Biomineralization of (magnesium) calcite and vaterite by
bacterial isolates has been known for quite some time. However, the
extracellular precipitation has hardly ever been linked to different
morphologies of the minerals that are observed. Here, isolates from
limestone-associated groundwater, rock and soil were shown to form calcite,
magnesium calcite or vaterite. More than 92 % of isolates were indeed
able to form carbonates, while abiotic controls failed to form minerals. The
crystal morphologies varied, including rhombohedra, prisms and pyramid-like
macromorphologies. Different conditions like varying temperature, pH or media
components, but also cocultivation to test for collaborative effects of
sympatric bacteria, were used to differentiate between mechanisms of calcium
carbonate formation. Single crystallites were cemented with bacterial cells;
these may have served as nucleation sites by providing a basic pH at short
distance from the cells. A calculation of potential calcite formation of up
to 2 g L
The processes of carbonate biomineralization by bacteria are usually linked
to an alkaline microenvironment enhancing the potential for carbonate
precipitation on small spatial scales (Hammes and Verstraete, 2002). Through
microbial production of CO
The occurrence of carbonate-forming bacteria has been investigated with respect to different environments (Andrei et al., 2017; Gray and Engels, 2013; Horath and Bachofen, 2009; Kang and Roh, 2016; Rusznyak et al., 2012). The process has generally been linked to changes induced in the direct microenvironment of the growing bacteria through metabolic features or to providing nucleation sites for crystallization in a supersaturated environment (Roberts et al., 2013; Seifan et al., 2017; Yan et al., 2017). Either microbiomes of a specific habitat or the physiological and biomineralization properties of a single isolate have been investigated. Thus, an investigation of more isolates from one environment seems indicated.
As a result of growing knowledge on carbonate biomineralization, applications
in concrete repair and formulation of self-healing cement have been derived
(Achal et al., 2009; Li et al., 2017; Seifan et al., 2016; Singh et al.,
2015). Additionally, carbonate formation for remediation purposes, including wastewater
treatment (Gonzalez-Martinez et al., 2017), has gained
interest (Kumari et al., 2016; Zhu et al., 2016). Basic scientific questions
addressed include the nature and rates of the biotic versus the abiotic nature of
calcite formation. For one species, 33 g L
We thus were interested in identifying different bacteria from a carbonate rock–water–soil system to address the question of how bacteria may contribute to different morphologies, even at a distance to the cells and how much calcium carbonate may be formed in vitro under conditions more likely to occur in nature than those previously used (Castanier et al., 1999). To obtain a variety of calcium-carbonate-forming bacterial isolates, we focused on marine Triassic limestones exposed in central Germany. We obtained gram-positive and gram-negative bacteria from the limestones, and rendzina soil developed on the limestones near the limestone quarry in Bad Kösen (Thuringia, Germany) and in groundwater from wells located nearby and probing these lithographies. The isolated bacteria represented major taxa present in the microbiomes, which were also investigated for taxonomic and physiological characterization (Meier et al., 2017). They were incubated in single culture or in cocultivation under different conditions to address the biomineralization relevant to carbonate formation. With our results, we can advance the background on microbially induced biomineralization with respect to different mechanisms.
The Muschelkalk quarry in Bad Kösen is characterized by Lower Muschelkalk (Jena Formation, Lower Wellenkalk core sampled) and Middle Muschelkalk (Karstadt Formation, upper Schaumkalk Formation sampled). The situation at the quarry allowed for direct access to the lithotypes by horizontally removing approximately 50 cm of stone and then manually coring to a depth of 20 to 30 cm to provide conditions not likely to contaminate the samples with cooling water that would be needed for drilling devices. The rock samples were taken into fresh and sterile plastic bags and brought to the laboratory, where surface sterilization by incubation in 70 % ethanol for 30 min was performed. Afterwards, the outer part was removed with a hammer and a sterile chisel at a clean-space working bench (Heraeus, Hanau, Germany) under sterile conditions.
