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Summer Research Fellowship Programme of India's Science Academies

Screening of biocementing potential of bacterial isolates from caves

Gokul G

3rd year BS-MS, IISER Bhopal, Bhauri, MP 462066

Prof. S.R Joshi

Department of Biotechnology and Bioinformatics, NEHU, Shillong 793022

Abstract

Microorganisms today are being exploited for various environmental aspects like reducing the carbon footprint, removal of toxins from water and soil, and they are also being extensively tried for degradation of plastic wastes which has management hurdles. Life on Earth would not be possible without bacteria. Microbes are present almost everywhere and have been shown to showcase diverse structural and functional aspects. With a record history of current emission of carbon dioxide that has reached 400 parts per million, it is increasingly important to minimise carbon footprint. With the current population explosion leading to rapid urbanisation, the contribution of urban-housing related carbon dioxide emissions has increased dramatically with a rise of around 1% per year since 2010. Various energy demands contributed by construction sector directly or indirectly and emissions from buildings acts as a major contributor to current carbon levels. Application of calcium carbonate precipitation induced by bacteria for improving the quality of cement used in construction sites has been recently reported. This phenomenon of mineral deposition is called biocementation or microbiologically induced calcite precipitation (MICP). Addition of bacterial suspension into cement changes its properties and because of such deposits it has emerged as an efficient binder for shielding and amalgamating various concreate structures. Urea degrading bacteria can convert urea into carbon dioxide which upon reaction with calcium chloride in presence of water forms calcium carbonate. This potential of bacteria has been exploited in the construction sector. The present investigation was undertaken to isolate and screen potential biocementing bacteria from cave samples. These bacterial cultures were isolated from Jaintia , Khasi hills and Garo hills of Meghalaya located in Northeastern part of India that are known for its complex cave systems hidden under its undulating hills, attributed to huge deposits of limestone. Bacterial cultures were characterised based on the morphological and biochemical parameters. Molecular analysis on the aforementioned bacterial isolates has been performed and the 16S rRNA gene sequencing identified the isolates as Bacillus toyonensis and Bacillus paramycoides. The optical microscopy and scanning electron microscopy detected the CaCO3 precipitation by the bacteria forming crystals. The present study is the first of its kind on the bacterial strains having the potential for biocementation. Further studies have to be performed using compressive strength test and water absorption test by the addition of bacterial suspension into the mortar. This work is a stepping stone in the field of biocementation which can be explored for construction sector arising in use biological process in conserving our mother Earth.

Keywords: carbon footprint, biocementation, calcium carbonate, bacterial culture

Abbreviations

Abbreviations
MICP  Microbiologically induced calcite precipitation
 TCA Trichloro Aceticacid
PCR Polymerase Chain Reaction 
 MRMethyl Red 
VP Voges-Proskauer 
 OD Optical Density
 BCG Bromcresol Green


INTRODUCTION

The potential of bacteria to show biocementation has been exploited for enhancing the mechanical properties of cementing material. This green technique has highly significance in the current world of development and modernisation (Charpe et al., 2018). Addition of potential biocementing bacterial suspension into the concrete mixture induces MICP thereby augmenting the strength of concrete structures. This property of bacteria helps to reduce the current carbon levels in the atmosphere (Montano-Salazar et al., 2018). Huge energy demands for the construction sector are contributing a major part of current carbon levels which leads to global warming. MICP is highly desirable, as this is a natural microbial activity free from contamination (De Muynck et al., 2010). This process is a promising technology in restoration of construction materials, metal remediation, soil reinforcement and CO2 sequestration. Moreover, MICP has the potential for the development of bacterial concrete, self-healing concrete and biomortar generated by the biologically induced binders (De Muynck et al., 2008; Montano-Salazar et al., 2018).

Caves are one of the unique ecosystem found on Earth which includes hydrological systems that are isolated from the surface and can support life that can resist harsh physical and chemical conditions in these habitat (Yasir, 2017). The dazzling architecture of caves are formed majorly by limestone and other calcareous rocks (Banerjee et al., 2013). The cave ecosystems are majorly dependent on allochthonous organic material for energy as the resources are limited due to the aphotic conditions (Poulson et al., 2000; Simon et al., 2003). Fauna which includes spider, millipedes, beetles and crustaceans ranging from tiny copepods to crayfish can be found in these Earth’s hidden habitat (Baskar et al., 2019). Insights into the cave microbiology have been emerged as a frontier field of research which includes the role of mineral precipitation by the bacterial communities in the past few decades (Banerjee et al., 2013). Microbial carbonates are important in cave environments and those crystals can be reproduced from organic calcium salts in laboratory with the help of bacteria which shows calcite precipitation (Northup et al., 1997; Banerjee et al., 2013). Several new members of the microbial world are nowadays been discovered from caves among which the major phyla includes Actinobacteria, Proteobacteria, Verrucomicrobia and Acidobacteria (De Mandal et al., 2017; Yasir, 2017). Noval strains gram-stain positive, rod shaped bacteria like Bacillus antri and Bacillus cavarnae had been discovered from caves of China recently (Feng et al., 2016; Rao et al., 2019).

