Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Calcium Carbonate Precipitation by Bacillus and Sporosarcina Strains Isolated from Concrete and Analysis of the Bacterial Community of Concrete

  • Kim, Hyun Jung (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Eom, Hyo Jung (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Park, Chulwoo (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Jung, Jaejoon (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Shin, Bora (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Kim, Wook (Department of Biotechnology, Korea University) ;
  • Chung, Namhyun (Department of Biotechnology, Korea University) ;
  • Choi, In-Geol (Department of Biotechnology, Korea University) ;
  • Park, Woojun (Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University)
  • Received : 2015.11.06
  • Accepted : 2015.12.14
  • Published : 2016.03.28
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Abstract

Microbially induced calcium carbonate precipitation (CCP) is a long-standing but re-emerging environmental engineering process for production of self-healing concrete, bioremediation, and long-term storage of CO2. CCP-capable bacteria, two Bacillus strains (JH3 and JH7) and one Sporosarcina strain (HYO08), were isolated from two samples of concrete and characterized phylogenetically. Calcium carbonate crystals precipitated by the three strains were morphologically distinct according to field emission scanning electron microscopy. Energy dispersive X-ray spectrometry mapping confirmed biomineralization via extracellular calcium carbonate production. The three strains differed in their physiological characteristics: growth at alkali pH and high NaCl concentrations, and urease activity. Sporosarcina sp. HYO08 and Bacillus sp. JH7 were more alkali- and halotolerant, respectively. Analysis of the community from the same concrete samples using barcoded pyrosequencing revealed that the relative abundance of Bacillus and Sporosarcina species was low, which indicated low culturability of other dominant bacteria. This study suggests that calcium carbonate crystals with different properties can be produced by various CCP-capable strains, and other novel isolates await discovery.

Keywords

Introduction

Biomineralization is a common biochemical process that represents the synthesis of inorganic crystalline or amorphous minerals containing calcium, silicon, iron, magnesium, and zinc [3,40,43]. A variety of microorganisms, including bacteria, fungi, algae, and lichens, may be responsible for the production of biominerals such as silicate, carbonate, and calcium phosphate [14,15,24]. In particular, calcium carbonate (CaCO3) precipitation (CCP) appears to have geobiochemical significance because it shapes soil structure by changing its porosity, compressibility, and shear strength [5,9]. CCP is linked to global carbon cycling and global warming because of CO2 capture during the CCP process [32,45]. Carbonate minerals such as calcite, aragonite, and vaterite depend on microbial metabolic processes, with calcite being the most common form of bacterial carbonate [3,11,12,45]. Emerging evidence also suggests that the physical and chemical compositions of the surrounding environment determine the efficiency of production, shape, and size of carbonate mineral polymorphs [9,10,34,45]. This phenomenon has been observed in many CCP-capable bacteria in the genus Bacillus, Sporosarcina, Pseudomonas, Cyanobacteria, Pantoea, Myxococcus, and Halobacteria, and sulfate-reducing bacteria. [4,8,45].

Many bacteria are known to promote extracellular CCP [11,12,17,45]. Microbial extracellular polymeric substances (EPS) consisting of polysaccharides, proteins, nucleic acids, and lipids serve as nucleation sites for CCP owing to their anionic chemical characteristics that attract the surrounding metal ions such as Ca2+ [3,47]. Many physiological activities such as photosynthesis, denitrification, sulfur reduction, ammonification, and ureolysis can produce ammonium and hydroxide ions, which create alkaline conditions, thus accelerating CCP [5,9,11,14,45]. On the other hand, intracellular CCP is widespread among cyanobacteria, and sulfur-oxidizing chemolithotrophic Achromatium cells can store CaCO3 granules inside the cells, although the physiological role of CCP remains unclear [27,35].

