Ultrastructural analysis of calcite crystal patterns formed by biofilm bacteria associated with cave speleothems

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Journal of Microscopy and Ultrastructure 2 (2014) 217–223

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Journal of Microscopy and Ultrastructure

jo ur nal homep age: www.els evier .com/ locate / jmau

Ultrastructural analysis of calcite crystal patterns formed bybiofilm bacteria associated with cave speleothems

Subhro Banerjee, S.R. Joshi ∗

Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University Shillong 793 022, Meghalaya,India

a r t i c l e i n f o

Article history:Received 26 March 2014Received in revised form 13 May 2014Accepted 5 June 2014Available online 16 June 2014

Keywords:UltrastructureCaCO3 crystalsSpeleothemsBiofilm bacteriaBiogenic

a b s t r a c t

The ultrastructural morphology of calcium carbonate (CaCO3) crystals precipitated byeight culturable speleothemic biofilm bacteria associated with not-so-far explored caves ofMeghalaya were studied using electron microscopy. The isolated bacteria under in vitro con-ditions precipitated CaCO3 in the media when supplemented with proper calcium sourcewhich was not the case in dead cells suggesting that calcification required metabolic activ-ity in the speleothemic environment. Scanning electron microscopy revealed the presenceof various polymorphs of calcite distinctly different from inorganic CaCO3 crystals. Thepolymorphic crystals ranged from filaments and rods associated in large clusters with min-eral crystals to spiky needle. The results endorse the hypothesis that the isolated bacterialspecies contribute to active biogenic influence in the process of cave formations in thehypogean environment.

© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Caves offer excellent habitats for studying biominer-alization processes because they are stable environmentswhere microbial fabrics can be preserved without exten-sive changes [1]. Biofilms, which are complex aggregationof microorganisms embedded in a self-produced matrix [2]can be found adhered to rock surfaces of such caves, com-monly referred as speleothems. There is also considerablespatial bacterial diversity associated with biofilm in thesehypogean (subterranean) environments revealing that themicrobial community associated with biofilm may be morediverse than previously thought [3].

Calcium carbonate speleothems dominate most knowncaves of the world and recent studies have identified someof the factors that control the contribution of microbes to

∗ Corresponding author. Tel.: +91 9436102171; fax: +91 3642550076.E-mail address: [email protected] (S.R. Joshi).

CaCO3 precipitation [4]. Laboratory experiments supportthat the microbial species isolated from these minerals canproduce similar crystals from organic calcium salts [5–7].Calcium carbonate precipitation is a common process insome bacterial species with a high percentage recorded onmedia containing calcium acetate [8].

India has a large number of unexplored caves and thecaves in Meghalaya are among the largest attributed tohuge deposits of limestone in soils and abundant rainfall[3]. The present study was aimed at isolating the calcify-ing bacteria inhabiting these caves and to analyze usingscanning electron microscopy the biogenic polymorphiccalcium crystals prevalent in the speleothems.

2. Materials and methods

2.1. Isolation of calcifying bacteria

The eight calcifying bacterial strains selected during thisstudy were isolated from four caves in East Khasi Hills

http://dx.doi.org/10.1016/j.jmau.2014.06.0012213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.


218 S. Banerjee, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 217–223

district (3 from Mawsmai cave, 2 from Mawjymbuin caveand 1 each from Mawmluh and Dam caves) and East JaintiaHills (1 from Labit cave) (Table 1).

The biofilm samples from the speleothems of the caveswere collected aseptically in sterile tubes and kept at 4 ◦Cuntil analyzed. One gram of the samples were asepticallytransferred into 9 ml sterile Ringer salt solution [SRL, India](1/4 strength) and vortexed briefly for 2 min. Sample dilu-tions ranging from 10−1 to 10−5 were plated on B-4 agarmedium (2.5 g L−1 calcium acetate, 4 g L−1 yeast extract,10 g L−1 glucose and 18 g L−1 agar) [9] and were spread uni-formly with a sterile spreader. All experiments were carriedout in triplicate with controls consisting of uninoculatedculture medium and medium inoculated with autoclavedbacterial cells as source of dead cells. The plates were incu-bated aerobically at 25 ◦C (to mimic cave temperatures) inan inverted position for 2 weeks. Each isolate was period-ically examined upto 25 days for the presence of crystals.Individual colonies were selected and purified by streakingon B-4 agar.

