• TABLE OF CONTENTS
HIDE
 Front Cover
 Title Page
 Panel on microbial processes
 Dedication
 Preface
 Table of Contents
 Introduction
 1. Raw materials for microbial...
 2. Food and animal feed
 3. Soil microbes in plant health...
 4. Nitrogen fixation
 5. Microbial insect control...
 6. Fuel and energy
 7. Waste treatment and utiliza...
 8. Cellulose conversion
 9. Antibiotics and vaccines
 10. Pure cultures for microbial...
 11. Future perspectives in...
 Regulations for packaging and shipping...
 Board on science and technology...
 Advisory committee on technology...
 Advertising
 Back Cover














Title: Microbial processes
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Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00082049/00001
 Material Information
Title: Microbial processes promising technologies for developing countries : report of an ad hoc panel of the Advisory Committee on Technology Innovation, Board on Science and Technology for International Development, Commission on International Relations, National Research Council
Physical Description: xii, 198 p. : ill. ; 23 cm.
Language: English
Creator: National Research Council (U.S.) -- Board on Science and Technology for International Development. -- Panel on Microbial Processes
Publisher: National Academy of Sciences
Place of Publication: Washington D.C
Publication Date: 1979
 Subjects
Subject: Industrial microbiology   ( lcsh )
Technological innovations -- Developing countries   ( lcsh )
Genre: federal government publication   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references.
 Record Information
Bibliographic ID: UF00082049
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 06637145
lccn - 79091534

Table of Contents
    Front Cover
        Front Cover
    Title Page
        Page i
        Page ii
    Panel on microbial processes
        Page iii
        Page iv
        Page v
        Page vi
        Page vii
    Dedication
        Page viii
    Preface
        Page ix
        Page x
    Table of Contents
        Page xi
        Page xii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
    1. Raw materials for microbial processes
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
    2. Food and animal feed
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
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        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
    3. Soil microbes in plant health and nutrition
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    4. Nitrogen fixation
        Page 59
        Page 60
        Page 61
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        Page 63
        Page 64
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        Page 78
        Page 79
    5. Microbial insect control agents
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
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        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
    6. Fuel and energy
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
        Page 119
        Page 120
        Page 121
        Page 122
        Page 123
    7. Waste treatment and utilization
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
        Page 129
        Page 130
        Page 131
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        Page 134
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
        Page 140
        Page 141
    8. Cellulose conversion
        Page 142
        Page 143
        Page 144
        Page 145
        Page 146
        Page 147
        Page 148
        Page 149
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        Page 151
        Page 152
        Page 153
        Page 154
        Page 155
        Page 156
        Page 157
    9. Antibiotics and vaccines
        Page 158
        Page 159
        Page 160
        Page 161
        Page 162
        Page 163
        Page 164
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    10. Pure cultures for microbial processes
        Page 177
        Page 178
        Page 179
        Page 180
        Page 181
        Page 182
        Page 183
        Page 184
        Page 185
    11. Future perspectives in microbiology
        Page 186
        Page 187
        Page 188
        Page 189
        Page 190
    Regulations for packaging and shipping viable microbial cultures
        Page 191
        Page 192
    Board on science and technology for international development
        Page 193
        Page 194
    Advisory committee on technology innovation
        Page 195
    Advertising
        Page 196
        Page 197
        Page 198
        Page 199
        Page 200
        Page 201
    Back Cover
        Back Cover
Full Text





















































































































































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MICROBIAL PROCESSES:
Promising Technologies for
Developing Countries







Report of an Ad Hoc Panel of the
Advisory Committee on Technology Innovation
Board on Science and Technology for International Development
Commission on International Relations
National Research Council











NATIONAL ACADEMY OF SCIENCES
Washington, D.C. 1979




















This report has been prepared by an ad hoc advisory panel of the Board on Science and
Technology for International Development, Commission on International Relations, Na-
tional Research Council, for the Office of Science and Technology, Bureau for Technical
Assistance, Agency for International Development, Washington, D.C., under Contract
No. AID/csd-2584, Task Order No. 1.













NOTICE: The project that is the subject of this report was approved by the Governing
Board of the National Research Council, whose members are drawn from the Councils of
the National Academy of Sciences, the National Academy of Engineering, and the Insti-
tute of Medicine. The members of the Committee responsible for the report were chosen
for their special competence and with regard for appropriate balance.
This report has been reviewed by a group other than the authors according to proce-
dures approved by a Report Review Committee consisting of members of the National
Academy of Sciences, the National Academy of Engineering, and the Institute of Medi-
cine.