The contribution of CCP-capable bacteria to atmospheric CO2 fixation and removal is substantial, and the use of CCP-capable bacteria for solving environmental and industrial problems is a promising approach. Therefore, research on CCP-capable bacteria has been a steadily growing field [10,20,29,31], and the following are some of the popular topics: plugging and increasing durability of soil, biosorption of calcium from water, filling cracks in concrete (self-healing concrete) [9,12,20,36], bioremediation of heavy metals [1,32], and biomining of metals [33]. Nonetheless, the reasons for mineralization by CCP-capable bacteria, regulation of the possible CCP-specific genetic system, and the role or effects of CCP-capable bacteria in ecosystems are still poorly understood. The purpose of this study was to isolate and characterize novel CCP-capable bacteria from concrete. We sampled concrete from two locations (Jingyo and Seoul, South Korea) and characterized them by field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectrometry (EDS) mapping, urease activity assays, and EPS quantification. We also performed bacterial community analysis of the concrete samples using pyrosequencing.

 

Materials and Methods

Isolation of CCP-Capable Bacteria from Concrete and Their Growth Conditions

Bacterial strains were isolated from two concrete walls: a 60-year-old concrete from Jingyo (35°00'29.9"N 127°53'02.3"E) and a freshly mortared concrete from Seoul (37°35'06.9"N 127°01'33.5"E), South Korea. The surface of the concrete samples was gently wiped with 70% ethanol for sterilization. Then, the inner parts were aseptically hammered and ground to isolate calcium carbonate-precipitating bacteria. The ground parts were washed with phosphate-buffered saline (PBS, pH 7.5) and spread first onto a minimal salts basal medium containing 0.1% cycloheximide to suppress the growth of eukaryotic organisms. The colonies were subcultured onto Difco nutrient broth with pH set to 11. The colonies that were capable of growing on the alkaline nutrient broth were selected as possible calcium carbonate-precipitating bacteria because the conditions under which concrete is manufactured are highly alkaline.

The 16S rRNA gene was amplified with primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-CGGTTACCTTGTTACGACTT-3’). Polymerase chain reactions (PCRs) were performed on a MyCycler Thermal Cycler (Bio-Rad, USA), and the cycling conditions were as follows: 94℃ for 90 sec, followed by 25 cycles consisting of 94℃, 60℃, and 72℃, 45 sec each. The extension/hold step lasted 5 min at 72℃. Taxonomic affiliation was determined by sequencing the 16S rRNA and by construction of phylogenetic trees via sequence similarity by means of EzTaxon [6]. The 16S rRNA sequences of the three isolates have been deposited in National Center for Biotechnology Information (NCBI) GenBank under the accession numbers KU168425, KU168426, KU168427 for strains JH3, JH7, and HYO08, respectively. The optimal liquid medium for strains JH3 and JH7 was Luria-Bertani broth, and for strain HYO08 was tryptic soy broth with 2% urea (w/v). The incubation temperature for each strain was 37℃. Various pH (pH range: pH 5, 7, 9, 10, and 11) and NaCl concentrations (0.5, 1, 2, 5, and 10% (g/v)) were used to test alkali tolerance and halotolerance.

FE-SEM and EDS Mapping Analysis

The strains JH3, JH7, and HYO08 were each incubated in a urea-CaCl2 medium (3 g of nutrient broth, 20 g of urea, 2.12 g of NaHCO3, 10 g of NH4Cl, 4 g of CaCl2·2H2O, per liter of distilled water, adjusted to pH 6) for 12 h. Depending on the opacity of the culture, up to 2 ml of the cells was harvested by centrifugation (1 min, 13,000 ×g) and the pellet was washed twice with PBS. Primary fixation was performed overnight at 4℃ (Karnovsky’s fixation method). After that, we performed three washes with 0.05 M potassium phosphate buffer for 10 min each at 4℃. The cells were additionally fixed with a mixture of 0.1 M potassium phosphate buffer and 2% osmium tetroxide at 4℃ for 2 h. Two additional washes with distilled water were conducted briefly at room temperature. Dehydration by increasing ethanol concentrations (30, 50, 70, 80, and 90%, and three times in 100% ethanol, 10 min each step) was performed at room temperature. The samples were coated with platinum prior to examination by FE-SEM (FEI, Japan). EDS mapping analysis was conducted on a NORAN System 7: Silicon Drift Detector (Thermo Fisher Scientific, USA). The mapping elements were depicted as counts.