Calcifying bacteria were preserved by various methods[10]. For short-term preservation, bacterial strains wereincubated at 32 ◦C for 2–5 days and then kept at 4 ◦C onslants of B-4 medium. For long-term maintenance, cultureswere incubated in B-4 liquid medium for different periods,depending on the growth rate of the culture, harvested bycentrifugation at 3000 rpm at 4 ◦C for 20 min, washed twicein NaCl (0.9%), resuspended in fresh B-4 medium and thenfrozen at −80 ◦C as bacterial suspensions in 17% glycerol.

2.2. Ultrastructural analysis of the crystals

Morphology and size characteristics of both the crys-tals and the microorganisms were studied by scanningelectron microscopy [JSM 6360 (JEOL); resolution, 3 nm;magnification, 8–300,000×; accelerating voltage, 1–30 kV].SEM samples were prepared as follows: agar blocks of cul-tures grown on B-4 medium were fixed onto aluminumstubs with two-way adherent tabs with conductive paint,and allowed to dry [11]. They were then gold-coated bysputtering for approximately 2–3 min dried at 37 ◦C, goldshadowed, and observed under the microscope [4]. Bacte-ria are generally nonconductive in nature and therefore thesurface of such samples needs to be coated with a thin metalfilm so that the surface has conductivity. Therefore in thepresent study gold sputtering is done (at a low vacuum ofabout 10 Pa) forming a uniform film on the surface.

2.3. Characterization of calcifying bacteria byamplification of 16S rRNA

Once calcification by the bacterial isolates was con-firmed, PCR amplification of the 16S rRNA gene sequencewas used to determine their identity. Genomic DNA iso-lation was done by HipurTM Bacterial Genomic DNAPurification Spin Kit (HiMedia, India) and the DNA bandswere visualized in 0.8% agarose gel stained with ethidiumbromide. Genomic DNA content and purity were estimatedusing a NanoVue Plus Spectrophotometer (GE Healthcare’sLife Sciences, Sweden). Bacterial 16S rDNA was ampli-fied using the universal bacterial 16S rDNA primers, 27F Ta

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S. Banerjee, S.R. Joshi / Journal of Microscopy and Ultrastructure 2 (2014) 217–223 219

[5′-AGA GTT TGA TCC TGG CTC AG-3′] and 1541R [5′-AAGGAG GTG ATC CAG CCG CA-3′] [12] under the following con-ditions in Gene AMP PCR system 9700 (Applied Biosystems,California, USA): initial denaturation for 5 min at 94 ◦C, fol-lowed by 35 cycles consisting of denaturation at 94 ◦C for1 min, annealing at 55 ◦C for 1 min, elongation at 72 ◦C for2 min and then cycling was completed by a final elonga-tion step for 5 min at 72 ◦C. A control tube containing sterile

water instead of DNA solution was used as a negative con-trol. PCR products were analyzed by electrophoresis in 1.5%(w/v) agarose gel in 1× TAE buffer with ethidium bromide(0.5 !g/ml). PCR products were purified using QIAquick GelExtraction Kit (Qiagen, Germany). Sequencing reactions ofthe 16S rDNA fragments were performed with the Big DyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosystems,USA) using the above forward and reverse primers. The

Fig. 1. Phylogenetic tree constructed for the isolates using neighbor joining method.



nucleotide sequences were then deposited in GenBank andaccession numbers obtained.

2.4. Phylogenetic analysis of bacteria

The 16S rRNA gene sequence of the isolates andtheir closest match were retrieved from EzTaxon server[http://www.eztaxon.org/] [13] and aligned using ClustalW with MEGA software version 4.1 [14]. Neighbor-joiningmethod was employed to construct the phylogenetic treewith 1000 bootstrap replications to assess nodal support inthe tree [15].

3. Results

The bacteria isolated from different speleothemicniches had different morphology. Isolation of bacteria fromthe biofilm in the laboratory showed varied species ofbacteria (not shown in the paper). Only the representativebacterial isolates which showed precipitation are men-tioned in the study. The duration for calcite precipitationby these bacteria under laboratory conditions ranged from12 to 25 days. A phylogenetic tree was also constructedfor all the isolates using neighbor-joining method with1000 bootstrap replications (Fig. 1). Majority of the calcify-ing bacteria isolated from the cave environment belongedto the genus Bacillus and Lysinibacillus whilst the leastbelonged to genera Acinetobacter, Kocuria and Brevibacillus(Table 1).

