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Wolfgang Heinen
July 11, 1928 - † June 30, 2006
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| "I will observe a humble attitude and the springs will tell me their own stories"(WH) |
A study on nanobes: introduction
In the last decennium with the availability of new powerful microscopic techniques the existence of life-forms smaller than 1 micrometer
(1 µm = 0.000 001 m = 1000 nm in which 1 micrometer is abbreviated to µm; and 1 nanometer becomes 1 nm) has become evident. With the discovery of these mitute organisms the impressively the discussion on the size limits for life has shown a revival. Meanwhiles cell-like structures with a size of a few decades of hundreds of nanometers, called nanobes, nano-bacteria, or nannobacteria, have been detected in almost all kinds of environments, ranging from common freshwater springs and thermal springs to carbonate deposits and sandstone deep below sea level. It has been proposed that nanobes present in the lithosphere (the outermost layer of the earth's crust) would be able to perform alterations of the chemical properties of neighbouring minerals, like is the case with
sandstone-building bacteria. Not only in stones but also in pathological tissues nanosized bacteria-like structures have been found; they are presumed to be involved in calcification processes ocurring in blood vessels and kidneys (
More about this topic).
Whether nanobes are truly independently living organisms, and no kristals, or remnants or parts (e.g. flagellae) of bacteria or other larger organisms, is still a matter of debate.
Herebelow electron microscopy (fesem) images of nanobes are presented that come from thermal springs in caves located in
Badgastein (Austria). These unpublished data come from the scientific heritage left by our dear colleague, Dr. Heinen, who passed away in June 2006. During many years, together with his assistent Anne-Marie Lauwers and electron microscopist Huub Geurts, Wolfgang studied a broad variety of microorganisms and putative nanobacteria (see colored structures in the SEM pictures hereabove) in biomats that had developped on pebbles or experimental substracts in the thermal water in the caves. Presently at the university of
Salzburg a research project is conducted in which the relationship between the presence of DNA material and these nano-sized structures is investigated. Besides there are plans to combine structure-analysis with the FESEM with element analysis in order to unravel whether the observed nano-shapes consist indeed of organic material.
Springs
Thermal springs, like those at Badgastein, represent a part of a water-circulating process, lasting in this particular case for about 3000 years. The cycle begins with the melting of water from the the nearby glaciers. This water trickles down through fissures and pores of the rocks to deep-down pockets and chambers. In the depth, the bottom waters are heated up by the local temperature and the relatively high radioactivity, due to the presence of uranium minerals, initiating an upward flux, which eventually forces the water to emerge from the springs. During this circulation process, especially in the way up through capillaries and cracks, microorganisms harbored in the rocks can be taken along and transferred to the surface.
| Thermal springs in Badgastein: sampling location and sampling |
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a Gate to the Franz-Joseph thermal spring in Badgastein (zoom)
b Entry sign of the Franz Josef spring (zoom)
c The Franz-Joseph gallery (zoom)
d a 15th-century print of the Bad Gastein thermal baths (zoom)
e Sampling of biofilms in a fissure from which hot water runs (zoom)
f Sampled quarts pebbles (zoom)
g Coverslips to sample microorganisms (zoom)
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One of the main springs of the radioactive thermal springs at Badgastein is the "Franz-Joseph-Quelle". This spring can be reached through a gallery which was horizontally driven more than 100 m into the rock. The main spring delivers some 8000 liters of thermal water (45.6 °C) per hour. The water contains high amounts of aluminium (Al) and silicium (Si), medium amounts of potasium (K) and iron (Fe), but also appreciable quantities of magnesium (Mg), phosphor (P), sulphur (S), further calcium (Ca), manganese (Mn) and copper(Cu). The rock contains also a variety of uranium -derived- minerals, like
radium (Ra-226) and radon (Rn-222). Several secondary springs with a much lower output (54 to 62 ml/min.) originate from cracks and fissures in the vertical rock surface next to the main spring. This location appears to be heavily colonized by lithobiontic microorganisms (and nano-organisms). Interaction with the substratum results in a weathering process, which can partly be followed by determining the leaching of silica and uranium from the colonized rock. In these springs water is delivered that has been in contact with the deep reservoirs inside the rock and numerous geological layers on the way to the surface. In this way elements and organisms from the rock-dwelling subsurface may have been transported to us by the hot water, like a space craft would do. The thermal springs thus provide a key to open a hatch to the hidden realm of the litho/biosphere.
Sampling for the FESEM analysis
For analysis in the scanning electron microscope (FESEM), samples were taken from the above-mentioned secondary springs, as well as from submerged quarts pebbles and fragments of rock (see
photographs of pebbles made with the binocular). In addition, numerous SEM views were taken from round coverglasses that had been placed in the stream of hot water that pours out of the fissures. These
coverglasses were clamped in a holder, so that one part of the glass surface that remained covered could serve as control, and the other would be left open to allow pebbles to be deposited and microorganisms to colonize the surface. Coverglasses were held for various time durations (days, weeks, months) in the water in order to investigate the colonization process overtime. As an additional indication for the presence of -combustible!- organic material the same FESEM samples were scrutinized before and after a short exposure into a flame; microorganisms, unlike resistent material like rock, was expected to be selectively eliminated by this treatment.
