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NOTES

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widely separated areas of earth. In addition, Geodermatophilus


was found in both areas, although it is still not known whether
those from the Sonoran Desert are manganese oxidizers (studies
of this are in progress now). The finding of similar taxa in desert
varnishes that are widely separated and distinct in higher flora
and fauna is supportive of a common role in varnish deposition.
The results of this study of Negev Desert varnishes add
further support to our previous view of the position of this
lithobiont community in the desert rock biome (Danin 1985,
1986). In more moist habitats or microhabitats the varnishassociated microorganisms cannot compete with other lithobiont communities that cover the rock surface or cause rapid
removal of the rock surface. In both cases lithobionts associated
with rock varnish cannot develop.

Acknowledgements

We thank D r . Edward Ishiguro for his assistance in identifying


the Geodermatophilus isolates. This work was supported in part
by a U. S . National Aeronautics and Space Administration
(NASA) grant (NASN 40 16 "Rock weathering and geobotany").
ALLEN,C. C. 1978. Desert varnish of the Sonoran Desert: optical and
electron probe microanalyses . J . Geol . 86: 743-752.
BROMF~ELD,
S. M. 1974. Bacterial oxidation of manganese ions as
affected by organic substrate concentration and composition. Soil
Biol. Biochem. 6: 383-392.
DANIN,A. 1983. Desert vegetation of Israel and Sinai. Cana
Publishing House, Jerusalem.
1985. Palaeoclimates in Israel: evidence from weathering
patterns of stones in and near archaeological sites. Bull. Am. Sch.
Orient. Res. 259: 33-43.
1986. Patterns of biogenic weathering as indicators of
palaeoclimates in Israel. Proc. R. Soc. Edinburgh Sect. B . Biol. 89:
243-253.
DELAPORTE,
B., and SASSON,
A. 1967. ~ t u d ede bactkries des sols
ardies du Maroc: Bacillus maroccanus n. sp. C. R. Acad. Sci.
(Paris), Ser. D, 264: 2344-2346.

DORN,R. I., and OBERLANDER,


T. M. 1981. Microbial origin of desert
varnish. Science (Washington, D. C.), 213: 1245- 1247.
1982. Rock varnish. Prog. Phys. Geogr. 6: 3 17-367.
ENGEL,C. G., and SHARP,R. P. 1958. Chemical data on desert
varnish. Geol. Soc. Am. Bull. 69: 487-518.
ERL~CH,
H. L. 1981. Geomicrobiology. Marcel Dekker, Inc., New
York, Basel.
FE~GEL,
F. 1958. Spot tests in organic analysis. Elsevier, Amsterdam.
GERHARDT,P.(Editor-in-chiefi. 1981. Manual of methods for
general bacteriology. American Society for General Microbiology,
Washington, D.C.
GREGORY,
E., PERRY,R. S., and Staley, J. T. 1980. Characterization, distribution and significance of Metallogenium in Lake Washington. Microb. Ecol. 6: 125- 140.
KRUMBEIN,
W. E., and JENS,K. 1981. Biogenic rock varnishes of the
Negev Desert (Israel): an ecological study of iron and manganese
transformation by cyanobacteria and fungi. Oecologia, 50: 25-38.
LOCKHEAD,
A. G., and BURTON,
M. 0 . 1955. Qualitative studies of
soil microorganisms. XII. Characteristics of vitamin-B I ;?-requiring
bacteria. Can. J. Microbiol. 1: 319-330.
PALMER,
F. E., STALEY,
J. T., MURRAY,
R. G. E., COUNSELL,
T., and
ADAMS,
J. B. 1986. Identification of manganese-oxidizing bacteria
from desert varnish. Geomicrobial. J. 4: 343-360.
PERRY,
R. S., and ADAMS,
J. B. 1978. Desert varnish: evidence for
cyclic deposition of manganese. Nature (London) 276: 489-491.
SCHWEISFURTH,
R. 1968. Untersuchungen iiber manganoxydierende
und reduzierende Mikroorganismen. Mitt. Int. Ver. Theor. Angew.
Limnol. 14: 179- 186.
, P. (Editor). 1986. Bergey 's manual of systematic bacteriolSNEATH
ogy. Vol. 2. Gram positive bacteria. Williams & Wilkins, Baltimore
MD.
STALEY,J. T., JACKSON,
M. J., PALMER,
F. E., BORNS,D. J.,
CURTISS,B., and TAYLOR-GEORGE,
S. 1983. Desert varnish
coatings and microcolonial fungi on rocks of the Gibson and Great
Victoria Deserts. BMR J. Aust. Geol. Geophys. 8: 83-87.
TAYLOR-GEORGE,
S., PALMER,
F., STALEY,
J. T., BORNS,D., J.,
CURTISS,
B., and ADAMS,
J. B. 1983. Fungi and bacteria involved in
desert varnish formation. Microb. Ecol. 9: 227-245.