Urease Activity

Urease activity was measured by a colorimetric assay [30]. Briefly, the strains JH3, JH7, and HYO08 and Escherichia coli MG1655 (negative control) were grown to the stationary phase. The cell suspensions were then diluted with PBS to adjust the optical density at 600 nm (OD600) to 0.5. The cells (20 μl) were inoculated into 200 μl of Stuart’s urea broth in a 96-well plate (total final volume: 220 μl). The plate was incubated at 28℃ for 24 h in a Sunrise microplate reader (TECAN, Austria), and OD550 was measured at 1 h intervals. During the incubation, the growth of the three strains was negligible; hence, their urease activity was measured in the same amount of cells.

EPS Quantification and Biofilm Formation

EPS quantification was conducted according to previous studies, with slight modifications [25]. One milliliter of cell culture was mixed with Congo red at the final concentration of 3.5 mg/l and was incubated for 30 min at 37℃ with inversion every 10 min. The culture was centrifuged (10 min, 13,000 ×g) and OD480 of the clear colored supernatant was measured. A standard curve of Congo red was plotted after measurement of OD480 at the concentration gradient from 0.5 to 15 mg/l. The fraction of Congo-red bound cells was calculated from the difference between Congo red remaining in the supernatant and Congo red in the medium without cells and from the obtained equation of the linear curve. The calculation results were normalized to OD600 of 1 ml of cell culture.

A biofilm formation assay was conducted by the crystal violet staining method [16]. Briefly, the four strains, including Escherichia coli MG1655 as a control, were incubated overnight with aeration and washed twice with PBS. Approximately 106 CFU/ml of each strain were inoculated into a 48-well microplate in a static state, and OD595 was measured on a multi-detection microplate reader (HIDEX Sense, Finland). OD595 was normalized to OD600.

Endospore Formation

The strains JH3, JH7, and HYO08 were incubated for 5 days to induce sporulation. They were also incubated on TYE medium [42] to verify endospore formation. The spores were either stained with malachite green or unstained for visualization under a light microscope (Zeiss, Germany).

Soil Aggregate Visualization

Strain HYO08 was incubated overnight, and 7 × 106 CFU/ml was inoculated into 50 ml of autoclaved soil with 25 mM urea and CaCl2 as sources for precipitation of calcium carbonate. The same volume of distilled water was inoculated as a negative control, every 5 days. The soil condition was visualized by FE-SEM after 10 days of incubation at room temperature.

Pyrosequencing Analysis

To analyze the bacterial community inside the concrete samples where strains JH3, JH7, and HYO08 were isolated, DNA was extracted using the NucleoSpin Soil kit (Macherey-Nagel, Germany). The DNA yield was quantified by a Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA). A sequencing library was prepared by amplification of the 16S rRNA gene using primers 27F-AMP (5’-Adapter-Barcode-GAG TTTGATCMTGGCTCAG-3’) and 518R (5’-Adapter-WTTACC GCGGCTGCTGG-3’) targeting the region V1 to V3. Sequencing was performed on a GS-FLX (Roche, Switzerland). All sequencing procedures were conducted by Macrogen (Republic of Korea). Raw sequencing data were filtered in the mothur software to remove short and low-quality sequences [37]. Classification and rarefaction curves were analyzed in RDPipeline of the Ribosomal Database Project (RDP Release 11) [44]. The NGS data for community analysis was deposited at NCBI under the accession number SRA312453.

 

Results

Isolation of CCP-Capable Bacteria from Concrete Samples

The concrete samples were obtained from two locations (Jingyo and Seoul, South Korea). The concrete sample from Jingyo (JC) was taken from a part of a 60-year-old building that had not experienced significant weathering. The sample from Seoul (SC) was freshly mortared concrete. Three possible CCP-capable isolates were selected on alkaline medium; two isolates from JC, named strains JH3 and JH7, and one isolate from SC, named strain HYO08. Based on their phylogenetic relationship (sequence similarity and a neighbor-joining tree), strains JH3 and JH7 were assigned to the genus Bacillus (Fig. 1). Strain JH3 shared 99.93% 16S rRNA gene similarity with alkali-tolerant B. aryabhattai, and 99.33% with the calcite-precipitating bacterium B. megaterium [25]. On the other hand, the close phylogenetically species of strain JH7 were B. toyonensis (99.98%) and B. thuringiensis (99.80%), respectively; both species are known to be alkali-tolerant calcium carbonate-biomineralizing polymorphs [12,18]. Strain HYO08 shared 98.83% 16S rRNA gene similarity with Sporosarcina soli and 98.54% with S. contaminans, both having an optimal pH of 9.0 and a well pronounced ability to precipitate calcium carbonate [23].