Calcium carbonate, the carbonic salt of calcium (https://pubchem.ncbi.nlm.nih.gov/compound/Calcium-carbonate) is a common substance found on Earth’s crust and the building blocks of shells and pearls of aquatic organisms (Montano-Salazar et al., 2018). The formation of natural rocks involves the chemical precipitation of CaCO3. The chemical precipitation is accompanied by biological process involving microbes that have the ability to precipitate calcite by the process of biocementation or MICP (Lopez-Garcia et al., 2005; Achal et al., 2009; Montano-Salazar et al., 2018). Microbiologically Induced Calcium carbonate Precipitation (MICP) is a biochemical process driven by bacteria which induces calcium carbonate precipitation upon interacting with a calcium containing media (Dhami et al., 2012; Anbu et al., 2016; Rajasekar et al., 2017; Montano-Salazar et al., 2018). The exploitation of MICP has been evolved as an eco-friendly, alternative approach to solve environmental problems like improving the quality of construction material and to remove heavy metals that contributes to carbon footprint (Anbu et al., 2016; Sharma et al., 2016). MICP can be induced by various mechanisms which includes hydrolysis of urea, reduction of sulphate and aerobic oxidation (Tittelboom et al., 2010; Sharma et al. 2016; Montano-Salazar et al., 2018). Because of the negatively charged cell wall of bacteria, the Ca2+ contained in the media will deposit on cell surface which subsequently reacts with CO3 2- ions (produced by urease activity) leading to calcite precipitation (Eqn.1 and 2) (Whiffin et al., 2007; Tittelboom et al., 2010).

Ca2+ + Cell → Cell-Ca2+ ........................... (Eq.1)

Cell-Ca2++ CO3 2- → Cell-CaCO3 ........................... (Eq.2)

Urease also called urea amidohydrolase, the first enzyme to be crystallized is synthesised by different kinds of microorganisms like bacteria, fungi (Sirko et al., 2000; Konieczna et al., 2012) and also in plants (LeVeen et al., 1994), but not mammals. The hydrolysis of urea to yield ammonia and carbamate is been catalysed by this enzyme (Ferrero et al., 1998). The activity of this enzyme has various significance including (i) as a nitrogen source for fertilizers (Steyert et al., 2012), (ii) recycling of nitrogenous waste of domestic livestock (Mobley et al., 1995) and nowadays been exploited to reduce carbon footprint (Bose et al., 2017; Castro et al., 2016) by the process of biocementation which involves the formation of calcium carbonate by MICP (Stocks et al.,1999; Rajasekar et al., 2017). The hydrolysis of urea by urease enzyme is a multiplex process. In the first step, urea is hydrolysed into one molecule of ammonia and carbamate. Further, carbamate in water solution spontaneously converted into carbonic acid and a molecule of ammonia is formed as byproduct (Fig.1.) (Mobley et al., 1995; Tittelboom et al., 2010; Konieczna et al., 2012; Castro et al., 2016). The hydrolysis of urea possess the highest calcite conversion rate induced by MICP (Sharma et al., 2016).

Capture_1.PNG
    Schematic diagram of urea hydrolysis

    The aim of this work is to isolate and characterise potential biocementing bacteria from the Mawsmai cave, Cherapunjee located in Meghalaya, India. Moreover this work was intended as step for conserving the planet by reducing the energy demands for construction sector thereby reducing global warming.

    MATERIALS AND METHODS

    Isolation of Biocementing Potential Bacteria

    The bacterial strains showing the potential for biocementation were isolated from caves in East Khasi Hills District (Mawsmai Cave , Location:- 25.2988N 91.7086E), North from the centre of Cherrapunje town in Mawsmai village (Fig.2.).