Second Printing, March 1981


Library of Congress Catalog Number 79-91534









Panel on Microbial Processes


J. ROGER PORTER, Department of Microbiology, University of Iowa, Iowa
City, Iowa, Chairman
ROBERT F. ACKER, American Society for Microbiology, Washington, D.C.
ARTHUR W. ANDERSON, Department of Microbiology, Oregon State Uni-
versity, Corvallis, Oregon
WINTHROP D. BELLAMY, Department of Food Science, Cornell University,
Ithaca, New York
WAYNE M. BROOKS, Department of Entomology, University of North
Carolina, Raleigh, North Carolina
MARVIN P. BRYANT, Department of Dairy Science, College of Agriculture,
University of Illinois, Urbana, Illinois
LEE A. BULLA, JR., U.S. Grain Marketing Research Laboratory, Federal
Research, North Central Region, Science and Education Administration,
U.S. Department of Agriculture, Manhattan, Kansas
JOE C. BURTON, NITRAGIN, Milwaukee, Wisconsin
STAFFAN DELIN, Berkeley, California
RICHARD D. DONOVICK, American Type Culture Collection, Rockville,
Maryland
EUGENE L. DULANEY, Merck Institute for Therapeutic Research, Rahway,
New Jersey
LLOYD R. FREDERICK (formerly, Department of Agronomy, Iowa State
University, Ames, Iowa), Senior Microbiologist, Office of Agriculture,
Tropical Soil and Water Management Division, Development Support
Bureau, Agency for International Development, Washington, D.C.
JAMES W. GERDEMANN, Department of Plant Pathology, University of
Illinois, Urbana, Illinois
CLARENCE G. GOLUEKE, Cal Recovery Systems Incorporated, Richmond,
California
RICHARD S. HANSON, Department of Bacteriology, University of Wiscon-
sin, Madison, Wisconsin
CLIFFORD W. HESSELTINE, Northern Regional Research Laboratory, U.S.
Department of Agriculture, Peoria, Illinois
GLADYS L. HOBBY (formerly, Chief, Special Research Laboratory [Infec-
tious Diseases], U. S. Veterans Administration, Cornell University Medical
College, New York, New York), Editor-in-Chief, Antimicrobial Agents and
Chemotherapy, Kennett Square, Pennsylvania
RILEY D. HOUSEWRIGHT, Committee on Toxicology, National Academy
of Sciences, Washington, D.C.
CARLO M. IGNOFFO, Biological Control of Insects Research Laboratory,
Entomology Resources Division, U.S. Department of Agriculture, Science
and Education Administration, Research Park, Columbia, Missouri









T. KENT KIRK, U.S. Forest Products Laboratory, U.S. Department of Agri-
culture, Forest Service, Madison, Wisconsin
ALLEN I. LASKIN, EXXON Research and Engineering Company, Linden,
New Jersey
JOHN H. LITCHFIELD, Battelle Memorial Institute, Columbus Laboratories,
Columbus, Ohio
DONALD H. MARX, Forestry Science Laboratory, U.S. Department of Agri-
culture, Forest Service, Athens, Georgia
WILLIAM J. OSWALD, Professor of Sanitary Engineering and Public Health,
University of California, Berkeley, California
BURTON M. POGELL, Department of Microbiology, School of Medicine, St.
Louis University, St. Louis, Missouri
DAVID PRAMER, Associate Vice President for Research, Rutgers University,
New Brunswick, New Jersey
DONALD W. ROBERTS, Boyce Thompson Institute for Plant Research,
Cornell University, Ithaca, New York
OLDRICH K. SEBEK, Infectious Diseases Research, The Upjohn Company,
Kalamazoo, Michigan
KEITH H. STEINKRAUS, Department of Food Science and Technology,
New York State Agricultural Experiment Station, Geneva, New York
DONALD K. WALTER, Urban Waste Technology, U.S. Department of En-
ergy, Washington, D.C.
DEANE F. WEBER, Cell Culture and Nitrogen Fixation Laboratory, U.S.
Department of Agriculture, Beltsville, Maryland
BERNARD A. WEINER, Northern Regional Research Center, U.S. Depart-
ment of Agriculture, Peoria, Illinois
WILLIAM E. WOODWARD, Program in Infectious Diseases and Clinical
Microbiology, University of Texas Health Science Center, Houston, Texas
OSKAR R. ZABO R SKY, National Science Foundation, Washington, D.C.


