 wt

%. The waters (spring, pool and dripping) had concentrations

ranging between 31 and 37 ppb Sr, 1.5–2.6 ppb Si,\1 ppb

Cd, 3–12 ppb Cr, 0 to\1 ppb Ag, 4–5 ppb Ba, 1–4 ppb Cu,

3–28 ppb Zn,\1–7 ppb Mn, 2.3–5.8 ppb Mg and 0–1 ppb V

(Table 3, Fig. 4a). MCIW-02 is a sample from Mawsmai

caves and the rest are samples from Krem Phyllut caves.

Table 2 Summary of strain informations of isolates obtained in this study and their in vitro precipitation abilities

Internal

strain name

Identified

strain name

Isolation source Isolation

medium

MTCC

number

Accession

number

Bacterial

nomenclatureeMineral

precipitated

in vitro

Minimum days

required for mineral

precipitation

(days)

MCI01 Strain 2 Speleothem (M) NA/B-4 MTCC

8776

EU495983 Bacillus cereus(a) Calcite 7


8777

EU495984 Micrococcusluteus(a)

Calcite 15

MCI03 Strain 8 Speleothem (KP) NA/B-4 –a EU495985 Bacillus cereus(a) Calcite 7


8915

–d Bacilluslicheniformis(b)

Calcite 12

MCI05 Strain 10 Speleothem (M) NA/B-4 –b –d Bacilluslicheniformis(b)

Calcite 12

MCI06 Strain 14 Speleothem (KP) NA/B-4 –b –d Bacilluslicheniformis(b)

Calcite 12

MCI07 Strain 13 Cave wall

deposit (KP)

SA/B-4 MTCC

8916

–d Bacillus mycoides(b) Calcite 15


deposit (KP)

SA/B-4 –c –d Bacillus mycoides(b) Calcite 15


deposit (KP)



deposit (KP)



deposit (KP)


MCI12 Strain 19 Speleothem (M) B-4 –d –d Actinomycetes Calcite 20

MCI13 Strain 20 Speleothem (M) B-4 –d –d Red pigment

forming

Streptomyces

Calcite 20


deposit (KP)

TS/SA –d –d Unidentified Gypsum 20


deposit (KP)



deposit (KP)



deposit (KP)


MCI18 Strain 25 Speleothem (KP) TS/SA –d –d Unidentified chained

cocci

Calcite 22

MTCC microbial type culture collection and Gene Bank, Imtech, India; NA nutrient agar; SA sulphite agar; TS thiosuphate agar; B-4 Boquet et al.

(1973); M Mawsmai cave; KP Krem Phyllut cavea Similar to strain 2 (since strains 2 and 8 are similar in all properties including 100% match in their partial sequences, MTCC deposited strain 2

only and did not give strain 8 an MTCC number though it was also sequenced and analysed for partial sequencing by MTCC)b Similar to strain 5 (based on similarities with strain 5 regarding general colony morphology, grams reaction, spore staining, cell shape and

arrangement observed under the microscope, biochemical tests performed)c Similar to strain 13 (based on similarities with strain 13 regarding general colony morphology, grams reaction, spore staining, cell shape and

arrangement observed under the microscope, biochemical tests performed)d Not deposited within the time frame of this studye Determined by (a) identification by phenotypical characteristics and 16S rRNA gene (b) identification by phenotypical characteristics

1174 Environ Geol (2009) 57:1169–1186

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MC2W-01, a drip water sample is enriched in Zn compared

to other samples. Geochemically, the speleothems contained

45–54.67% Ca, 0.12–1.69% Fe2O3, 0.31–0.91% MgO,

0.002–0.131% Na2O, 0–3.24% Al2O3 and Sr 46–501 ppm

(Table 4, Fig. 4b). The pool sediments of the sampled caves

have 19.8% iron and are basically pyrite (Table 4). XRD

revealed calcite as the dominant mineral and traces of

dolomite, aragonite, iron titanium hydrate, copper minerals,

tin telluride, palladium hydride, quartz, and iron minerals.

