All previous studies of the pyritization of soft parts have analyzed vegetal and animal fossils. Our paleomicrobiological analysis provides new information, and a substantial part of these data do not support some postulates and general affirmations about pyritization. In all observed and analyzed microbial fossils, permineralization of cells seemed to be the main mode of pyritization. It is stated that in a permineralized tissue, pyrite occurs inside the boundary between the organism and the surrounding sediment [5]. In (macro)fossils, preservation by this mechanism usually involves the replacement by pyrite of more degradable components, such as the cell walls of plants. The poorly biodegradable tissues, such as cellulose or chitin, may be preserved by the precipitation of pyrite in their pore spaces or infilling of cellular cavities [32]. However, the pyritization of labile tissues (for example, skin and muscle fibres) has not been documented [4, 33]. Experimental induction of pyritization in microalgae resulted in the infilling of Chlorella with iron sulphides and a limited disruption of cells [34]. This is the first time that it has been possible to analyze the pyritization pattern of a unicellular organism, including the internal architecture. The protist cortical structures, irrespective of their organization (cortex, plasmatic membrane, cell wall) and composition, are important centres of pyritization and they appear infilled by microcrystals of pyrite. This does not correspond to a mineral coat, since the pyrite does not occur in a thin and well-defined zone, immediately outside the organism/cellular boundary. Another cellular structure that is preferentially replaced by pyrite is the nucleus or nuclei, despite being composed of easily biodegradable macromolecules (DNA and proteins). It is remarkable that the pyrite texture of the nuclear envelope was different to that of the nucleoplasm. This pyritization pattern is different from those reported in plants, where the pyrite is precipitated in fluid-filled spaces in the plant, from cell-sized voids to spaces between fibrils in the cell wall [7, 34, 35]. Furthermore, there is no evidence for iron sulphides penetrating the cells during experimental pyritization [34].
Different cell wall composition in these two types of biological systems, as well as the absence of this structure in some protists (for example, ciliates), might explain the different patterns observed in protists and plants. When the protist has a skeleton, such as Cymbella, the siliceous frustule is replaced by a pyritized skeleton made of microcrystals. Dispersive X-ray analysis has shown the complete replacement of silica by pyrite. However, we cannot observe the degree of ornamentation preservation because we have only obtained longitudinal sections. Numerous assemblages of marine pyritized diatoms from the Tertiary (Palaeocene-Eocene) have been described [36–38]. Two different modes of pyritization have been reported; a change of the siliceous frustule by a well-preserved pyritized skeleton and the crystallization of pyrite in internal cavities of the diatoms, preserving only the external morphology.
The most frequent pyrite textures found in our fossilized protists were microcrystalline and framboidal. However, outside cells but inside the amber and also in the sediments surrounding the amber deposits, we found mainly octahedral or subhedral pyrite in clusters of different sizes. In studies of fossil plants from the Eocene London Clay, Grimes et al reported different textures in adjacent cells of the same vegetal tissue. They stated that there is no exclusive relationship between cell type and a particular texture or combination of textures [7]. This fact has been interpreted as evidence of the development of isolated chemical micro-environments and the progressive changes in pore water chemistry as a result of microbial activity and reactant availability during burial [7]. Our observations of pyritized plant debris from inside Cretaceous amber nuggets and the adjacent sediments showed some differences. We found no framboidal pyrite within the lumen of xylem vessels, a common occurrence in plant fossils from London Clay. On the contrary, the texture found in the lumina of xylem vessels is subhedral. In secondary xylem, microcrystalline pyrite infilled the lignified cell walls.
These results do not agree with the four-stage model proposed to explain the pyritization of fossils from London Clay [7], which assumed two affirmations which are very difficult to support from a microbiological point of view. First, it was proposed that pyrite nucleation may have been facilitated by the availability of oxidizing agents or bacterial biofilms. Sulphate-reducing bacteria, the microorganisms involved in biological formation of pyrite, are obligate anaerobes. Microbial biofilms favour the existence of anaerobic environmental conditions that are incompatible with the presence of oxidizing agents. Second, the existence of distinct pyrite textures in two adjacent cells is explained as a consequence of microhabitats [7]. The existence of microhabitats in biofilms and microbial mats, in which very different physiological groups of bacteria are distributed according to their physiological requirements, is well known. However, it is difficult to assume that there are diverse chemical micro-environments in the same eukaryotic microbial cell. The pyritized cell in Figure 6 is only around 12 μm in diameter and it showed at least two different pyrite textures.
