Bacterial Degradation of Lignified Wood Cell Walls in Anaerobic Aquatic Habitats

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1983, p /83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 46, No. 3 Bacterial Degradation of Lignified Wood Cell Walls in Anaerobic Aquatic Habitats D. M. HOLTV* AND E. B. G. JONES2 Pira, Leatherhead, Surrey,1 and Department of Biological Sciences, Portsmouth Polytechnic, Portsmouth, Hampshire,2 United Kingdom Received 22 April 1983/Accepted 13 June 1983 Test blocks of beech (Fagus sylvatica) and Scots pine (Pinus sylvestris) were buried in fresh, brackish, and seawater anaerobic muds for periods ranging between 1 and 18 months. At appropriate time intervals the test blocks were recovered and examined for changes in weight and for bacterial attack of lignified wood cell walls. Only small weight losses occurred. Scanning electron microscopy studies revealed that there was extensive superficial bacterial erosion of beech wood cell walls. The decay patterns are illustrated by micrographs and discussed in relation to other types of bacterial attack. One of the major problems of studying wood degradation by bacteria is the inability of research workers to isolate the organisms responsible and monitor the different stages of the decay process in the laboratory. Although recent reports indicate that pure cultures of actinomycetes can degrade wood fibers, Douglas fir phloem, and various lignocellulosic preparations (3, 4, 10, 11, 33), experiments with monocultures of nonfilamentous bacteria show that lignified tissues are not attacked unless they have been chemically or biologically pretreated (20-22, 29-32). Consequently, the descriptions of bacterial degradation are mainly derived from observations of timber exposed under natural conditions (5, 6, 16, 17, 27). The various decay patterns reported are often regarded with scepticism because there is always the possibility that fungi were involved in their formation. This is especially relevant when timber has been exposed for long periods, as prior invasion and attack by other microorganisms cannot be totally discounted. Bacterial degradation of beech and Scots pine is extensive in test blocks submerged in a freshwater river and the sea, as shown in an earlier investigation (20a). Two distinct types of attack were observed in regions devoid of fungal activity, and it was possible to identify different stages in the decay process. However, to reduce the interference from fungi even further we decided to expose wood under anaerobic conditions. Anoxic muds provide an ideal environment for studying bacterial colonization and degradation of lignocellulosic materials. They remain relatively stable for long periods, and fungi exist only in small numbers (25). Although previous studies on timber taken from such situations 722 indicate that the decay process is extremely slow (14, 28), the data presented in this paper demonstrate that superficial bacterial degradation may occur during very short exposure periods. The different types of attack observed are illustrated and described in detail. MATERIALS AND METHODS Exposure and retrieval of test blocks. The methods used for exposing wood in anaerobic muds are based on those of Jones (23) and Fazzani et al. (15). Strings of test blocks, containing six of beech (Fagus sylvatica) and six of Scots pine (Pinus sylvestris), were prepared for each sample period. The test blocks (5 by 2.5 by 0.5 cm) were centrally drilled with 2-mm holes, oven dry weighed (105 C overnight), and threaded onto terylene whipping twine. Five blocks of each wood were required to monitor changes in weight, and one was used for microscopic examination. Three sites were selected for the studies: (i) the edge of a slow-moving stream in Wickham, Hampshire; (ii) a brackish bog at Lymington, Hampshire; and (iii) the low-tide zone of Bosham harbour, West Sussex. At each site ph and Eh values were determined using standard meters. Test blocks were then buried in the black sulfide layer of mud deposits at a depth of 10 cm. They were exposed in May 1978 and recovered after 1, 3, 6, 9, 12, and 18 months. At each sampling time, strings of test blocks were removed from the three sites together with a small amount of mud to prevent dessication. They were conveyed to the laboratory in plastic bags, scraped, washed with sterile distilled or seawater, and then examined immediately for weight change (oven dried at 105 C) and visual signs of decay. For scanning electron microscopy studies, sections of the test blocks (up to 1 mm thick) were cut by hand with Everready single-edged razor blades. These were fixed with 5% glutaraldehyde in 0.05 M sodium cacodylate buffer (ph 7.2) for 3 h at room temperature. They were then postfixed overnight in similarly buff- Downloaded from on September 17, 2018 by guest

