(2) Microbial Silicification
Silica precipitation is an important geological process in many modern geothermal systems. Early studies considered it to be an entirely passive inorganic process. However, attempts to reproduce inorganic conditions in the laboratory usually generate precipitation rates which are orders of magnitude slower than those recorded in nature. This discrepancy may be the result of microbial activity, and many recent studies at hot springs have shown that microbes facilitate silicification over a range of temperatures. During silicification, some cells become so encrusted that their organic residues remain intact after lysis, i.e., as microfossils. As part of my early research programme while still at the University of Leeds, I was interested in better understanding the process by which microbes became fossilised in a siliceous matrix. In this regard, a first step was to ascertain the sites of silicification. Led by my former PhD student (Vernon Phoenix, now a Professor at the University of Strathclyde), we artificially subjected the cyanobacterium, Calothrix sp., to silica supersaturated solutions. Examination by transmission electron microscopy (TEM) revealed mineralisation of intact cells only occurred upon the extracellular sheath. No intracellular mineralisation was observed (Phoenix et al., 1999). In addition, polycationised ferritin (a probe 11 nm in diameter) labelled the sheath's outer surface but failed to penetrate the sheath matrix, indicating its impermeability to particles of this size and greater. We subsequently proposed a model to explain the restriction of silicification to the outer surface of the sheath. We suggested that cyanobacterial photosynthesis creates a moderately alkaline environment (pH 7-9) adjacent to the sheath, causing silica to form colloids. These colloids are too large to penetrate the sheath, and hence mineralisation is restricted to the outer surface. It was further proposed that the partial diffusion barrier created by the sheath allows very high photosynthetically induced pH levels (> pH 10) to build up inside the sheath matrix. Under these conditions, silica is both highly soluble and in its monomeric state and is thus less able to bind to the sheath matrix. To further test if the cells survived silicification, we measured the autofluorescence of the encrusted cells and found that their pigmentation remained intact. Their viability was confirmed by oxygen electrode analysis, which showed that the mineralised colonies were photosynthetically active. Moreover, they exhibited comparable rates of photosynthesis to the non-mineralised colonies, implying that mineralisation was not notably detrimental to the microorganism (Phoenix et al., 2000).
(Left Figure) Model for cyanobacterial silicification. Light microscopy image (Top Right Figure) and fluorescent image (Bottom Right Figure) of Calothrix filaments after silicification. The fluorescence indicates that mineralised cells are pigmented and still viable. Scale bar is 75 microns. X is filament. From Phoenix et al. (2000).
These observations have important consequences for the Precambrian. Many models of atmospheric evolution predict that the Archean atmosphere contained insufficient oxygen to form an effective ozone screen. Under such unfavourable conditions, early life forms must have used several different lines of defence, including vertical migration strategies, the production of shielding pigments or a variety of photo-repair mechanisms. However, we proposed a new alternative, i.e., that benthic bacteria naturally precipitated iron-silicate minerals, which fortuitously afforded them with UV protection (Phoenix et al., 2001). This experimental study made two exciting and completely novel observations. First, it showed that mat-forming cyanobacteria could survive the processes of iron-silicate biomineralisation, even when some filaments were encrusted in a mineral coating several micrometres thick. Second, we documented that the mineral coatings provided them with nearly 100% protection against UV-C; unmineralised cells quickly died. More recently, a former PhD student (Aleksandra Mloszewska, now a PDF at the University of Toronto) performed UV-C irradiation experiments to examine the growth of the planktonic marine cyanobacterium Synechococcus sp. PCC 7002 in Fe- and Si-rich media designed to mimic Archean seawater (Mloszewska et al., in review). Cultures grown in the presence of Fe and Si sustained less genetic damage than those grown in media either supplemented with Si alone or without either. Nanometer-sized Fe(III)-Si colloids effectively absorbed up to ~99% of incoming UV-C over a 1 cm path length. These findings have two significant implications. First, it suggests that such colloids may have been important in the earliest microbial colonization of the shallow marine littoral and open marine photic zones. Second, if marine cyanobacteria were protected from UV-C irradiation, then an alternate cause must exist to explain the hundreds of millions of years time gap between their evolution and the onset of atmospheric oxygenation at 2.45 Ga, the so-called Great Oxidation Event.
