Paper II: Biological Study

1. Abstract

2. Introduction

In recent years, stable isotope records in speleothems (i.e., calcium carbonate deposits found

in caves) have become more and more important as proxies of past climate variability (e.g.,

Spotl and Mangini (2002), Fleitmann et al. (2004), Harmon et al. (2004)). Speleothems, whiczare found in most continental areas provide high resolution records and can be precisely dated by U-series (Scholz and Hoffmann (2008)). The stable isotope signals of carbon δ13 C and δ18 O recorded in stalagmites are the most widely used proxy to reconstruct past climate changes. The isotopic composition of the drip water which feeds the stalagmites is influenced by several climate dependent processes occuring i) above the cave (e.g., geographical position, amount and type of vegetation, rainfall amount, temperature), ii) in the soil/karst zone (e.g., flow path of the solution, pCO2, host rock dissolution occuring under open or closed conditions iii) in the cave and on the surface of the speleothems (e.g., drip rate, pCO2). Inside the cave calcite precipitation results from the difference in pCO2, leading to a progressive degassing of CO2 from the drip water and hence, to an increase in supersaturation with respect to calcite. The isotopic composition of the precipitated calcite depends on the isotope value of the drip water and on isotope fractionation processes between the different species involved in calcite precipitation.

A recent study by Mickler et al. (2006) indicated that most of the investigated stalagmites

show evidence for calcite precipitation under kinetic conditions. To interpret the stable

isotope signals of such stalagmites in terms of past climate variability, it is necessary to

understand the chemical, biological and physical reactions occuring in the drip water during calcite precipitation.

In recent years, numerical models have been developed describing the evolution δ13 C and δ18 O in the drip water in dependence of several parameters such as temperature, drip

rate, and initial saturation state of the drip water (Mickler et al. (2006), Romanov et al.

(2008), Dreybrodt (2008)). Most of these models are based on a Rayleigh fractionation process. The model results strongly depend on the rate constants used to describe the chemical reactions and the fractionation factors (equilibrium and kinetic). Some of these rate constants have not been determined precisely yet.

In addition, the models do not take into account the possible influence of microbial induced crystal growth mechanisms (Kunz and Stumm (1984), Lebr´on and Su´arez (1998), Fernandez- Diaz et al. (2006)), which depend on the degree of supersaturation and/or the presence of other ions in the drip water such as organic material or trace elements.

Laboratory experiments are, thus, necessary to quantitatively determine these parameters.

Furthermore, they allow to detect processes not yet considered in theoretical models.

Within this work laboratory experiments with synthetic carbonates were performed under

controlled conditions to improve the understanding of isotope fractionation of oxygen and

carbon stable isotopes in speleothems.

2a. Intention

Calcite precipitation in caves is influenced by several parameters such as temperature, drip rate, solution composition and pCO2. Low temperature, high drip rates and low super saturation of the solution with respect to calcite preferentially lead to equilibrium isotope fractionation between the calcite and corresponding drip water. In this case the fractionation factor only depends on temperature and could theoretically and experimentally be determined for the different species involved in the calcite precipitation process. However, for the most natural cave systems this is only valid to some extent. More often evidence exist for kinetic fractionation between calcite and drip water indicated by the enrichment of both δ18O and δ13C along a single growth layer and by a positive correlation between these isotopes along a growth layer, respectively (Hendy (1971)). In this case interpretation of the isotope data in terms of climate change is much more complicated because kinetic isotope fractionation not only depends on temperature, but also on various parameters, which can be temperature- dependent themselves.

By now several model calculations have been carried out to describe the evolution of the δ18O and δ13C value of the bicarbonate in the solution (e.g., Mickler et al. (2006), Romanov et al. (2008), Dreybrodt(2008), Scholz et al. (2009), Muhlinghaus et al. (2009). All model calculations depend on the different chemical reaction rates included in the calcite precipitation mechanism such as calcite precipitation rate, HCO3 -CO2-conversion rate, or the CO2-hydration and CO2- hydroxilation rates. The latter are important in the case of modeling the evolution of δ18O, which is much more complicated than δ13C because of the

influence of the water reservoir, which buffers the oxygen isotope composition.