NAS Steering Committee
J. ROGER PORTER, Department of Microbiology, University of Iowa, Iowa
City, Iowa
ROBERT F. ACKER, American Society for Microbiology, Washington, D.C.
RILEY D. HOUSEWRIGHT, Committee on Toxicology, National Academy
of Sciences, Washington, D.C.


Study Staff

B. K. WESLEY COPELAND, Board on Science and Technology for Inter-
national Development, Commission on International Relations, National
Academy of Sciences-National Research Council, Washington, D.C.









M. G. C. McDONALD DOW, Board on Science and Technology for Inter-
national Development, Commission on International Relations, National
Academy of Sciences-National Research Council, Washington, D.C.
E. GRIFFIN SHAY, Board on Science and Technology for International
Development, Commission on International Relations, National Academy
of Sciences-National Research Council, Washington, D.C.


Contributors
MARTIN ALEXANDER, Department of Soil Science, Cornell University,
Ithaca, New York
LARRY L. ANDERSON, Department of Mining & Fuels Engineering, Uni-
versity of Utah, Salt Lake City, Utah
DURWARD BATEMAN, President, Plant Pathology Society, Cornell Univer-
sity, Ithaca, New York
L. JOE BERRY, Department of Microbiology, University of Texas, Austin,
Texas
JOHN J. BOLAND, Department of Geography and Environmental Engineer-
ing, The Johns Hopkins University, Baltimore, Maryland
HENRY R. BUNGAY, Department of Chemical and Environmental Engineer-
ing, Rensselaer Polytechnic Institute, Troy, New York
ROBERT H. BURRIS, Department of Biochemistry, University of Wisconsin,
Madison, Wisconsin
R. R. COLWELL, Director, Sea Grant Program, University of Maryland, Col-
lege Park, Maryland
R. JAMES COOK, Regional Cereal Disease Research Laboratory, USDA,
Washington State University, Pullman, Washington
THOMAS M. COOK, Department of Microbiology, University of Maryland,
College Park, Maryland
DON L. CRAWFORD, Department of Bacteriology & Biochemistry, College
of Agriculture, University of Idaho, Moscow, Idaho
CONSTANT C. DELWICHE, Department of Land, Air and Water Resources,
University of California, Davis, California
RAYMOND N. DOETSCH, Department of Microbiology, University of Mary-
land, College Park, Maryland
LOUIS A. FALCON, Department of Entomology, University of California,
Berkeley, California
RICHARD A. FINKELSTEIN, Department of Microbiology, University of
Texas, Dallas, Texas
E. M. FOSTER, Director, Food Research Institute, University of Wisconsin,
Madison, Wisconsin
HARLYN O. HALVORSON, Director, Rosenstiel Basic Medical Sciences
Research Center, Brandeis University, Waltham, Massachusetts