Understanding the mechanisms and salient physico-chemical features associated with bacterial silicification may also lead to a better understanding of what species comprised Earth's earliest microfossil assemblages (Konhauser et al., 2003). A case in point is the 1.88 billion-year-old "Gunflint-biota" that are preserved as carbonaceous and hematite filaments and spheres within microcrystalline chert, and whose taphonomy has been argued to be either cyanobacteria or Fe(II)-oxidizing bacteria (see Shapiro and Konhauser, 2015). In order to contribute to our understanding of what microbes comprised Precambrian microfossils, two areas of active research are (i) ascertaining how different microbes silicify and, ultimately fossilise, and (ii) can modern hot spring systems be used as analogues for determining what may have comprised Precambrian mat assemblages? Silicification potentially permits excellent preservation of the cells, as well as the microbial community in their life position. The small size of the initial silica precipitates (10s of nanometres in diameter) often facilitates a high fidelity moulding of cell shape and preservation of detail, such as septa separating two cells, internal cellular components, and details of the cell wall (e.g., Schultze-Lam et al. 1995; Konhauser and Ferris, 1996; Phoenix et al., 2000; Jones et al., 2004; Jones et al., 2005). By contrast, without fossilisation, microorganisms typically degrade within a few days of death, leaving unidentifiable remnants of the previous cells. The quality and preservation of silica cell moulds appear to depend on the rapidity of silica precipitation and complete entombment of microbial structures by silica. Fossilised microorganisms, or their moulds, may also be subject to post-depositional modification in shape, structure or size depending on the conditions of fossilisation and characteristics of the microorganisms. Therefore, if the identification of microbial taxa from the moulds is possible, it must be done with extreme care and complete knowledge of the possible preservational biases.
In two recent studies, my former Postdoctoral Fellow (Francois Orange, now a PDF at the University of Bordeaux) employed an experimental approach to simulate the formation of microbial moulds, using silica gel in presence of the cyanobacterium, Synechococcus elongatus. In the first study (Orange et al., 2013a), we showed that environmental constraints, such as temperature and time of drying, were crucial to obtaining accurate and strengthened moulds, as well as the ability of the microorganisms to cope with these constraints. However, our experimental conditions also systematically created preservational biases (size changes, additional structures) that can be misleading and make the identification of the microorganisms uncertain. In the second study (Orange et al., 2013b), silica solutions of different concentrations were repeatedly allowed to evaporate in both the presence and absence of the cyanobacterium Synechococcus elongatus. Without the cyanobacteria, consecutive silica additions led to the formation of well-laminated deposits comprised entirely of aggregated silica particles with average sizes in the 10s of nanometer range. By contrast, when bacteria were present, they acted as reactive surfaces for heterogeneous silica particles nucleation. Instead of forming a laminated structure, the deposits were either porous with a mixture of silicified and unmineralised cells, or they formed a denser structure with a complete entombment of the cells by a thick silica crust; which deposit formed depended on the initial silica concentrations. The deposits obtained experimentally showed numerous similarities in terms of their fabric to those previously reported for natural hot spring, demonstrating the complex interplay between abiotic and biotic processes involved in silica sinter growth.
(Left Figure) SEM and (Middle Figure) TEM micrographs of a cluster of well preserved and silicified Calothrix filaments. Photo courtesy of Vernon Phoenix. Scale bar is 5 microns. (Right Figure) TEM image of both extracellular and intracellular silicification of an unidentified bacterium from an Icelandic hot spring. From Konhauser and Ferris (1996).