    The cave samples were collected in sterile tubes and kept at 4℃ till the samples were analysed. One gram of samples were dissolved in 9ml of saline buffer and sample dilutions ranging from 10-1 to 10-4 were plated on nutrient agar media (1gL-1 beef extract, 2gL-1 Yeast extract, 5gL-1 Peptone, 5gL-1 NaCl and 15gL-1 agar). The plates were incubated aerobically for overnight and colonies were selected and purified by streaking on nutrient agar. The biocementing potential bacteria were further safe guarded by various methods (Cacchio et al.,2003; Banerjee et al., 2014; Peristiwati et al., 2018). The bacterial strains were grown on Nutrient agar slants for short term maintenance at 4℃. For long term preservation 500µl of bacterial cultures grown on nutrient broth were resuspended in 500µl of 70% glycerol stock and then frozen at −20℃.

    Biochemical Parameters

    Gram staining for bacterial strains

    Bacterial strains were characterised based on gram staining technique (Beveridge et al., 2001). Bacteria that retain the purple colour (crystal violet stain) are classified as “gram positive” and those which shows red (safranin stain) are said to be “gram negative”.

    Oxidase and catalase test

    The catalase activity were determined by addition of 3% H2O2 to bacterial strain cultured in nutrient broth (Bascomb et al., 1998; Drancourt et al., 2000; Cappucino et al., 2007). The formation of effervescence due to release of presence of O2 shows a positive test. Oxidase test is performed to check the presence of cytochrome oxidase. Bacterial suspensions were added to oxidase disc and a colour change from white to purple shows the presence of enzyme (Jurtshuk et al., 1976; Cappucino et al., 2007) (Fig.6).

    Simmon’s Citrate test for Bacterial Growth

    Bacterial cultures grown on nutrient agar media were streaked onto citrate agar slants (0.2gL-1 MgSO4, 1gL-1 ammonium dihydrogen phosphate, 1gL-1 dipotassium phosphate, 2gL-1 sodium citrate, 5gL-1 sodium chloride, 0.08gL-1 bromothymol blue, 15gL-1 agar) (Ishiguro et al., 1978) and was incubated overnight at 37℃. This is used to determine the bacteria’s ability to utilize citrate. A colour change from green to blue shows a positive result (Fig.4).

    MR-VP test

    The MR test characterises the bacteria according to their ability to perform mixed acid fermentation. The bacterial strains were inoculated in MR-VP broth and were incubated for 24 hrs at 37℃ with continuous shaking. Red colour formation after the addition of methyl red shows a positive result for MR test (Cappucino et al., 2007; Vashist et al., 2013).
    VP test is used to check the presence of acetoin in bacterial broth media. Bacterial strains were grown on MR-VP broth incubated at 37℃ overnight. 5ml of Barritt’s reagent A followed by Barritt’s reagent B were added to the broth. Formation of a pink – red colour at the surface indicates the presence of acetoin in the broth (Cappucino et al., 2007; Vashist et al., 2013).

    Determination of proteolytic activity

    To check the protease activity, the bacterial strains were streaked on casein agar plates (5 gL-1 peptic digest of animal tissue, 1.5gL-1 beef extract, 1.5 gL-1 yeast extract, 5gL-1 NaCl, 15 gL-1 agar, 10gL-1 casein and 0.0015% (w/v) BCG) (Vijayaraghavan et al., 2013; Rautela et al., 2017). The agar plates were incubated overnight at 37℃. The proteolytic activity was detected by a clear zone around the streaked region (Fig.9).

    Urease activity

    For examining the urease activity, the bacterial strains were grown on Christenson’s Urea Agar slants and was incubated for 24hrs at 37℃. A colour change from yellow to red (hydrolysis of urea to ammonia raises the pH) shows positive result (Christensen, 1946; Montano-Salazar et al., 2018) (Fig.8).

    Quantification of urease enzyme

    The urease activity was performed by mixing 0.8ml urea concentration of 20mM, 1ml of assay buffer (50mM Tris HCl) and 0.2mL of urease enzyme in a sample tube. The solution was kept for incubation at room temperature for 1hr. 1ml of TCA (Trichloro acetic acid was added inorder to stop the activity of the enzyme. 0.1 ml of the above mentioned solution was aliquoted in fresh tubes containing 0.1ml of Nessler reagent and 4.8 ml of distilled water inorder to make the final volume 5ml. The absorbance was taken using UV-V spectrometry at 405nm and the estimation was done by comparing with standard ammonia curve (Fig.11) (Krom et al., 1980; Zusfahair et al., 2018; Cordero et al., 2019).