The groundwater wells installed for monitoring purposes near Bad Kösen,
in Stöben (Hy Camburg 13/198; 4478864N, 5660183E; Lower Muschelkalk
sampled at 34 m depth) and Wichmar (Hy Camburg 121/1988; 4478030N, 5655906E;
120 m; Middle Muschelkalk sampled at 17 m) were sampled with the help of an
electric pump MP1 (Grundfos, Bjerringbro, Denmark) after reaching constant pH
and temperature. All samples were stored at 6
Soil was sampled for rendzina at 40 cm depth at 15 sampling points in the Bad Kösen quarry on the limestone bed within a radius of about 1 km, and it was then homogenized.
For the isolation of bacteria from the limestones, Std I (Carl Roth, Roth,
Germany; supplied with NaCl at 3, 5, 7 % if indicated by sample
chemistry), minimal AM (Amoroso et al., 2002) and oligotrophic R
For rock samples, 5 g of powdered rock sample was added to 45 mL sterile 0.9 % NaCl, followed by vortexing for 20 min. Subsequently, sonication was applied for 15 min and filtered (Sartorius, Göttingen, Germany) supernatant was plated. In addition, a dilution of 0.9 % NaCl was prepared without filtering and cultured in a liquid medium, or particles were directly placed on nutrient agar to account for different amounts of bacteria in samples.
A total of 2.5 L of groundwater samples was filtered before culturing using
0.2
Independently of the origin of samples, pure cultures were obtained by serial
plating. Soil extracts from 100 g mixed soil samples, dried at
40
For strain identification, genomic DNA (DNeasy Power Soil kit, Qiagen,
Hildesheim, Germany) was extracted from pure cultures and 16S rDNA was
amplified (primers 27F and 1492r at 100 mM, 0.02 U Dream Taq polymerase,
1 x Dream Taq buffer, 100 mM deoxynucleotide triphosphate mixture,
1
To study effects on carbonate mineralization, B-4 agar plates or liquid
cultures were incubated at 28 or 10
Using the strains of one habitat (Table S1), co-inoculation plates were produced to test for competition or the induction of biomineralization by streaking out the strains crossing each other on B-4 agar. Two- or four-strain interactions were tested using sympatric isolates from the same habitat.
Solid products were visualized with a stereomicroscope (Zeiss, Jena, Germany)
and sampled into 0.2 mL reaction tubes under the binocular using sterile tweezers with as few bacteria as
possible attached. A powder X-ray
diffractometer (Bruker D8 Advance; Bruker, Ettlingen, Germany) with Cu
K
Scanning electron micrographs (SEMs; Quanta 3D FEG; FEI) were taken from samples placed on a sample holder and sputtered with carbon or gold without additional preparation and then imaged in secondary (SE) or back-scattered electron mode (BSE) at an acceleration voltage of typically 10 kV. Semi-quantitative chemical analysis was conducted using an energy-dispersive X-ray (EDX) spectrometer (EDAX, Mahwah, NJ, USA).
Liquid medium inoculated with
For the calculation of yearly biomineral formation, we used the amount produced in our cultures (approx. 100 mg) during the time of incubation (3 weeks) to calculate how much this would make in 52 weeks, a full year. The result of such an approximate calculation for new mineral formation during a year would be 1700 mg. This is an underestimation, since the nucleation time is needed only once.
This study is focused on 138 bacterial isolates belonging to Proteobacteria (7 Alpha-, 7 Beta-, 35 Gammaproteobacteria), Bacteroidetes (5 isolates), Actinobacteria (47 strains) and Firmicutes (37 isolates; Table S2) from two different limestones – Lower Muschelkalk (LM) and Middle Muschelkalk (MM) – and three compartments for each lithotype representing rock (r), groundwater (gw) and soil (s). We used the total of 138 isolates to test whether they are able to form biominerals under laboratory conditions. Only 10 of these isolates (7.2 %) formed no crystals when tested on five solid and one liquid medium and at two temperatures. There were no obvious differences between isolates obtained from the six habitats (Figs. 1 through 6). This shows that in a carboniferous environment, strains forming carbonates are highly enriched.
Mineral formation by all strains tested from rock of Lower
Muschelkalk. The media used were B-4 with calcium acetate or B-4 with calcium
carbonate (B4-CO) or calcium phosphate (B4-CP), all tested at 28 or
10
Mineral formation by all strains tested from rock of Middle Muschelkalk. See legend of Fig. 1 for details.