Another area of previous research was directed at determining the kinetics and physico-chemical processes associated with microbial silicification (Konhauser et al., 2004). Preliminary laboratory studies conducted by my former Postdoctoral Fellow (Nathan Yee, now a Professor at Rutgers University) showed that the polymerisation of silica-supersaturated solutions occurs rapidly and that microbial biomass does not exert a significant effect on its rate (Yee et al., 2003). In fact, at high silica levels, there is such a strong chemical driving force for homogeneous nucleation and silica precipitation that there is no obvious need for microbial catalysis. Therefore, biogenic silicification at hot springs occurs simply because microbes grow in a polymerising solution where silicification is inevitable. With that said, there are species-specific patterns of silicification. Different microbes are certainly capable of being silicified with different degrees of fidelity. This is not at all surprising given that the actual mechanisms of silicification rely, in part, on the microorganisms providing reactive surface ligands that adsorb silica from solution, and accordingly, reduce the activation energy barriers to heterogeneous nucleation. This means that cell surface charge may have a fundamental control on the initial silicification process. For example, Phoenix et al. (2002) showed that the sheath of Calothrix is electrically neutral at pH 7, comprising predominantly of neutral sugars, along with smaller amounts of negatively charged carboxyl groups and positively charged amine groups, in approximately equal proportions. On the one hand, the low reactivity of Calothrix’s sheath gives the cells hydrophobic characteristics that facilitate their attachment to solid submerged substrata, i.e., siliceous sinters. On the other hand, this same property makes the sheath material less inhibitive to interaction with the colloidal silica fraction in solution. In this case, silicification subsequently occurs through hydrogen bonding between the hydroxy groups associated with the sugars and the hydroxyl ions of the silica (Benning et al., 2002; Benning et al., 2004). In contrast, the highly anionic nature of Bacillus subtilis limits silicification from occurring on its cell wall, likely as a result of electrostatic charge repulsion between the organic ligands and the negatively charged silica colloids (Phoenix et al. 2003). For silicification to proceed, cation bridging (e.g., Fe3+) is required. Subsequently, in the first study ever to artificially silicify a hyperthermophilic bacterium (Sulfurihydrogenibium azorense), my former PhD student (Stefan Lalonde) demonstrated that the magnitude of silica adsorption was dependent on the chemolithoautotrophic pathway being utilised by the cells during their metabolism (i.e., H2 versus S oxidation). It was also interesting to note that silica adsorption took place through a novel mechanism, that being an electrostatic interaction between silica and protein-rich biofilms that contain cationic amino groups (Lalonde et al., 2005).
(Left Figure) TEM image of Calothrix sp. These cyanobacteria produce extremely thick sheaths that can often double to triple the size of the cell. (Center Figure) Electrophoretic mobility measurements performed on Calothrix sp. at pH 5.5. The very electronegative peak at −2.5 μm /(s-1V-1cm-1) is characteristic of phosphate groups. The peak around +0.1 is isolated sheath material, made up predominantly of polysaccharides. The broader peak at around −0.3 is likely a composite of wall and sheath material. From Phoenix et al. (2002). (Right Figure) TEM images of Sulfurihydrogenibium azorense (grown by H2 oxidation after 50 h in a silica supersaturated solution. Note the nanoscale silica spheres have been restricted from the immediate cell surfaces. From Lalonde et al. (2005).
The goals for the future are to better understand the mechanisms underpinning silicification, and search for specific morphologies and/or chemical compositions of silicification to use as “biosignatures”. Major questions being sought are:
What are the primary environmental factors leading to microbial silicification?
Do the sites of silicification vary amongst species? In other words, are there species-specific patterns of silicification?
How do natural microbial communities survive silicification, and do the microbes change their surface reactivity or produce extracellular layers to cope with silicification?
Do the modern microbes benefit from being silicified? Did pre-ozone microbes use silica biominerals as an ultraviolet shield?
What is the fidelity of cellular preservation during silicification, and can modern silicified cells be used as analogues for ancient silicified microfossils?