Electron microscopic observations

SEM revealed most strikingly a significant number of

microcrystalline needle fiber crystals, large micro-rod cal-

cites, dendritic calcite crystals, nano scale to microscopic

sized calcite crystals (Fig. 5a, b). All these structures may

possibly have been induced by the metabolic activities of

the associated bacterial communities. The presence of

needle, fibrous, micro-rod calcites, highly weathered and

disintegrated crystals of calcite, organic inclusions and

lithified structures that resemble stromatolitic microbial

mats indicate that they may have been formed by microbial

activities (Melim et al. 2001; Northup et al. 2000; Chafetz

1986; Northup and Lavoie 2001). The classification of

micro-rod calcite has been earlier reported, and its origin

has been attributed to rapid precipitation at high super-

saturation states during evaporation in soils or to calcifi-

cation of bacilliform bacteria (Loisy et al. 1999). The

present study also revealed the presence of micro-rods and

can be classified as follows: microcrystalline needle fiber

crystals, 0.1 m wide, 1–2 lm long; large needle fibers,

2–20 lm wide (Fig. 5a–c), 50–100 lm long; calcified fil-

aments 5–50 lm long (Fig. 5c), spiky calcite, rounded

balls of calcite, a number of bacterial ligaments, filaments

in the speleothems (Fig. 5d, e) (Verrecchia and Verrecchia

Fig. 3 a, b Stromatolitic

laminations. c Clotted chocolate

brown blebs of peloidal material

Table 3 Water analysis (ICP-MS), East Khasi Hill caves, Meghalaya

S.no. Mn Cu Zn Cd Ba Sr Cr Ag Mg Si V

MC1W-02 7 4 8 \1 4 37 12 0 5.8 1.7 1

MC2W-01 1 2 28 \1 4 35 6 0 2.3 1.5 \1

MC2W-07 \1 1 3 \1 4 31 6 \1 3.7 2.1 0

MC2W-08 \1 2 3 \1 4 31 6 \1 3.9 1.7 0

MC2W-03 \1 2 3 \1 5 32 5 0 3.7 2.6 0

MC2W-05 \1 1 4 \1 5 33 6 0 4.1 1.9 0

MC2W-04 \1 2 2 \1 4 32 4 0 3.8 1.7 0

MC2W-09 \1 2 5 \1 5 32 3 0 3.7 1.9 0

MC2W-10 \1 2 4 \1 5 31 3 0 3.7 1.9 0

Values are in ppb. Dripping waters MC1W-02, MC2W-01. Pool

waters MC2W-03, MC2W-04, MC2W-05, MC2W-07, MC2W-08.

Spring waters MC2W-09, MC2W-10

Environ Geol (2009) 57:1169–1186 1175

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1994; Loisy et al. 1999; Melim et al. 2001). Similar mi-

crofabrics have been observed in marine environments and

cave speleothems elsewhere suggestive of microbial

involvement in speleothem precipitations (Chafetz 1986;

Melim et al. 2001; Baskar et al. 2007).

Microbiological investigations

Based on the aerobic isolation procedure described earlier,

around 30 purified aerobic, heterotrophic isolates were

obtained from both caves. The dominating bacterial iso-

lates obtained were identified by a combination of

conventional and molecular based tools (all results sum-

marised in Tables 2, 5) as: Bacillus cereus (two strains—

MCI01, MCI03); Bacillus mycoides (five strains—MCI07,

MCI08, MCI09, MCI10, MCI11); Bacillus licheniformis

(three strains—MCI04, MCI05, MCI06); Micrococcus

luteus (one strain—MCI02), Actinomycetes (three strains—

MCI12); a few strains of unidentifiable cocci (MCI18); and

of some unidentifiable strains of gypsum precipitating

bacteria (MCI14, MCI15, MCI16, MCI17). Comparison of

the retrieved partial 16S rRNA gene sequences of three of

these isolates with gene sequences available on the NCBI

website, showed high (99–100%) similarities to other cul-

turable organisms (Tables 2, 5). Additional bacterial

isolates which could be tentatively identified based entirely

on morphology were red-pigmented strains similar to

Streptomyces (MCI13). All the bacteria mentioned above,

as well as Actinomycetes-like bacteria were able to pre-

cipitate calcium minerals in the laboratory, and the type

and shape of the mineral varied with each bacterial strain

(Fig. 6a–d). In addition, the number and size of crystals

increased with time, reaching optimum level of growth

between 25 and 32�C (after 15 days for Bacilli strains);

