Table Of ContentTHERMOPHILIC MOULDS IN BIOTECHNOLOGY
THERMOPHILIC MOULDS
IN BIOTECHNOLOGY
Edited by
B. N. JOHRI
Department of Microbiology, CBSH,
G.B. Pant University of Agriculture & Technology,
Pantnagar, India
T. SATYANARAYANA
Department of Microbiology,
University of Delhi South Campus,
New Delhi, India
and
J. OLSEN
Department of General Microbiology,
University of Copenhagen,
Copenhagen, Denmark
Sp ringer-Science+ Business Media, B. V.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-90-481-5315-2 ISBN 978-94-015-9206-2 (eBook)
DOI 10.1007/978-94-015-9206-2
Printed on acid-free paper
All Rights Reserved
© 1999 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1999.
Softcover reprint of the hardcover I st edition 1999
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owner.
Contents
Forward vii
Preface ix
List of contributors xi
1. Introduction
B.N Johri, J. Olsen and T. Satyanarayana
2. Ecology and Distribution 13
A. Subrahmanyam
3. Thermophilic Fungi: Present Taxonomic Concepts 43
J. Mouchacca
4. Physiology and Biochemistry 85
T. Satyanarayana and B.N Johri
5. Amylases and their Industrial Potential 115
B. Jensen and J. Olsen
6. Cellulolytic Thermophilic Fungi 139
G. Canevascini
7. Xylanolytic Enzymes 169
A. Archana, A. Sharma and T. Satyanarayana
8. Proteolytic Enzymes 191
SK. Garg and B.N Johri
9. Lipases 219
B.N Johri and S Ahmad
10. Miscellaneous Enzymes 245
B. Jensen and J. Olsen
11. Composting and Solid State Fermentation 265
T. Satyanarayana and W. Garjek
12. Spoilage of Stored Products 289
P.e. Jain
13. Molecular and General Genetics 317
Mo. Azevedo, MSS Felipe and T. Satyanarayana
14. Future Perspectives 343
B.N Johri, T. Satyanarayana and J. Olsen
v
Foreword
It is a privilege to be invited to write a foreword for this book reviewing the progress of
work on thermophilic fungi since the publication in 1964 of Cooney and Emerson's
monograph, Thermophilic fungi: An account of their biology, activities and classification.
Perhaps this honour has been given to me because of my own fascination for the
thermophilic fungi, aroused in 1962 by my major Professor, Dr. Paul J. Allen of the
University of Wisconsin, and by listening to a talk on thermophilic fungi by Dr. Ralph
Emerson. During the war years, with the objective of producing rubber, Allen and Emerson
had studied the retting of the rubber-bearing guayule plant, Parthenium argentatum.
During the retting of the chopped plant material, the temperature of the mass rose. Allen
and Emerson,_ then working at the Guayule Rubber Extraction Research Unit of the United
States Department of Agriculture in Salinas, California, isolated several species of
thermophilic fungi from the guayule mass that were capable of reducing the resinous
contaminant and effect an improvement in the recovery and the quality of the extracted
rubber. In so far as I know, this was the first investigation of thermophilic fungi from a
'biotechnological' point of view, almost fifty years ago (Allen, P.J. and Emerson, R. 1949.
Guayule rubber, microbiological improvement by shrub retting. Industrial and Engineering
Chemistry 41,346-365). Later, based on the isolates of thermophilic fungi obtained from
the retting guayule shrub and the collections of all the known species of thermophilic fungi
from other investigators and culture collections, Donald Cooney and Ralph Emerson
published a monograph in which 13 species were described. In addition to the taxonomic
descriptions of species known up to the time of their writing, the authors gave a lucid
account of the general biology of thermophilic fungi. This book called attention of
biologists to thermophilic fungi, which had generally remained unknown. Since the
publication of their book, some more species of thermophilic fungi have been described
(and with more taxonomic confusion!). Research in the last 35 years has increased our
knowledge of the biology of thermophilic fungi as they have evoked considerable interest
as potential sources of commercially valuable enzymes and antibiotics. This book,
Thermophilic Moulds in Biotechnology, gives an account of all this and of other aspects of
the biology of the thermophilic fungi.