Ultrastructural studies using scanning electronmicroscopy showed that under laboratory conditions,the isolates were capable of forming crystalline CaCO3at 25 ◦C in B-4 medium containing calcium acetate. Thisobservation confirms that under appropriate conditions,especially in carbonate-rich environments such as a karstcave (large areas of limestone deposition with distinctivesurface and underground geomorphological features),many bacteria can form CaCO3 crystals. However, nocrystals were detected on control plates inoculated with

heat-killed (autoclaved) cells indicating the calcifyingbacterial strains as heterogeneous nucleus for the pre-cipitation of carbonates through their metabolic activity.The CaCO3 precipitation phenotype on B-4 medium formany isolates was lost when they were cultivated on othermedia. The calcification process could be restored by priorpassage of the bacteria on media containing either calciumacetate or CaCO3 powder.

The SEM observations revealed that both size and shapeof the newly formed crystals varied with the bacterialstrains selected for the calcification studies. Bacillus strainswere the most common bacteria found among the calcify-ing isolates which led us to hypothesize that Bacillus spp.may play a major role in carbonate deposition in natu-ral habitats [5]. The size and shape of the microbial rodsof Bacillus thuringiensis, isolated from Labit cave can beobserved on the surface or around the crystals (Fig. 2A).Bacillus cereus, isolated from Mawjymbuin cave formedspiky needle fiber crystals of calcite (Fig. 2B) whereaslarge micro-rod filaments of calcite could be observed incase of Bacillus halodurans, isolated from Mawmluh cave(Fig. 2C). The classification of micro-rod calcite and itsorigin has been attributed to rapid precipitation at highsuper-saturation states during evaporation in soils or tocalcification of bacilliform bacteria [16]. Our observationsrevealed that although all the identified Bacillus strainswere capable of depositing calcium carbonate, there weresharp variations in the quantity and crystal shape of theprecipitated mineral. The presence of these highly var-ied structures of calcite indicated their formation throughmicrobial activities [17,18].

The two Lysinibacillus spp. (Lysinibacillus macroides andLysinibacillus parviboronicapiens) isolated from Mawsmaiand Mawjymbuin caves, respectively, were found to beconsistently precipitating in the medium. The morpholog-ical analysis of calcite precipitated by L. macroides showedthe predominance of spherulites as isolated spheres orgroups of a few spherulites were seen covered by smoothlayers (Fig. 3A). Precipitation by L. parviboronicapiens was

Fig. 2. Morphotypes of crystals and their close association with bacterial cells isolated from the Labit, Mawjymbuin and Mawmluh caves, respectively (barsdenote the magnification in the figure). (A) Bacterial prints of Bacillus thuringiensis isolated from a stalagmite associated with the precipitated crystal ofLabit cave. (B) Spiky crystals of Bacillus cereus isolated from cave wall deposit of Mawjymbuin cave. (C) A filament of calcite formed by Bacillus haloduransisolated from cave wall deposit in Mawmluh cave.



Fig. 3. Morphotypes of crystals and their close association with bacterial cells isolated from the Mawsmai and Mawjymbuin caves (bars denote themagnification in the figure). (A) Spherical crystals of Lysinibacillus macroides isolated from a column of Mawsmai cave. (B) Large aggregate of individualmicrostone precipitated by Lysinibacillus parviboronicapiens isolated from a column of Mawjymbuin cave. (C) Association of rhombic crystals with newlayers of biolith of Acinetobacter johnsonii isolated from cave wall deposit of Mawsmai cave. (D) Spiky crystals of Kocuria rosea isolated from a stalactite ofMawsmai cave.

observed as a large aggregate of the individual biolithsbinding with non-globular carbonate bridges in between(Fig. 3B). SEM observations of Acinetobacter johnsonii,isolated from Mawsmai cave showed rhombic crystals ofcalcite in association with layers of bioliths (Fig. 3C), whichcorroborates to the observations of Ferrer et al. [19]. Thebioliths, as observed in Fig. 3C also contained numerousridges and depressions that included structures whichwere indicative of cell growth and division. Kocuria rosea,

isolated from the same cave formed similar spiky needlefiber crystal pattern (Fig. 3D) as that of B. cereus. Similarmicrofabrics have been observed in marine environmentsand cave speleothems elsewhere which is suggestiveof microbial involvement in speleothem precipitations[20,21].