however, the number of crystals decreased at higher tem-

peratures (Table 2). Our observations revealed that

although all the identified Bacillus strains were capable of

depositing calcium carbonate, the observed differences

were merely based on variations in the quantity and crystal

shape of precipitated calcium carbonate on the agar plate

colonies. The bacterially precipitated calcite differed

entirely in crystal structure from the pure inorganic calcite

crystal shape. Similar types of bio-minerals could also be

observed in the speleothem samples and, thus this resem-

blance suggests additional support for biogenic influence

on the cave formations at Meghalaya.

Gypsum and cave wall deposits

Spring waters emerge at three locations in the cave and

form a small stream that flows through the length of the

cave. Gypsum stalagmite mounds and gypsum deposits on

cave walls were encountered in the aphotic zone of the

Krem Phyllut caves. Interestingly, these deposits covered

5 m (length) and 2 m (high) of the cave wall. The water

that emerges from the cave springs is located in the aphotic

zone, pH *7.3. The cave walls are coated with gypsum as

identified by XRD and contained 94.08% CaO, 0.19–

0.32% Fe2O3, 0.074–1.54% P2O5, 1.30–1.54% SiO2

(Table 4). The different morphotypes observed by SEM

include rod-shaped microorganisms of different lengths

(\1 lm in length, long rods [1–2 lm), filamentous bac-

teria, cocci, vibrio and spirilla (Fig. 7a–d). Gypsum

crystals were precipitated in vitro from the cave wall

deposit samples that were cultured using sulphite and

thiosulphate agar (Fig. 7a–d). The so far unidentified

strains precipitated gypsum after 20 days of incubation

(Table 2). The EDX of the precipitated bio-mineral sug-

gests that it is composed predominately of carbon, calcium

and sulphur (Fig. 7e–i). The high concentration of carbon

as evidenced by EDX in the original cave wall and cultured

samples may possibly reflect the visible microbial coloni-

sation of the cave walls.

0

5

10

15

20

25

30

35

40

Mn

Elements

Co

nce

ntr

atio

n (p

pb)

MC1W-02

MC2W-01

MC2W-07

MC2W-08

MC2W-03

MC2W-05

MC2W-04

MC2W-09

MC2W-10

0

10

20

30

40

50

60

70

80

90

100

Na2O

Elements

Co

nce

ntr

atio

n (

wt%

)

MC1R12

MC1R18

MC2R02

MC2R08

MC2R11

MC2R13

MC2R15

MC2R23

MC2R25

VSiMgAgCrSrBaCdZnCu

TOCFe2O3MnOTiO2CaOK2OP2O5SiO2Al2O3MgO

A

B

Fig. 4 a Variations in trace elements in the cave waters. b Variations

in major elements in the speleothems

1176 Environ Geol (2009) 57:1169–1186

123

Table 4 Speleothem rock geochemical analysis (WDXRF), East Khasi Hill caves, Meghalaya