I would like to join hands with other fungal biologists in congratulating the editors and the
publishers for this timely book on thermophilic fungi. I am sure this book will serve the
scientific community as its predecessor. In particular, it should stimulate many young
readers in discovering new species of thermophilic fungi, in investigations of their
physiology, ecology and biochemistry, and of course, in finding applications of
thermophilic fungi in biotechnology.
28th May, 1999 Ramesh Maheshwari
Department of Biochemistry Professor
Indian Institute of Science
Bangalore-560 012, India
vii
Preface
When Cooney and Emerson compiled their research on thermophilic fungi in a monograph
in 1964, it was not in sight that some of these organisms shall serve as experimental
systems of choice nor were their technological potentialities truly in sight. However, this
had set an area of research demarcated, which during the last three decades or more has
seen a great deal happen by way of taxonomy, diversity and especially the exocellular
enzymes of thermophilic fungi which has prompted us to put together these developments
in one place for the benefit of teachers, researchers and practitioners. After all, thirty-five
years is a long time to bring forth another volume in this fascinating discipline! The
foundation for bringing together this volume were laid during the 4th International
Mycological Congress held at Regensburg in 1990, but concretised through yet another
workshop "Thermophilic Fungi Miehe to Emerson" at the 5th IMC at Vancouver in 1994.
The present volume comprises of chapters by workers involved in thermophilic fungal
research both at the fundamental as well as the applied level, and carries with it tenets of
the latest developments. The distribution and ecology of the group by Subrahmanayam
traces very early to recent work on their presence in both, natural and man made
environments and describes how this small fungal group has become the focus of attention
on account of ecological adaptations. Mouchaccha puts together the taxonomic status in the
right perspective with changes in species epithets, as also those forms are doubtful
belonging to this small group. The physiological features of thermophilic fungi with their
distinct nutrition and growth behaviour are discussed by Satyanarayana and Johri, wherein
comparative assessment with mesophilic counterpart has been stressed to bring about the
salient points. A major component of this volume describes the production, regulation and
application of polys accharase and other hydrolytic enzymes of thermophilic fungal species.
Olsen and Jensen discuss the developments pertaining to amylases and their industrial
applications to highlight the potentiality of the key organism, Thermomyces lanunginosus.
Canevascini discusses the cellulolytic system of thermophilic fungi to bring out the
potentiality of Sportrichum thremophile and other species and limitations that are implicit
in their large scale use. The associated hemicellulolytic enzymes especially the xylanases
are described by Archana, Sharma and Satyanarayana. Garg and Johri discuss proteolytic
enzymes to describe recent developments that have taken shape with Rhizomucor miehei
protease on account of its applicability in cheese making. The most sought after enzymes
of biotechnological significance from thermophilic fungal species are the lipases. Humicola
lanuginosa protein was first genetically engineered moiety to hit the world market over a
decade ago. On the other hand, lipase from Rhizomucor miehei has been used for a variety
of trans - and inter-esterification reactions. These developments and future scenario for
lipases is put together by Ahmad and Johri. Other enzymes of limited significance but
which are catching up now are discussed by Olsen and Jensen. In the context of waste
management including their exceedingly interesting contribution to mushroom composting,
Grajek and Satyanarayana have brought together the role of thermophilic fungi in both
liquid and solid systems including protein enrichment. On the other hand Jain highlights
ix
x Preface
the contributions of these species in spoilage of stored products with emphasis on
agricultural produce wherein exothermic reactions can lead to buildup localised 'hotspots'.
In the fmal chapter, Felipe, Azevedo and Satyanarayana bring together the current
information in the context of molecular biology and its implications in the future scenario
of biotechnological developments. It has been the endeavour of various authors to bring the
subject matter from the viewpoint of both, fundamentals and applications and point out
future areas of research and development.