The amber nuggets from Peñacerrada had frequent inclusions of vegetal debris, including root fragments, suggesting the microbial and vegetal materials were included in the resin in the soil, before their transport. The sedimentology of the amber deposits, including the type of mineralization found in the layer which was the most productive source of amber lumps, is representative of the early diagenesis caused by diverse populations of anaerobic bacteria that result from the deposition of organic matter, which impedes oxygen diffusion across the sediment [39]. The formation and accumulation of pyrite during the early diagenesis is controlled by a number of environmental factors, including availability of iron, sulphate and organic carbon, anoxic conditions, pyrite oxidation and hydrodynamics [10]. These factors are crucial for the optimal growth and metabolism of sulphate-reducing bacteria, the main microbial group involved in biological pyritization. With regard to the taxonomic aspects of the amber deposit, according to Alonso et al [25], amber lumps from nearby forests were transported in suspension by rivers and deposited in low-energy areas as floodplains and swamps. In this concentration process, well-preserved lumps and altered ones come together.
It is important to assign a temporal sequence to both pyritization and trapping in amber fossilization processes that protists underwent more than 114 My ago. Microanalysis of sediments adjacent to the amber deposits indicated that plant debris were pyritized, so the pyritization seems to be a prior and independent process to cellular inclusion into the resin. However, the pyritization may have continued for some time after the cells were embedded in the resin. Two reasons support this hypothesis. Organic matter degradation (decay) and mineralization (pyritization) represent two competing forces during early fossilization, and the amount of detail preserved in a biological fossil is usually a reflection of the end result of the relative timing of these two polar forces [1, 6]. Trapping in amber preserves tissues and cells because it arrests organic degradation; however, mineralization is also occurring. It is therefore possible to observe some details of cellular morphology of the protists in amber. Secondly, chemical analysis from Peñacerrada amber demonstrated anoxic conditions inside fossilized resin [40].
At present, microbial involvement in both pyrite precipitation to form sediments and pyritization (mineralization) of organisms, tissues and cells, is the subject of intense controversy. Authors favouring a chemical origin support their hypothesis with two main facts; pyrite framboids have been synthesized in some laboratories (see [41]), and microfossils of bacteria (in sediments, amber and so on) are relatively rare, exhibiting very low cellular concentrations although bacterial concentrations are, at present, usually high in many environments [42, 43]. However, more evidence supports early diagenetic pyrite forms, natural pyrite framboids and biological pyritization being a result of microbial processes, although some abiotic chemical reactions might also be involved. First, successful experimental synthesis of framboids has been achieved in only a few instances in which high temperature (150 to 300°C) and the addition of SO2 were employed, or by increasing the redox potential of the systems [41]. Second, fossilized bacteria with distinct morphologies have been observed in pyrite-containing sediments and oncoids by SEM and transmission electron microscopy [42–45]. Third, the results from very diverse experimental pyritization assays with plants show that anoxic sediments or cultures of sulphate-reducing bacteria are needed, that oxidizing conditions inhibits pyritization and that biomineralization of plants cells is rapid (24 to 80 days), which contributes to cell preservation [8, 34, 46]. Finally, it must be added that the chief physiological group of bacteria that lead pyritization, the sulphate reducers, appears to be a very ancient and diverse group of microorganisms [47]. Moreover, there is isotopic evidence of microbial sulphate reduction more than 2.7 Gy ago, in the early Archaean era [48].
Additional valuable information is provided by the characteristics of sediments located around the amber deposits where pyritized (micro/macro) fossils appeared. In the detailed description of fossiliferous amber deposits in New Jersey (Upper Cretaceous) by Grimaldi et al [49], where two pyritized insects were reported, the amber was buried in sediments that were highly reducing, as revealed by strong sulphurous gases during excavations, and by the abundance of pyrite and marcasite. A similar reducing environment, with H2S emission, fragments of carbonized microcrystalline wood (lignite) and octagonal crystals of pyrite crusts has been described in Peñacerrada sediments [25]. This type of mineralization is representative of deposition and degradation of organic matter by anaerobic bacteria [39]. For all of the above reasons, we think that the anaerobic sulphate-respiring bacteria play a key role in the biomineralization (pyritization) of Cretaceous protists and other microorganisms found in amber from Peñacerrada.
