Natural Science Forum / Biology / Paleontology / July 2005

Have you tried CALCALGAE  and the IFAA  (International Fossil Algae
Association)?
http://paleopolis.rediris.es/petralga/
<http://paleopolis.rediris.es/petralga/links.html>

http://www.ku.edu/~ifaa/m-whatsnew.html
<http://www.ku.edu/%7Eifaa/m-whatsnew.html>

Fay (carbonita@geologist.com)

Carnets de Géologie / Notebooks on Geology: Letter 2004/03
(CG2004_L03)

Contents

[Introduction] [Materials and methods] [Results] [Discussion]
[References] [Figures] and ... [Video]

Laboratory cultures of calcifying biomicrospheres generate ooids - A
contribution to the origin of oolites

Ulrike Brehm

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany
Ulrike.Brehm@uni-oldenburg.de

Katarzyna A. Palinska

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany

Wolfgang E. Krumbein

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany

Manuscript online since June 20, 2004

Abstract

The in vitro production of ooid-like structures as possible precursors
of oolites has been observed in laboratory cultures of spherical
microbial communities isolated from the Wadden Sea (North Sea). The
microbial spherulites consist of aggregated benthic diatoms (Navicula
perminuta) enveloped by layers of filamentous cyanobacteria of the
genus Phormidium and a halo-like biofilm of heterotrophic bacteria. The
development of the structures takes several months and these
configurations appear to be stable, before they calcify. The
precipitation starts on the surface of the spheres as clouds of small
scattered crystals, which later increase in size and aggregate to form
hollow spheres around the microbial assemblage. Here we report for the
first time carbonate precipitation in defined spherical microbial
communities.

Key Words

Carbonate precipitate; cyanobacteria; diatoms; ooids; spherulites;
microbial association.

Citation

Brehm U., Palinska K.A., Krumbein W.E. (2004).- Laboratory cultures of
calcifying biomicrospheres generate ooids - A contribution to the
origin of oolites.- Carnets de Géologie / Notebooks on Geology,
Maintenon, Letter 2004/03 (CG2004_L03)

Zusammenfassung

Ooidbildung durch carbonatfällende Biomikrosphären -
Laboruntersuchungen zur Entstehung von Oolithen.- Die in vitro
Entstehung von ooidähnlichen Strukturen als möglichen Vorläufern der
Oolithe, konnte in sphärischen mikrobiellen Gemeinschaften im Labor
beobachtet und dokumentiert werden. Die Biomikrosphären wurden aus dem
Wattenmeer (Nordsee) isoliert. Die Sphären bestehen aus einer
Ansammlung von benthischen Diatomeen (Navicula perminuta), die mit
mehreren Lagen fädiger Cyanobakterien der Art Phormidium und einer
äußeren Hülle von heterotrophen Bakterien umgeben sind. Die
Strukturentwicklung zieht sich über mehrere Monate hin und scheint
stabil zu sein, ehe die Calcifizierung eintritt. Die Kristallisierung
beginnt auf der Oberfläche der Sphären in Form von vereinzelten
kleinen Kristallansammlungen, die sich später vergrößern,
zusammenwachsen und damit eine Hohlkugel um die mikrobielle
Gemeinschaft bilden. Wir berichten hier zum ersten Mal über die
Carbonatausfällung in sphärischen mikrobiellen Gemeinschaften.

Stichworte

Carbonatausfällung; Cyanobakterien; Diatomeen; Ooide; Sphären;
mikrobielle Gemeinschaft.

Résumé

Production d'ooïdes à partir de cultures en laboratoire de
biomicrosphères se calcifiant - Une contribution à la genèse des
oolithes.- La production in vitro de structures semblables aux ooïdes
a été observée à partir de cultures d'associations microbiennes
sphériques récoltées en mer de Wadden (Mer du Nord). Les sphérules
microbiennes sont des agrégats de diatomées benthiques (Navicula
perminuta) entourés de couches de cyanobactéries filamenteuses du
genre Phormidium et d'une auréole de bactéries hétérotrophes. Le
développement de ces structures prend plusieurs mois et ces
organisations sont stables avant qu'elles ne se calcifient. La
calcification débute à la surface des sphères sous forme de nuages
de petits cristaux isolés qui vont ensuite se développer et
s'agglomérer pour constituer des sphères creuses autour des
associations microbiennes. Il s'agit de la première observation de
calcifications au sein de communautés microbiennes de forme
sphérique.