Degradation of lignocellulosics by unique tunnel-forming bacteria


GEOFFREYF. DANIEL'AND T . NILSSON
Department of Forest Products, Swedish University of Agricultural Sciences, Box 7008, S-750 07 Uppsala, Sweden
AND

A . P. SINGH
Forest Research Institute, Private Bag, Rotoura, New Zealand
Received April 13, 1987
Accepted June 24, 1987
DANIEL,
G. F., NILSSON,
T., and SINGH,A. P. 1987. Degradation of lignocellulosics by unique tunnel-forming bacteria. Can.
J. Microbiol. 33: 943-948.
Transmission electron microscopic observations on a wide range of decaying wood samples obtained from both field situations
and laboratory exposure tests have confirmed bacteria to have a capacity to degrade intact highly lignified substrates including
preservative-treated and naturally durable woody tissues. Studies have shown a number of bacterial forms to be involved and
have provided morphological evidence for in situ lignin degradation confirming I4c-labelled experiments with synthetic and
natural lignins. A unique type of bacterial attack (tunnelling) characterized by the development of tunnels containing peculiar
cross-tunnel wall secretions has been recognized. Cytochemical studies have shown the extracellular tunnel secretions to contain
negatively charged constituents, while transmission electron microscopic energy-dispersive X-ray spectroscopy has shown these
tunnels to bind heavy metals during decay of timbers treated with metal-containing preservatives. The tunnelling bacteria are
motile nonflagellated Gram-negative rods. These bacteria have highly plastic cell envelopes which can produce vesicles. Our
studies provide evidence that tunnelling bacteria can remove total cell wall material, including lignin, and show a relationship
between their motility and the unique tunnels they produce.
' ~ u t h o rto whom reprint requests should be addressed.

Printed in Canada I Irnpnrne au Canada

943

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944

CAN. J. MICROBIOL. VOL. 33, 1987

DANIEL,
G. F., NILSSON,T., et SINGH,A. P. 1987. Degradation of lignocellulosics by unique tunnel-forming bacteria. Can.
J. Microbiol. 33 : 943-948.
Des observations ont CtC faites en rnicroscopie Clectronique a transmission sur une grande variCtC d'Cchantillons de ligneux
pourrissants qui provenaient de situations de champs ainsi que de tests d'exposition en laboratoire. Ces Cchantillons ont permis
de confirmer que es bactCries ont la capacitC de ddgrader des substrats intacts hautement lignifiCs, incluant des ligneux traitCs de
fason prkventive et d'autres durables naturellement. Des Ctudes ont dCmontrC qu'un certain nombre de formes bactdriennes sont
impliquCes dans ce processus et ont fourni I'Cvidence morphologique de la dkgradation de la lignine in situ, confirmant les
espkriences du marquage de lignines synthktiques et naturelles auI4c. Un type unique d'attaque bactkrienne caractCrisC par le
dkveloppement de tunnels ("tunnelling") a CtC reconnu; des sCcrCtions pariCtales particulieres occupent ces tunnels. Des Ctudes
cytochimiques ont dCmontrC que les sCcrCtions extracellulaires des tunnels contenaient des substances chargdes nkgativement,
alors qu'en rnicroscopie Clectronique a transmission 1'Cnergie de dispersion analysCe par spectromCtrie de rayons X a montrC que
ces substances servaient a lier les mktaux lourds au cours du pourrissement de poutres traitCes avec des agents de conservation
contenant des mktaux. Les bactkries qui forment des tunnels sont des bitonnets non-flagellCs, mobiles et Gram nCgatifs. Ces
bactCries ont des enveloppes cellulaires hautement plastiques qui peuvent produire des vCsicules. Les prksents travaux
fournissent l'kvidence que les bactkries qui forment des tunnels peuvent dCgrader tout le matkriel pariCtal cellulaire, incluant la
lignine, et qu'il existe une relation entre leur mobilitC et les tunnels uniques qu'elles produisent.
[Traduit par la revue]