ROBERT P. HANSON, Department of Veterinary Science, University of Wis-
consin, Madison, Wisconsin
CARL-GO RAN HEDEN, Karolinska Institute, Stockholm, Sweden
DAVID HENDLIN, Senior Director, Developmental Microbiology, Merck
Sharp & Dohme Laboratories, Rahway, New Jersey
H. HEUKELEKIAN, New York, New York
WILLIAM N. HUBBARD, JR., President, The Upjohn Company, Kalamazoo,
Michigan
ARTHUR E. HUMPHREY, Dean, College of Engineering and Applied Sci-
ence, University of Pennsylvania, Philadelphia, Pennsylvania
J. W. M. LA RIVIERE, International Institute for Hydraulic and Environ-
mental Engineering, Delft, The Netherlands
RAYMOND C. LOEHR, Director, Environmental Studies Program, Riley-
Robb Hall, Cornell University, Ithaca, New York
CLAYTON W. McCOY, Institute of Food and Agricultural Sciences, Univer-
sity of Florida, Lake Alfred, Florida
WALSH McDERMOTT, Robert Wood Johnson Foundation, Princeton, New
Jersey
ROSS E. McKINNEY, N. T. Veatch Professor of Environmental Health,
Department of Civil Engineering, University of Kansas, Lawrence, Kansas
ROBERT A. MAH, Division of Environmental & Nutritional Studies, Univer-
sity of California School of Public Health, Los Angeles, California
STAN M. MARTIN, National Research Council of Canada, Ottawa, Canada
EMIL M. MRAK, Chancellor Emeritus, University of California, Davis, Cali-
fornia
DANIEL J. O'NEIL, Engineering Experiment Station, Georgia Institute of
Technology, Atlanta, Georgia
H. PEPPLER, White Fish Bay, Wisconsin
ELWYN T. REESE, Food Sciences Laboratory, U.S. Army Natick Research
and Development Command, Natick, Massachusetts
MARTIN H. ROGOFF, Senior Staff Scientist, Hazard Evaluation Division,
Office of Pesticide Programs, U.S. Environmental Protection Agency,
Washington, D.C.
JAMES P. SAN ANTONIO, Science and Education Administration, U.S.
Department of Agriculture, Beltsville, Maryland
WILLIAM D. SAWYER, Chairman, Department of Microbiology and Im-
munology, School of Medicine, Indiana University, Indianapolis, Indiana
RICHARD SOPER, Acting Research Leader, Insect Pathology Research Insti-
tute, Boyce Thompson Institute, U.S. Department of Agriculture, Ithaca,
New York
KENNETH V. THIMANN, Thimann Laboratories, University of California,
Santa Cruz, California
GEORGE T. TSAO, Laboratory of Renewable Resources, Purdue University,
West Lafayette, Indiana








D. M. UPDEGRAFF, Chemistry and Geochemistry Department, Colorado
School of Mines, Golden, Colorado
ABEL WOLMAN, The Johns Hopkins University, Baltimore, Maryland
B. C. WOLVERTON, National Space Technology Laboratories, NSTL Sta-
tion, Mississippi
LUNG-CHI WU, Campbell Institute for Agricultural Research, Napoleon,
Ohio























Roger Porter, who directed the organization and
preparation of this report, died on May 24, 1979.
Dr. Porter will be remembered for his unflagging dedication
to the use of science for the benefit of mankind and for
his warm and gracious manner in pursuing this purpose.













Preface






The National Academy of Sciences-National Research Council, through
the Commission on International Relations and its Board on Science and
Technology for International Development (BOSTID), has investigated
scientific and technological advances that may be applicable to the less-
developed regions of the world. BOSTID's Advisory Committee on Tech-
nology Innovation (ACTI) has reviewed a number of technologies in the
United States and elsewhere to assess their potential for contributing to the
economic and social well-being of those in developing countries. It is in this
context that this study of microbiological processes was carried out.
An ad hoc panel of ACTI convened in August 1977 to select a group of
microbial processes with promise for wider use in the developing world. To
make the selection process more manageable, a steering committee of the
panel chose ten subject areas they felt were most important to developing
countries. In each subject area, specialists from the panel were asked to
analyze the responses from a questionnaire sent to approximately 25,000
biological scientists and engineers. Each subpanel selected a small number of
examples of microbial processes that met either of the following criteria:

The process can be beneficially employed in developing countries
or
The process has sufficient potential for developing countries to merit
research and development for future use.

Because of the unique conditions in each country where the processes may
be used, no attempt has been made to quantify economic feasibility. Depend-
ing on indigenous needs and resources, a process appropriate for one country
may be inappropriate for another.
Assistance in reaching technical or economic conclusions concerning the
various processes may be solicited from the individuals and institutions cited
throughout the report.






PREFACE


For the convenience of the reader, each process is presented in a separate
chapter, giving the following information:

Methodology
Potential value of the process
Special needs and limitations
Research and development requisites
Suggested readings
Sources for obtaining microorganisms.

In addition, the Introduction provides a nontechnical summary of the
processes described in each chapter and characterizes the organisms and their
general physical and nutritional needs.
The panel wishes to thank the many scientists who contributed informa-
tion. Special appreciation is expressed to Marcia A. Duncan, research assis-
tant; Mary Jane Engquist, staff assistant; and to Dorothy M. Woodbury and
Cicely Henry, who served as administrative secretaries, for preparing draft
documents for the meeting and for producing the final manuscript of this
report; they have been most helpful. The panel also acknowledges the help of
Diosdada DeLeva, Maryalice Risdon, and Wendy D. White for bibliographic
editing and Harry Hatt, of the American Type Culture Collection, for stan-
dardizing the nomenclature of microorganisms.
The final report was edited and prepared for publication by F. R. Ruskin,
for whose assistance the panel is grateful.