Mots-Clefs

Calcification ; cyanobactéries ; diatomées ; ooïdes ; sphérulites
; association microbienne.

Introduction

Precipitation of calcium carbonate is widespread in microbial
communities forming biofilms and microbial mats. The laminated
structure of these communities consists of layers of carbonate which
outlast the microbial colony that produced them. Fossilized remains of
these communities in which particles of other sediment are also
included are known as stromatolites. They have a long fossil record
since early Proterozoic and still flourish in particular in the reefs
of the Bahamas and Australia (e.g. Visscher et alii, 2002).

The typical stromatolitic structure is laminated. Each lamina
represents a horizon of former microbial biofilm or mat (Kalkowsky,
1908). Associated mineral particles (precipitates and detrital grains)
are overgrown and sometimes entirely coated by microbial assemblages
(Riding and Awramik, 2000). Small (mm size), spherical to oval
concentrically laminated carbonate bodies or aggregates, which form in
shallow tropical seas are called ooids and are known to become
consolidated into rocks called oolitic limestones (oolith, Rogenstein;
Kalkowsky, 1908). The genesis of ooid grains is still enigmatic. The
alternative explanations are confronted along the lines of
predominantly abiotic vs. biogenic origin of ooid grains and the
associated carbonate precipitates.

The principal biochemical processes that have been recognized to affect
the degree of carbonate saturation and therefore may cause carbonate
precipitation include:

environmental carbon depletion by autotrophs during photosynthesis (and
possibly chemolithotrophy),
deamination of amino acids in the course of bacterial proteolysis,
anaerobic bacterial dissimilatory sulfate reduction. All result in an
increase in pH and/or alkalinity, which promote carbonate precipitation
(Browne et alii, 2000).
In this study we show the microbially induced formation of ooids in the
laboratory.

Materials and methods

Filamentous cyanobacteria of the genus Phormidium, diatoms (Navicula
perminuta) and heterotrophic bacteria were isolated from Wadden Sea
(North Sea) microbial mats. Species identification was based on both
morphological features and the sequencing of the 16Sr RNA gene fragment
(data not shown).

The isolates were grown on artificial seawater medium ASNIII solidified
with 1% of Bacto Agar, prepared according to Rippka et alii (1979). The
Petri Dishes were maintained at 18ºC, and 120 µmol photons m-2 s-1
(Osram tungsten light tubes) and with a light/dark cycle of 12/12 h.
For the control experiments, the same medium and conditions of
incubation were used, without organisms.

The cultures used in our experiments were not axenic. We worked on
microbial community consisting of cyanobacteria, diatoms, and bacteria.
However, filamentous cyanobacteria were always the dominant species
regulating the development of the spheres.

In order to accelerate bacterial activity the experiments were set up
with signal substance BHL (Butyroyl-Homoserinlacton) in a final
concentration of 10 mM (Brehm et alii, 2003).

Light microscopy was performed on an inverted microscope (Zeiss
Axiovert).

Samples for TEM were prepared as described previously (Palinska and
Krumbein, 2000).

Results

Distinct spherical structures (Fig. 1 ) developed in culture by
aggregation of cells of filamentous cyanobacteria (Phormidium sp.),
heterotrophic bacteria and benthic diatoms (Navicula perminuta),
persist for an extended time and may suggest a symbiotic relationship
(Brehm et alii, 2003). Biomicrospheres isolated from a microbial mat of
the Wadden Sea (German Bight) have now been cultured and systematically
transferred in the laboratory for more than four years (Brehm et alii,
2003). Interestingly, the same type of biomicrospheres has also been
repeatedly observed and isolated from fresh, microbial mat samples.
Invariably after a cultivation period of two to three weeks a community
of one cyanobacterium species (Phormidium sp.), several well defined
heterotrophic species of bacteria and a diatom (Navicula perminuta)
created biomicrospheres 40-400 µm in diameter. Under laboratory
conditions the first step in the formation of biomicrospheres is the
appearance of a thin 1-3 µm thick envelope. This spherical envelope is
always observed and documented using light- and transmission electron
microscopy (Fig. 2 ) and is probably produced by the heterotrophic
bacteria. When the spheres appear they are recognized by filamentous
cyanobacteria that rapidly approach and forcefully penetrate into them
(see Video file ). All trichomes arrange themselves in the shape of a
thin spherical film inside the biomicrosphere. N. perminuta eventually
sneaks in with the Phormidium trichomes and by massive multiplication
fill the whole interior of the sphere (frustules and EPS). After twelve
weeks the peripheral part of the cyanobacterial coating of the sphere
turn "sclerotic", i.e. tiny scleres or sclera form a layer at or near
the outer surface.

