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Exploitable results
The significance of bacterial decay for piling constructions became obvious after 1990 in the Netherlands and in the last decade a general impression was achieved of the actual state of the Dutch foundation constructions. It became clear that within 100 years piling constructions, which were always underneath the groundwater table could be severely degraded and that some cities are more susceptible than others. Although wooden foundation constructions are well known in Sweden and Venice, the problems appear to be not so prominent in both areas. In Sweden bacterial decay in wooden piles was already recognise in 1970 but it was regarded as of minor importance for the quality of the buildings. However Venice has to deal with severe settlement problems, but bacterial degradation was not seen a possible cause of this problem.
In Great Britain and Germany wooden piling construction are of less importance, although some locations are known with buildings on wooden piles. Beside some fundamental research was carried out on archaeological wood, bacterial degradation is not an item in the archaeology, in any of the five countries. However all available information shows that bacterial degradation is the major threat to the wooden cultural heritage stored under water or in soils. As under fungal and animal activity wood is degraded fast and if the environment allows these types of degradation no wood is conserved after 100 years and no archaeological wooden remainders will be found. There are records of wooden remaining over a long period of more then 2000 years. For archaeologists the state of the wood sample is important and is related to the possibility to recognise the original sample shape and size. In wooden remains degraded by bacterial, the outline of the original sample is often intact and although it is soft its wood quality is regarded as good. It is known that after drying the wood structure will collapse and specify treatments are developed to remove the water and retain the original sizes.
Based on all experiences and fundamental research the following statements on bacterial degradation can be made:
In general
If wood is well conserved (no bacterial degradation appears) it can last for centuries and retain its strength properties;
It is believed that in combination with fungal activity, bacterial wood degradation is more aggressive;
The significance of bacterial degradation for piling constructions and archaeological wood is underestimate in Germany, Great Britain and Italy.
Related to wooden foundations:
All wooden foundation piles that have been in service for about 100 years and that are situated under the groundwater level show bacterial decay at least in their outermost layer;
The degradation process starts from the out-side and gradually decreases towards the centre of the pile;
Spruce seems to have a higher resistance against bacterial degradation than pine sapwood and alder. Pine heartwood seems to be quite resistant;
In contrast to the general trend, situations were recorded where 100 years old Spruce piles were severely degraded and in other situations 100 years old Pine piles were without any degradation;
The degree of bacterial degradation depends on the location. There are Dutch cities were the sapwood of the pine piles is fully degraded in a period of 70 years. Whereas in other cities the pine sapwood is less degraded in the same period. The degraded peal in Spruce piles is much more limited and in about 100 years this peal is less than 5mm in some cities or can be as wide as 20m in other cities.
Polluted environment with nitrogen / phosphorus seems to increase the degree of degradation and a clear relationship was found between the degree of degradation and nitrogen / phosphorus concentration in the wood;
Oxygen levels around the piles are supposed to be low;
Severe bacterial attack seems to appear more in permeable soils (such as sand), whereas the attack is less in non-permeable soils such as clay and peat;
Boron treatment is used to conserve piles but no result is available on its efficiency.
Archaeology
In the archaeology Pine and oak are regard as most durable species in contrast to alder, willow, beech, birch, and lime.
In contrast to the situation around piles, archaeology wood underneath the groundwater surface, degradation seems to be less fast in sandy soils than in peat or clay soils.
In archaeological wood there seems to be no relation between degree of attack and age;
With increasing excavation depth the degree of decay seems to be decreasing.
Although excavated wood seems often sound, this is not case because precautions are almost always necessary in order to avoid collapse of the wood structure and excavated wood seems to be susceptible for fungal attack.
It can be concluded that the impact of bacterial wood degradation in Europe is underestimated especially in the archaeology.
Molecular analyses of original BACPOLES wood samples showed a large number of different bacteria species to be present in the wood material.
Wood degradation of BACPOLES samples by bacteria was observed in the laboratory and these bacteria were successfully isolated and purified. It was proposed that these bacteria belonged to the CFB complex (Cytophaga-Flavobacterium-Bacteriodes), based on:
- Their common occurrence in BACPOLES samples;
- literature surveys that described the CFB group as abundant in very diverse environments in nature, including anoxic sediment;
- Morphological studies showing the isolates to have similarities with bacteria from the CFB group (gram negative rod shaped bacteria, motile by gliding, absence of flagellae, and size);
- Subsequent molecular analysis confirmed that the isolates did belong to the CFB complex and that these bacteria were common in many samples.
Erosion bacteria were isolated from quite varying environments and the different molecular sequences that were identified suggested that several different species of erosion bacteria probably exist, even within the CFB complex. The identity of active erosion bacteria must therefore be documented on a site-by-site basis before any treatment can occur. Further, if phages are to be used as a biological control for wood degradation then a number of phages will have to be produced.
Future work is necessary to find the most common erosion bacteria and also to gain more knowledge on the individual species and their physiological requirements for culturing under laboratory conditions. With increased access to more pure cultures and isolates it will be possible to take a closer look at their general physiology but also the enzymatic systems required for the degradation of lignocellulosic materials. As yet these systems are not understood.
