Reflections on Cellulolysis
Bernard Dixon
Henry Tribe of Wolfson College, Cambridge, United
Kingdom, was lifting a cardboard box containing a bottle of Turkish
liqueur, called Yeni Raki, from his minicellar when the bottle fell
through the bottom of the box. He was relieved not to lose any of the
contents. Nevertheless, Tribe was surprised when he discovered that the
lower side of the box had been colonized and degraded by, a fungus,
although the cardboard was very strong and had a glossy, water-repellent
surface. This was obviously an aggressive organism.
Being a mycologist, Tribe decided to investigate, and
quickly found that a low-temperature fungus was to blame. As he and
Roland Weber now report in Mycologist (16:1, 2002),
"the interior of the bottom part of the box was softened and
covered with the ascomata of Myxotrichum chartarum. These were
massed together in an essentially pure stand which was so dense that it
formed a continuous chocolate-brown crust accompanied by very little
aerial mycelium."
Although this is the first report of M. chartarum
attacking such a tough target, the organism was discovered and named as
long ago as 1832. Christian Nees von Esenbeck, a German botanist who
devoted most of his life to the study of fungi and algae, found it on
writing paper, and described its capacity to degrade this as a
substrate. Its appearance in Henry Tribe's wine cellar was no doubt
associated with the fact that his so-called minicellar was in fact
simply the space under the floorboards—other authors have reported
finding the organism in rich organic soil, plant debris and dung from
herbivores.
Although intolerant of high temperatures, M.
chartarum can proliferate slowly at 5-7șC—probably close to the
temperature in the aerated underfloor space where it was well
established. The fungus was subsequently recovered from another of
Tribe's cardboard boxes, this one containing a bottle of Israeli Sabra.
Clearly, paper/cardboard is a prime substrate for M.
chartarum. One survey of the fungal order to which it belongs, the
Onygenales, showed that paper was by far its most favoured source of
carbon (R. S. Currah, Mycotaxon 24:1, 1985). Further work on
Tribe's isolate, cultured on various simple media, has shown that the
best of these is mineral salts agar with a sheet of cellophane as the
only available carbon.
Because cellophane is an ideal carbon source for
cellulolytic fungi, Tribe and Weber include in their report a reminder
of the need for appropriate precautions when it is used as a drying film
for electrophoresis gels, and as a seal for glass jars containing jams
and other preserved foods. The material should first be immersed in a
jar of distilled water and autoclaved thoroughly to remove any
plasticizers.
I did not have a wine cellar and I knew nothing whatever
about onygenales or M. chartarum when I did my own first,
frustrating experiment on soil organisms and paper over 50 years ago.
But I was keenly interested in microbiology, and one day I learned from
a book addressed to "boy scientists" about what seemed to be
an enticing experiment.
The author advised readers to tear a small piece out of
a newspaper, lay it on some damp soil in a plant pot, and keep it moist
and warm for several weeks. The paper would gradually disintegrate, he
said, and eventually disappear completely as it was attacked by
animalcules from the soil. These would probably be the myxobacterium Cytophaga
together with Cellvibrio and other cellulolytic
microorganisms. We were not to worry our young heads with complexities
of that sort, however, but simply watch what happened.
Following the instructions diligently, I observed the
experiment every day. But nothing happened. Even with different
newspapers and different soils, the paper remained disappointingly
resistant to biodegradation. So I asked my science teacher. "You've
made the mistake of using glossy paper," he said. No, I hadn't.
"Then you have let the soil dry out." No, I hadn't. "You
must be doing something wrong," the teacher concluded angrily.
"Think for a moment. If microorganisms in the soil did not break
down paper, we would be ankle-deep in the stuff by now. Anyway, I'm
busy. Go away."
Not the most helpful encounter, you may feel, with a
young lad keen to learn about the natural world. I was certainly
dismayed at the time. The situation was retrieved, however, several
months later when I came across virtually the same experiment in another
book, but this time with scraps of filter paper instead of newsprint.
This time it worked a treat. In less than two weeks, the
piece of Whatman no. 1 was virtually invisible. Why the difference? The
answer came about eight years ago from Stephen Cummings and Colin
Stewart at the Rowett Research Institute in Aberdeen, Scotland (J. Appl.
Bacteriol. 76:196, 1994). It emerged from their study of the
degradation of municipal solid waste, 28 million metric ton of which are
generated in the United Kingdom every year. Much of this finds its way
into landfill sites in remote parts of the country. Just under a third
of the material consists of paper and cardboard, and the Rowett
researchers were trying to determine the effectiveness of various
landfill microorganisms in breaking down newspaper in particular. They
worked with five cellulolytic bacteria (two species of Eubacterium and
two of Clostridium) isolated from a landfill site. The organisms
attacked cellulose robustly when inoculated and grown anaerobically on
Whatman no. 1 filter paper, which consists of comparatively refined
cellulose. But the bacteria performed far less spectacularly when they
were presented with newsprint—generously provided by Aberdeen Journals
Ltd, publishers of the Aberdeen Evening Express.
Even the most powerful strains, which were extremely
active when attacking a diet of filter paper, failed to rise beyond the
mediocre when offered Scottish newsprint instead. Part of the
explanation for the disparity was the presence of printing ink in the
paper. Although not directly toxic to the bacteria, the ink masked the
surface of the paper, stopping the organisms from adhering to the
cellulose fibers.
The main problem, however, was that as much as 24% of
the newspaper consisted of lignin, a polymer of high molecular weight.
This prevented the microorganisms from using their cellulases to tear
apart cellulose and other inherently susceptible polymers.
Using gas chromatography, electron microscopy, and
spectrophotometry, Cummings and Stewart had demonstrated, under
anaerobic conditions, something which I had discovered by a considerably
more simplistic aerobic approach half a century previously. Both
experiments are in accord with a third report (E. J. Booth, The American
City 80:26,1965) of newspapers, deeply buried in a landfill site,
which were still legible when they were recovered 25 years later.
Sadly, my unhelpful school teacher is long deceased, so
I have not been able to inform him of this work, nor of Henry Tribe's
wine cellar and M. chartarum.
Nor, come to think of it, can I bring the same man up to
date with a whole series of topics upon which he regaled science classes
with what even then appeared to be imprudent certitude. He insisted, for
example, that gaps would always be essential in railroad tracks,
allowing them to expand on warm days. Yet all over the world today,
trains run quite safely on continuous welded rails.
Likewise, we were assured that it would never be
practicable to build skyscrapers in London like those of New York and
other American cities, because of the soft underlying London clay. Not
so, as we now know. So too with my science teacher's calculations which
established the sheer impossibility of a spacecraft attaining the escape
velocity required to leave the Earth.
Given this trio of erroneous assurances about big
technology, it's hardly suprising that he was wrong about animalcules.
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