Vernon R. Phoenix

Kinetics of calcite precipitation induced by ureolytic bacteria at 10 to 20°C in artificial groundwater

The kinetics of calcite precipitation induced in response to the hydrolysis of urea by Bacillus pasteurii at different temperatures in artificial groundwater (AGW) was investigated. The hydrolysis of urea by B. pasteurii exhibited a temperature dependence with first order rate constants of 0.91 d−1 at 20°C, 0.18 d−1 at 15°C, and 0.09 d−1 at 10°C. At all temperatures, the pH of the AGW increased from 6.5 to 9.3 in less than 1 d. Dissolved Ca2+ concentrations decreased in an asymptotic fashion after 1 d at 20°C and 15°C, and 2 d at 10°C. The loss of Ca2+ from solution was accompanied by the development of solid phase precipitates that were identified as calcite by X-ray diffraction. The onset of calcite precipitation at each temperature occurred after similar amounts of urea were hydrolyzed, corresponding to 8.0 mM NH4+. Specific rate constants for calcite precipitation and critical saturation state were derived from time course data following a second-order chemical affinity-based rate law. The calcite precipitation rate constants and critical saturation states varied by less than 10% between the temperatures with mean values of 0.16 ± 0.01 μmoles L−1 d−1 and 73 ±3, respectively. The highest calcite precipitation rates (ca. 0.8 mmol L−1 d−1) occurred near the point of critical saturation. While unique time course trajectories of dissolved Ca2+ concentrations and saturation state values were observed at different temperatures, calcite precipitation rates all followed the same asymptotic profile decreasing with saturation state regardless of temperature. This emphasizes the fundamental kinetic dependence of calcite precipitation on saturation state, which connects the otherwise dissimilar temporal patterns of calcite precipitation that evolved under the different temperature and biogeochemical regimes of the experiments.

The effect of cyanobacteria on silica precipitation at neutral pH: implications for bacterial silicification in geothermal hot springs

In this study, we performed silica precipitation experiments with the cyanobacteria Calothrix sp. to investigate the mechanisms of silica biomineralization. Batch silica precipitation experiments were conducted at neutral pH as a function of time, Si saturation states, temperature and ferrihydrite concentrations. The experimental results show that in solutions undersaturated with respect to amorphous silica, the interaction between Si and cell surface functional groups is weak and minimal Si sorption onto cyanobacterial surfaces occurs. In solutions at high Si supersaturation states, abiotic Si polymerization is spontaneous, and at the time scales of our experiments (1–50 h) the presence of cyanobacteria had a negligible effect on silica precipitation kinetics. At lower supersaturation states, Si polymerization is slow and the presence of cyanobacteria do not promote Si–solid phase nucleation. In contrast, experiments conducted with ferrihydrite-coated cyanobacteria significantly increase the rate of Si removal, and the extent to which Si is removed increases as a function of ferrihydrite concentration. Experiments conducted with inorganic ferrihydrite colloids (without cyanobacteria) removes similar amounts of Si, suggesting that microbial surfaces play a limited role in the silica precipitation process. Therefore, in supersaturated hydrothermal waters, silica precipitation is largely nonbiogenic and cyanobacterial surfaces have a negligible effect on silica nucleation. D 2003 Elsevier Science B.V. All rights reserved.

Molecular characterization of cyanobacterial silicification using synchrotron infrared micro-spectroscopy

Synchrotron-based Fourier-transform infrared (SR-FTIR) micro-spectroscopy was used to determine the concentration-dependent response of the organic structure of live cyanobacterial cells to silicification. Mid-infrared (4000–600 cm−1) measurements carried out on single filaments and sheaths of the cyanobacteria Calothrix sp. (strain KC97) were used to monitor the interaction between a polymerizing silica solution and the organic functional groups of the cells during progressive silicification. Spectra of whole-cells and sheaths were analyzed and the spectral features were assigned to specific functional groups related to the cell: lipids (-CH2 and -CH3; at 2870–2960 cm−1), fatty acids (>C=O at 1740 cm−1), proteins (amides I and II at 1650 and 1540 cm−1), nucleic acids (>P=O 1240 cm−1), carboxylic acids (C-O at 1392 cm−1), and polysaccharides (C-O between 1165 and 1030 cm−1). These vibrations and the characteristic vibrations for silica (Si-O between 1190 and 1060 cm−1; to some extent overlapping with the C-O frequencies of polysaccharides and Si-O at 800 cm−1) were used to follow the progress of silicification. Relative to unsilicified samples, the intensity of the combined C-O/Si-O vibration band increased considerably over the course of the silicification (whole-cells by > 90% and sheath by ∼75%). This increase is a consequence of (1) extensive growth of the sheath in response to the silicification, and (2) the formation of thin amorphous silica layers on the sheath. The formation of a silica specific band (∼800 cm−1) indicates, however, that the precipitation of amorphous silica is controlled by the dehydroxylation of abiotically formed silanol groups.