FISH (Fluorescent in situ hybridisation) appears to offer a valuable technique for fast in situ observation and identification of potential erosion bacteria prior to phage-treatment. However, further refinement of this technique is required.
It was hard to compare the 27 sites sampled because of their large variety in soil type, groundwater level and age. It can be concluded that wood degrading bacteria exist in a wide range of environmental conditions and is active in timbers from different ages ranges from 65 - 3000 years. Although the process of degradation is not fully understood it is clear that velocity is environmental and species depended. From the long-term measurements site we learn that in two different Amsterdam environments (one more active then the other one) pine sapwood is not degraded by bacteria within a period of 18 months. Whereas degradation in a lower level is found only after 300 years under salty conditions in site 12 or after 600 years in Venice.
Regarding the foundation sites only, the following general trends can be seen:
1. There seems to be no relation between the degree of decay and the surrounding environment;
2. There seems to be a relation between the nitrogen content in the wood (and phosphors content) and the degree of degradation;
3. The transport of nitrogen (and phosphorus) to the inside of the wood seems to be crucial for active bacterial wood degradation. The origin of nitrogen could be explained by (or a combination of) A) already available in the wood before installation of the piles; B) active accumulation by bacteria (e.g. nitrogen fixating bacteria); C) water flux through the piles; D) diffusion
4. Because bacterial wood degradation is found under oxygen free circumstances and without a "continuous" stream of oxygen supply, it seems that oxygen is not a crucial factor to initiate and support the process of bacterial wood degradation. However an increasing oxygen concentration seems to speed up the process of degradation as showed in the microcosm experiment (chapter 4. But this could be caused by secondary wood degrading fungi, which feed themselves with bacterial debris and stimulate in this way the bacterial activity. Wood under these circumstances shows more "colonising fungi", which could be actually secondary wood degraders. Therefore the role of oxygen is unclear, but it is probably of minor importance while high degradation velocities were found in deep soil layers, where no oxygen can be expected.
5. It is hypothesed that a water-flux through the wood supports the process of bacterial wood degradation because of the processes as explained at point 3 and 4. The higher degree of degradation in pine compared to spruce under the same conditions (e.g. site 3 Amsterdam) can be explained by the flux theory. But also the high degradation in relative short pine piles, in situations without a difference over longer time in water pressure between the deeper and shallower groundwater, shows that specific events (e.g. temporary lower groundwater, heavy rain) causes a water flux in the wood which is probably enough to support bacterial wood degradation.
6. The degradation velocity within Spruce piles is lower than that of pine but within 100 years a peal of 1-20mm is weakly degraded by bacteria. These differences are clear by comparing the degree of bacterial wood degradation of those sites were whole piles were extracted. The pine-line: In Amsterdam (site 3, pile length 11m), Zaandam (site 6 pile length (6) and Haarlem (site 4, pile length 5m) pine piles were extracted and all piles are degraded over the whole length and only in the sapwood. Haarlem: severe degradation both at the head and at the tip; Amsterdam severe to weak degradation both at the head and at the tip; Zaandam severe to weak degradation at the head and weak to sound at the tip. The Spruce line: Amsterdam (site 3, pile length, 11m) weakly degraded in the outmost 20mm in both the head and the tip; Rotterdam (site 5, length 16m) weakly degraded in the outermost 0-10mm in both the head and the tip.
Regarding the archaeological sites site only, the following statements can be made:
1. Because of the large diversity no specific conclusions could be made;
2. Because the sites were chosen with the expectation that bacterial wood degradation would occurs, sites with oxygen were ignored. If oxygen in measurable concentrations occurs fungal activity will appear and destroy wood structures in relative short periods. At all sites, except for Bryggen, the main cause of the degradation was bacterial activity, although often-colonising hyphes were observed;
3. Almost all samples, ranging in age from 300 - 2000 years, were over full diameter degraded by bacteria. Although the degree degradation is age independent, in most of the samples the wood degrading bacteria were still active. This means that not the infection, but the velocity of bacterial wood degradation is regulated by the conditions under which the wood is storied. As no environmental (soil chemistry) dependency was found it is also here supposed that the presence of a water flux could be crucial.
Bacterial wood decay was induced under laboratory conditions and was more intense when oxygenated water was circulated through the wood containing sediment. Even in the nitrogen gassing treatment providing anoxic conditions bacterial wood decay was present. The CO2 production from the sediment was found to correlate with bacterial wood decay although the underlying CO2 producing processes are divers and not fully understood.
The nitrogen and phosphate addition to the sediment did not promote bacterial wood decay. On the contrary the bacterial wood decay intensity could be interpreted as being negatively related to the sediment nitrogen concentration. The nutrient addition to the sediment revealed a sediment pH dependency of the bacterial wood decay intensity. In sediment with sulphate addition, bacterial wood decay was not found after 155 days. The addition of glucose to sediment addition repressed bacterial decay intensity. In sediment with glucose and sulphate addition nearly no bacterial decay was found. Although no strong evidence was presented there are hints that the sulphate addition to sediment might as least for 155 days inhibit bacterial wood decay. When glucose is added to the sulphate containing sediment nearly no bacterial decay was found.