The biological degradation of lignified tissues, particularly


is in contrast to previous reports where lignin has been
wood, plays a major role in the recycling of an important carbon
considered to constitute a barrier and hinder attack (Liese 1970).
resource to the environment. While fungi have always been
We describe here a unique pattern of wood cell wall
considered the major progenitors of the process, it is now
degradation where a certain type of bacteria which have
realised that bacteria may also play an important role. Until
ligninolytic activity penetrate wood cell walls by localised
recently evidence for the ability of bacteria to degrade lignified
substrate dissolution and then attack the remaining wall by a
tissues has come from observations on wood placed, or
novel tunnelling action.
occumng, under normal or contrived natural situations and has
For TEM, thin sections cut from small wood pieces exposed
under laboratory conditions or from samples (e.g., stakes) from
been based on either light or scanning electron microscopic
investigations (Harmsen and Nissen 1965; Greaves 1968; D. M.
in-service situations were fixed for 3 h in 3% (v/v) glutaraldeHolt. 1981. Ph.D. thesis, Portsmouth Polytechnic, Portsmouth,
hyde in 0.1 M sodium cacodylate buffer (pH 7.2) at room temU. K.). Such observations have often been considered fortuiperature (RT). After rinsing in buffer, postfixation was carried
tous and thought to represent secondary colonization by
out in 2% (w/v) osmium tetroxide in 0.1 M sodium cacodylate
bacteria after prior fungal attack and modification of the
for 3 h also at RT. After subsequent washing in 3 changes of
substrate. This opinion has been somewhat strengthened by the
deionized water, samples were dehydrated in a graded ethanol
weak ability of known cellulolytic bacteria under controlled
series to propylene oxide and flat embedded in Spun's (1969)
laboratory conditions to degrade intact lignified cell walls (Holt
resin.
and Jones 1978; Schmidt 1978; 0. Schmidt. 1980. Proc. Int.
Selected material was sectioned using an LKB ultratome I
Biodeterior Symp. 4th, 1978).
fitted with a diamond knife. Sections were stained with either
Investigations over the last few years using transmission
lead citrate (Reynolds 1963) and uranyl acetate or 1% aq.
electron microscopy (TEM) (G. Daniel and T. Nilsson. Inter(w/v) potassium permanganate.
national Research Group on Wood Preservation (IRG). DocuInformation on the cytochemical nature of the cross-tunnel
ment nos. IRG/WP/ 1260 (1985) and IRG/WP/ 1283 (1986)~) wall secretions and other organic material associated with the
have yielded evidence that certain bacteria have the ability to
bacteria was carried out by a preincubation of samples in
degrade a diverse range of timbers from coniferous and decidbuffered (pH 7.2) glutaraldehyde fixative, and postosmication
uous trees as well as natural fibres, irrespective of the type
(as above), all in the presence of 0.15% (w/v) ruthenium red
(i.e., guaiacyl or syringyl-guaiacyl) or levels of lignins and
according to Luft (1971). Final examination took place in a
extractives present. Similar results have also been obtained
Philips 201 TEM operated at 60 kV.
with both nonligniiied substrates (e .g ., cellophane) and bleacFor TEM X-ray microanalysis, samples of decayed material
hed pulped fibres containing reduced (ca. 3%) lignin levels.
were fixed in 3% (w/v) glutaraldehyde in 0.1 M phosphate
TEM has provided the only effective means of assessing what is
buffer (pH 7.2) for 3 h at room temperature. Postosmication
happening in the wood samples, since pure cultures of the
was omitted with samples dehydrated directly. Thereafter the
bacteria have not so far been obtained for taxonomic assesssamples were processed as above. Gold sections (120- 150 nm)
ment. TEM has made it possible to observe the intricate patterns
were cut and collected on carbon-stabilized, "par1odion"-coated
which are produced during the breakdown of wood cell walls by
aluminium grids. Sections held in a beryllium specimen holder
certain groups of bacteria and to confirm that the various
were viewed unstained in a Philips 400T TEM (equipped with a
patterns previously recognised in marine, aquatic, and terresiield emission gun) using an accelerating voltage of 60 kV and a
trial environments are indeed caused by single-celled bacteria
spot diameter of 50 nm. Analyses were performed in the TEM
and not actinomycetes as previously thought (Eaton and Dickinmode (specimen tilt at 24') using an EDAX Edith 71 1
son 1975). In addition, it has also been possible to obtain
energy-dispersive system over a live time of 100 s. To obtain
evidence that certain wood-degrading bacteria have an ability to
semi-quantitative results without the use of standards, the weight
degrade lignin in situ in addition to the carbohydrate componfraction ratios of the elements of interest were determined using
ents (i .e., cellulose and hemicelluloses) of wood cell walls. This
the EDAX "direct element ratio model routine" developed for
thin sections. Computations were performed using an EDAX
silent 700 ASR electronic data terminal.
2~vailablefrom IRG Secretariat, Mr. Ron Cockcroft, Drottning
Samples for scanning electron microscopy (SEM) were fixed
Kristinas vag 47 C, S- 114 28 Stockholm, Sweden.