S.no. Na2O MgO Al2O3 SiO2 P2O5 K2O CaO TiO2 MnO Fe2O3 Sr TOC

MC1R12 0 0.2 0.06 0.19 0.07 0 53.7 0 0.01 0.13 52 1.3

MC1R18 0.01 0.22 0.55 1.49 0.16 0 94 0 0.01 0.28 322 1.521

MC2R02 0 0.77 0.05 0.12 0 0 53.9 0 0 0.07 123 1.716

MC2R08 0.12 0.44 0.29 1.3 0.07 0 94.1 0 0.01 0.19 228 1.892

MC2R11 0.02 0.92 1.01 6.32 0.05 0.2 49.1 0.1 0.01 0.75 336 1.463

MC2R13 0 0.29 3.24 62 0.15 0.2 3.18 0.9 0.03 19.8 ND 1.984

MC2R15 0.13 0.22 0.53 1.54 0.16 0 94.1 0 0.01 0.32 399 1.933

MC2R23 0.04 1.14 2.85 11.7 0.07 0.3 45.4 0.3 0.03 1.69 501 1.23

MC2R25 0 0.31 0 0.05 0.02 0 54.7 0 0 0.12 46 1.521

Concentrations wt %

Fig. 5 a Needle calcite, fibrous

calcite, micro-rods in the

speleothems: note the highly

weathered nature of calcite

(MC2R-02). b Enormous nano-

scale calcites, micro-rods as

small as 0.5–1 lm, needle

calcites, fossilised filaments

(MC1R-18). c Large needle

fibres, micro-rod calcites,

calcified filaments (MC2R-23).

d, e Spiky calcite, rounded balls

of calcite, a number of bacterial

ligaments, filaments in the

speleothems (MC2R-25;

MC1R-18)

Environ Geol (2009) 57:1169–1186 1177

123

Postulation of the biogenic role of microbes on the cave

formations in Meghalaya

The geological processes that are mediated by microbial

metabolism appear to be fundamentally related to the dis-

tribution of microbes throughout the cave (Jones 2001). It

is generally assumed that metabolic diversity is a reflection

of the different electron flow paths which are employed by

various microorganisms. Mineral surfaces provide periodic

arrays of reactive sites that absorb protons, water, metal

ions and organic molecules. Recent studies on stromatolite

forming microbial mats indicate that microbial activity in

Fig. 6 a, b Tentatively

identified as Actinomycetes like

bacteria, unidentified cocci, a

variety of tentatively identified

Bacillus like bacteria

precipitating calcite in in vitro

culture experiments: note the

different types and shapes of

crystals precipitated by different

strains of bacteria (MC2R-23,

MC2R-25). c, d Calcite

precipitated by tentatively

identified Bacilli like bacteria

in vitro (MC1R-18, MC2R-02)

Fig. 7 a, b Unidentifiable

bacteria precipitating gypsum

crystals in in vitro culture

experiments (bacteria cultured

from MC2R-08). c, d Different

strains unidentifiable bacteria

precipitating gypsum (bacteria

cultured from MC2R-15).

e Gypsum precipitated in

culture experiments. f EDX

showing composition of 2-K1 in

e. g EDX showing composition

of 2-K2 in e. h EDX showing

composition of 2-K3 in e. iSpectrum of precipitated

crystals of gypsum by

unidentifiable lab cultures

1178 Environ Geol (2009) 57:1169–1186

123

caves can result in: (1) calcification of individual microbes

by metabolic activities, (2) creation of an environment by

communities of microbes in which precipitation occurs,

and (3) production of extra cellular organic compounds that

control precipitation in the absence of microbes (Mark

et al. 2001).

Biogenic influences on speleothem carbonates

Examination of the speleothems in SEM showed a large

number of nano-scale calcites presumably to be inter-

preted as precipitated or calcified microorganisms in

association with different stages of mineralisation. The

highly weathered nature of the calcite crystals in the

speleothems suggests that they have been etched by

microbially mediated processes. The minerals precipi-

tated in vitro were found to concentrate within, or

around, dense matrices of the bacterial colonies and their

mucilaginous exopolysaccharides. Specific microenviron-

ments with geochemical conditions favorable for

microbe–mineral precipitation may thus develop in the

cave deposits due to invasion, followed by establishment

of bacterial microcolonies and subsequent bacterial

metabolic activities. In addition, since the bacterial

extracellular polymers may bind ions from solution, they

may, therefore, provide nucleation sites for mineral

2-K 1 HV: 15.0 kV Puls th.: 2.65 kcps

2-K 2 HV: 15.0 kV Puls th.: 3.26 kcps 2-K 3 HV: 15.0 kV Puls th.: 4.06 kcps

Spectrum: 2-K 1 El AN Series unn. C norm. C Atom. C Error

[wt.-%] [wt.-%] [at.-%] [%] C 6 K-series 33.53 33.53 44.17 10.9 Ca 20 K-series 15.75 15.75 6.22 0.5 S 16 K-series 4.12 4.12 0.55 0.1 O 8 K-series 49.60 49.60 49.06 16.4