As to the means of inducing mineralization, the influence of pH might be tested. While 89 strains did not change medium pH, 43 produced an alkaline environment supportive of carbonate formation (Figs. 1 through 6). However, two of those alkalinity-producing strains did not produce biominerals (rMM21, see Fig. 2, and W_5.3a, see Fig. 4); all other non-producers did not change the pH. In addition, crystal formation was observed with all strains which actually lowered pH (Figs. 1 through 6). The temperature clearly had an effect, although there was no clear correlation. While on one medium, a higher temperature may have induced formation of crystals not observed at a low temperature, the opposite effect was visible on the next medium (compare, e.g., rLM4.3, Fig. 1). Any combination of traits was observed. This indicates that differences among strains, rather than a mere general influence on the micro-environmental conditions leading to abiotic carbonate formation, were observed.
Biomineral identification by powder X-ray diffraction and EDX analyses from strains obtained from groundwater (gw), rock (r) and soil (s) samples from Lower (LM) and Middle Muschelkalk (MM) and grown on B-4 agar plates.
The highest incidence of mineral production was observed with soil isolates
obtained from soil developed on Middle Muschelkalk (Fig. 6).
Biomineralization potential was not associated with phylogeny. To give an
example, eight isolates of the genus
Mineral formation by all strains tested from groundwater in Stöben, Lower Muschelkalk. See legend of Fig. 1 for details.
From each habitat, the four most prevalent isolates as judged by colony
morphology during isolation were selected for further study (Table 1). Of
these 24 strains, 17 were gram positives, representing the taxa
Mineral formation by all strains tested from groundwater in Wichmar, Middle Muschelkalk. See legend of Fig. 1 for details.
Powder X-ray diffraction and EDX analyses identified mostly calcite and less
frequently vaterite and magnesium calcite (see Table 1). Calcite formed on
cultures from rock of Lower Muschelkalk
Mineral formation by all strains tested from soil on Lower Muschelkalk. See legend of Fig. 1 for details.
The color and morphology of crystal aggregates were highly variable; the colors ranged from colorless to brownish or purple, and morphologies ranged from individual rhombohedral crystals and round or acicular aggregates or rosettes to laminated crusts. Aggregates located within the agar mostly formed small rhombohedra or spheres.
Mineral formation by all strains tested from soil on Middle Muschelkalk. See legend of Fig. 1 for details.
Crystal morphologies and distribution shown for selected strains.
From Middle Muschelkalk soil,
Biominerals were on top of or below the biomass; for some strains the
crystals always formed at a certain distance from the colonies, which might
indicate a zone of change in pH around the culture (Fig. 7). Indicator plates
with bromothymol blue mainly indicated a change to basic pH
Mineral morphology is changed by growth temperature.
A recurring morphological feature was subparallel intergrowth of micrometer-
to sub-micrometer-sized crystallites. In simple cases, the resulting
aggregates resembled the morphology of a single crystal; in other cases, more
complex aggregates were found (Fig. 8; for further morphologies of other
isolates, see Fig. S1 in the Supplement). Crystal aggregates produced by
Phenotypic differences in crystal morphology were readily observed, dependent
also on temperature (Fig. 8). To show two examples,
On plates containing calcium carbonate or calcium phosphate, more bacterial
growth but less biomineralization was observed. On the medium with
CaCO
Growth and biomineralization of pairwise cocultured bacterial isolates from groundwater (gw), rock (r) and soil (s) samples from Lower (LM) and Middle Muschelkalk (MM).
The bacterial influence was visible also by direct associations.
Quantification of calcite precipitation by isolates from rock (r) and soil (s) of Lower (LM) and Middle Muschelkalk (MM). Similar cell counts were obtained. The dry weight of crystals was measured (see “Material and methods”).
Scanning electron micrographs for crystal morphologies of minerals
formed by
Two or four strains of one community were cocultivated to determine their
interactions. There was either no interaction, inhibition leading to lack of
growth or parasitism visible by overgrowing the competing strain(s).