2 VOL. 46, 1983 TABLE 1. Eh and ph measurements of the three test sitesa Test site Eh ph Bosham -400 ± ± 0.02 Wickham -408 ± ± 0.10 Lymington -420 ± a Results are the means of five replicates + standard errors. ered 2% osmium tetroxide at 4 C. The fixed material was dehydrated in a graded ethanol series, 15 min in each 10% step. This was followed by a gradual substitution of the alcohol with acetone before critical point drying the specimens in a Polaron E3000 apparatus, which uses liquified carbon dioxide as the drying agent. Dried sections were mounted on aluminium stubs with double-sided adhesive tape (Scotch), gold coated (60-nm thickness) in a Polaron E5000 diode sputtering system, and observed in a JEOL T20 scanning electron microscope. DEGRADATION OF LIGNIFIED WOOD CELL WALLS 723 RESULTS Physical parameters. All three test sites had a low Eh and ph around neutrality (Table 1). There was also a strong odor of hydrogen sulfide when the muds were disturbed. Changes in weight of test blocks. Very small percentage weight losses were detected over the 18-month exposure period; this is consistent with the view that wood is degraded slowly under anaerobic conditions (Table 2). These losses are considered insignificant, except for specific instances such as beech at the Wickham site. The changes cannot be attributed solely to the activities of bacteria, as weight losses may also occur by leaching or chemical degradation of the wood. Scanning electron microscope observations. Test blocks were examined primarily for bacterial attack of lignified wood cell walls, and the results were almost identical for the three test sites. The degradation of Scots pine was superficial and occurred infrequently, and many of the decayed areas were devoid of bacteria. In comparison, the surfaces of beech were severely degraded, and a variety of bacteria and actinomycetes were observed. Fungal hyphae were occasionally seen in all test blocks, but no visible decay was apparent. Consequently, the descriptions of cell wall colonization and degradation are limited to data obtained for beech because of the low microbial activity in Scots pine. Bacteria colonized the test blocks within the first month, and a wide range of species was observed throughout the exposure period. It is not feasible to describe the individual organisms, but worthy of note are the spirochaetes and spore formers, which are rarely found in aerobic environments. Bacterial degradation was extensive in the surface layers of all of the beech test blocks. Degradation was observed after 1 month of exposure at Wickham and 3 months of exposure at Bosham and Lymington. The decay is initiated from within the wood cell lumen and appears to be confined to the activities of rod-shaped bacteria. Both fibers (Fig. la and b) and vessels (Fig. lc and Fig. 2a, b, and c) are susceptible to this type of attack, and several species of bacteria may be involved. An erosion of the S3 layer produces the small troughs around each organism (Fig. 2a and c) which are characteristic of this decay pattern. With further degradation there is a gradual removal of the S3 layer, and the bacteria then proceed to utilize the S2 layer (Fig. 2a). A major feature of the attack is the formation of erosion troughs along a specific axis of the wood cell wall (Fig. 1 and 2). There is even a change in direction to accommodate for the structure of the pits (Fig. lc). This indicates that the orientation of the wood microfibrils may influence the initial alignment of the bacteria. DISCUSSION The data presented clearly demonstrate that bacteria can rapidly colonize and degrade the lignified cell walls of timber buried in anoxic muds. Beech test blocks were readily attacked by a variety of rod-shaped bacteria which caused an erosion type of degradation. By comparison, the microbial activity in Scots pine test TABLE 2. Percent change in weight of test blocks exposed in anaerobic muds at three test sitesa o% Gain or loss after exposure period of (months): Test site Wood Bosham Beech 1.50G ± G ± L ± L ± L ± 0.37 Scots pine 2.44G ± G ± G G L Wickham Beech 0.98L ± L ± L ± L ± L Scots pine 1.03L ± L ± L ± L ± L ± 0.31 Lymington Beech 0.38G ± G ± L ± L ± L Scots pine 1.92G ± G ± L ± L ± G ± 0.23 a Results are the means of five replicates ± standard errors. G, Gain; L, loss. Downloaded from on September 17, 2018 by guest