Spectrum: 2-K 2 El AN Series unn. C norm. C Atom. C Error

[wt.-%] [wt.-%] [at.-%] [%] C 6 K-series 28.41 28.41 38.42 9.3 Ca 20 K-series 16.91 16.91 6.85 0.5 S 16 K-series 5 .12 5.12 0.57 0.1 Na 11 K-series 0.69 0.69 0.49 0.1 O 8 K-series 52.87 52.87 53.67 16.9

Spectrum: 2-K 3 El AN Series unn. C norm. C Atom. C Error [wt.-%] [wt.-%] [at.-%] [%]

C 6 K-series 20.49 20.49 28.40 6.8 Ca 20 K-series 17.09 17.09 7.10 0.5 S 16 K-series 3.89 3.89 0.46 0.1 O 8 K-series 61.53 61.53 64.04 19.3

0 2 4 6 8 10 12 14keV

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

cps/eV

Ca

Ca

Sc Sc Cl

Cl S S

Na O

0 2 4 6 8 10 12 14keV

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

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cps/eV

Ca

Ca

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0 2 4 6 8 10 12 14keV

0.0

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3.0

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Ca

Ca

I I Cl Cl

S S P

E F

G H

I

Fig. 7 continued

Environ Geol (2009) 57:1169–1186 1179

123

crystallisation and growth. Bacteria may also act as

highly reactive geochemical interfaces (Beveridge1989;

Fein et al. 1997) and their extracellular polymers are

especially effective at binding ions from solution and

serving as nucleation surfaces for mineral formation

(Ferris et al. 1987; Beveridge 1989; Fortin et al. 1997).

Their metabolic activities can, therefore, induce localised

conditions that are favorable for mineral precipitation

(Thompson and Ferris 1990; Merz 1992; Fortin et al.

1997; Barker and Banfield 1998). Furthermore, it has

now become established that also biologically produced

exopolysaccharides (EPS) influence calcite precipitation

rates considerably.

Bacterially induced precipitation of calcium carbonate,

the so-called carbonatogenesis (Kowalchuk et al. 1997),

has drawn much attention in recent decades because of its

numerous implications. For example, certain strains of

Bacillus and Actinomycetes have been reported to precip-

itate calcium minerals (Cacchio et al. 2003; Rivadeneyra

et al. 1993; Baskar et al. 2006). The strain Bacillus cereus

is used as a conservation treatment for ornamental stone

(Kowalchuk et al. 1997), and the strain Bacillus mycoides

has been reported to be closely associated with carbonate

speleothems (Ehrlich 1996).

Interestingly, our isolation efforts yielded indeed some

strains (MCI01, MCI03—Bacillus cereus) that showed

nearly 100% identities to some different Bacillus strains

with at least some of the above-mentioned capacities.

Further physiological characterisation of some of the

metabolic capabilities of these strains, showed that strains

MCI01, MCI03, MCI04, MCI05, MCI06, MCI07, MCI08,

MCI09, MCI10, MCI11 are facultatively anaerobic, pro-

duce acid, follow the ammonification and nitrate reduction

metabolic pathway (Table 5) and are able to precipitate

calcite in vitro. This is a heterotrophic pathway that

involves the ammonification of amino acids and nitrate

reduction, and is often observed in geological sediments

(Fujita et al. 2000). These involve the production of met-

abolic CO2 and ammonia (NH3) which in the presence of

calcium ions lead to precipitation (Hammes and Verstraete

2002). Interestingly, it has indeed been reported that

Bacillus cereus precipitates calcite through the ammonifi-

cation and nitrate reduction metabolic pathway (Castanier

et al. 1999).

Two of our other isolates were identified as Bacillus

licheniformis and Micrococcus luteus. Since it has been

reported that Bacillus licheniformis is facultatively anaer-

obic and also reduces nitrate with/without N2 gas

production, these strains could, therefore, possibly also

precipitate calcite through the ammonification and nitrate

reduction metabolic pathway (Castanier et al. 1999).