Results
Because there is no way to preclude whether nano-sized organisms will partake in all stages of the build-up of a microbial mat, all possible phases of biofilm formation were explored by microscopy. From such structural studies it is impossible to determine which type of organisms are populating the thermal springs. However, what could be clearly put into evidence was that a broad variety of microbes and nanobes was present in young as well as mature stages of colonization of the natural rocks and pebbles and experimental glass surfaces (illustrated here below in the figures
1 to
4. Variations in shape and size can be observed with a virtual magnifier in
5). When the bio-films were inspected under the scanning electron microscope at very high magnification, intricate honeycombed ribbon-structure were discovered, which are shown in part
6):
- 1 Surfaces with early stages of colonization
- 2 Surfaces with a developing biofilm
- 3 Small mineral particles surrounded by, or embedded in a covering mat
- 4 Mature biofilms adhering to each other with to the matrix
- 5 Shapes and sizes
- 6 honeycombed ribbon-structure
- Detailed figures article #1 (items 1 to 6) and figures article #2 (item 6)
| Results of investigations on nanobes in thermal springs |
| 1. "Nanobes" in early stages of colonization |
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a. Biomat with a.o. filaments lined up by beads with sharp edges (possibly inorganic crystalline 'excretates').
b. A multitude of beads (20-60 nm diameter) on a mineral surface, which locally align to short rods (colorized).
c. Spirilla (60-110 nm diameter) and small rods (<400nm x <200nm). |
| 2. "Nanobes" in a developing biofilm |
 | Filaments: Long but narrow filaments (70, 100, 140 nm diameter) and short rods (600-700 x 340 nm) with pili (arrows). Also "normal-sized" cells are present (not shown here).
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| 3. Mineral particles surrounded by a mat |
 | Beads and chains: Beads (bar = 100 nm !) of 40 - 45 nm diameter forming normal and branched rows which together combine to a huge cluster. |
| 4. Mature biofilm |
 | Overview of the variety: Rods, single beads and beads in a row, filaments of various diameter, spirils, networks of regular-shaped structures build together a dense biomat.Pseudo-color display |
| 5. Shape and size of nanobes in mature bio-films |
 Move the mouse over the picture to enlarge the view. If the magnifying function is lost, it can be reactivated by pressing the (F5) key for refresh in your browser.
Magnifier javascript: http://valid.tjp.hu/zoom
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Details of the variety : in the biofilm spheric forms with a diameter of about 700 nm, long filaments with diameters varying from 180 to 220 nm, rods with micrometer-sizes and also the following smaller featureswere observed (see example here below):
1 Small cocci with diameters of 140-200 nm
2 Tiny Spirillae (70 nm diameter)
3 "Unidentified spherical structure with a "top" (710 nm diameter)
4 Very small beads (35 nm diameter)
5 Three cells forming a filament, each 280 x 145 nm
6 Very small rods (210x36 nm)
7 Filaments (180-210 nm diameter)
8 Ultrathin filaments (60 nm diameter and about 4500nm long)
9 Small rod
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| 6. Honeycombed ribbon-structure at nanoscale |
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Ribbon-shaped honeycombed web: It is composed of small beads, which align to strings, merge to segments, and combine to tri-, tetra- and hexagons, which in turn arrange to the final ribbon structure. This intricate honeycombed ribbon-structure apparently represents an integral part of the mat community, because it was observed at many sites of the individual samples collected from various locations of the primary and secondary springs. Apparently the ribbons consist of organic matter, because they vanish after incineration. (scale bar = 100 nm!).
There are few examples for the existence of comparable features. One is a microbial fossil of undetermined identity dubbed "microcholla" (from its resemblance to the lignified skeletons of cholla cacti), detected in the now dry paleopools of Hidden Cave, NM, (Boston et al., 2001; Astrobiol. 1, 25-55). Scientific article #2 by Heinen et al. entirely dedicated to these structures.
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Scheme of the putative formation of above-shown honeycomb ribbon-shaped webs: The basic units for the web are beads of ca. 30 nm diameter (1), with the potential to merge to strings or segments. They can vary considerably with respect to the compounds they contain. Depending on which types of beads (A, B, C) align and merge, the resulting segments may differ greatly with regard to their properties (2). The two terminal spheres in a row of five are only by one third integrated into the unit, while the other two thirds belong as constitutional sections to the segments which branch off at both ends with a 45 degree angle (3, 3a, 4). This produces an intermediate Y-shaped structure (5), and delivers an upside-down-mirrored "Y" (6), which can further associate to hexagons (6). These in turn assemble horizontally (7) up to the width of the web (approximately 800 - 1000 nm), and apparently indefinitely in vertical direction, to accomplish the honeycomb-structure of the ribbon (8).