Fig. 1.A neighbor-joining tree based on the 16S rRNA gene sequence of the bacterial strains capable of c-alcium carbonate precipitation. The strains are depicted in boldface. Numbers at nodes indicate bootstrap values for 1,000 replicates.

FE-SEM/EDS Mapping Analysis of CCP

To test whether the isolated strains can precipitate crystals in a calcium carbonate-inducing medium, all three strains were visualized by phase contrast microscopy (data not shown) and FE-SEM (Fig. 2). The cells of each strain were aggregated with crystals. Further EDS mapping confirmed that those crystals are composed of calcium carbonate (Fig. 3, Fig. S1). It is worth noting that both Bacillus and Sporosarcina species formed extracellular calcium carbonate and their crystals were morphologically distinct (Fig. 2).

Fig. 2.Field emission scanning electron microscopy (10,000×magnification) of calcium carbonate crystals produced by the strains JH3, JH7, and HYO08 after 12 h of incubation in a urea-CaCl2 medium. (A) Strain JH3. (B) Strain JH7. (C) Strain HYO08.

Fig. 3.Energy dispersive X-ray spectrometry mapping of the strains JH3, JH7, and HYO08. Two calcium ion peaks indicate the presence of calcium in biomineralized crystals produced by these calcium carbonate precipitation-capable strains. (A) Strain JH3. (B) Strain JH7. (C) Strain HYO08.

Alkali Tolerance and Halotolerance of the Strains

The ability to tolerate high pH, temperature, and a high-salt environment is crucial for CCP-capable bacteria because concrete is exposed to many environmental conditions and is made under these conditions [20]. The optimal growth temperature of each strain was 37℃. The strains JH3, JH7, and HYO08 showed alkali tolerance and halotolerance near their optimal growth temperature (25-37℃); however, HYO08 was the only strain that preferred alkaline conditions (up to pH 11) at all temperatures because it showed smaller differences in the lag phase and maximum growth at high pH levels. Nonetheless, strains JH3 and JH7 also grew well at 30℃ and pH 9 and 10, respectively (Fig. S2). On the other hand, strain JH7 was the most halotolerant because it was capable of growing at the NaCl concentration of 5%. In addition, the only strain that could not tolerate high temperature (40℃) was strain HYO08.

Physiological Characteristics of the CCP-Capable Bacteria: Urease Activity, EPS Production, and Endospore Formation

Ureolysis is a key process for accelerating major CCP [5]. The urease activities of the three strains using the same number of cells were measured. The colorimetric changes of phenol red revealed that all three strains showed urease activity (Fig. 4). Strain HYO08 had the strongest urease activity, followed by strains JH7 and JH3. Strain HYO08 started to increase urease activity after 1 h of incubation, with the maximum activity being within 7 h. Strain JH7 showed urease activity after 4 h with a double-sigmoid pattern. The reason for this pattern is not straightforward. The urease activity of strain JH3 appeared after 17 h and increased slowly. EPS might be an important key factor for CCP because it can bind to calcium ion, which facilitates CCP [3]. It has been known that the EPS production and biofilm formation are often co-related [25]. EPS and biofilm formation were quantified using Congo red and crystal violet, respectively. Strain JH3 produced the greatest amount of EPS (1.001 mg/l) and showed higher biofilm formation (Fig. S3).

Fig. 4.Urease activity of strains JH3, JH7, and HYO08 according to a colorimetric assay in Stuart’s Broth. Escherichia coli MG1655 was used as a negative control.