Micrococcus luteus is a strict aerobe, and oxidizes carbo-

hydrates into CO2 and water and produces the enzyme

catalase. This CO2 production helps in the mediation of

carbonate precipitation.

Ten of the characterised isolates (MCI01, MCI03,

MCI04, MCI05, MCI06, MCI07, MCI08, MCI09, MCI10,

MCI11) may through the ammonification and nitrate

reduction metabolic pathways (Hammes and Verstraete

2002), induce an overall pH increase, which can in turn

shift the bicarbonate equilibrium. This, results in the for-

mation of carbonate ions which, in the presence of soluble

calcium ions, precipitate CaCO3, as summarised by the

reactions listed below:

CO(NH2Þ2 þ H2O! CO2 þ NH3

2NH3 þ CO2 þ H2O! 2NHþ4 þ CO2�3

HCO�3 þ Hþ þ 2NHþ4 þ OH� $ CO2�3 þ 2NHþ4 þ 2H2O

CO2�3 þ Ca2þ $ CaCO3:

As the above mentioned reactions indicate, microbial

carbonate precipitation always depend on metabolic CO2

production/CO2 consumption and the relative insolubility

of calcium carbonate, and their metabolic activities are

proven to participate at carbonate mineral precipitation

(Ehrlich 2002).

Although our aerobic isolation efforts on nutrient-rich

media have probably only retrieved a minor and possibly

even biased fraction of the total existing prokaryotic

diversity in the caves investigated in this study, we have

nevertheless obtained some isolates that seem to be at least

theoretically able to participate at precipitation reactions

that may take place in the cave. Thus, together with our

petrographic observations and SEM studies, these obser-

vations seem to add additional support to the generally

formed hypothesis about the biogenic influence of the

speleothem genesis.

Biogenic influence on gypsum and cave wall deposits

Microbes may play an active part in changing the nature of

limestone rocks, both by dissolving it as well as by con-

tributing to the creation of a range of deposits on the cave

walls. The amount of light entering the cave and the type

of microbe inhabiting each part of the cave system

may, however, differ. For example, the microbial biota

inhabiting totally dark zones are most likely mainly

chemolithotrophs (Northup et al. 1997). Since we did not

attempt to isolate chemolithotrophs in the present study, we

were not able to identify presumably chemolithotrophic

CaSO4 precipitating bacteria in the complete aphotic zone

where gypsum is precipitated on the cave walls. However,

the heterotrophic isolates that we obtained (MCI07,

MCI08, MCI09, MCI10, MCI11—one of the dominant

species in the microbial community obtained by the

1180 Environ Geol (2009) 57:1169–1186

123

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Environ Geol (2009) 57:1169–1186 1181

123

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1182 Environ Geol (2009) 57:1169–1186

123

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Environ Geol (2009) 57:1169–1186 1183

123

traditional aerobic isolation effort), showed high similarity

to Bacillus mycoides, which has been shown to be able to

produce H2S (Tables 2, 5). Thus microbial H2S oxidation

to sulphuric acid followed by subsequent reaction of the

sulphuric acid with limestone to precipitate gypsum could

indeed take place in the cave, as suggested by the following

chemical reactions:

H2Sþ O2 $ Hþ þ HSO�4 $ 2Hþ þ SO2�4

2Hþ þ SO2�4 þ CaCO3 $ Ca2þ þ SO2�

4 þ H2Oþ CO2

Ca2þ þ SO2�4 þ H2O! CaSO4 � 2H2O:

Furthermore, as previously mentioned, we observed by

SEM also unidentifiable strains of presumably CaSO4-

precipitating bacteria; this group could thus contribute as

an additional possible factor for gypsum precipitates. Thus,

biological activity can significantly accelerate the oxidation

of H2S, causing the production of H2SO4 as a by-product of

their metabolism. Therefore, cave gypsum precipitates may

also in some cases be considered as a consequence of

bacterial activity (Galdenzi et al. 1999). Pyrite FeS2, which

was identified in our pool sediments and was interspersed

with the limestone deposits, could also serve as another

oxidation source to sulphuric acid by the local bacterial

community, which in turn then may react with limestone to

form gypsum.