The spheres promote calcification in the surface layers and ultimately
produce ooid-like hollow carbonate structures (Fig. 3 A-F ). In control
runs without microorganisms no carbonate precipitation was observed.

The calcium carbonate precipitates in many forms: microscopic carbonate
needles, wheat seed-shaped grains, small rods, dumbbells, simple balls
and joined balls (Fig. 4 ). Fractal growth influences not only the size
of the fractals but also the inclination of the next generation
(Krumbein, 1983; Busch et alii, 1999). All stages from sticks to balls
can exist at the same time. The solids merge mutually and build shells
and complex structures. In the laboratory these precipitates of
carbonate are closely connected with the appearance of structured
cyanobacterial assemblages (Fig. 5 , 6 ). After two or three months of
cultivation the spheres appear as multilayered circular assemblages in
which several belts of carbonates are precipitated (Fig. 7 ).

Discussion

The term "oolite" was introduced and defined by Brueckmann (1721) using
material collected a few kilometer away from the type locality where
the terms "stromatolite" and "ooid" were introduced and defined more
than 175 years later by Kalkowsky (1908). Interestingly Brueckmann
(1721), Kalkowsky (1908) and Ludwig and Theobald (1852) suggested
identical conditions of formation for ooids and stromatolites. However,
ooids would often be washed out of stromatolitic microbial mats and
deposited elsewhere. The formation of a typical ooid is connected with
a nucleation center, which can be of biotic or abiotic origin (Kühl et
alii, 2003). Here we supply laboratory evidence that the aforementioned
authors correctly analyzed the situation despite the vast literature on
calm water benthic stromatolites contrasted with agitated water
planktonic ooids and oolites. The formation of microbial mat derived
ooids and oolites has never before been demonstrated in laboratory
experiments.

Our studies were focused on carbonate precipitation in spherical
microbial communities (Fig. 1 ): assemblages of cyanobacteria
(Phormidium sp.), heterotrophic bacteria and diatoms (Navicula
perminuta) (Brehm, 2001; Brehm et alii, 2003). Precipitation occurs
where masses of bacteria are enclosed and concentrated in spherical
envelopes.

The envelopes are permeable only for cyanobacteria; other organisms
cannot penetrate them. Within the sphere diatoms accumulate in the
centre and cyanobacteria surround them. In this way a lamination is
established, comparable to the lamination of common benthic biofilms. A
similar phenomenon of microsphere formation has already been reported
by Fox et alii (1959). They demonstrated that when placed in water
certain proteins spontaneously self-organize into structures, known as
microspheres , that resemble primitive cells and proposed that
microspheres might represent a significant early stage in precellular
evolution.

Calcium carbonates precipitate in the laminations formed by
cyanobacteria and associated heterotrophs following the form of the
organism's organization: in stromatolites as horizontal laminations and
in the biomicrospheres studied here as concentric layers. The geometry
of the biofilm determines the shape and size of the carbonate layer.
Subsequently a calcisphere or spherulite, composed of numerous small
calcite crystals will form; the first step in the development of an
ooid (Fig. 8 ).

This structure is comparable to that of fossil oolites and suggests a
common genesis for calcispheres and oolites.

Oolites are always built up in several carbonate layers in which each
layer represents a separate population of organisms. These fossilized
concentric oolitic layers are preserved in carbonate rocks. The main
processes in the development of the aggregates studied in the
laboratory are limited to diffusion and/or cluster-cluster mechanisms.
The aggregates range from 10 to 40 µm in diameter. In sparsely
populated areas sparsely disseminated carbonates precipitate. They may
be the result of several discrete chemical reactions.

About three months elapse before the first carbonates crystallize in
the Petri dishes. The calcification of the biomicrospheres was always
observed to start on the spherulitic surface in the form of numerous
discrete calcite crystals. Ongoing precipitation leads to the covering
of the surface by carbonates. At the same time our control runs without
microorganisms never showed any precipitation.

Our investigations indicate that the building of oolites is
biologically induced and external nuclei are not necessary to create
spherulites. The microorganisms create the basis for their structures
in complete independence.