Endospores were observed in all three strains because the genera Bacillus and Sporosarcina can form endospores [41,42]. Strain JH3 formed subterminal endospores, whereas strains JH7 and HYO08 produced central endospores (Fig. S4). Among the three strains, strain JH3 showed the fastest spore formation cycle and as strain JH3 showed mature spore state (seed-like spores) much earlier than other strains within the same time interval (data not shown). The soil-aggregation effect of strain HYO08 was analyzed because this strain was found to be the most alkali tolerant and had the highest urease activity. As shown in Fig. S5, a large amount of minerals were drawn to and surrounded the cell wall of strain HYO08 in the presence of urea and CaCl2, whereas sporulation occurred when there were no CCP sources available. Soil aggregation assay under the two tested conditions showed that formation of the aggregated strain HYO08-soil particle complex occurred in the presence of urea and calcium chloride, whereas there were no signs of soil aggregation without CCP.

Bacterial Community Analysis in the Concrete Samples

The DNA yield from the duplicate samples of JC and SC was 2.60 ± 2.78 a nd 0 .1 1 ± 0 .01 ng/mg ( mean ± SD), respectively. Different DNA yields from the two samples suggest that there could be substantial variation in the community size among concrete samples. The large standard deviation of the DNA yield from JC was suggestive of significant variation in bacterial colonization depending on the concrete structure, such as cracks and pores. The bacterial community of JC and SC showed similar structure, with Actinobacteria, Proteobacteria, and Cyanobacteria being the major phyla (Fig. 5). The three phyla accounted for >70% of cells in the two samples. Although the relative abundance of Actinobacteria was similar in the two communities (40.33% in JC and 37.7% in SC), Proteobacteria showed different relative abundance in the two samples (23.1% in JC and 34.7% in SC). Cyanobacteria was the third dominant phylum, accounting for 11.9% and 8.8% of cells in JC and SC, respectively. There were a large proportion of unidentified bacteria, pointing to unexpectedly high bacterial diversity inside the concrete. The community structure at the genus level was substantially different between the two samples. JC did not contain a genus with relative abundance >10% and the predominant genus was Blastococcus (9.6%), whereas the predominant genus of SC, Methylobacterium, showed a relative abundance of 23.5%. In JC, a number of taxonomically ambiguous groups of bacteria were noticed, such as Gp1, Gp8, unidentified Rhodobacteraceae, unidentified Geodermatophilaceae, and unidentified Micrococcaceae. Another characteristic of the two communities was that many prevalent genera in the SC community had low relative abundance in JC. Those genera included Methylobacterium, Sphaerobacter, Propionibacterium, Truepera, Pseudomonas, Lactobacillus, and Microbacterium.

Fig. 5.Bacterial community analysis of two concrete samples: Jingyo concrete (JC) and Seoul concrete (SC). Phyla and genera with relative abundance less than 1% were included as well. (A) Phyla. (B) Genera.

 

Discussion

Application of biomineralization to fields such as environmental and civil engineering and geology [4,8,10] is now a popular ecofriendly strategy; biomineralization is also known to participate in the carbon cycle because this process sequesters a large amount of carbon [3,4,7,29,31]. In fact, this phenomenon is a natural process that occurs almost ubiquitously; it exists in terrestrial and aquatic habitats such as shells, coral reefs, and sands [7,11,12] and even in the human body as mineralo-organic forms [46]. Biomineralization usually involves metabolic activities of microorganisms and can occur in a biologically controlled, biologically influenced, or biologically induced manner (or a combination of the three) [8,9,32]. Utilization of this natural process could be a turning point in the field of environmental engineering; therefore, it is important to select bacterial strains with high efficiency of CCP.

The ability to exist in such an alkaline, high-salt environment is crucial for calcium carbonate-precipitating bacteria because concrete itself represents such environments [9,26]. Therefore, the three strains, Bacillus sp. JH3, Bacillus sp. JH7, and Sporosarcina sp. HYO08, could be promising candidates for ecofriendly industrial applications.