The experiments, help to investigate and quantify the effects induced by biological and kinetic isotopic fractionation under varying parameters such as temperature, drip rate or solution composition. In addition, these (2) sets of experiments provides information about the general aspects of calcite precipitation under varying parameters for long duration (40 days). In these experiments, the biological and kinetic fractionation pattern between calcite and nutrient solution is studied by enrichment of both δ13 C and δ18 O for 40 days. The positive correlation between these isotopes along growth layer will be used as evidence for biological precipitation.

2b. Summary of previous studies

Several laboratory experiments with synthetic carbonate were carried out under varying aspects (e.g. Fantidis and Ehhalt (1970), Kim and O’Neil (1997), Huang and Fairchild (2001), Fernandez- Diaz et al. (2006), Wiedner et al. (2008)). Kim and O’Neil (1997) studied the effect of different initial concentrations of calcium- and bicarbonate-ions on the oxygen isotope composition by bubbling N2 through a saturated CaCO3-solution. This promotes supersaturation and hence, calcite is precipitated through removal of CO2. They concluded that the oxygen isotope fractionation factor strongly depends on the initial concentration of the calcium and bicarbonate-ions and presented a new oxygen isotope equilibrium fractionation factor resulting from appropriate conditions of precipitation’. Huang and Fairchild (2001) simulated stalagmite growth by pumping CaCl2- and NaHCO3-solutions through a tube, where the solutions are mixed. Afterwards the solution drips out of a natural or glass straw (analogous to a stalactite) onto a natural stalagmite or a precoated convex glass plate. The intention of their experiments was to determine of the temperature dependent partitioning coefficients for Sr2+ and Mg2+.Fernandez-Diaz et al. (2006) investigated the influence of divalent cations on calcite crystal morphology using a column of porous silica hydrogel, in which calcite precipitated during a period of one year.

The laboratory experiments in this study( Drip Experiment) are based on the set-up of Fantidis and Ehhalt (1970), who used 1.5 m long semicircular glass channels with a diameter of 15mm, which were slightly inclined. On the channels a steadily flowing bicarbonate solution with an adjustable flow speed led to the precipitation of calcite by degassing of CO2. This set-up, which is described in detail in the PhD-thesis of Daniela Polag (2009), mimics the flow of solution along a stalagmite. Thus the isotopic evolution of the precipitated calcite can be investigated with the main focus on kinetic and biological isotope fractionation.

In recent years, Wiedner (2004), Gewies (2003) and Marz (2006) modified and improved the set- up of Fantidis and Ehhalt (1970) and carried out laboratory experiments to investigate the fractionation processes of δ18O and δ13C isotopes within synthetic carbonates.

Wiedner (2004) studied the fractionation processes in stagnant water as well as in experiments using a flowing solution under varying parameters such as temperature, solution composition, reservoir and flow velocity.

In contrast to the experiments of Daniela Polag (2009), the other experiment set (Bottle Experiment) is designed to study the change in calcite precipitation rate and isotopic fractionation pattern due to the biological precipitation.

2c. Comparison between cave system and laboratory set-up

Fig. BB shows the comparison between a natural cave system and the laboratory setup. Both systems simulate similar conditions essential for calcite precipitation. They offer a gradient in pCO2 levels and the sloped channel represents an analogue for the stalagmite where the solution flows along the side forming the single growth layers

1) Super saturation in the cave drip water with respect to calcite mainly results from the sudden loss of CO2 when the dripwater impinges on the stalagmite tip. The laboratory experiments do not simulate this process due to the small distance between the drip capillaries and the glass fiber strips causing the drops to slide over the filter instead of falling.

2) However the initial solution within the experiment is already supersaturated with respect to calcite comparable to the values of the drip water after impinging. In addition, the solution is far from CO2equilibrium when reaching the channel system. For the cave drip water it is not really clear to what degree the equilibrium with the cave pCO2 has reached after the impinging. Dreybrodt (2008) for example states that after the impinging the drip has equilibrated with the cave pCO2, but quantitative measurements are lacking.

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Figure BB: Comparison between the cave system (left) and the laboratory set-up (right)

3. Study area and site description

Located in Northeastern part of India lies Meghalaya covering an area of about 22,429 square Km (Figure AA). In Meghalaya caves are developed in an almost 300km long belt of diverse sedimentary rocks occurring along the southern and southeastern borders of Meghalaya and Mikir Hills, where more than 1,000 caves have been spotted. Krem Syndai lies in the western area of Jaintia Hills (25º09"; 25º15" North and 92º00"; 92º15" East), 1500mts above sea level and is bounded by Umtapoh, Nongtalang,Taarablang and Laremshiap.