Cyanobacterial viability during hydrothermal biomineralisation

The cyanobacterium Calothrix sp., an isolate from the Krisuvik hot spring, Iceland, was mineralised in both silica and iron–silica solutions. After 12 days of incubation, many filaments in the silica solution developed extensive mineral crusts up to 5 μm thick. Mineralisation of filaments in the Fe–Si solution was more rapid; in 12 days, the entire colony became totally encased within a mineralised matrix. Examination by transmission electron microscopy (TEM) revealed mineralisation of intact cells only occurred upon the extracellular sheath; no intracellular mineralisation was observed. Additionally, mineralisation was predominantly restricted to the sheaths outer surface. Analysis of the mineralised bacteria by autofluorescence revealed the mineralised cells were intact and therefore likely viable. The viability of these cells was confirmed by oxygen electrode analysis, which showed that the mineralised colonies were photosynthetically active. Moreover, the mineralised colonies exhibited comparable rates of photosynthesis to the non-mineralised colonies, suggesting mineralisation was not notably detrimental. It is thus proposed that mineralisation can occur on living microorganisms, providing it is restricted to extracellular material such as the sheath. We further suggest that the sheath may be necessary in enabling some microorganisms to survive mineralisation, by both acting as an alternative mineral nucleation site (preventing cell wall and cytoplasmic mineralisation) and by providing a filter against colloidal silica.

The Microbial Role in Hot Spring Silicification

Abstract Recent experimental studies indicate that microorganisms play a passive role in silicification. The organic functional groups that comprise the outer cell surfaces simply serve as heterogeneous nucleation sites for the adsorption of polymeric and/or colloidal silica, and because different microorganisms have different cell ultrastructural chemistry, species-specific patterns of silicification arise. Despite their templating role, they do not appear to increase the kinetics of silicification, and at the very most, they contribute only marginally to the magnitude of silicification. Instead, silicification is due to the polymerization of silicasupersaturated hydrothermal fluids upon discharge at the surface of the hot spring. Microorganisms do, however, impart an influence on the fabric of the siliceous sinters that form around hot spring vents. Different microorganisms have different growth patterns, that in turn, affect the style of laminations, the primary porosity of the sinter and the distribution of later-stage diagenetic cementation.

The dynamics of cyanobacterial silicification: an infrared micro-spectroscopic investigation

Abstract The dynamics of cyanobacterial silicification was investigated using synchrotron-based Fourier transform infrared micro-spectroscopy. The changes in exo-polymeric polysaccharide and silica vibrational characteristics of individual Calothrix sp. filaments was determined over time in a series of microcosms in which the microbially sorbed silica or silica and iron load was increased sequentially. The changes in intensity and integrated area of specific infrared spectral features were used to develop an empirical quantitative dynamic model and to derive silica load-dependent parameters for each quasi-equilibrium stage in the biomineralization process. The degree of change in spectral features was derived from the increase in integrated area of the combined silica/polysaccharide region (Si-O/C-O, at 1150–950 cm−1) and the Si-O band at 800 cm−1, the latter representing specific silica bonds corresponding to hydrated amorphous SiO4 tetrahedra. From the degree of change, a two-phase model with concurrent change in process was derived. In the first phase, a biologically controlled increase in thickness of the exo-polymeric polysaccharide sheath around the cell was observed. In phase two, a transition to an inorganically controlled accumulation of silica on the surface of the cyanobacterial cells was derived from the change in integrated area for the mixed Si-O/C-O spectral region. This second process is further corroborated by the synchronous formation of non-microbially associated inorganic SiO4 units indicated by the growth of the singular Si-O band at 800 cm−1. During silicification, silica accumulates (1) independently of the growth of the sheath polysaccharides and (2) via an increase in chain lengths of the silica polymers by expelling water from the siloxane bonds. IR evidence suggest that an inorganic, apparently surface catalyzed process, which leads to the accumulation of silica nanospheres on the cyanobacterial surfaces governs this second stage. In experiments where iron was present, the silicification followed similar pathways, but at low silica loads, the iron bound to the cell surfaces slightly enhanced the reaction dynamics.

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

ABSTRACT The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pKa] between 5.0 and 6.2) and amine groups (mean pKa of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pKa spectra similarly dominated by carboxyls (pKa of 4.6 to 6.1) and amines (pKa of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pKa values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10−5 and 12.3 × 10−5 mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.

Role of biomineralization as an ultraviolet shield: Implications for Archean life

Cyanobacteria, isolated from the Krisuvik hot spring, Iceland, were mineralized in an iron-silica solution and irradiated with high levels of ultraviolet light. Analysis of the rates of photosynthesis, chlorophyll-a content, and phycocyanin autofluorescence revealed that these mineralized bacteria have a marked resistance to UV compared to nonmineralized bacteria. Naturally occurring sinters composed of iron-silica biominerals collected from

A field and modeling study of fractured rock permeability reduction using microbially induced calcite precipitation.

Microbially induced calcite precipitation (MICP) offers an attractive alternative to traditional grouting technologies for creating barriers to groundwater flow and containing subsurface contamination, but has only thus far been successfully demonstrated at the laboratory scale and predominantly in porous media. We present results of the first field experiments applying MICP to reduce fractured rock permeability in the subsurface. Initially, the ureolytic bacterium, Sporosarcina pasteurii, was fixed in the fractured rock. Subsequent injection of cementing fluid comprising calcium chloride and urea resulted in precipitation of large quantities (approximately 750 g) of calcite; significant reduction in the transmissivity of a single fracture over an area of several m(2) was achieved in around 17 h of treatment. A novel numerical model is also presented which simulates the field data well by coupling flow and bacterial and solute reactive transport processes including feedback due to aperture reduction via calcite precipitation. The results show that MICP can be successfully manipulated under field conditions to reduce the permeability of fractured rock and suggest that an MICP-based technique, informed by numerical models, may form the basis of viable solutions to aid pollution mitigation.

Ocean acidification impacts mussel control on biomineralisation

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.