Concerning biomineralization, the cocultivation had either no effect or it
enhanced precipitation, producing larger crystals in the contact zone
(Table 2). The biotic interactions showed an impact on biomineralization,
e.g., with more crystals being produced in the interaction zone between
The quantification of crystals formed resulted in similar cell counts ranging
from 2.06
Microbially induced calcite precipitation has been known as a general phenomenon since the 1970s and has found application in different fields such as the cementation of cracks, mostly through ureolytic bacteria, in historical memorials, buildings or sculptures made of limestone (van Tittelboom et al., 2010; Wong, 2015; Zhu and Dittrich, 2016). The general availability of bacterial isolates from different environments associated with limestones, however, was not accessed to the full (Gonzalez-Martinez et al., 2017; Seifan et al., 2017). Here, two lithotypes of Lower and Middle Muschelkalk were assessed for the prevalence of carbonate-precipitating bacteria.
Calcite, magnesium calcite and vaterite could be formed by the bacteria growing on standard laboratory media. Since vaterite occurs in aqueous, supersaturated solutions, high water content in the precipitates might be explained with water available from the agar. Magnesium calcite was likely formed when the bacterial cell surfaces accumulated the element (see also Cui et al., 2015; Rusznyak et al., 2012). Few trace elements, such as sulfur, chloride and phosphorus probably originating from cell components or salts included in the medium, have been detected (see also Rivadeneyra et al., 2000).
Different crystal aggregates with macromorphologies such as rhombohedra, rosettes and spheres were detected, occurring either in the medium at a distance to the inoculated bacteria or below or on top of the cultures. Morphological variations of crystal shapes revealed a microbial impact on mineral precipitation (compare Branson et al., 2016). Crystal colors by impurities incorporated into the lattice such as salts or secreted pigments were clearly observed, as calcite can incorporate metal ions in its crystal structure (Kang et al., 2014).
With respect to the alkaline pH favorable for calcium carbonate precipitation, a probable process might be the ammonification of amino acids, deriving from yeast extract added to the medium. By degrading amino acids, ammonia develops, which increases the pH to alkaline conditions. So far, mostly urease activity has been implied for a pH increase in biogenic calcite formation (Bachmeier et al., 2002; Okyay et al., 2016; Wei-Soon et al., 2012). However, our results with strains acidifying the medium and still precipitating calcium carbonate clearly show that other mechanisms are involved as well.
Biotic effects were investigated by cocultivation. Several interspecies
reactions might influence the outcome of biomineral production. For an easy
way to interpret interactions, growth should be considered. For example,
growth inhibition of a sympatric strain could be a result of secreted
antimicrobial compounds or competition (Hibbing et al., 2009). Growth
promotion, like with
Biogenic calcite precipitation may contribute to limestone sedimentation
(compare García et al., 2016). With 0.104 g L
Bacteria are known to contribute to the growth of carbonate stalactites that
grow by a few millimeters per year (Genty et al., 2011). Comparing these
observations to our experiment, bacterial isolates may exert a meaningful
impact on limestone deposition. In our experiment, 104 mg L
As a main result of our investigation of 138 isolates of two lithotypes of limestone in Germany, we can conclude that (magnesium) calcite and vaterite production can be induced through medium alkalinity and through direct surface interaction for nucleation visible in close associations but also in acidified media and a distance apart from the growing bacteria. This indicates that within the microbially induced calcium carbonate precipitation, mechanistically different routes of biomineralization are possible. Specifically, the control of morphologies at a distance to the colony seems interesting. We propose that molecules secreted by the bacteria, e.g., specific proteins, might lead to preferential crystal growth at different mineral surfaces due to coating. This clearly warrants further, more molecular studies.
All obtained sequences were made available via NCBI GenBank (accession numbers KX527662-KX527725, KX536502-KX536520, KX570902-KX570911, KX573089-KX573101; see Table S1 in the Supplement; see Meier et al., 2017). The strains are deposited with the Jena Microbial Resource Collection (Jena, Germany).
The authors declare that they have no conflict of interest.
The authors would like to thank Falko Langenhorst, Dirk Merten, Thomas Wach, Hans-Martin Dahse and Justus Linden for help with measurements. The International Max-Planck Research School ”Global Biogeochemical Cycles” and the Jena School for Microbial Communication (GSC124) are thanked for financial support. Erika Kothe wishes to acknowledge DFG-CRC 1127. Edited by: Denise Akob Reviewed by: two anonymous referees