Contents





INTRODUCTION 1

1 RAW MATERIALS FOR MICROBIAL PROCESSES 10
Typical Raw Materials 11
Underutilized Raw Materials 15

2 FOOD AND ANIMAL FEED 18
Food Preservation 19
Improving Nutritional Value 24
Production of Meat-Like Flavors 28
Koji Method of Producing Enzymes 30
Indonesian Tempeh 32
Single-Cell Protein Production 37

3 SOIL MICROBES IN PLANT HEALTH AND
NUTRITION 47
Mineral Cycling by Soil Microorganisms 48
Mycorrhizal Fungi 51
Biological Control of Soil-Borne Pathogens 55

4 NITROGEN FIXATION 59
Symbiotic Systems 61
Asymbiotic Fixation 71

5 MICROBIAL INSECT CONTROL AGENTS 80
Development of Bioinsecticides 80
Bacteria 84
Viruses 89
Protozoa 94
Fungi 98

6 FUEL AND ENERGY 107
Ethanol 108
Utilization of Cellulose 111
Methane 111






CONTENTS


Methanol 116
Hydrogen 117
Bacterial Leaching 119

7 WASTE TREATMENT AND UTILIZATION 124
Algal-Bacterial Systems 125
Composting 131
Anaerobic Lagoons 133
Recycling Animal Waste by Aerobic Fermentation 136
Recycling Animal Waste by Anaerobic Fermentation 138

8 CELLULOSE CONVERSION 142
Volvariella Species 143
Lentinus edodes 144
Pleurotus Species 148
Thermoactinomyces Species 149
Phanerochaete chrysosporium 151
Trichoderma reesei 152
Other Species 153

9 ANTIBIOTICS AND VACCINES 158
Antibiotics 160
Vaccines 169

10 PURE CULTURES FOR MICROBIAL PROCESSES 177
Major Pure Culture Collections 178
World Data Center and Microbiological Resource Centers 178
Preservation Methods 180
Mixed Microbial Cultures 184
Patenting of Processes Involving Microorganisms 184

11 FUTURE PERSPECTIVES IN MICROBIOLOGY 186

REGULATIONS FOR PACKAGING AND SHIPPING
VIABLE MICROBIAL CULTURES 191

Board on Science and Technology for International
Development 193
Advisory Committee on Technology Innovation 195














Introduction






Microorganisms have simultaneously served and assaulted man throughout
history. Man is totally dependent on some microbes for life processes, while
remaining subject to the destructive capacities of others in diseases not yet
conquered.
The study of microorganisms and microbial processes has provided a vari-
ety of benefits. For instance:

World health has been improved through the discovery of the microbial
causes of most human, animal, and plant diseases, leading to the development
of vaccines, antibiotics, and chemical agents to combat many of these dis-
eases.
Foods have been improved in quality and protected from spoilage to
enable wide distribution and storage against times of need.
Sewage treatment methods have been developed to break the chain of
disease transfer through waterborne pathogens. Microorganisms also enhance
the water quality of rivers and lakes by degrading naturally occurring organic
matter.
Farming practices have been improved through recognizing and capital-
izing on the role of soil microorganisms; microbes have been used to break
down nonedible crop residues for reuse by new crops. Nitrogen-fixing micro-
organisms have been used to inoculate legumes.
Microbial fermentation processes have provided foods, beverages, medi-
cines, and chemicals for human use.

Microbes, as organized systems of enzymes, can often perform these func-
tions more efficiently than purely chemical processes, and current environ-
mental and economic constraints make the potential contribution of
microbes increasingly attractive.
From these examples it is clear that microbes can be marshaled to aid in
solving many important global problems including food shortages, resource
recovery and reuse, energy shortages, and pollution. Microbiology is particu-
larly suited to make important contributions to human needs in developing







MICROBIAL PROCESSES


countries, yet it has received comparatively little attention. The range of
possible applications covers uses by individuals and industries in rural settings,
villages, and cities.
This report covers examples of microbial processes that may be useful in
developing countries. Although many of these processes may not have a
direct and immediate use, their scope and diversity should serve to indicate
the strong potential for microbial applications.
Above all, the report highlights the pervasiveness and importance of mi-
crobes, along with the increasing need to train microbiologists and to support
their research and development activities. A group of well-trained micro-
biologists with adequate support can make valuable contributions to social
welfare.


