Bacteria adhere strongly to the holocellulose component characteristic of Sphagnum mosses, and this interaction may form an important part of the remarkable preservative nature of peat bogs.
Figure 1. The Tollundmannen is one of the most famous proofs of the preservation efficiency of peat bogs. After being buried in a Danish peat bog for 2200 years, the body was recoved remarkably intact (Turner, R. C and Scaife, R. G. (1995). Bog Bodies - New Discoveries and New Perspectives. London: British Museum Press.)
Painter, T. J. (1983 a). Residues of d-lyxo-5-hexosulopyranuronic acid in Sphagnum holocellulose, and their role in cross-linking. Carbohydrate Research 124, C18 - C21.
Painter, T. J. (1983 b). Carbohydrate origin of aquatic humus from peat. Carbohydrate Research. 124, C22 - C26.
Painter, T. J. (1991 a). Lindow Man, Tollund Man and other peat-bog bodies: the preservative and antimicrobial action of sphagnan, a reactive glycuronoglycan with tanning and sequestering properties. Carbohydrate Polymers 15, 123 - 142.
Painter, T. J. (1991 b). Preservation in peat. Chemistry. & Industry. (London), 421 - 424.
Painter, T. J. (1998). Carbohydrate polymers in food preservation: an integrated view of the Maillard reaction with special reference to discoveries of preserved foods in Sphagnum - dominated peat bogs. Carbohydrate Polymers 36, 335-347.
Painter, T. J. (2000). Preservation in Sphagnum – dominated peat bogs. In: Briggs, D. E. G., Crowther, P. R. (Eds.). Palaeobiology II. Oxford: Blackwell Science.
Børsheim, K. Y., B. E. Christensen, T. J. Painter (2001) Preservation of fish by embedment in Sphagnum moss, peat, or holocellulose: experimental proof of the oxopolysaccharidic nature of the preservative substance and its antimicrobial and tanning action. Innovative Food Science and Emerging Technologies. 2, 63-74.
K. Y. Børsheima,*, B. E. Christensenb,$ and T. J. Paintera,¶
aDepartment of Biotechnology and bNorwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology, N-7491 Trondheim, Norway
Peat bogs formed of Sphagnum mosses have remarkable capacity to preserve organic materials. Objects which under other circumstances are easily mineralized by heterotrophic organisms, have been found to be preserved in peat bogs. Archaeologists have collected documentation of this phenomenon, including household utensils holding materials like butter, and various well preserved bodies including human specimens. During a study of the preservation processes that occur in peat bogs, we discovered an interaction between the Sphagnum moss polysaccharides and bacteria, that may play an important role in peat bog preservation. We have elsewhere described other important preservative effects, basically consisting of chemical reactions involving highly reactive carbonyl groups in Sphagnum holocellulose, binding ammonium and amino acids (Painter 1983a,b, Painter 2000). In this poster we suggest one more mechanism, which consists of direct binding of bacterial cells to the Sphagnum holocellulose.
Peat bogs consists mainly of dead sphagnan plant material, which seems to be close to resistant to biological degradation. Foreign organic material incidently introduced into the interiour of peat bogs may also be preserved for considerable amounts of time (Figure 1).
Aims and objectives
To test the effect of Sphagnum holocellulose on the concentration of various types of bacteria in suspension.
Material and Methods
Sphagnum holocellulose was prepared from leaves of peat moss (Sphagnum palustre). The leaves were first washed with acetone to remove pigments etc., and hot extraction with 1% sodium chlorite was used to remove lignin. This procedure leaves the structural carbohydrate from the plant, which appears as a white, amorphous substance which is almost insoluble in water. The structure of the holocellulose is not yet completely resolved, but 5-keto-d-oxymannurose (5KMA) constitutes approximately 20% of the molecule (Painter 1991a,b, Børsheim et al. 2001). The pure holocellulose has been shown to act as a strong preservative (Børsheim et al. 2001). In preservation experiments we used holocellulose which had been reduced by borohydride as a control substance. This treatment removes the reactive carbonyl groups on the 5KMA, which also turns off the preservative effect. As a double nonpreservative control we used wood cellulose.
Various cultures of bacteria were diluted in sterile buffer, and holocellulose was added. For practical reasons the cellulose was enclosed in a kind of ”teabag” which was sewed from plankton gauze of 60mm mesh size. Bacteria abundance was measured before and after the addition of the cellulose using plate counts.
The pure Sphagnum holocellulose removed a very large portion of the bacteria, regardless of bacterial type among the strains tested (Table 1). Some removal of bacteria can be expected in porous materials, but only a small part because the controls, both the borohydride treated holocellulose and the wood cellulose, removed only a few percent of the plate counts compared to the holocellulose.
We have previously shown that the carbonyl groups on the 5KMA residues in the holocellulose reacts with amino groups, and binds covalently to a variety of primary amines including proteins and pure ammonium (Painter 1998). Since proteins are abundant on bacterial surfaces, it is not surprising that also bacterial cells are immobilised by the holocellulose reactivity. The ability to arrest bacteria irreversibly may form a part of the amazing preservative capacity of peat bogs.
Escherichia coli < 0.4
Bacillus sp. < 0.25
Micrococcus sp. <10
*The strain was isolated from a preservation experiment.
The preserving properties of Sphagnum mosses are probably mediated by several mechanisms acting in concert. We have previously shown the effect of three separate mechanisms:
· Exoenzymes formed by microorganisms which are necessary to hydrolyse organic polymers are immobilized and inactivated because they rapidly form Schiff’s bases with the reactive carbonyl groups abundantly present in the Sphagnum holocellulose.
· The Sphagnum holocellulose reacts with organic material in general and the reaction products are inaccessible to normal enzymatic degradation. These reactions are superficially the same as the reactions taking place under industrial tanning of leather, and they lead to similar products.
· By forming Schiff’s bases with free ammonium, bonded nitrogen is removed from the bog water, consequently the microbes in the system experience severe nutrient limitation. (references: Børsheim et al. 2001, Painter 1991a,b, Painter 1998)
When we performed experiments with pure cultures of bacteria, we found that all bacterial species investigated adhered strongly to Sphagnum moss, peat and pure holocellulose from Sphagnum moss. The adhesion was strong, and occurred regardless of cell wall type such as inferred from the Gram reaction. We are planning to investigate how the strong adhesion of the bacterial cells to the plants influences the metabolisms, viability and survival of the bacteria. Presently we are confident that solid surfaces in natural peat bogs have a large capacity to remove bacterial cells from the aqueous phase of the environment, by reactions between highly reactive carbonyl groups in the Sphagnum holocellulose, and the bacterial surfaces.