Generally, sulphur oxidizers retrieve their energy by

oxidising sulphur and acquiring their carbon from the air,

thus converting carbon dioxide into organic material. In the

dark aphotic cave ecosystems, bacteria, tentatively postu-

lated to have a ‘‘chemosynthetic’’ metabolic lifestyle may

thus possibly form the basis of the food chain in this habitat.

We anticipate that a wide range of different bacterial groups

may be found on the cave walls and springs in the dark,

pitch black aphotic zones, such as sulphate-reducing bac-

teria and sulphur-oxidising bacteria, with species such as

Thiothrix, Thiovulum, Desulfovibrio. These organisms may

utilize sulphate, thiosulphate (S2O3-), sulphite (SO3

-), or

other reducible sulphur-containing ions as terminal electron

acceptors in their respiratory metabolism. Based on these

metabolic activities, sulphur-containing ions can, therefore,

be reduced to hydrogen sulphide. The hydrogen sulphide

thereafter mixes with oxygen to form sulphuric acid, which

dissolves the limestone rock, leaving behind a deposit of

gypsum. A number of bacteria are known to function in the

transition zone between aerobic and anaerobic environ-

ments, particularly in the cave wall biofilms associated with

elemental sulphur and formation of gypsum crystals. The

sources of gypsum in caves differ depending on the specific

caves.

The proposed postulations in this study are based

on geochemical observations in the field, coupled with

laboratory experiments and drawing on the general

knowledge about cave environments and microbial activi-

ties. Based on our geochemical and initial microbiological

observations, at least some of the isolated bacteria seem to

be able to contribute to the proposed mineral precipitation

reactions, thus, confirming the role of microorganisms in

the mineral formations. However, since culture indepen-

dent molecular techniques have shown that only a minor

fraction of the total microbial diversity is culturable (see

e.g. Amann et al. 1996), future studies will be employed to

implement other tools, such as culture-independent tech-

niques to unravel further the hidden biodiversity of the

caves investigated in this study. This will expand our

knowledge considerably about the overall microbial pro-

cesses and influences in the cave, and confirm as well as

expand the basic hypotheses that we have laid in this paper.

Conclusions

In this paper, we present further evidences that support

current general hypotheses on the bacterial involvement in

speleothem cave formations, exemplified with novel

investigations of a so far non-explored cave, the Meghalaya

caves, in India. Our laboratory experiments confirm that (1)

carbonate precipitation occurs during microbial CaCO3

precipitation in a closely related group of bacteria fol-

lowing the ammonification/nitrate reduction pathway; and

(2) gypsum precipitates are formed by microbial mediated

geochemical reactions.

Several processes appear to be acting in tandem induc-

ing the precipitation of carbonates and gypsum observed in

the cave systems studied. This combination of microbio-

logical and inorganic transformation processes may explain

the biogenic involvement in the formation of the speleo-

thems and cave wall deposits in these and in other caves

systems. The observations suggest that the microbes and

their metabolic activities have definitely influenced the

formation of these cave deposits. Further studies, based on,

e.g. stable isotope chemistry and culture independent

molecular techniques will be employed to cast further light

on the mechanism and extent of the microbial role in the

speleothem genesis of the cave areas investigated in this

study, to confirm and expand the basic hypotheses laid in

this study.

Acknowledgments S. Baskar and R. Baskar thank the Wadia Insti-

tute of Himalayan Geology, Dehradun (WIHG), for laboratory and

library facilities (ICP-MS, XRD, SEM-EDX) and Institute of Micro-

bial Technology (IMTECH), Chandigarh for bacterial identification.

R. Baskar thanks the World Bank for financial assistance to attend the

FISH training programme in connection with this project at Technical

University, Munich, Germany. R. Baskar thanks UGC, New Delhi for

financial assistance in the form of Major research project.

1184 Environ Geol (2009) 57:1169–1186

123

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Documents

Speleothems from Mawsmai and Krem Phyllut caves, Meghalaya, India: some evidences on biogenic activities