The impetus for research on thermophilic fungal systems to the senior author of the book
(BNJ) was provided through lectures on the subject by late Prof. Ralph Emerson and
personal interactions. That this legacy has culminated in bringing together the present
volume, is a life time ambition as a personal satisfaction but also a joint effort to knit
together the developments in comprehensive manner for the benefit of active researchers
and practitioners.
An exercise of this nature could not have materialised but for active cooperation of various
authors, student researchers as also our friends across the globe who have participated in
this project. The editors wish to thank Mr. S. M. Noorwez and Dr. M. S. R. Sastry for their
help in reading and correcting the proofs. Special thanks go to Kluwer Academic
Publishers for accepting this proposal for publication and editorial support. While every
effort has been made to put together the volume with utmost care, the responsibility for
errors falls on us.
B. N. Johri
T. Satyanarayana
J. Olsen
LIST OF CONTRIBUTORS
s.,
1. Ahmad, School of Biochemistry, Faculty of Science and Technology, La Trobe
University, Bundura, Victoria, Australia 3083.
2. Archana, A., S.S. College, Alipur, Delhi-lI0036, India.
3. Azevedo, Mo., Department of Cellular Biology, University of Brasilia, DF, Brazil.
4. Canevascini, G., Department of Plant Biology and Phytochemistry, University of
Fribourg, CH-1700 Fribourg, Switzerland.
5. Felipe, s.s., Department of Cellular Biology, University of Brasilia, DF, Brazil.
6. Garg, s.K., Department of Microbiology, RML Avadh University, Faizabad-224 001
(U.P.), India.
7. Grajek, W, Department of Biotechnology and Microbiology, Agricultural University, ul,
Mazowiecka 48, 60-623 Poznan, Poland.
8. Jain, P.e., Department of Microbiology and Biotechnology, Dr.H.S.Gaur
Vishwavidhyalaya, Sagar-470 003 (M.P.), India.
9. Johri, B.N, Department of Microbiology, CBSH, G.B.Pant University of Agriculture &
Technology, Pantnagar-263145 (U.P.), India.
10. Mouchacca, M, Cryptogamy Laboratory, National Museum of Natural History, 12 Rue
Buffon, 75005 Paris cedex, France.
11. Jensen, B., Department of General Microbiology, University of Copenhagen, Solvgade
83H, DK-1307 Copenhagen K, Denmark.
12. Olsen, J., Department of General Microboilogy, University of Copenhagen, Solvgade
83H, DK-1307 Copenhagen K, Denmark.
13. Satyanarayana, T., Department of Microbiology, University of Delhi South Campus,
New Delhi-I 10021, India.
14. Sharma, A., Department of Microbiology, University of Delhi South Campus,
New Delhi-ll0021, India.
15. Subrahmanyam, A., Department of Botany, Kakatiya University, Vidyaranayapuri,
Warangal-506 009 (A.P.), India.
Xl
INTRODUCTION
B. N. JOHRI, 1. OLSEN and T. SATYANARAYANA
Temperature is one of the key physical variables that determine sustenance ofliving beings
on earth. Microorganisms are known to have arrived on the scene nearly 4 billion years ago
at a time when temperatures were likely to be in extremes. That this indeed was true has
been shown by the discovery of microorganisms from geothermal areas around the world
and has pushed up our viewpoint concerning the uppermost temperature for existence of
life forms to above boiling water. All microbes recovered until now from such extremes are
prokaryotes suggesting distinct limit for eukaryotic life forms, which currently extends to
62°C. However, notwithstanding the drastic difference between the upper temperature
limits for the existence of prokaryotes and eukaryotes, it is definitely of some consequence
that proteins from the latter have been found to be stable upto 95°C or higher (46).
It is interesting that the eukaryotes can grow only upto 60°C or so, herein too the
extremes are shown by a small number of fungal species (47). Other eukaryotes exhibit
upper temperature limits between 38 to 50°C with the lower forms in the group depicting
larger temperature variations.