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NOTES

and processed as for conventional TEM, but with additional


acetone dehydration after ethanol. Thereafter samples -were
critical-point dried in a Polaron E 3000 critical-point drier using
liquid C 0 2 as the substitution fluid. After coating with gold
using a Polaron E 5000 diode sputtering device, the samples
were observed using a Cambridge S150 scanning electron
microscope at 20 kV.
Mixed populations of wood-degrading bacteria were obtained
by initial inoculation of naturally bacterial degraded wood pieces
into Erlenmeyer flasks containing either pine wood splinters or
small pieces of birch and 25 mL of a nutrient solution (NaN03,
0.2 g; K2HP04,0.4 g; (NH4)2S04,0.2 g; MgS04.7H20, 0.1 g;
CaC03, 0.5 g; KCl, 0.05 g; FeS04. 7H20, 0.01 g; trace element
solution 5 mL; casein hydrolysate, 0.2 g; yeast extract, 0.2 g;
and deionized water to 1000 mL). Fugal development was prevented by using Delvocid (0.2 g - ~ - ' , Delvocid Instant,
Gist-Brocades, Delft, Holland). By repeated aseptic transfer of
splinters or sections from the newly attacked wood pieces into
further flasks, or by streaking onto similar medium solidified
with agar, mixed cultures of active wood-degrading bacteria
showing various patterns of attack have been obtained. Cultures
were maintained in the dark at 22C.
To assess the bacteria's ability to degrade lignin, three
different 14c-labelled lignin preparations were used. These
were labelled in the ring, side-chain, and methoxyl groups,
respectively. The lignin was added to bacteria growing in flasks
containing pine wood shavings and 25mL of the nutrient
medium. Each flask received labelled lignin corresponding to a
specific activity of around 50 000 dpm (1 dpm = 0.0 167 Bq).
The flasks were supplied with flushing rings and incubated as
stationary cultures at ambient room temperature (approx.
22C). The flasks were flushed with sterile air after 2 and 5 days.
Later flushings after 7 , 9 , 11, and 16 days, were done with pure
sterile oxygen. The 14c02
evolved was collected in scintillation
vials that contained 10 mL of scintillation fluid. The 14c02
activity was determined with a Packard 3003 Tri-Carb liquid
scintillation spectrometer.
After substrate colonization, decay by tunnelling bacteria is
normally initiated by the cells penetrating the S3 wall layer and
then the underlying S2 layer of wood fibre walls (Figs 1a , 1b).
The presence of either a layer of extractives or preservatives
does not appear to form a barrier to these bacteria. The bacteria
adhere to the cell wall with the help of an extracellular
capsularlike structure which appears similar to the extracellular
layers produced at the tunnel entrance at the time of bacterial
penetration into the fibre wood cell wall (Figs. 1a , 1b). These
layers protrude into the lumen and serve to seal the tunnel entrance (Fig 1b, 1c). At the time of penetration the bacteria have
a "pearlike" outline with the more pointed end contained within
the wood cell wall (Fig. lb). On reaching the S2 layer the
bacteria change direction and form tunnels perpendicular to the
original entrance (Fig. 1c); the latter always containing conspicuous cross-tunnel walls which are concentric (Figs. 1c and 2a).
The bacteria then multiply within the S2 layer by repeated
divisions and form colonies characteristic of species. The
characteristic nature of the attack process gives rise to a distinct
microbial decay pattern which is dependent upon the chemical
composition of the wood cell walls and whether they have been
chemically modified. Eventually, bacteria penetrate and tunnel
all layers of the wood cell wall including the highly lignified
middle lamella, the former often being reduced to a ramifying
association of decay tunnels.
The overall shape of the decay tunnels indicates that the

