Enzymatic phosphatization of fish scales-a pathway for fish fossilization

Abstract

Phosphatized fish fossils occur in various locations worldwide. Although these fossils have been intensively studied over the past decades they remain a matter of ongoing research. The mechanism of the permineralization reaction itself remains still debated in the community. The mineralization in apatite of a whole fish requires a substantial amount of phosphate which is scarce in seawater, so the origin of the excess is unknown. Previous research has shown that alkaline phosphatase, a ubiquitous enzyme, can increase the phosphate content in vitro in a medium to the degree of saturation concerning apatite. We applied this principle to an experimental setup where fish scales were exposed to commercial bovine alkaline phosphatase. We analyzed the samples with SEM and TEM and found that apatite crystals had formed on the remaining soft tissue. A comparison of these newly formed apatite crystals with fish fossils from the Solnhofen and Santana fossil deposits showed striking similarities. Both are made up of almost identically sized and shaped nano-apatites. This suggests a common formation process: the spontaneous precipitation from an oversaturated solution. The excess activity of alkaline phosphatase could explain that effect. Therefore, our findings could provide insight into the formation of well-preserved fossils.

Figure 1

Phase diagram of calcium phosphate species in water. The position of present seawater is marked and lies within the apatite stability. (Ca concentration after, pH in equilibrium with 423 ppmV CO₂ = 2023).

Figure 2

SEM image (SE) of experiment FAV 1_e. A fish scale with 50 µl AP, and 100 mM NPP in seawater buffer for 1 week. (a) shows the whole scale, where the growth rings can be seen. (b) shows a detailed view and that the scale is covered with newly formed apatite. (c) shows that the newly formed apatite replicates the primary physiological information (growth rings). Note that the whole scale is covered in newly formed apatite and the original surface cannot be seen anymore.

Figure 3

SEM images (SE) of different experiments where small apatite crystals were precipitated. These experiments were performed using pasteurized bacterial biomass as substrate (see “Methods”). The crystals appeared as small apatite grains (size ~ 50 µm) on top of the target material (fish scale) as can be seen in (a) and had an angular shape with clear crystal surfaces (b). The composition was verified by EDS. (c) Different grain from a replicate experiment.

Figure 4

SEM image (SE) of a larger (~ 800 µm) apatite. Please note: The apatite appears to be broken in half and could have been even larger before breaking. Its shape resembled the curvature of the fish scale. The composition was verified by EDS.

Figure 5

SEM image of the largest (~ 1 mm) apatite found in the experiments. This grain resulted from an experiment with the same composition as described above (bovine AP, M. luteus as substrate, lysozyme, and TRIS buffer). This grain was later analyzed by TEM.

Figure 6

EDX analyses of the Gyrodus sample. Calcium, phosphorus, and oxygen are typical for apatite. Fluorine is typical in natural apatites from sediments. (A) Overview. Two marked sections of the fossils were examined in (B) and in (C).

Figure 7

Results from the TEM analysis of the fossil apatite from Gyrodus in Solnhofen. (a,b) show bright-field images of the fish fossil. The apatite here is composed of ~ 20 nm large single crystals. Dark contrast crystals are those with a low-indexed zone axis oriented parallel to the electron beam. HREM (high-resolution TEM) is used for (c) to show the amorphous pore space between the individual nanocrystals with lattice fringes visible thus indicating crystallinity.

Figure 8

Results from the TEM analysis of the experimental apatite. (a) shows the position the sample was taken from the grain of apatite. (b) shows a bright-field image of the nanocrystals that make up the larger grain. Crystals with dark contrast are oriented with a low-indexed zone axis in parallel to the electron beam. (c) shows the HREM results and demonstrates that the crystals in the experimental apatites are separated by amorphous material from each other.