Organisms Involved in Microbial Processes

The organisms responsible for the microbial processes discussed in this
report are an integral, all-pervasive part of the biological world. Although
they are rarely seen (the larger fungi, mushrooms, are perhaps the most
visible), it is estimated that microorganisms make up about one-quarter of the
biomass-the total weight of living organisms in the world-with animals
and plants accounting for the remainder. Microorganisms occur everywhere,
and extraordinary aseptic measures are required to exclude them from
places where their presence would be harmful, such as the operating room
of a hospital or a food-processing plant. Even then, these measures are not
always successful.
The bulk of microorganisms reside in the soil, where they are responsible
for the predominant biological activity. Others are located in the upper layers
of the oceans and in fresh and brackish waters, as well as on the surfaces
above ground, in the air, and of course inside larger organisms, both plant and
animal.
A number of microorganisms are harmful, or pathogenic, to humans and
animals. Although the terms microbe or germ initially were used to describe
any minute microorganism, they tend to be used especially to connote harm-
ful organisms. Yet most microorganisms are either harmless or essential for
the maintenance of the biological cycles on which all life depends.
Microorganisms comprise the following classes of organisms:

Bacteria
Fungi (yeasts and molds)
Algae
Protozoa
Viruses.







INTRODUCTION


Their classification, characteristics, and harmful and beneficial effects are
shown in Table 1.


Physicochemical Factors Affecting Microbial Growth

A number of physical factors affect the growth or retardation of micro-
organisms, including temperature, osmotic pressure, acidity or alkalinity, the
presence of oxygen or light, and the degree of agitation. Although no species
of microbes can survive over the complete range of conditions found in
nature, there are varieties that thrive in hot springs, polar wastes, acidic bogs,
and highly saline waters like the Dead Sea.



Temperature
Most microorganisms grow within a temperature range of 300C. Individual
species have well-defined upper and lower temperature limits and optimum
temperatures for growth.
Microorganisms are usually divided into three groups with respect to their
most favored temperature range. Psychrophiles grow best between about
0C and 300C. These organisms occur in cold areas and are frequently associ-
ated with refrigerated food spoilage. Mesophiles grow best between about
200C and 500C. Most disease-causing bacteria are in this group. Thermophiles
grow best from 400C to 700C. This division into three groups is convenient
but somewhat arbitrary, since the dividing lines are not sharp. Further, not
every organism can grow over the entire range indicated for its group.



Acidity and Alkalinity
Taken as a whole, microorganism species can tolerate a wide range of
acidity and alkalinity. Some thrive under highly acidic conditions (pH 1-3)
and others in alkaline environments (pH 9-10). However, most microorgan-
isms grow best at neutral pH (pH 7).

Oxygen
Microorganisms can be divided into three major groups with respect to
their oxygen requirements. Obligate aerobes have a requirement for oxygen
and grow best when oxygen is continuously available. Obligate anaerobes
grow in the absence of free oxygen. The requirement for oxygen reflects the
metabolic pathways the organisms use to obtain energy. Aerobes break down










TABLE 1 Microorganisms: Characteristics, Problems, and Uses


Organisms Characteristics Problems Uses


Bacteria:
Schizomycetes or
Protista



Fungi:
Plants devoid of
chlorophyll





Algae:
Thallophyta
(undifferentiated
plants)


Protozoa:
Microscopic animals




Viruses:
Submicroscopic forms;
considered intermedi-
ate between living and
nonliving.


Single-celled; spherical rod
and spiral forms. Most are
saprophytes (use dead matter
for food).


Variety of forms; microscopic
molds, mildews, rusts, and
smuts; larger mushrooms
and puffballs.




Single cells, colonies, or filaments
containing chlorophyll and
other characteristic pigments.
No true roots, stems, or leaves;
aquatic.

Single-celled or groups of similar
cells, found in fresh and sea
water, in soil, and as parasites
in animals, man, and some
plants.

Infective agents, capable of mul-
tiplying only in living cells;
composed of proteins and
nucleic acids.


Some forms are pathogens for
plants, humans, and
animals.



Rot textiles, leather, harvested
foods, and other products;
cause important plant and
animal diseases.




