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 uniqu....

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

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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