    Calcite Production and Estimation

    The bacteria’s ability to precipitate calcite were identified by growing the bacterial strains in B4 agar media (2.5gL-1 calcium acetate, 4gL-1 yeast extract, 10gL-1 glucose and 18gL-1 agar) (Banerjee et al., 2014) and were kept for incubation for 2 weeks at 30℃. The formation of white precipitation shows the calcite production. To estimate the amount of calcite produced, the isolates were cultured in nutrient broth for 24 hrs and subcultured onto BPU broth (3gL-1 beef extract, 5 gL-1 peptone, and 20 gL-1urea at pH 7) at 30 degree Celsius for 72 hrs with continuous shaking. 500 µl of bacterial broth were transferred into 2ml tubes and 500ul of 350 mM calcium chloride dihydrate solution was added. The mixture was centrifuged at 12000rpm for 5 min at 25℃ to collect the precipitate. Finally, the precipitate was dried at 50℃ for 24 hrs prior to being weighed (Ho Kang et al., 2016).

    Molecular Characterisation

    Two bacterial isolates were selected based on the ability to perform urease activity. Genomic DNA isolation was done using Hipure TM Bacterial Genomic DNA Purification Spin Kit (HiMedia, India) and the bands were visualized in 0.8% agarose gel. The amplification of the bacterial 16SrDNA was done using universal primers [27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5' GGTTACCTTGTTACGACTT3'] (Banerjee et al., 2012; Chen et al., 2015) with the following cyclic conditions (initial denaturation for 5min at 94℃, denaturation at 94℃ for 1min, annealing at 55℃ for 1min and elongation at 72℃ for 2 min for 35 cycles followed by the final elongation step for 5min at 72℃). Amplification of the PCR product were done using PCR system 9700 (Applied Biosystems, USA). PCR products were run on 1.2% agarose gel in 1xTAE buffer with ethidium bromide. The sequencing were performed (Microgen Hygeine Pvt.Ltd), analysed and compared using EzTaxon (https://www.ezbiocloud.net).

    SEM for Ultrastructure Analysis of Crystals

    The ability of bacterial strains to precipitate were studied by scanning electron microscope [JSM 6360(JEOL)] (Stocks et al., 1999; Leveille et al., 2000; Banerjee et al., 2012; Montano-Salazar et al., 2018). Bacterial strains were grown on Christenson urea agar containing 1mM CaCl2 (24.5g urea agar in 950 ml distilled water, 50 ml 40% urea and 50ml 1mM CaCl2). The growth of the bacterial strains were also analysed in various concentration of CaCl2 (1M, 2M, 3M, 4M, 5M and 6M). The bacterial culture broth were incubated at 37℃ for 1 week and OD was calculated at 600nm. After the incubation period, 3-4 pieces of agar containing the precipitate ranging 2-3mm were taken in 2ml tubes and were prefixed by immersing in 3% Glutaraldehyde for 24hrs. They were then washed in 0.1M Sorenson’s buffer for 15 min at 4℃ for 3 changes. The substrate were then dehydrated twice in acetone solution (30%, 50%, 70%, 80%, 90%, 100%) for 15min each. The specimens were then immersed in TMS for 5-10 min and were dried. The samples were fixed onto aluminium stubs, gold plated for 3-5min and were observed under scanning electron microscope [JSM 6360 (JEOL); resolution, 3nm; magnification, 8–300,000×; accelerating voltage, 1–30 kV ].

    RESULTS AND DISCUSSION

    A total of 9 bacterial strains were isolated from cave samples out of which 2 strains were selected based on their ability to perform urease activity (Fig.3.). The bacterial strains were named as CD-3 and CA-1 based on the caves from which they were isolated. The colonies of the isolate CD-3 had white colour, irregular shape, flat elevation and opaque whereas CA-1 had creamy white colour, irregular and flat elevation. Gram staining determination showed both the isolates as gram positive rods (Fig.5.). However morphology in itself was not adequate to differentiate the strain, because of that conventional microbiological biochemical characterisation was also performed on both the strains. The results of the biochemical tests are mentioned in Table 1.

    The ability of bacterial strains to precipitate calcite were analysed by growing the strains in B4 agar medium (Fig.7.). The CA-1 strain much more precipitation compared to CD-3. Quantitative estimation of the amount of calcite precipitated also shows a similar result (CA-1 precipitated 0.02g/0.5ml bacterial broth of calcite whereas CD-3 - 0.012g/0.5ml bacterial broth).

    The genomic DNA bands were visualized using Gel Documentation unit by running 1kb DNA ladder (Fig.10.). Based on the 16S rRNA gene sequence analysis, the strain CD-3 shared 100% similarity with Bacillus toyonensis and strain CA-1 shared 97.1% similarity with Bacillus paramycoides (Liu et al. 2017) (Table.2.). The ability of Bacillus toyonensis to precipitate CaCO3 had already reported (Kim et al. 2016) but this is for the first time reporting in Bacillus paramycoides.