3 724 HOLT AND JONES APPL. ENVIRON. MICROBIOL. Downloaded from FIG. 1. Scanning electron micrographs of beech fibers (a and b) and vessels (c and d) illustrating bacterial cell wall erosion found in test blocks buried in freshwater anaerobic muds. Various rod-shaped bacteria caused erosion of the S2 cell wall layer. Note the alignment of the bacteria around the pit in panel c. Exposure period: a, c, and d, 6 months; b, 1 month. Bar, 2 plm. on September 17, 2018 by guest blocks was low with only limited superficial cell wall attack even after 18 months of exposure. Fungal growth was rarely observed, confirming reports that these organisms are inactive in conditions of low oxygen concentration (5, 6, 14, 25) Ṫhe resistance of Scots pine to bacterial degradation may be attributed to the presence of known inhibitory substances such as lignin or resinous compounds. Differences in the structure and content of lignin in beech and Scots pine probably have a major effect on the susceptibility of each wood to bacterial attack. It may take many years (obviously more than 18 months) before these inhibitors are chemically decomposed or leached from the test blocks, but ultimately the wood will become susceptible to bacterial attack. Several research workers have shown that Scots pine and other softwood species suffer considerable decay after long-term exposure (over 40 to 50 years) in water-saturated muds (5, 6, 14, 18, 19); these authors suggest

4 Downloaded from on September 17, 2018 by guest FIG. 2. Scanning electron micrographs of beech vessels illustrating bacterial cell wall erosion found in test blocks buried in freshwater (a and b) and marine (c) anaerobic muds. Rod-shaped bacteria caused erosion of the S3 and S2 wall layers. The arrows in a and c indicate an early stage of attack where individual bacteria produce small erosion troughs in the S3 layer. Note also the copious amounts of mucilage in panel b and the different types of microorganisms in panel c. The exposure period was 6 months. Bar, 3,um. 725

5 726 HOLT AND JONES that this is due to the cumulative effect of bacterial or actinomycete activity (or both) over many years. Boutelje and Goransson (6) isolated both aerobic and anaerobic cellulose-degrading bacteria from submerged timbers and found that some species were capable of utilizing wood sections in the laboratory. Thus, it is possible that once decay of softwood begins, the process continues, albeit extremely slowly. Several different kinds of bacterial attack on lignified wood cell walls occur in timbers exposed in aquatic situations (5, 24, 26, 27). The two major types, cell wall erosion and cavity formation within the S2 layer, have recently been described in detail (D. M. Holt, Ph.D. thesis, Portsmouth Polytechnic, Portsmouth, England, 1981). Similar decay patterns have also been observed in studies on the breakdown of forage tissues in the rumen of herbivores, an activity that depends on anaerobic cellulolytic bacteria and actinomycetes (1, 2, 7, 8, 13). In the present investigation only cell wall erosion was detected. The absence of other types of degradation may be a feature peculiar to the totally anaerobic environment. Cavity formation is frequently encountered in watersoaked timbers exposed under aerobic or microaerophilic conditions (12, 24, 26; T. Nilsson and D. M. Holt, Holzforschung, in press). It is also probable that several early reports on the decay of submerged pilings refer to an attack indicative of cavity formation. Harmsen and Nissen (18, 19) described small conical depressions extending from the cell lumen into the secondary wall and then spreading irregularly in the S2 layer. Scheffer et al. (28) observed bacteria in elongated cavities, some of which were orientated along the woods microfibrils. However, only a brief exposure to aerobic conditions may have allowed the cavity-forming bacteria to develop. In conclusion, the results presented confirm, at the scanning electron microscopic level, earlier reports that bacteria can actively decay the lignified cells of timbers submerged in aquatic situations. Softwoods (Scots pine) appear to be resistant to attack over short-term exposure periods, but hardwoods (beech), especially the superficial layers, are rapidly degraded by rodshaped bacteria. ACKNOWLEDGMENTS We thank S. T. Moss and G. Bremer for help with the scanning electron microscopy and E. Hawton for preparing the photographs for this publication. LITERATURE CITED 1. Akin, D. E Ultrastructure of rigid and lignified forage tissue degradation by a filamentous rumen microorganism. J. Bacteriol. 125: Akin, D. E Evaluation by electron microscopy and APPL. ENVIRON. MICROBIOL. anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls. Appl. Environ. Microbiol. 39: Antai, S. P., and D. L. Crawford Degradation of softwood, hardwood, and grass lignocelluloses by two Streptomyces strains. Appl. Environ. Microbiol. 