Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Summary

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Abstract

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS) were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Introduction

Sporosarcina pasteurii is a gram-positive bacterium able to survive in highly alkaline environments (pH~10)¹ and is one of the bacterial species that can become a causative agent of a phenomenon called Microbiologically Induced Calcite Precipitation (MICP)^2-4. MICP is a process wherein precipitation of calcium carbonate is induced by certain microbes under suitable environmental conditions. S. pasteurii has assumed importance in recent years due to its identification as a possible agent for inducing significant volumes of MICP under certain conditions. This possibility stems from the fact that S. pasteurii has the unique ability to secrete copious amounts of the enzyme urease. This enzyme acts as a catalyst, promoting an accelerated lysis of urea (a naturally occurring biochemical compound with widespread and abundant supply) in the presence of water molecules. Through a cascade of reactions, this process ultimately leads to the generation of negatively charged carbonate ions. These ions, in turn, react with positive metal ions like calcium to finally form precipitates of calcium carbonate (calcite); hence the label MICP^5-9.

The process of MICP has been known and studied for several decades^10,11. Over the past few years, MICP has been investigated for a wide range of engineering and environmental applications including bottom-up green construction¹², enhancement of large-scale structures^13,14 and carbon sequestration and storage^15,16.

For example, Cunnigham¹⁷ et. al designed a high pressure moderate temperature flow reactor containing a Berea sandstone core. The reactor was inoculated with the bacteria S. fridgidimarina and under conditions of high-pressure supercritical carbon dioxide injection, a massive accumulation of biomass inside the pore volumes was observed, which led to more than 95% reduction in permeability. Jonkers and Schlangen¹⁸ studied the effect of certain special strains of bacteria on the self-healing process in concrete. External water transported into the pore network entering through the surface pores is expected to activate the dormant bacteria which in turn help structural strength via MICP. Tobler¹⁹ et al. have compared the ureolytic activity of S. pasteurii with an indigenous groundwater ureolytic microcosm under conditions favoring large-scale MCIP and found that S. pasteurii has a consistent capability to improve calcite precipitation even when the indigenous communities lacked prior urease activity. Mortensen²⁰ et.al have studied the effects of external factors like soil type, concentration of ammonium chloride, salinity, oxygen concentration and lysis of cells on MICP. Their demonstration that the biological treatment process is very robust with respect to a wide variation in parameter space substantiates the fitness of this process for various large-scale remediation applications provided a proper enrichment process to reinforce the bacteria is undertaken. Phillips²¹ et. al designed experiments to study the changes in permeability and strength of a sand column and a sandstone core after being injected with S. pasteurii cultures. They found that while the permeability decreased 2 - 4 times while the fracture strength increased three times.

S. pasteurii and its role in MICP are topics of active research and several issues relating to the mechanism of chemical precipitation are still not fully understood. In light of this, it is very important to have a set of consistent standardized protocols to accurately culture a suitably enriched stock of S. pasteurii to achieve MICP. Here, we outline a rigorous protocol that will ensure repeatability and reproducibility. This manuscript describes the detailed protocols for culturing S. pasteurii and suitably enriching the culture medium to induce precipitation. The process is investigated through various microscopic techniques such as optical and Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS). The focus of the manuscript is on the process of MICP. Procedures like SEM and SIMS, being well-established standard protocols, are not described separately.

Protocol

NOTE: Perform the experimental protocols in the order described below. The bacterial culture protocol is discussed in Section 1 (Also see Figure 1). Section 2 describes the protocol for enriching the culture medium using external additives. Section 3 describes the protocols for multi-mode microscopy. Weights of all the individual components may be measured using an analytical balance. Volume of each solution may be measured using a volumetric cylinder.

NOTE: Proper biosafety protocols must be followed for Sections 1 - 2. Consult the institutional safety office for details.