Phagen production
Successful isolation and identification of bacteria are prerequisites for specific phage isolation and hence for the development of a phage-based wood preservatives. The elusive wood degrading bacteria appeared difficult to isolate and cultivate in the laboratory environment. Due to these difficulties the objective to develop a phage-based wood preservative was delayed.
Another aim in the project was to investigate effects of phages in the microcosm experiments. The phage isolation procedure and phage techniques proved successful. Phages were isolated from several samples from the microcosms. However, the general microflora of the microcosms was not investigated, nor were specific wood degrading bacteria successfully identified by other Bacpoles partners. The lack of identification of the bacteria of interest limited possible conclusions. Phagen isolated a large number of various bacteria from the Bacpoles wood samples. The bacteria may now be employed in the 16S rRNA assays used by UoP to assess the identity of living organisms in the samples. In addition, all isolated bacteria constitute potential reservoirs of suitable phages to be used as phage-based preservatives once the wood degrading bacteria have been identified.
From the tests three main results can be deduced but it has to be realised that because of the restricted amount of replica these results can only be regarded as indicative:
1. The water permeability of wooden stems is species dependent and restricted to the axial direction. The highest permeability is found for pine sapwood, less permeable are alder over the whole diameter and oak sapwood. Visible heartwood is not permeable. Spruce and the sapwood of larch and Douglas have an intermediary permeability.
2. Water pressure on the top stem surface is positively correlated with water flow through the stem.
3. Blue stain infection seems to decrease the water flow through the stems
If a water flow through the wood is related to the process of bacterial wood degradation, these results offer possibilities to stop this process. Sealing of the cross section is one possibility and using blue stain infection could be another one.
Possible preservation or conservation strategies
The actual information on chemical-based preservatives is mainly related to fungal wood degradation, and their efficiency against bacterial degradation was never reliably proven. As it has to be used under water-saturated conditions, water-soluble non-fixating products are not suited. Moreover, a possible preservative should fit within the strict regulations for soil and groundwater minimum toxic levels. Therefore toxic water-soluble preservatives are not regarded as realistic to use against bacterial wood decay.
There are three promising approaches defined and each of them starts with a full description of the area to be treated. The site hydrology as well as the identification of the bacteria consortium, which causes bacterial degradation, is most important. Based on these inquiries specific mixtures of phages can be made. However field tests have to be carried out in order to get more knowledge whether a mono-phage-preservative should be used which is effective against the present bacteria consortium only, or whether it is possible to prepare a mixed-phage-preservative which is effective on a wider range of locations.
A second approach is related to the hydrology. It became clear that bacterial wood degradation is active only when there is a water flux in the wood. In order to create a static situation, either the hydrology can be manipulated or the wood structure can be closed by impregnation in the field. Probably a combination of both strategies is most efficient and could improve in addition the strength of the wood.
A third approach is based on a non-toxic active product, which affects the already weak competition position of wood degrading bacteria by promoting others. The result of this treatment should be that the number of wood degrading bacteria is diminished over longer time.
Future work and missing knowledge
Although populations of erosion bacteria in water-saturated wood make up a wide variety of species, the emergence of the CFB (Cytophaga-Flavobacterium-Bacteroides) group as an important component of the micro-flora requires further investigation for definite identities of pure cultures. Use of FISH (fluorescent in-situ hybridisation) techniques will pinpoint known bacterial types within a consortium. This will also provide a starting point for understanding the ecology and physiology of these bacteria. Particularly the conditions suitable for demonstrating attack on wood and kapok fibres, their carbon and nitrogen requirements, their respiration/fermentation and their response to different levels of oxygen, carbon dioxide and hydrogen sulphide, plus the effects of pH and temperature on their activity have to be further investigated. Such investigations will be tied to studies on cellulases, hemicellulases and ligninase enzymes produced by these bacteria in order to identify the optimum conditions for decay. At the same time the longevity and ecology of bacteriophages specific to isolated bacterial strains needs to be determined in natural environments.
One of the important results arising from the present work has been our improved understanding of water flow within wood. This has particular relevance to the movement of virus particles and bacterial cells in wood and raises questions about the need for a better understanding of the dynamics of water pressure in the ground, the permeability of different soil layers, the velocity of ground water flow and soil water analyses along the whole length of piles. This latter aspect of work to be done has special significance with respect to nitrogen availability and its importance in wood decay by erosion bacteria bearing in mind the limited amount of nitrogen naturally present in wood. Future work requires the establishment of field trials alongside laboratory experimentation. In the case of nitrogen, the use of 15N to monitor uptake into bacteria in laboratory microcosms and the use of radioactively spiked wood to monitor uptake are just two avenues of investigation. Physiological studies of this type will determine the value of changing environmental conditions in order to inhibit/control the activity of erosion bacteria by using for instance lime-milk to increase pH. Similar studies using laboratory microcosms can also be set up under very low oxygen tensions to determine the effect on erosion bacterial activity, or to measure the effect of selected phages on bacteria artificially introduced into wood samples.
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