Acknowledgements

The content of this article was presented originally at the 8th
International Symposium on Fossil Algae organized by J.C. Braga and J.
Aguirre in Granada, Spain (September, 2003). The authors are most
grateful to N.J. Sander (USA) for improvements in the English of the
preliminary version. In addition they thank S. Golubic and two
anonymous reviewers for their comments and suggestions. The DFG project
Pa 842/1-1 is acknowledged as well.

References

Brehm U. (2001).- Untersuchungen zur mikrobiologisch induzierten
Strukturbildung-rezente und fossile Beispiele im Vergleich.- Ph.D.
Thesis, Oldenburg, 132 p.

Brehm U., Krumbein W.E., Palinska K.A. (2003).- Microbial spheres: a
novel cyanobacterial-diatom symbiosis.- Naturwissenschaften, Berlin,
90, pp. 136-140.

Browne K., Golubic S., Seong-Joo L. (2000).- Shallow marine microbial
carbonate deposits. In: Riding E.D., Awramik S.M. (Eds.): Microbial
Sediments.- Springer, Berlin, pp. 233-249.

Brueckmann F.E. (1721).- Specimen physicum exhibens historiam
naturalem, oolithi seu ovariorum piscium & concharum in Saxa.-
Mutatorum, Helmestadii, Salomoni & Schnorrii, 21 p.

Busch S., Dolhaine H., DuChesne A., Heinz S., Hochrein O., Laeri F.,
Podebrad O., Vietze U., Weiland Th., Kniep R. (1999).- Biomimetic
morphogenesis fluorapatite-gelantin composites: Fractal growth, the
question of intrinsic electric fields, core/shell assemblies, hollow
spheres and reorganization of denatured collagen.- European Journal of
Inorganic Chemistry, Weinheim, pp. 1643-1653.

Fox S.W., Harada K., Kendrick J. (1959).- Production of spherules from
synthetic proteinoid and hot water.- Science, Washington, 129, p. 1221.

Kalkowsky E. (1908).- Oolith und Stromatolith im Norddeutschen
Buntsandstein.- Zeitschrift der Deutschen Geologischen Gesellschaft,
Stuttgart, 60, pp. 84-125.

Krumbein W.E. (1983).- Microbial Geochemistry.- Blackwell, Oxford, 330
p.

Kühl M., Fenchel T., Kazmierczak J. (2003).- Growth, structure and
calcification potential of an artificial cyanobacterial mat. In:
Krumbein W.E., Paterson D.M., Zavarzin G.A. (Eds.): Fossil and recent
biofilms.- Kluwer, London, pp. 77-102.

Ludwig R., Theobald G. (1852).- über die Mitwirkung der Pflanzen bei
der Abscheidung des kohlensauren Kalkes.- Annalen der Physik und
Chemie, Leipzip, 87, pp. 91-107.

Palinska K.A., Krumbein W.E. (2000).- Perforation patterns in
filamentous cyanobacteria.- Journal of Phycology, Corvallis, 36, pp.
139-145

Riding R.E., Awramik S.M. (2000).- Microbial sediments.- Springer,
Berlin, 331 p.

Rippka R., Deruelles J,. Waterbury J.B., Herdman M., Stanier R.Y.
(1979).- Generic assignments, strain histories and properties of pure
cultures of cyanobacteria.- Journal of General Microbiology, Reading,
Vol. 111, pp. 1-61.

Visscher P.T., Reid R.P., McKenzie J.A., Vasconcelos C. (2002).-
Geomicrobial mechanisms of carbonate precipitation: Novel insights from
laminated structures.- Abstract, 12th Annual Goldschmidt Conference
2002, Davos, Switzerland.- Geochimica et Cosmochimica Acta, Oxford, 66
(15A), Suppl. 1, p. 808.

Figures

Click on thumbnail to enlarge the image.

Figure 1: Microbial biosphere. Assemblage of filamentous cyanobacteria,
heterotrophic bacteria and diatoms within a sphere. Diameter of the
sphere 166 µm.

Click on thumbnail to enlarge the image.

Figure 2: Transmission electron microscopy of the outer part of the
sphere. The envelope consisting of heterotrophic bacteria and their
excretes form the surface of the sphere. Below the envelope two
filaments of Phormidium sp. and one diatom (lower right side) are
documented. The space between organisms is filled by EPS (Extracellular
Polymeric Substances).