Krem Syndai is located in Syndai village128 km from Shillong, the capital of Meghalaya which lies on a peninsular outlier of the plateau proper and about 500m above Muktapur village, a former railhead on the Bangladesh border.Syndai can be reached via Dwaki or via Jowai. Abundant limestone deposits in the west Jantia Hills are found around the Krem Syndai , Krem Shuki and Krem Rupasor. The cave is about 400 m long, an obvious track down the slope from the village to a point where it steepens and immediately below this, a small path heads of through the forest to the lift of some 50 m into a depression, where the cave entrance lies. The cave entrance is of stooping height but once inside the passage immediately opens up into impressive proportion of 25 m high and 30 m wide. The cave contains limestone formations in form of stalagmites, stalactites and lot of flow stones.

Cave air temperature of 20 deg C at the sampling location. Various locations with dripping water were found in Krem Syndai, and drip rates were generally slow, with less than 2-3 drip per minute. The natural vegetation above the cave consists of shrubs and trees. All stalagmites were collected in a small chamber at the end of an approximately 30m long, narrow passage which is about 40 mts from cave entrance.

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Fig.AA:- Location map of Krem Syndai, Inset shows its position in India

4. Methods

4a. Sampling and analysis

Using aseptic techniques, samples were collected with sterilized disposable gloves and forceps and then placed into sterilized zip lock sachets. Fresh samples were collected in December 2009 brought to IISER Kolkata and Guru Jambheshwar University, Hisar. They were immediately processed for different analyses and stored at 4◦C and -20◦ C. The pH, humidity and temperature were determined in situ using portable instruments.

4b. Microbiological Analysis

Isolation of bacteria strain

Attempts to isolate bacteria from stalagmites and solid deposits were carried out for identifying the culturable and aerobic heterotrophic microbial communities. In addition, microorganisms capable of precipitating carbonate minerals were also evaluated. The stalagmite samples (1 g) were aseptically powdered in a sterilized mortar and pestle, suspended in sterile 0.9% saline solution and vortexed. This solution was diluted with 9 ml autoclaved distilled water and later followed by successive dilutions.

Media used for isolating the heterotrophs included nutrient rich and nutrient poor media. Nutrient agar (NA) (peptone 5 g l-1, NaCl 5 g l-1, beef extract 1.5 g l-1, yeast extract 1.5 g l-1, agar 15 g l-1) (Hi-Media), Triple B-4 agar (calcium acetate 2.5 g l-1, yeast extract 4 g l-1, glucose 10 g l-1, agar 18 g l- 1)(Boquet et al., 1973), Kliger iron agar (KIA) (peptone 23 g l-1, yeast extract 3 g l-1,glucose 1 g l-1, lactose 10 g l-1, Iron (II) sulfate 0.2 g l-1, NaCl 5 g l-1, sodium thiosulphate 0.3 g l-1, phenol red 0.05 g l-1, agar 7 g l-1) (Hi-Media), sulfite agar (SA) (tryptone 10 g l- 1,sodium sulfite 1 g l-1, agar 20 g l-1) (Na2S2O3.5H2O 5 g l-1, KH2PO4 3 g l-1, (NH4)2SO4 0.4 g l-1, MgSO4.7H2O 0.5 g l-1,CaCl2.2H2O 0.25 g l-1, FeSO4.7H2O 0.01 g l-1) starch-casein agar (SCA) (soluble starch- 10 g l-1, potassium phosphate dibasic 2 g l-1, potassium nitrate 2 g l-1, NaCl 2 g l-1, casein 0.3 g l-1, MgSO4. 7H2O 0.05 g l-1, CaCO3 0.02 g l-1, FeSO4. 7H20 0.01 g l-1, agar 15 g l-1) (Hi-media), minimal acetate medium (MAM) and Mn agar (MNA) (beef extract 1 g l-1, yeast extract 0.075 g l-1, manganous carbonate 2 g l-1, ferrous ammonium sulfate 0.15 g l- 1) were used to isolate the different bacterial strains.