945

bacteria are able to change their cellular morphology at will and


can degrade in any direction. Accordingly, the cross-tunnel
walls correspond to and reflect the positioning of the bacteria
during a prior stage of degradation. The peculiar concentric
form of the cross walls result from the aggregation of fibrillar
materials (ca. 5-8 nm in diameter, Fig. 2b) released or sloughed
off from the outer bacterial cell wall during tunnelling action. In
some cases it is possible to observe almost an entire layer being
sloughed off from the outer bacterial cell wall (Fig. 3). The
fact that cross-tunnel walls may be formed laterally or at the
distal end of the motile bacteria gives rise to the varied patterns
recognized in both TEM and SEM (Figs. 2a, 3, and 4). In some
cases, one end of the bacteria seems to remain stationary while
the rest of the cell continues to degrade in a lateral manner
causing the bacteria to rotate 180". This not only allows a
change in direction but also affords degradation of all the
available wood substance in the bacteria's immediate vicinity.
The periodicity of cross-tunnel wall development is variable for
different wood types and even within the same wood cell wall.
Generally, in woods containing a high concentration of lignin,
e.g., Homaliumfoetidum (Fig. 2a), very compact cross-tunnel
wall layers are formed, while in pure cellulosic substrates, such
as cotton fibres and cellophane, .thelayers can form a continuous
lamellated path behind the bacteria. Pronounced tunnel cross
wall layers may, therefore, represent an aggregation of the
continuous lamellations, the degree of their compaction presumably being related to the time spent by the bacteria at any
one location. Density of the substrate and degree of lignification
may be factors which impose restrictions on the rapidity of the
attack process. Production of similar secretions onto surfaces
and in pure cellulose substrates (e.g ., cotton, cellophane)
further indicates that the cross walls are formed during bacterial
motility. The strong reactions and conjugation of the secretions
with ruthenium red (not shown) indicate a negatively charged
polymer of polysaccharides-proteins.
A further feature of the chambered tunnels produced in
copper-chrome-arsenic preservative treated wood is their
association with the heavy metals released during the decay
process (Fig. 5). Semiquantitative TEM energy-dispersive
X-ray spectroscopy studies performed on samples from Pinus
radiata stakes treated at a retention of 24.7 kg/m3 Tanalith
NCA preservative showed the majority of the metals to remain
and even be concentrated (compared with adjacent S2 cell wall
areas) in the extracellular secretions, especially Cr and As
(Table I). Analyses conducted on bacteria contained within
tunnels showed only significant copper and very low levels of Cr
and As to have entered the cells. In this respect, the bacteria
appear to tolerate the immediate high levels of the preservative
by binding it both extra- and intra-cellularly. This may partly
explain the bacteria's ability to degrade the preservative-treated
P . radiata wood which was resistant to other forms of biological
attack because of its high metal loading.
A further characteristic of the decay process concerns the
large numbers of membrane-bound vesicles released from the
bacteria during tunnelling (Fig. 3). Certain cellulolytic rumen
bacteria (Forsberg et al. 1981) and wood-degrading erosion
bacteria (G. Daniel and T. Nilsson. International
Research Group on Wood Preservation. Document no.
IRG/WP/ 1283 ( 1986. See footnote 2)) produce similar
vesicles, which were shown to cany both cellulases and hemicellulases in the former type.
A major adaptive feature of tunnelling bacteria is their
pleomorphic nature, a characteristic which seems to reflect