Click on thumbnail to enlarge the image.

Figure 3: Schematic biomicrosphere development stages. Cyanobacteria
are represented as long filaments, diatoms as rice-shaped grains, and
hetrotrophic bacteria as small dots. Five different stages of carbonate
crystallization are shown at the right, lower edge. The final stage of
the carbonate crystallization appears in E and F. A-B Initial stage;
Cyanobacteria passing through the spherical, bacterial envelope and
encompassing the diatoms. C Densely packed biomicrosphere with diatoms
concentrated in the centre and cyanobacteria surrounding them. D Late
stage of the biomicrosphere. Diatoms and some cyanobacteria abandon the
sphere. E-F Carbonate precipitation following the emplacement of
cyanobacteria. F Calcified biomicrosphere.

Click on thumbnail to enlarge the image.

Figure 4: Different stages of carbonate deposition in biomicrospheres.
In the background filaments of cyanobacteria are visible.

Click on thumbnail to enlarge the image.

Figure 5: Calcium carbonate precipitate, composed of numerous
spherically arranged layers of small calcite crystals. Intergrown
crystals are connected to cyanobacterial filaments.

Click on thumbnail to enlarge the image.

Figure 6: Concentric layers of calcium carbonate following the
cyanobacterial orientation.

Click on thumbnail to enlarge the image.

Figure 7: Late stage of calcification. Two coalescent spheres of
carbonate grains.

Click on thumbnail to enlarge the image.

Figure 8: Spherulitic surface of the biomicrosphere. Numerous, discrete
calcite crystals are visible.

Video

Click on thumbnail to play a low-resolution video file (wmv = 762 KB)
or click on the icon  to play a high-resolution video file (mpg = 8,606
KB)
or click on the icon  to download another high-resolution video file
(avi = 4,774 KB)

Video File: Cyanobacterial movement inside a biomicrosphere.

Carnets de Géologie / Notebooks on Geology: Letter 2004/03
(CG2004_L03)

Contents

[Introduction] [Materials and methods] [Results] [Discussion]
[References] [Figures] and ... [Video]

Laboratory cultures of calcifying biomicrospheres generate ooids - A
contribution to the origin of oolites

Ulrike Brehm

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany
Ulrike.Brehm@uni-oldenburg.de

Katarzyna A. Palinska

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany

Wolfgang E. Krumbein

Carl von Ossietzky Universität Oldenburg, Institute for Chemistry and
Biology of the Marine Environment, Arbeitsgruppe Geomikrobiologie, PO
Box 2503, D 26111 Oldenburg, Germany

Manuscript online since June 20, 2004

Abstract

The in vitro production of ooid-like structures as possible precursors
of oolites has been observed in laboratory cultures of spherical
microbial communities isolated from the Wadden Sea (North Sea). The
microbial spherulites consist of aggregated benthic diatoms (Navicula
perminuta) enveloped by layers of filamentous cyanobacteria of the
genus Phormidium and a halo-like biofilm of heterotrophic bacteria. The
development of the structures takes several months and these
configurations appear to be stable, before they calcify. The
precipitation starts on the surface of the spheres as clouds of small
scattered crystals, which later increase in size and aggregate to form
hollow spheres around the microbial assemblage. Here we report for the
first time carbonate precipitation in defined spherical microbial
communities.

Key Words

Carbonate precipitate; cyanobacteria; diatoms; ooids; spherulites;
microbial association.

Citation

Brehm U., Palinska K.A., Krumbein W.E. (2004).- Laboratory cultures of
calcifying biomicrospheres generate ooids - A contribution to the
origin of oolites.- Carnets de Géologie / Notebooks on Geology,
Maintenon, Letter 2004/03 (CG2004_L03)

Zusammenfassung

Ooidbildung durch carbonatfällende Biomikrosphären -
Laboruntersuchungen zur Entstehung von Oolithen.- Die in vitro
Entstehung von ooidähnlichen Strukturen als möglichen Vorläufern der
Oolithe, konnte in sphärischen mikrobiellen Gemeinschaften im Labor
beobachtet und dokumentiert werden. Die Biomikrosphären wurden aus dem
Wattenmeer (Nordsee) isoliert. Die Sphären bestehen aus einer
Ansammlung von benthischen Diatomeen (Navicula perminuta), die mit
mehreren Lagen fädiger Cyanobakterien der Art Phormidium und einer
äußeren Hülle von heterotrophen Bakterien umgeben sind. Die
Strukturentwicklung zieht sich über mehrere Monate hin und scheint
stabil zu sein, ehe die Calcifizierung eintritt. Die Kristallisierung
beginnt auf der Oberfläche der Sphären in Form von vereinzelten
kleinen Kristallansammlungen, die sich später vergrößern,
zusammenwachsen und damit eine Hohlkugel um die mikrobielle
Gemeinschaft bilden. Wir berichten hier zum ersten Mal über die
Carbonatausfällung in sphärischen mikrobiellen Gemeinschaften.