When one considers thermophily in the fungal world it also becomes apparent that
most species depict a rather broad minima, maxima and optima which usually extends right
from 20 to upward of 50°C (14). Thus, the group could be defined as eurythermal (8). A
truly thermophilic fungus has therefore been considered to show no growth below 20°C but
good growth beyond 50°C (20). This definition has been utilised as a working model as
opposed to others including that of Crisan (16) who described all species growing above
40°C as thermophilic. However, this definition is fraught with problems because certain
fungi such as Aspergillus fumigatus can grow upto 10-12°C (minima) but also frequently
occupy a niche with maxima of 50°C. Such species are more appropriately grouped under
thermotolerants but are treated in this book because they act as markers of a pattern that
hinges between meso-and thermo-phily.
It is appropriate to define here thermoduric species which produce highly resistant
propagules such as ascospores, chlamydospores or sclerotia to overcome stressed
environmental conditions but usually grow best in the mesophilic range. It should also be
stressed here that even amongst the thermophilic fungi, cultural and morphogenetic
behaviour of some species show temperature-dependent variations. Thus, Chaetomium
thermophile and Thermoascus aurantiacus generally produce fertile ascocarps at 40-45°C
but only sterile ascocarps or vegetative mycelia at higher growth temperatures. However,
major changes in cardinal temperatures as a result of alterations in chemical environment
have not so far been reported for these fungi. But it is interesting to note the ubiquity of
B. N Johri, T. Satyanarayana and J. Olsen (eds.) Thermophilic Moulds in Biotechnology, 1-11.
© 1999 Kluwer Academic Publishers.
2 Johri et at .
thermophilic fungi as a consequence of their inherent ability to occupy a temperature niche
that most other fungi can not exploit successfully.
1. Early History
The discovery of earliest thermophilic fungal species was a result of chance contamination
of organic materials incubated at elevated temperatures. This included Mucor pusil/us from
bread (34) and Humicola lanuginosa from potato disk (51). However, real early beginnings
are attributed to Meihe who made exhaustive study of fungi occurring in self-heating hay
that resulted in publication of a book "Die Selbsterhitzung des Heus". Two important
species described by him were Malbranchea pulchella var. sulfurea known for production
of penicillin, and Thermoascus aurantiacus with strong hemicellulolytic machinery.
Another important species, Talaromyces dupontii was described by Griffon and Maublanc
(24) and represented, until recently, the only thermophilic member of the genus
Penicillium. Description of Chaetomium thermophile by La Touche (33) and its cellulolytic
nature ushered in new interest in thermophilic fungi and intensive research of the group
began culminating in the appearance of a complete monograph by Cooney and Emerson in
1964. In the last nearly three decades, there have been further additions to new
thermophilic fungal species but the pace has been much slower for two reasons. One, the
interest lay now in understanding their nutritional and physiological behaviour and, two,
some commercially suitable strains were in sight.
2. Geographical Distribution and Habitat
Thermophilic fungi are world wide in distribution and most species do not show any
geographical restrictions. However, tropical sites favour recovery as a consequence of
elevated temperatures and more competitive microbial environment. Thus they have been
reported from a wide variety of habitats including different soil types and places where
self-heating of plant material results in high temperatures. These include compost, wood
chip piles shavings, plant straw, nesting materials of birds and animals, municipal refuge,
stored grains and dung of animals. They may occupy these habitats as either resting
propagules or as active mycelia. Besides these natural self-heated habitats, thermophilic
fungi have also been recovered from man-made habitats such as cooling towers, effluents
from nuclear power reactors and ducts employed for thermal insulation (32). Such wide
ranging habitats suggest their wide dissemination machinery through vegetative or
reproductive propagules to places of man-made activities. That this dissemination can be
wide is borne out by the fact that thermophilic fungi have been recovered from even
Antarctic soils (46); the dispersal in this habitat is likely to occur through droppings of the
birds.
3. Survival Strategies and Regulation in Nature
In spite of the thermal habitats serving as reservoirs for growth and survival of
thermophilic fungi, their population in soils from tropical environment does not appear to