The agar plates were inoculated with sample dilutions ranging from 101to 107 and incubated under aerobic and aphotic conditions at 37°C. The experiments were designed to mimic the cave temperature and light conditions. Controls consisting of autoclaved distilled water and 0.9% saline solution were also incubated. Individual colonies were selected (based on differences in color and colony morphotypes) and further purified by Triple B4 agar and 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.

A total of 10 purified isolates were obtained. The isolates were smeared separately on slides for Gram stain in order to separate the Gram positive and negative strains. The stained slides were viewed with an Axiophot optical microscope (ZeissAAAAAx magnification). Some basic morphological (Gram stain), physiological were done on the isolates to facilitate their identification. In order to assess and quantify the overall In order to assess and quantify the overall abundance of total culturable microorganisms, serial dilutions with plate counts for viable culturable microorganisms was done in different media. The visible colonies on plates were counted after 1 week of incubation.

Mineral Precipitation Capability of the Isolated Strains

Bacterial isolates were cultured separately in triplicate and were incubated aerobically and aphotically between 37 ◦ C for 40 days. These isolates were examined periodically by optical microscopy for the presence of carbonate minerals. The controls consisted of uninoculated culture medium.

Direct amplification, cloning and sequencing of 16S rDNA molecules

A two-stage PCR procedure was used to amplify 16S rDNA molecules from melt water. All reagent transfers were undertaken within a sterilized laminar flow hood. Before use, all reaction tubes and micropipette tips were autoclaved and all solutions, except for the AmpliTaq Gold DNA polymerase (cat. no. N808-0240; Perkin-Elmer Biosystems), were passed through Microcon

YM-100 filters (Millipore). Nuclease free water was added directly to PCR mixtures (PE buffer, 4 mM MgCl2) that contained 5 pmol of the forward primers 21F (complementary to bacterial 16S rDNA; Reysenbach and Pace,1995) or 27F (complementary to bacterial 16S rDNA; Lane,

1991) and 2mM of the universal reverse primer 1465R(Lane, 1991). Reaction mixtures were placed at 958C for 10 min and then subjected to 36 cycles of PCR amplification by incubation for 1 min at 95°C, 1 min at 56°C and 1 min at 72°C, thermally activated AmpTaq Gold DNA polymerase (Fermantas). An aliquot (2 ml) of the resulting product was used as the template in the second PCR, with the universal forward primer 515F combined with either reverse primer or the universal reverse primer 1492R. Samples of the PCR products were subjected to agarose gel electrophoresis and visualized by ethidium bromide staining. DNA molecules of the expected length (1465 bp) were amplified using the bacterial 16S rDNA primers (27F with 1525R, and 515F with 1392R or 1492R), but not when the archaeal-specific 21F primer was used, consistent with the observations of Priscu et al. (1999).Individual DNA molecules were cloned from the =1465 bp populations into pGEM-Teasy (Promega), and both DNA strands were sequenced using primers that annealed the flanking T7 and SP6 promoter sequences. The sequences obtained were aligned and analysed for phylogenetic relationships using the software package and the beta-4a version of PAUP 4.0 (Swofford, 1999).

16S rDNA molecules from bacterial isolates

Cells from a single colony of each isolate were resuspended, lysed and the region of their 16S rDNAs corresponding to nucleotides 27-1525 of the Escherichia coli 16S rDNA was amplified and both DNA strands sequenced (Zeng and Kreitman, 1996). The sequences obtained were subjected to phylogenetic evaluation using the ARB software.

4c. Bottle Experiments - Laboratory Setup and working

The bottle experiment was set up to determination biotic calcite precipitation rate. This experiment was designed simultaneously with another biotic calcite precipitation experiment (Drip Experiment) focusing on varying drip rate of saturated carbonate solution .The experiment was performed in bottles inoculated with bacteria isolated from stalagmite samples involved in calcite precipitation in Syndai cave. Controls were setup to detect the isotopic change that could be used as evidence for biological precipitation over a period of 40 days.

1.7 L of B4 medium was prepared according to (Boquet et al., 1973) [calcium acetate 2.5 g l-1, yeast extract 4 g l-1, glucose 10 g l-1 and agar 18 g l- 1 ].The medium was equally divided between

16 conical flasks (100ml each), Another 10 ml of the medium was poured in a culture tube, plugged well and sterilized by autoclaving it at 121° C and 15 psi pressure for 15 mins.