Stichworte

Carbonatausfällung; Cyanobakterien; Diatomeen; Ooide; Sphären;
mikrobielle Gemeinschaft.

Résumé

Production d'ooïdes à partir de cultures en laboratoire de
biomicrosphères se calcifiant - Une contribution à la genèse des
oolithes.- La production in vitro de structures semblables aux ooïdes
a été observée à partir de cultures d'associations microbiennes
sphériques récoltées en mer de Wadden (Mer du Nord). Les sphérules
microbiennes sont des agrégats de diatomées benthiques (Navicula
perminuta) entourés de couches de cyanobactéries filamenteuses du
genre Phormidium et d'une auréole de bactéries hétérotrophes. Le
développement de ces structures prend plusieurs mois et ces
organisations sont stables avant qu'elles ne se calcifient. La
calcification débute à la surface des sphères sous forme de nuages
de petits cristaux isolés qui vont ensuite se développer et
s'agglomérer pour constituer des sphères creuses autour des
associations microbiennes. Il s'agit de la première observation de
calcifications au sein de communautés microbiennes de forme
sphérique.

Mots-Clefs

Calcification ; cyanobactéries ; diatomées ; ooïdes ; sphérulites
; association microbienne.

Introduction

Precipitation of calcium carbonate is widespread in microbial
communities forming biofilms and microbial mats. The laminated
structure of these communities consists of layers of carbonate which
outlast the microbial colony that produced them. Fossilized remains of
these communities in which particles of other sediment are also
included are known as stromatolites. They have a long fossil record
since early Proterozoic and still flourish in particular in the reefs
of the Bahamas and Australia (e.g. Visscher et alii, 2002).

The typical stromatolitic structure is laminated. Each lamina
represents a horizon of former microbial biofilm or mat (Kalkowsky,
1908). Associated mineral particles (precipitates and detrital grains)
are overgrown and sometimes entirely coated by microbial assemblages
(Riding and Awramik, 2000). Small (mm size), spherical to oval
concentrically laminated carbonate bodies or aggregates, which form in
shallow tropical seas are called ooids and are known to become
consolidated into rocks called oolitic limestones (oolith, Rogenstein;
Kalkowsky, 1908). The genesis of ooid grains is still enigmatic. The
alternative explanations are confronted along the lines of
predominantly abiotic vs. biogenic origin of ooid grains and the
associated carbonate precipitates.

The principal biochemical processes that have been recognized to affect
the degree of carbonate saturation and therefore may cause carbonate
precipitation include:

environmental carbon depletion by autotrophs during photosynthesis (and
possibly chemolithotrophy),
deamination of amino acids in the course of bacterial proteolysis,
anaerobic bacterial dissimilatory sulfate reduction. All result in an
increase in pH and/or alkalinity, which promote carbonate precipitation
(Browne et alii, 2000).
In this study we show the microbially induced formation of ooids in the
laboratory.

Materials and methods

Filamentous cyanobacteria of the genus Phormidium, diatoms (Navicula
perminuta) and heterotrophic bacteria were isolated from Wadden Sea
(North Sea) microbial mats. Species identification was based on both
morphological features and the sequencing of the 16Sr RNA gene fragment
(data not shown).

The isolates were grown on artificial seawater medium ASNIII solidified
with 1% of Bacto Agar, prepared according to Rippka et alii (1979). The
Petri Dishes were maintained at 18ºC, and 120 µmol photons m-2 s-1
(Osram tungsten light tubes) and with a light/dark cycle of 12/12 h.
For the control experiments, the same medium and conditions of
incubation were used, without organisms.

The cultures used in our experiments were not axenic. We worked on
microbial community consisting of cyanobacteria, diatoms, and bacteria.
However, filamentous cyanobacteria were always the dominant species
regulating the development of the spheres.