    In our study, Urease activity is the major accelerating factor for MICP. The quantification of urease activity using standard ammonium chloride curve showed that Bacillus paramycoides (1.35μg/ml) shows a stronger urease activity compared to Bacillus toyonensis (0.82μg/ml).

    To check whether the bacterial strains can precipitate crystals in a CaCO3 inducing media, both the strains were visualized by optical microscope and SEM. Both the strains were aggregated with crystals (Fig.12,13,14.). Bacillus toyonensis and Bacillus paramycoides species formed extracellular CaCO3 after an incubation period of 1week and shows more precipitation. Both the bacterial strains showed growth in 1M and 2M CaCl2 but the growth was restricted in higher concentrations of CaCl2 (3M, 4M, 5M and 6M).

    cave.JPG
      Mawsmai Cave.(A)(B)-Interior of Mawsmai Cave, (B)(D) - Stalactite deposition
      plates.JPG
        . Bacterial cultures streaked on nutrient agar plates. (A) – CD-3, (B) – CA-1.
        citrate.JPG
          (A) Bacterial strain CA-1 showing negative result for citrate test. (B) CD-3 showing citrate positive
          gram.JPG
            Optical microscopic images of gram positive rods; (A)-CD-3, (B)-CA-1
            oxi.JPG
              (A) CD-3 showing oxidase negative, (B) CA-1 showing oxidase negative (no colour change to purple),(C) CA-1 showing negative result for catalase test, (D) CD-3 showing catalase negative (no formation of bubbles)
              b4.JPG
                B4 agar media; (A),(B):CD-3 and CA-1 bacterial strains showing calcite precipitation respectively
                ureas.JPG
                  (A)&(B); CD-3 and CA-1 showing urease activity in Christensen’s Urea Agar slants
                  pro.JPG
                    CD-3 and CA-1 showing clear zone around the streaked region in casein agar plates (protease positive)
                    dna.JPG
                      Agarose gel (0.8%) image showing the integrity of the isolated genomic DNA
                      graph.PNG
                        Standard graph for Ammonium Chloride for urease quantification
                        opt.JPG
                          Optical microscopy images of CaCO3 crystals.(a)CaCO3 crystals precipitated in the presence of [Ca2+] = 1M and 40% Urea solution by the CD-3 strain. (b) CaCO3 crystals precipitated in the presence of [Ca2+] = 1M and 40% Urea solution by the CA-1 strain. 
                          sem1.JPG
                            SEM images showing CaCO3 precipitation by CA-1 strain. (A) CaCO3 crystals deposited by the urease activity of CA-1 bacterial strain. (B) Aggregation of CaCO3 crystals. (C) rod shaped bacteria. (D) Rod –shaped bacterial colonies surrounded by CaCO3 flakes.
                            sem2.JPG
                              SEM images of CaCO3 precipitation by CD-3 bacterial strain. (E) Aggregates of CaCO3 crystals. (F) Aragonite surrounded by spike shaped Vaterite. (G) Spherical shaped Vaterite. (H) Rod shaped bacteria embedded inside the crystal deposit due to high rate of precipitation
                              table1.JPG
                                The results of biochemical test on isolated bacteria
                                tabl2.JPG
                                  Bacteria Identified

                                  CONCLUSION

                                  In conclusion, two bacterial strains have been found to have the potential for biocementation. The potential of Bacillus toyonensis to induce calcite precipitation has been already reported (Kim et al. 2016) and shows a good urease activity. The aforementioned species along with Bacillus paramycoides are highly potential for future biocementing applications in the construction sector and a key stone for reducing current carbon levels. Furthermore, this study contributes new information to the field of MICP related biotechnological advances. This is also an area in which few studies are carried out especially from cave samples.

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                                  ACKNOWLEDGEMENTS

                                  I am greatly thankful to IAS-INSA-NASI for giving a great opportunity to work under Prof. S.R.Joshi, Dept. of Biotechnology, North-eastern Hill University, Shillong. I am extremely thankful to Prof. S.R.Joshi who has given me the great opportunity to work on this project. He has been a great source of inspiration and his timely support and guidance has helped in the successful completion of the same. I am extremely grateful to Lily Shylla for guiding me throughout the course of this project and for lending me a helping hand whenever I was in need of one. Also, I express my sincere thanks to Bishal, Niramla, Upashna, Welfare, Augustine, Macmillan, Aawaj and Akanksha of Microbiology Laboratory, Dept. of Biotechnology & Bioinformatics, NEHU, Shillong who were always with me for the successful completion of this project. I thank IAS-INSA-NASI, INSPIRE, DST India for the contingency and scholarship.

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