CAN. 1. MICROBIOL. VOL. 33, 1987

TABLE1. Relative count ratesa (K,) (background corrected) and ratios between the major elements
detected in the bacterial secretions and in the adjacent wood cell wall

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Elements and ratiosb


Area of analysis

Ca

Cu

Cr

As

Bacterial tunnel wall


Bacterial cross-tunnel wall
Bacterial intracell ppts.'
S2wall layer
Middle lamella
Control analysis, resin only

4866(2.82)
1406(1.06)
467 l(2.87)
269(0.36)
1223(0.63)

7847(3.15)
1089(0.57)
396(0.16)
756(0.70)
4554(1.64)
72(0.16)

2475(1.OO)
1899(1.00)
2332(1.00)
1067(1.OO)
2768(1 .OO)
441(1.00)

14090(5.29)
4416(2.16)
103(0.04)
3 164(2.75)
927 l(3.11)

7265(3.42)
3 159(1.94)
62(0.03)
2278(2.49)
4520(1.90)

'Count rates represent mean values obtained from energy-dispersive X-ray spectroscopy point analyses (100 s) of 10 different
regions for each category. Spot size for analysis was kept constant at 50nm.
bRatios are given in parentheses.
'ppts., precipitates.

whether the cells are constrained by the substrate. When


constrained, they may be rodlike closely fitting the diameter of
the tunnel (Fig. 2a). When less closely applied various shapes
may be recognized, suggesting considerable cell wall flexibility
and an ability to modify themselves to their surrounding
substrate. The motile ability of the bacteria is indicated by their
capacity to tunnel within their substrate, although the mechanism by which this is carried out is still unknown at this stage.
There seems little doubt, however, that the tunnel cross walls
are related in some way to motility which presumably takes
place by some form of stop-start action, possibly involving
cellular contraction and expansion. To date no distinct organelles of motility such as flagella have been observed associated
with tunnelling bacteria actively involved in degradation.
Ultrastructurally, the bacteria have a Gram negative type wall
structure (Fig. 2a). The cytoplasm often contains a variety of
vesicular bodies and fibrillar elements in the nuclear region.
The ability of these bacteria to attack and actively degrade
woods containing high concentrations of lignin suggests in itself
that the cells possess ligninolytic activity, since purely cellulolytic forms are unable to degrade such substrates. In addition,
TEM examination of the tunnels indicates that the majority of
the wood cell wall components have been removed and
assimilated. Some staining precipitates adhering to tunnel
secretions can be recognized after osmium tetroxide and also
after potassium permanganate staining, both reagents known to
react with lignin and its breakdown products (Bland et al. 197 1;
Messner et al. 1985), but overall the tunnels remain largely
empty of gross precipitates or anything approaching a residual
lignin skeleton (Fig.3).
Electron microscopic evidence for active bacterial decay in