1. Bacterial Culture

1. Culture Bacteria - Agar Plate Medium Preparation
   1. Assemble equipment and ingredients such as Petri dishes, flask, Tris-base, HCl, agar, Millipore water, pH-meter etc.Sterilize all containers by autoclaving at 121 °C before use.
   2. Prepare 1 L of 0.13 M aqueous solution of Tris-buffer by mixing 15.75 g Tris-base with 1 L of Millipore water. To lower the pH level of the original solution (pH 10.4) add 2,800 µl of HCl (50% concentration). Check continuously using a pH-meter to set pH = 9.
   3. Divide the 1 L buffer solution into two parts as follows:
      1. Take 800 ml of this solution. Divide it equally into two parts of 400 ml each. Dissolve 8 g (NH₄)₂SO₄ to one solution and 16 g yeast extract to the other solution.
      2. Take the remaining (200 ml of) solution and divide it again into two parts of 100 ml each. Mix 2 g (NH₄)₂SO₄ to one. Add 4 g yeast extract and 4 g agar to the other.
   4. Autoclave the 4 solutions separately after wrapping the respective flasks in Al foil and sticking autoclave tapes.
      NOTE: If a benchtop autoclave unit is used, the volume should be set to 500 ml (temperature and pressure automatically specified as a function of volume).
   5. After taking them out from the autoclave, set the two 400 ml solutions aside for step 1.3.1 (below). Mix the two 100 ml solutions to have a 200 ml solution. Pour the mixture into 10 - 12 Petri dishes.
2. Culture Bacteria - Agar Plate Sample Preparation
   1. Remove the bacterial stock from freezer (-80 °C) and allow it to thaw. After thawing properly, place the bacterial stock and the agar plate inside a biosafety hood.
   2. Select the micropipette of smallest available dimension (0.5 - 10 µl is a good choice) to infest the tip with the Sporosarcina pasteurii stock. Streak an agar plate with the micropipette tip. Place the streaked agar plate inside a non-shaking incubator at 31 °C for 48 hr.
   3. After 48 hr, remove the plate from the incubator and visually examine for the existence of single colonies. If there are no single colonies, then place it in the incubator for another 24 hr.
   4. Repeat the process until single colonies are detected. Do not exceed 7 days of trial.
      NOTE: If single colonies do not appear even after a week, then it is concluded that the steps have not been followed properly and the entire process must be repeated from step 1.
3. Culture Bacteria - Final Sample Preparation
   1. Mix the two 400 ml solutions (Tris buffer + (NH₄)₂SO₄ and Tris buffer + yeast extract (1.5.1) together to obtain an 800 ml solution. Transfer 125 ml of this solution into a flask.
   2. Perform a visual examination of the surface of the agar plate to identify regions with high concentration of single colonies. Gently nudge and break one of the colonies with a micropipette tip.
   3. Dip the same micropipette tip into the 125 ml flask and stir it thoroughly to ensure that sufficient number of cells for robust multiplication get transferred. Place the flask in a shaking incubator at 150 rpm, 30 °C for 2 - 3 days. After 2 - 3 days, remove the flask from the incubator.
4. Culture Bacteria - Final Cell Count
   1. Perform serial dilution of the non-diluted culture solution using PBS to attain a dilution of at least ten million (10^-7) to ensure countable single colonies appear. Draw seven parallel equidistant lines on one of the agar-plates.
      1. Do this by drawing bold lines on the bottom surface of the Petri dish, prominent enough to be visible from top. Drop 3 little drops of non-diluted solution into one segment. Add 1 ml of non-diluted solution to 9 ml of Phosphate Buffered Saline (PBS) to obtain a 1:10 dilution.
   2. Take a small aliquot (~ 0.1 ml) of this newly diluted solution with a pipette and drop 3 more small drops on the next segment. Transfer the newly diluted solution to a new flask and further diluted ten times (10x) by adding PBS. This brings down the dilution to 10^-2 or 1:100.
      1. Use this 1:100 solution in the next segment. Repeat this he process with small volumes of the freshly diluted solutions by successively transferring them to new flasks and continuously diluting ten-fold (10x) in tandem with PBS to obtain more and more dilute samples from 10^-3 or 1:1,000 all the way down to 10^-7 or 1:10 million into the last segment.
   3. Perform Colony-Forming Unit (CFU) plate count to count the number of cells present in the agar plate after incubating the plate for 1 - 2 days at 31 °C. This gives a quantitative measure of the bacterial count in the undiluted sample.
      NOTE: The CFU value is measured based on the ability of the system to give rise to colonies under the specific conditions of nutrient medium, temperature and time assuming that every colony is separate and founded by a single viable microbial cell.
   4. Seal the Petri dishes with self-sealing film and store remaining items in a refrigerator for future use.

2. Protocol for Enrichment of Nutrient to Accelerate Precipitation

1. Transfer 9 ml of the prepared culture liquid (cells + medium) into several sterilized centrifuge tubes, each of 10 ml volume.
2. Prepare a 100 ml stock solution of the external enrichment consisting of four components in the following concentrations in fresh medium: Urea: 2 g/L, Ammonium chloride: 1 g/L, Sodium bicarbonate: 212 mg/L, Calcium chloride: 280 mg/L. Carefully measure all the ingredients using an analytical balance and mix all but urea with fresh medium in a beaker and place in the autoclave (121 °C, 15 psi, 15 min).
   1. Following autoclave, mix the requisite amount of urea (2 mg) with 1 ml of fresh medium and pass through a syringe fitted with 0.22 µm syringe filter to complete the process of enrichment.
   2. Add 1 ml of this enrichment medium with additives to the sterilized centrifuge tubes containing 9 ml of the prepared culture liquid (cells + medium) (see step 2.1).
      NOTE: Of all these components, urea being degradable at elevated temperatures, cannot be sterilized in an autoclave. Hence, after the other components have been autoclaved (121 °C, 15 psi, 15 min), urea is added last through a 0.22 µm syringe filter.
3. Vortex each tube thoroughly using a mechanical vortexer. Place the enriched liquids (cells + medium + additives) in a non-shaking incubator at 30 °C. Monitor all the units regularly for initiation of precipitation. Using a light microscope, begin microscopic observations once onset of precipitation is detected with the naked eye. This is usually between 30 - 36 hr after start of experiments.