In order to accelerate bacterial activity the experiments were set up
with signal substance BHL (Butyroyl-Homoserinlacton) in a final
concentration of 10 mM (Brehm et alii, 2003).

Light microscopy was performed on an inverted microscope (Zeiss
Axiovert).

Samples for TEM were prepared as described previously (Palinska and
Krumbein, 2000).

Results

Distinct spherical structures (Fig. 1 ) developed in culture by
aggregation of cells of filamentous cyanobacteria (Phormidium sp.),
heterotrophic bacteria and benthic diatoms (Navicula perminuta),
persist for an extended time and may suggest a symbiotic relationship
(Brehm et alii, 2003). Biomicrospheres isolated from a microbial mat of
the Wadden Sea (German Bight) have now been cultured and systematically
transferred in the laboratory for more than four years (Brehm et alii,
2003). Interestingly, the same type of biomicrospheres has also been
repeatedly observed and isolated from fresh, microbial mat samples.
Invariably after a cultivation period of two to three weeks a community
of one cyanobacterium species (Phormidium sp.), several well defined
heterotrophic species of bacteria and a diatom (Navicula perminuta)
created biomicrospheres 40-400 µm in diameter. Under laboratory
conditions the first step in the formation of biomicrospheres is the
appearance of a thin 1-3 µm thick envelope. This spherical envelope is
always observed and documented using light- and transmission electron
microscopy (Fig. 2 ) and is probably produced by the heterotrophic
bacteria. When the spheres appear they are recognized by filamentous
cyanobacteria that rapidly approach and forcefully penetrate into them
(see Video file ). All trichomes arrange themselves in the shape of a
thin spherical film inside the biomicrosphere. N. perminuta eventually
sneaks in with the Phormidium trichomes and by massive multiplication
fill the whole interior of the sphere (frustules and EPS). After twelve
weeks the peripheral part of the cyanobacterial coating of the sphere
turn "sclerotic", i.e. tiny scleres or sclera form a layer at or near
the outer surface.

The spheres promote calcification in the surface layers and ultimately
produce ooid-like hollow carbonate structures (Fig. 3 A-F ). In control
runs without microorganisms no carbonate precipitation was observed.

The calcium carbonate precipitates in many forms: microscopic carbonate
needles, wheat seed-shaped grains, small rods, dumbbells, simple balls
and joined balls (Fig. 4 ). Fractal growth influences not only the size
of the fractals but also the inclination of the next generation
(Krumbein, 1983; Busch et alii, 1999). All stages from sticks to balls
can exist at the same time. The solids merge mutually and build shells
and complex structures. In the laboratory these precipitates of
carbonate are closely connected with the appearance of structured
cyanobacterial assemblages (Fig. 5 , 6 ). After two or three months of
cultivation the spheres appear as multilayered circular assemblages in
which several belts of carbonates are precipitated (Fig. 7 ).

Discussion

The term "oolite" was introduced and defined by Brueckmann (1721) using
material collected a few kilometer away from the type locality where
the terms "stromatolite" and "ooid" were introduced and defined more
than 175 years later by Kalkowsky (1908). Interestingly Brueckmann
(1721), Kalkowsky (1908) and Ludwig and Theobald (1852) suggested
identical conditions of formation for ooids and stromatolites. However,
ooids would often be washed out of stromatolitic microbial mats and
deposited elsewhere. The formation of a typical ooid is connected with
a nucleation center, which can be of biotic or abiotic origin (Kühl et
alii, 2003). Here we supply laboratory evidence that the aforementioned
authors correctly analyzed the situation despite the vast literature on
calm water benthic stromatolites contrasted with agitated water
planktonic ooids and oolites. The formation of microbial mat derived
ooids and oolites has never before been demonstrated in laboratory
experiments.

Our studies were focused on carbonate precipitation in spherical
microbial communities (Fig. 1 ): assemblages of cyanobacteria
(Phormidium sp.), heterotrophic bacteria and diatoms (Navicula
perminuta) (Brehm, 2001; Brehm et alii, 2003). Precipitation occurs
where masses of bacteria are enclosed and concentrated in spherical
envelopes.