42: Baecker, A. A. W., and B. King Decay of wood by Actinomycetales, p In T. A. Oxley, G. Becker, and D. Allsopp (ed.), Biodeterioration, Proceedings of the 4th International Symposium, Berlin, August-September Pitman Publishing Ltd., London. 5. Boutelje, J. B., and A. F. Bravery Observations on the bacterial attack of piles supporting a Stockholm building. J. Inst. Wood Sci. 4: Boutelje, J. B., and B. 0. Goransson Decay in wood constructions below the ground water table. Swed. J. Agric. Res. 5: Cheng, K. J., D. E. Akin, and J. W. Costerton Rumen bacteria: interaction with particulate dietary components and response to dietary variation. Fed. Proc. 36: Clarke, R. T. J The gut and its micro-organisms, p In R. T. J. Clarke and T. Bauchop (ed.), Microbial ecology of the gut. Academic Press, Inc., London. 9. Courtois, H., and J.-J. Erasmy Bakterienangriff auf die Zellwande von Eichen-und Buchenholz wahrend einer Wasserlagerung. Holz Roh. Werkst. 34: Crawford, D. L Lignocellulose decomposition by selected Streptomyces strains. Appl. Environ. Microbiol. 35: Crawford, D. L., and J. B. Sutherland The role of actinomycetes in the decomposition of lignocellulose. Dev. Ind. Microbiol. 20: Cundell, A. M., and R. Mitchell Microbial succession on a wooden surface exposed to the sea. Int. Biodeterior. Bull. 13: Dinsdale, D., E. J. Morris, and J. S. D. Bacon Electron microscopy of the microbial populations present and their modes of attack on various cellulosic substrates undergoing digestion in the sheep rumen. Appl. Environ. Microbiol. 36: Eslyn, W. E., and J. W. Clarke Appraising deterioration in submerged piling. Mater. Org. Beiheft 3: Fazzani, K., S. E. J. Furtado, R. A. Eaton, and E. B. G. Jones Biodeterioration of timber in aquatic environments, p In D. W. Lovelock and R. J. Gilbert (ed.), Microbial aspects of the deterioration of materials. Academic Press, Inc., London. 16. Greaves, H Micromorphology of the bacterial attack of wood. Wood Sci. Technol. 3: Greaves, H The bacterial factor in wood decay. Wood Sci. Technol. 5: Harmsen, L., and T. V. Nissen Der bacterienangriff auf Holz. Holz Roh. Werkst. 23: Harmsen, L., and T. V. Nissen Timber decay caused by bacteria. Nature (London) 206: Holt, D. M The effect of pre-conditioning wood with fungi on its subsequent degradation by cellulolytic prokaryotes. Mater. Org. 17: a.Holt, D. M Bacterial degradation of lignified wood cell walls in aerobic aquatic habitats: decay patterns and mechanisms proposed to account for their formation. J. Inst. Wood Sci. 9: Holt, D. M., and E. B. G. Jones Bacterial cavity formation in delignified wood. Mater. Org. 13: Holt, D. M., E. B. G. Jones, and S. E. J. Furtado Bacterial breakdown of wood in aquatic habitats. Rec. Ann. Conv. Br. Wood Preserv. Assoc., p Jones, E. B. G The ecology and rotting ability of marine fungi, p In E. B. G. Jones and S. K. Eltringham (ed.), Marine borers, fungi and fouling organisms of wood. Organization for Economic Cooperation and Development, Paris. 24. Kohlmeyer, J Bacterial attack on wood and cello- Downloaded from on September 17, 2018 by guest

6 VOL. 46, 1983 DEGRADATION OF LIGNIFIED WOOD CELL WALLS 727 phane in the deep sea, p In T. A. Oxley, G. Becker, and D. Allsopp (ed.), Biodeterioration, Proceedings of the 4th International Symposium, Berlin, August- September Pitman Publishing Ltd., London. 25. Laurent, M Experiment investigation of cellulolysis in mud, p In Proceedings of the 4th Conference on Water Pollution Research, Prague. 26. Leightley, L. E., and R. A. Eaton Mechanisms of decay of timber by aquatic micro-organisms. Rec. Ann. Conv. Br. Wood Preserv. Assoc., p Liese, W., and H. Greaves Micromorphology of bacterial attack, p In W. Liese (ed.), Biological transformation of wood by microorganisms. Springer- Verlag, Berlin. 28. Scheffer, T. C., C. G. Duncan, and T. Wilkinson Condition of pine piling submerged 62 years in river water. Wood Preserv. News 47: Schmidt, On the bacterial decay of the lignin cell wall. Holzforschung 32: Schmidt, Laboratory experiments on the bacterial activity towards the woody cell wall. p It T. A. Oxley, G. Becker. and D. Allsopp (ed.), Biodeterioration, Proceedings of the 4th International Symposium, Berlin, August-September Pitman Publishing Ltd.. London. 31. Schmidt, Uber den bakteriellen Abbau der chemisch behandelten verholzten Zellwand. Mater. Org. 15: Schmidt, Zum Verhalten von Bakterien gegenuber der verholzten Zellwand. Mitteilungen der Bundesforschungsanstalt fur Forst-und Holzwirtschaft. Nr M. Wiedebusch, Hamburg. 33. Sutherland, J. B., R. A. Blanchette, D. L. Crawford, and A. L. Pometto Breakdown of douglas-fir phloem by a lignocellulose-degrading Streptomxvces. Curr. Microbiol. 2: Downloaded from on September 17, 2018 by guest