3. Protocol for Multi-mode Microscopy

1. Transfer a small volume (~ 1 ml) of fluid to a high-magnification microscopy-chambered coverglass system to perform initial observations with bright field mode. To begin with, start all observations with the lowest magnification (4X). which provides a wide area of coverage and hence global information about the distribution of precipitates.
2. Select particular areas with significant volumes of precipitations and zoom in (20X, 40X, 60X) by progressively increasing the magnification.
   NOTE: Since it's impossible to properly resolve the cells from the Extracellular Polymeric Substance (EPS) under bright-field (due to very close refractive indices), switch on phase-contrast mode whenever necessary. In this mode, just the outlines of the cell membranes (being a different phase than the bulk cell) are visible, thus enabling visual differentiation.
3. Finally, stain some chambers with Live/ Dead stain that selectively dyes the live components as green while coloring the dead components in red. Refer to standard protocols²⁸.
4. Switch on fluorescent mode to properly distinguish between the red and green regions. Red (Red filter cube with Excitation wavelength of 540 - 580 nm, Dichroic mirror of 595 nm and Barrier filter of 600 - 660 nm) and Green (GFP Long-pass Green filter cube with Excitation wavelength of 440 - 480 nm, Dichroic mirror of 505 nm and Barrier filter of 510 nm) filter cubes are used to facilitate fluorescent micrographs.
5. Once all multi-mode data have been gathered, select the best images and manually overlay (individual brightfield, phase-contrast and fluorescent images) to obtain the best possible contrast and clarity.
6. Perform SEM on the dried solid samples to understand crystal structures. This is a well-established and standard procedure.²² Perform XPS to identify chemical signatures of functional groups (carbonates). This is a well-established and standard procedure as well.²³

Results

S. pasteurii being an alkaliphile²⁴ can survive relatively harsh conditions. When the above mentioned culture protocol is followed, and S. pasteurii is grown inside a chamber, the bacteria leads to the precipitation of calcium carbonate over time (Figure 2A). Figure 2 (b) shows a phase-contrast optical microscopic image of the bacterial cell population within the culture medium. Individual cells can be clearly distinguished, w...

Discussion

Critical Steps: This manuscript describes in detail the protocols for culturing a viable sample of S. pasteurii. Once the culture has been readied, it must be suitably enriched. This is a key step vital to the success of the experiment because a failure to provide the proper chemical environment leads to either very long time scales of precipitation or a complete lack thereof. S. pasteurii is quite sensitive to several external agencies and must be cultured with a high degree of care an...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petridish	Fisher Scientific	FB0875712	Petridishes being used as Agar plate
Pyrex Flasks	Fisher Scientific	S63268	Corning Erlenmeyer
Tris-Base	Promega	H5133	being used to make Tris-Buffer
Hydrochloric Acid	Sigma-Aldrich	H9892	1.0 N, Bioreagent, suitable for cell culture
Agar Powder	Sigma-Aldrich	A1296	microbiology tested, plant cell culture tested, cell culture tested, powder
Ammonium Sulphate	Sigma-Aldrich	A4418	for Molecular Biology
Yeast extract powder	Sigma-Aldrich	51475
Measuring Cylinder	Cole-Parmer	CP08559GC	Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC; 1,000 ml,1/Pk
Analytical Balance	OHAUS	AX124E	being used to measure weight of reagents
Autoclave	Brinkmann	58619000
Autoclave Tape	VWR	52428864
Aluminum Foil	Sigma-Aldrich	Z185140	being used to seal the flask before placing it in Autoclave
Bacterial Stock	Cedarlane	11859	-80 °C stock of S. pasteurii, ATCC No. is mentioned against Cat. No.
Mline Single-Channel Mechanical Pipettors, Variable Volume	Biohit	725010	Marketed by VWR under catalog number 14005976
Micropipette Tip	Fisher Scientific	212772B	Used for scratching Agar plates
Incubator	Binder	80079098	Microbiology Incubator,BF Series
Shaking Incubator	VWR	14004300	VWR Signature Benchtop Shaking Incubators
Phosphate Buffer Saline (PBS)	Sigma-Aldrich	P7059
BD Falcon Express Pipet-Aid Pipetting Device	BD Biosciences	357590	Marketed by VWR under catalog number 53106220
Parafilm	Sigma-Aldrich	P7793	Being used to seal Agar plates
Urea	Sigma-Aldrich	U1250	Enrichment for nutrient medium
Sodium Bicarbonate	Sigma-Aldrich	S8875	Enrichment for nutrient medium
Calcium chloride	Sigma-Aldrich	C1016	Enrichment for nutrient medium