The parameters of a bacterial culture environment such as temperature and aeration can influence the types of calcium carbonate produced by CCP-capable bacteria [11,17,26]. Because all three strains were incubated under the same conditions, the different structure (shape) of the resulting precipitated crystals could be due to differences in their innate mechanisms of utilization of calcium. Therefore, each strain probably has a distinct optimal microenvironment to induce CCP, such as a specific pH level and production of (or the level of expression of) a specific protein of EPS [11,13,21]. Thus, the properties of EPS associated with the strains may also be a factor in the distinct morphology of calcium carbonate crystals [13,40]. The magnitude of EPS production and the composition and electrical charge of glycoproteins and proteins may differ among CCP-capable strains. The composition of EPS may affect the efficiency of collection of calcium cations and the efficiency of binding of calcium ions to carbonate [3,11]. In fact, the magnitude of CCP by the three strains was altered when we replaced the calcium sources in the original urea-CaCl2 medium with calcium lactate, calcium acetate, or calcium sulfate (data not shown). Understanding the distinct mechanisms by which Bacillus and Sporosarcina strains utilize different calcium sources could be the key to the production of crystals of specific shape.

Many studies have suggested that the cell wall is a main location of CCP because of its nucleation effect for crystal formation [5,9,25]. It seems likely that the strains JH3, JH7, and HYO08 generate a special macro- or microenvironment for precipitation of calcium carbonate regardless of nucleation. The cells and crystals were aggregated, but none of the strains had calcium carbonate on their cell wall because whole cells were clearly visible (Fig. S5). Although the nucleation effect of CCP-capable bacteria is still a hypothesis [3,13], the exact location triggering CCP in our three strains needs further research.

Formation of calcium carbonate crystals requires a high concentration of calcium and carbonate sources. In a habitat abundant with calcium sources such as a cave, limestone, and soil [7,8,9,31], utilization and production of carbonates through metabolic activities such as urease-driven hydrolysis may be the cornerstone of mineralization of calcium carbonates [2]. Here, all three strains exhibited urease activity that drove the biologically influenced and biologically induced CCP under optimal alkaline environmental conditions. Recent studies suggest that urea is abundant in CCP environments because of input from various sources: mammal urine, water infiltration from the surface, human activities, and agricultural waste [19,31]. Therefore, these findings suggest that the concrete samples from which we isolated the CCP-capable bacteria might have contained urea that the isolates could use for metabolism and formation of minerals.

Our results of community analysis are consistent with a previous community analysis of a sample from a deteriorated monument containing bacteria harboring carbonatogenic activity [21]. Geodermatophilaceae is a family of stone-dwelling bacteria containing the genera Blastococcus, Geodermatophilus, and Modestobacter, all of which were detected in our analysis. Proteogenomic analysis of Blastococcus, Geodermatophilus, and Modestobacter revealed their characteristic stress resistance-related features such as starvation-inducible, biofilm-related, and DNA-protecting proteins; they could be an important physiological requirement for an endolithic lifestyle [39]. The presence of Cyanobacteria and free-living nitrogen-fixing Actinobacteria such as Arthrobacter and Propionibacterium may play an important role by supplying nutrients to a concrete-dwelling community because an endolithic lifestyle involves extreme starvation [15,38]. For practical applications of CCP-capable bacteria, the relation between the capacity for CCP and other physiological features of CCP-capable bacteria as well as the interaction of CCP-capable bacteria with other species should be studied in more detail.

The results of community analysis also showed that the relative abundance of Bacillus and Sporosarcina was low, even though we successfully isolated those strains. This discrepancy could be due to a culturing bias that possibly provided favorable conditions for Bacillus and Sporosarcina, or due to our focused screening strategy targeting alkali-tolerant strains. Nevertheless, identification of CCP-capable bacteria from other taxa such as Acinetobacter and Myxococcus is to be expected, as shown in one study on bioconsolidation of historical limestone [22].

Thus, we analyzed three alkali- and halotolerant CCP-capable bacterial strains, Bacillus sp. JH3, Bacillus. sp. JH7, and Sporosarcina sp. HYO08, isolated from two concrete samples. Our data on urease activity, EPS production, biofilm formation, and soil aggregation suggest that our CCP-capable strains are promising candidates for the development of self-healing concrete. The bacterial community analysis showed that for industrial and environmental applications, researchers should take into account the interaction between CCP-capable bacteria and the indigenous community.

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