Previously isolated bacterial strains grown in B 4 medium agar plates were used as inoculum. A single colony was picked up and 10 ml of B 4 medium was inoculated, it was then incubated at 37° C overnight.

This seed culture was further used as an inoculum for closed system. 1ml of the culture was added aseptically to 8 flasks whereas the controls had no bacterial inoculum. All 16 closed bottles were incubated at 37 °C.

Growth and pH analysis

Every fifth day one bottle along with control was checked for change in pH, bacterial growth (spectrophotometrically) and calcite precipitation using CCCCC.

XRD analysis

Minerology of the biologically precipitated calcite was studied at the 40th day using XRD . XRD spectra were obtained using an RIGAKU XXX with a Cu anode (40 kV and 30 mA) and scanning from 5° to 85° 2theta. The components of the sample were identified by comparing them with standards established by The International Center for Diffraction Data.

Isotopic analysis

Calcite precipitated was further purified by removing the cell debris by chemical oxidation with 1:10 ratio of alkaline H2O2 solution and 0.5M NaOH, followed by repeated washing with milli Q water.

The carbonates collected every fifth day was analyzed for δ18O and δ13C isotopes. H3PO4 acid was added in vials containing the sample and flushed with Ar for 10 minutes. After contact between the acid and sample, CO2 gas released in the sealed container was left to equilibrate for 24 h. The gas was transferred via a Thermo Gasbench II unit into a Finnigan MAT 252 isotope ratio mass-spectrometer for analyses of stable C and O isotopes (Debajyoti et al., 2006). Various international standards like NBS18, NBS19, IAEA-CO-1, and IAEA-CO-8 were used for standardization. The results were reported versus the Vienna Peedee Belmenite for both 13C and 18O.

4e. Drip Experiments -Laboratory Setup and working

The experiment was carried out under stable room conditions. All the experimental setups were autoclaved before use to avoid contamination. In two separate glass vessels pure salt solution of NaHCO3 (5mol/L) and CaCl2(2.3mol/L) were dissolved in milli-Q water and equilibrated by bubbling humidified air for 48 hrs. The microbial colonies were cultured in plastic tubes for 4-5 days. Later dense colonies were selected and transferred along with the nutrient media into the U-shaped glass channel at an interval of 10 cm. Zero air was passed through a combination of UV lamp and 0.2µm air filters and circulated throughout the glass chambers for maintaining stable sterile experimental condition. In the experiment a Whatman microfiber filter paper was used a as substrate for calcite precipitation. The filter paper was cut into thin strips of 10cm and placed in the glass channel (Refer FigAA).

The salt solutions were pumped into the glass chamber using a peristaltic pump using Tygon-LFL tubes. The mixed solution dripped out of steel capillaries and formed single droplets before dripping down the channel and flowing over the Watman filter paper strips. It was important to maintain the mixing of the solution before dripping into the channel to prevent prior calcite precipitation. The glass chamber was constantly flushed with sterile and humidified air. To avoid evaporation the gas was bubbled through a Plexiglas-bottle filled with milli-Q water. At the end of the glass channel the solution and enriched air are pumped out. A holding device was used for 4 glass chamber. So, that all 4 experiments could be carried out simultaneously.

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FigAA: Shows the laboratory experimental setup

5.Results And Discussion

5a.Microbiological analysis

Gram Characterization

The isolates were smeared separately on slides for Gram stain in order to separate the Gram positive and negative strains. The stained slides were viewed with an Axiophot optical microscope ( Carl Zeiss AAA-40x magnification). The S4 isolates strains were Gram positive and coccus shaped.

Colony isolations

Single-colony isolates were obtained from these enrichment cultures by plating on B4 agar media aerobically at 37°C. The S4 isolate are most closely related phylogenetically to the to XXXXXXXXXX (Table 1 Fig. KK), and all the single-colony isolates investigated.

Identification of strain by 16S rRNA gene characterization

Populations of small-subunit rDNA molecules were amplified directly from S4 isolate by using universal and Bacteria-specific primers. Individual DNA molecules were cloned from these populations and sequenced, revealing the presence of bacterial 16S rDNAs from different phylogenetic lines of descent (Table 2; Fig. 3). Sequence NNNNN originated from an a-proteobacterium whose nearest cultured neighbour was