intact wood cell walls has been supported by biochemical


experiments using both 14C-labelled natural and synthetic
~
lignin
~
~ analyses
lignins (Table 2) and by 1 3 ~ and- Klason
(T. Nilsson and G. Daniel. 1986. Proceedings of the 3rd International Symposium on Biotechnology in the Pulp and Paper
Industry, Stockholm, June 16-19, 1986. Swedish Forest Products Research Laboratory, Stockholm. pp. 54-57).
Degradation of I4C-labelled dehydrogenative polymer lignins, labelled in side chain, aromatic rings, or methoxyl groups,
indicates an active lignin-degrading capacity by tunnelling
bacteria and supports the observation that tunnels formed in the
highly lignified woody tissues tend to lack conspicuous lignin
residues. Similar studies with wood-degrading erosion bacteria
have also shown these bacteria to have a capacity for lignin
degradation (Table 2). With respect to the enzyme system
involved, preliminary TEM cytochemical studies suggest localized peroxidase activity, which is an interesting observation in
view of the recently discovered ligninase (peroxidases)
enzymes used by white rot fungi for lignin degradation (Tien
and Kirk 1983). While the capability of bacteria to degrade
isolated and synthetic lignin derivatives is well known (Crawford and Crawford 1980), the same isolates have not been
shown to have a proven ability to attack and actively degrade
lignin in woody substrates. In this context the enzymic capacity
of tunnelling bacteria would also seem unique.
In conclusion, tunnelling bacteria would seem to be highly
adapted for the degradation of lignocellulosic materials, not
only by way of their ligninolytic capacity but also by their motile
and pleomorphic nature which allows both initial invasion of the
substrate and subsequent reorientation and movement of the cell
in the tunnels during the attack process. The peculiar cross-

FIGS.1-5. Electron micrographs showing features of tunnel formation and reaction with preservatives. Figs. 1-3, and 5. TEM. Fig. 1.
Stages in bacterial penetration of the fibre cell wall of Laurelia novae-zelandiae;a tropical hardwood. (a) Initial settlement and adhesion of an
encapsulated bacterial cell (B) to the fibre lumen S3layer. (6) Penetration of the fibre wall by a characteristic pear-shaped bacterium (B) and the
development of characteristic cross-tunnel secretions (CT) into the lumen. ( c )Change in direction of decay tunnel on entry to the secondary S2
fibre wall layer. Continuity between extracellular material sealing the entrance and that forming the cross-tunnel walls (CT) is apparent (arrow).
Fig. 2. Tunnel formation in a vessel wall from the hardwood Homalium foetidum (lignin content, ca 32.0%). (a) Compact concentrically aligned
cross-tunnel wall (CT) secretions are developed periodically behind the bacteria during degradation. (6) High magnification to show the fibrils
which form the wall secretions (arrowhead). Fig. 3. Lateral tunnel formation in Pinus radiata (lignincontent, ca. 28%) and release of vesicles (V)
and fibrillar materials to form the cross wall (CT) secretions. Fig. 4. SEM showing the pointed ends of both tunnels (arrowheads)and bacteria (B)
and the characteristic 3-D nature of cross-tunnel secretions (CT) produced in Pinus sylvestris (lignin content, ca. 27%). Fig. 5. Tunnels formed
within the S2wall layer of Tanalith NCA treated Pinus radiata showing aggregation of electron-dense preservative (Cu, Cr, As) particles with the
cross-tunnel (CT) and tunnel wall (TW) extracellular secretions. Unosmicated unstained preparation as used for energy-dispersive X-ray analysis
TEM. (Bars = 0.5 Fm, except in Fig. 26,O. 1 Fm).

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NOTES

947

948

CAN. J. MICROBIOL. VOL. 33, 1987

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TABLE2. 14c02
produced by cultures containing tunnelling or erosion
bacteria from synthetic lignins labelled in the side chain, aromatic ring,
or methoxyl groups
Days
Culture
no.