The envelopes are permeable only for cyanobacteria; other organisms
cannot penetrate them. Within the sphere diatoms accumulate in the
centre and cyanobacteria surround them. In this way a lamination is
established, comparable to the lamination of common benthic biofilms. A
similar phenomenon of microsphere formation has already been reported
by Fox et alii (1959). They demonstrated that when placed in water
certain proteins spontaneously self-organize into structures, known as
microspheres , that resemble primitive cells and proposed that
microspheres might represent a significant early stage in precellular
evolution.

Calcium carbonates precipitate in the laminations formed by
cyanobacteria and associated heterotrophs following the form of the
organism's organization: in stromatolites as horizontal laminations and
in the biomicrospheres studied here as concentric layers. The geometry
of the biofilm determines the shape and size of the carbonate layer.
Subsequently a calcisphere or spherulite, composed of numerous small
calcite crystals will form; the first step in the development of an
ooid (Fig. 8 ).

This structure is comparable to that of fossil oolites and suggests a
common genesis for calcispheres and oolites.

Oolites are always built up in several carbonate layers in which each
layer represents a separate population of organisms. These fossilized
concentric oolitic layers are preserved in carbonate rocks. The main
processes in the development of the aggregates studied in the
laboratory are limited to diffusion and/or cluster-cluster mechanisms.
The aggregates range from 10 to 40 µm in diameter. In sparsely
populated areas sparsely disseminated carbonates precipitate. They may
be the result of several discrete chemical reactions.

About three months elapse before the first carbonates crystallize in
the Petri dishes. The calcification of the biomicrospheres was always
observed to start on the spherulitic surface in the form of numerous
discrete calcite crystals. Ongoing precipitation leads to the covering
of the surface by carbonates. At the same time our control runs without
microorganisms never showed any precipitation.

Our investigations indicate that the building of oolites is
biologically induced and external nuclei are not necessary to create
spherulites. The microorganisms create the basis for their structures
in complete independence.

Acknowledgements

The content of this article was presented originally at the 8th
International Symposium on Fossil Algae organized by J.C. Braga and J.
Aguirre in Granada, Spain (September, 2003). The authors are most
grateful to N.J. Sander (USA) for improvements in the English of the
preliminary version. In addition they thank S. Golubic and two
anonymous reviewers for their comments and suggestions. The DFG project
Pa 842/1-1 is acknowledged as well.

References

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Figures

Click on thumbnail to enlarge the image.

Figure 1: Microbial biosphere. Assemblage of filamentous cyanobacteria,
heterotrophic bacteria and diatoms within a sphere. Diameter of the
sphere 166 µm.

Click on thumbnail to enlarge the image.

Figure 2: Transmission electron microscopy of the outer part of the
sphere. The envelope consisting of heterotrophic bacteria and their
excretes form the surface of the sphere. Below the envelope two
filaments of Phormidium sp. and one diatom (lower right side) are
documented. The space between organisms is filled by EPS (Extracellular
Polymeric Substances).

Click on thumbnail to enlarge the image.

Figure 3: Schematic biomicrosphere development stages. Cyanobacteria
are represented as long filaments, diatoms as rice-shaped grains, and
hetrotrophic bacteria as small dots. Five different stages of carbonate
crystallization are shown at the right, lower edge. The final stage of
the carbonate crystallization appears in E and F. A-B Initial stage;
Cyanobacteria passing through the spherical, bacterial envelope and
encompassing the diatoms. C Densely packed biomicrosphere with diatoms
concentrated in the centre and cyanobacteria surrounding them. D Late
stage of the biomicrosphere. Diatoms and some cyanobacteria abandon the
sphere. E-F Carbonate precipitation following the emplacement of
cyanobacteria. F Calcified biomicrosphere.

Click on thumbnail to enlarge the image.

Figure 4: Different stages of carbonate deposition in biomicrospheres.
In the background filaments of cyanobacteria are visible.

Click on thumbnail to enlarge the image.

Figure 5: Calcium carbonate precipitate, composed of numerous
spherically arranged layers of small calcite crystals. Intergrown
crystals are connected to cyanobacterial filaments.

Click on thumbnail to enlarge the image.

Figure 6: Concentric layers of calcium carbonate following the
cyanobacterial orientation.

Click on thumbnail to enlarge the image.

Figure 7: Late stage of calcification. Two coalescent spheres of
carbonate grains.

Click on thumbnail to enlarge the image.

Figure 8: Spherulitic surface of the biomicrosphere. Numerous, discrete
calcite crystals are visible.

Video

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Video File: Cyanobacterial movement inside a biomicrosphere.