Brevibacillus agri, isolated from Dam cave formed twotype of crystals: large rounded balls of calcite and calcifiedfilaments. The large ball of calcite seemed highly weathered

Fig. 4. (A) Large calcitic ball, and (B) numerous calcitic filaments formed by Brevibacillus agri isolated from a cave wall deposit of Dam cave.



and fibrous in nature with sponge like non-globular bridges(Fig. 4A). Numerous calcitic filaments can also be seen sim-ilar to that precipitated by B. halodurans. Rod shaped cell ofBr. agri can also be seen in close association with the biolith(Fig. 4B).

4. Discussion

Bacterial consortia in caves acquire energy by variouspathways (transforming aromatic compounds, fixing gases,and oxidizing reduced metals within rocks) and by theirinteraction with minerals. This plays an important role inreshaping the mineral environment of caves and helps formfeatures such as stalactites, stalagmites and various cavewall deposits [1,4].

The present study showed that the biofilm bacteriapresent in the calcareous speleothem fragments from thecaves could be cultivated on B-4 medium in the labora-tory mimicking the ideal cave conditions and parameters.This demonstrated a close association between the bac-terial cells as biofilms growing in the cave walls and thenature of polymorphic crystals. Killed cells were unable toprecipitate CaCO3 crystals in B-4 media, suggesting thatcells need to be metabolically active for calcification andthat cell structure alone is not sufficient to promote theprocess. Such calcifying bacteria might promote passivecarbonate precipitation in the studied caves, by modifyingthe medium as a result of the physiological capabilities [22].The production of such CaCO3 particles is known as pas-sive carbonatogenesis [23]. The role of microbial speciesin the development of secondary carbonate deposits incaves has remained controversial for quite some time [24],with mostly anecdotal evidence of microbial associationwithin speleothems [7] and the lack of a cause-and-effect rationale [25]. Irrespective of the pathway, bacterialmetabolic activity in these environments appears to leadto the precipitation of various polymorphs of CaCO3. Thepresent observation corroborating earlier reports [8,26]suggests that bacterial metabolism plays a dominant rolein calcification process. Furthermore, it has now becomeestablished that biologically produced exopolysaccharides(EPS) too influence calcite precipitation rates considerably[4]. Within the microbial biofilms, microcrystals may formwhen the conditions (pH and concentrations of ions) aresuitable for mineral precipitation. These crystals, depositedon the cave walls, may form a layer which when becomethicker and heavier, may detach and accumulate, formingthick deposits. If the microcrystals are generated withinbiofilm formations hanging from the walls and ceilings,visible crystals can be seen developing on their surface [27].

The ultrastructural revelations of the polymorphs ofcrystals suggest that the identified strains were capable ofdepositing CaCO3, with distinct variations in the quantityand crystal shape of precipitated CaCO3 under culture con-ditions. The bacterially precipitated calcite differed entirelyin crystal structure from the pure inorganic calcite crystalas reported earlier [4]. Similar types of bio-minerals couldalso be observed in the speleothem samples and thus thepresent work supplements evidences for biogenic influ-ence on the cave formations.

5. Conclusion

The study produced concrete proof based on SEM stud-ies that support the involvement of biofilm bacteria inspeleothem formations through calcite precipitations incaves, when investigated for lesser known caves of Megha-laya. The nature of carbonate crystals produced by thestudied bacterial strains was found to be strictly calcitecrystals as they developed in media supplemented withcalcium source. The bacterial precipitation of CaCO3 hasbeen overlooked by some researchers as a passive one,as information is scarce to link calcium metabolism withmicrobial energetics. Therefore, further studies based oncalcium ion sequestration, X-ray diffraction studies andstable isotope chemistry could shed further light on thebiogenic role in speleothems formation.

Conflict of interest

The authors declare that there is no conflict of inter-est and confirm that this manuscript does not infringe anyother person’s copyright or property rights. All authorshave contributed equally to the manuscript and agreed topublication of the work.

Acknowledgements

The study was accomplished within the framework ofa research project supported by Department of Electronics& Information Technology (DeitY), Government of India.Authors thank Sophisticated Analytical Instrument Facil-ity (SAIF), NEHU, Shillong for providing the SEM services.SB would also like to acknowledge the financial assistancereceived from DeitY and DST-FIST-PURSE programme ofNEHU, Shillong and the help rendered by K. Bhattachar-jee, A. Lamare, M. Rynghang and V. Joshi in collection ofsamples from the sites.

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Ultrastructural analysis of calcite crystal patterns formed by biofilm bacteria associated with cave speleothems