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Discussion and conclusions
Extracts from the
scientific article #1 by Heinen et al. on "
Putative nanobacteria in alpine thermal springs":
Our concept of the extension of the biosphere is again in a state of change since we have learned that "bacteria can penetrate rock", and that an entire realm of our planet, which had been considered to be sterile, is in fact teeming with life, representing a substantial part of the "unseen majority". By now we have at least some awareness of "the biosphere below", and begin to comprehend the consequences, including the concept that the nano-sized members of the community presumably represent structural-evolutionary steps towards the organizational level of present-day bacterial cells.
Realizing the spatial dimensions of sub-surface environments (the holes, pores, cracks and fissures in the rocks), it is conceivable that the smallest forms of life would fit in most conveniently. The observation that Triassic and Jurassic rock from 3400 to 5100 m below the seafloor is anything but sterile, instead heavily colonized by nano-sized organisms, is one of many convincing examples for this concept. The nano-scale representatives of the biosphere also fit into such environments so well because they are obviously involved in quite a variety of
geochemical processes.
In this respect, these springs represent an entry giving access to the biosphere below. Our results show, that the water brings up almost all the nano-sized features observed by other investigators /.../ It demonstrates that a remarkable fraction of the rock-dwelling community seems to be small-sized, and reflects the abundance, diversity and versatility, as well as the ubiquity of these subsurface nano-scaled forms of life. These observations also demonstrate that the search for possible life on planets or moons of our solar system should concentrate on the collection of subsurface samples instead of just scratching the surface.
Points of discussion from the scientific article #2 by Heinen et al. on " A honeycombed web from microbial mats of a thermal spring, a conceivable model for the structural evolution of microbial entities via self-assembly of nano-structures? ":
The "theoretical minimal size for a viable cell", in which all the indispensable ingredients (DNA, enzymes and other elements) can be harbored in a 20% to 70% water environment, is about 140 nm in diameter and 1.44 x 10-3 µm3 in volume (Maniloff 1997, Science 276, 1777). The majority of the nano-sized units presented in this paper are below this limit. The strategic solution to reach the level of a viable organism could be achieved by associating structural (sub-)units (in this case beads, segments and hexagons) to a coordinated and cooperating entity - probably an association of constituents with different properties. Is the "infinite web", a microbe composed of self-assembling nanobes (or at least nano-sized units), a conceivable model for what we define as "structural evolution"?
Download
Science:
- Full scientific paper #1 (pdf format) by Wolfgang Heinen, Huub Geurts and Anne-Marie Lauwers, entitled "Putative nanobacteria in biofilms from an alpine thermal spring"
- Detailed figures article #1
- Full scientific paper #2 (pdf format) by Wolfgang Heinen, Anne-Marie Lauwers and Huub Geurts, entitled "A honeycombed web from microbial mats of a thermal spring, a conceivable model for the structural evolution of microbial entities via self-assembly of nano-structures?"
- detailed figures article #2
- Abstract of paper by Weidler et al., entitled "Communities of Archaea and Bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps and evidence for ammonia oxidizing Crenarchaeota", accepted in Applied and Environmental Microbiology
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Wallpapers with nanobes (1024x768 px):
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Reference card :
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Links to further information on nanobes
Colophone
Scientific research: Wolgang Heinen and Anne Marie Lauwers (Department of Microbiology, University of Nijmegen).
Scientific collaboration: Helga Stan-Lotter (University of Salzburg) and co-workers (Weidler et al.; abstract of paper in Applied and Environmental Microbiology)
Electron microscopy: Wolgang Heinen, Huub Geurts and Geert-Jan Janssen (Department of GI)
Web-structure and lamascripts: Remco Aalbers
Webpages: Liesbeth Pierson
Contact: Email
Magnifier javascript: http://valid.tjp.hu/zoom
Acknowledgement
In his manuscript 'Nanobes 1', Wolfgang Heinen wrote: "A.M.L. and W.H want to express their gratitude to all officials at Badgastein, responsible for the attendance and maintenance of the thermal springs, who for years supported our activities at the Franz-Joseph-Quelle, especially Wassermeister Knoll and his coworkers (Wasserwerke). We are also very grateful to Dr. Alexandra Sänger (Dept. of Zoology, University of Salzburg, Austria) in her function as coordinator of the Forschungsinstitut Badgastein/Tauern region and as our guide to Austrian soul and spirit and other essentials. I (W.H.) also want to thank Professor Celestina Mariani, and all members of the Dept. of Experimental Botany, and the Dept. for General Instrumentation, Faculty of Science, University of Nijmegen, for their unwavering perpetual support, infinite patience and kind hospitality." 
![[Heet water bronnen in Badgastein en monstername][Thermal springs in Badgastein and sampling](/new/images/fesem_nanobes-thermalspringsoverview.jpg)