Type of
bacteria

Label

Tunnelling
Tunnelling
Tunnelling
Tunnelling
Tunnelling
Tunnelling
Erosion
Erosion

Ring
Ring
Side chain
Side chain
Methoxyl groups
Methoxyl groups
Ring
Ring

11

16

"Data are given as percent of total radioactivity recovered as I4CO2.

tunnel wall secretions produced as a consequence of cell


motility during tunnel formation appear unique and have not, to
the author's knowledge, been reported earlier to be involved in
bacterial motility.
Whether the bacteria possess similar ligninase enzymes as
those recently described for white rot fungi remains to be
elucidated, as does their potential in biotechnological processes
involving lignin bioconversion. Our attempts to establish pure
cultures of tunnelling bacteria have not so far been successful,
so their true taxonomic affiliation is still unclear. However, the
many ultrastructural and general features they exhibit, as well as
their general environmental occurrence, strongly suggests that
they may be gliding bacteria belonging to either the Myxobacteriales or Cytophagales, both groups which contain well
known cellulolytic forms (Reichenbach and Dworkin 1981).
From our studies on a very large number of wood samples from
collaborative field decay test trials on timbers (both
preservative-treated and nontreated), it would seem that tunnelling bacteria have a world-wide and ubiquitous distribution and
can, therefore, be encountered in decaying wood under most
situations.

Acknowledgements
This research was supported partly by the Swedish Board for
Technical Development (STU) and by a fellowship (to T .
Nilsson) from the Organization for Economic Co-operation and

Development (OECD) (Co-operative Research Project on Food


Production and Preservation). We are also indebted to Professor
T. Kent Kirk for facilities made available at the Forest Products
Laboratory, Madison, during fulfillment of the OECD award.
BLAND,D. E., FOSTER,R. C., and LOGAN,A. F. 1971. The
mechanism of permanganate and osmium tetroxide fixation and the
distribution of lignin in the cell wall of Pinus radiata. Holzforschung, 25: 137- 143.
D. L., and CRAWFORD,
R. L. 1980. Microbial degradation
CRAWFORD,
of lignin. Enzyme Microb.Techno1. 2: 11-22.
EATON,R. A., and DICKINSON,
D. J. 1976. The performance of
copper-chrome-arsenic treated wood in the marine environment.
Mater Org. (Berlin), ll(Supp1.): 521-529.
T. J., and HELLSTROM,
A. 1981.
FORSBERG,
C. W., BEVERIDGE,
Cellulose and xylanase release from Bacteroides succinogenes and
its importance in the rumen environment. Appl. Environ. Microbiol.
42: 886-896.
GREAVES,
H . 1968. Occurrence of bacterial decay in copper-chromearsenic treated wood. Appl . Microbiol . 16: 1599- 1601.
HARMSEN,
L., and NISSEN,T. V. 1965. Timber decay caused by
bacteria. Nature (London), 206: 3 19.
HOLT,D. M., and JONES,
E. B. G . 1978. Bacterial cavity formation in
delignified wood. Mater Org. (Berlin), 13: 13-30.
LIESE,W. 1970. The action of fungi and bacteria during wood
deterioration. Rec. Annu. Conv. Br. Wood Preserv. Assoc. 1970:
81-97.
LUFT,J. H. 1971. Ruthenium red and violet. I. Chemistry, purification,
methods of use for electron microscopy and mechanism of action.
Anat. Rec. 171: 347-368.
MESSNER,
K., FOISNER,
R., STACHELBERGER,
H., and ROHR,M.
1985. Osmiophilic particles as a typical aspect of brown and white
rot systems in transmission electron microscope studies. Trans. Br.
Mycol. Soc. 84: 457-466.
REICHENBACH,
H., and DWORKIN,
M. 1981. Introduction to the
gliding bacteria. In The prokaryotes. Vol. 1. Springer-Verlag, New
York. pp. 315-327.
REYNOLDS,
E. S. 1963. The use of lead citrate at high pH as an electron
opaque stain in electron microscopy. J. Cell Biol. 17: 208-2 12.
SCHMIDT,
0 . 1978. On the bacterial decay of the lignin cell wall.
Holzforschung, 32: 2 14-2 15.
SPURR,
A. R. 1969. A low viscosity embedding medium for electron
microscopy. J. Ultrastruct. Res. 26: 3 1-43.
TIEN,M., and KIRK,T. K. 1983. Lignin-degrading enzyme from the
hymenomycete Phanerochaete chrysosporium. Science (Washington, D.C.),221: 661-663.