Cover pond surfaces, producing
scum and unpleasant odor and
taste (in drinking water); absorb
02 from ponds and some pro-
duce toxins.

Responsible for serious human and
animal diseases-malaria, sleeping
sickness, dysentery, etc.



Cause of variety of diseases in
humans (measles, influenza,
pneumonia, poliomyelitis),
animals (foot and mouth, canine
distemper), and in plants.


Break down organic matter and
assist soil fertility, waste dis-
posal, and biogas production;
source of antibiotics and
other chemicals.

Assist in recycling cellulose,
lignin, and other complex
plant constituents; mushrooms
and yeasts are important in
food and nutrition; many are
also used in chemical and
pharmaceutical industries.
Red and brown seaweeds are im-
portant foods in Asia and Poly-
nesia. Red algae produce agar.
Some blue-green algae fix nitro-
gen. Major food for ocean fish.
Assist in breakdown of organic
matter such as cellulose in
ruminant nutrition.



Important as carriers of genetic
information. Also cause dis-
eases in insects and other pests,
and research is directed to their
use in biological pest control.







INTRODUCTION


their nutrients by a sequence of enzyme reactions that require oxygen. An-
aerobes utilize a pathway for metabolism that does not require free oxygen,
and in fact they may be inhibited by it.
The third group of organisms are the facultative anaerobes. These can use
either metabolic pathway, depending on the presence or absence of oxygen.


Osmotic Pressure

The osmotic pressure across a cell wall depends on the relative concentra-
tion of dissolved substances within the cell and outside it. For example, most
bacteria can grow over a broad range of salinity because their cells are capable
of maintaining a relatively constant internal salt concentration. But if salt
concentrations outside the cell become too high, water is lost from the cell
and growth is inhibited. This is the basis for food preservation by salt. Sugars
and other substances also influence osmotic relationships between cells
and their environment.



Nutritional Requirements for Microbial Growth


All microorganisms require water to grow and water can be considered the
single most important component in their growth.
Microorganisms can be divided into two groups based on the source of
carbon they convert into their cell components. Heterotrophic organisms
utilize organic compounds as a source of carbon for both synthesis and en-
ergy. Autotrophic organisms utilize carbon dioxide as their major source of
carbon for synthesis and obtain energy either from the sun (through photo-
synthesis) or by metabolizing inorganic compounds. The inorganic com-
pounds that can be used by various autotrophic organisms include ammonia,
hydrogen, reduced iron, manganese and other minerals, and hydrogen sulfide.
Heterotrophs can utilize a wide variety of organic materials as sources of
carbon. In fact, there are probably no biologically generated materials in the
environment that cannot be degraded by some species of microorganism.
In addition to carbon, all organisms require sources of the other elements
found in cell components. These include nitrogen, sulfur, phosphorus, and
potassium. Both heterotrophs and autotrophs require certain inorganic salts
for optimum growth and reproduction.
Most microorganisms cannot utilize (fix) atmospheric nitrogen and require
nitrogen in the form of an ammonium or nitrate salt or in an organic form.
Sulfur is usually obtained from sulfate salt and phosphorus from salt of
phosphoric acid.






MICROBIAL PROCESSES


Raw Materials for Microbial Processes

A variety of materials have been used in microbial processes in industrial-
ized countries. For less-developed nations, however, it is not necessary to
restrict usage to these substances; indigenous raw materials, for example
agricultural residues, may be much more appropriate.

Food and Animal Feed

Microorganisms have long been used to produce certain foods, beverages,
condiments, and animal feeds. Recently, several new commercial microbial
processes have been developed. These include the production of single-cell
protein to supplement animal feeds; mushrooms for human food from agri-
cultural wastes; microbial rennet for cheese making; enzymes such as glucose
isomerase; meat-like flavorings using the Chinese soy sauce and Japanese miso
processes; xanthan and amino-, hydroxy-, and keto-acids, and vitamins,
among other products.
There are many potential ways for utilizing microorganisms in food pro-
duction, from the household and village level to full-scale commercial opera-
tions. The need continues for better food preservation and methods to reduce
postharvest food spoilage.


Soil Microbes in Plant Health and Nutrition

The region where the roots of plants make contact with the soil is called
the rhizosphere. This is a complex biological area in which the microbial
population is considerably higher and its activity greater than in root-free soil.
Growth of microorganisms in the rhizosphere is undoubtedly enhanced by
nutritional substances released from the roots, and growth of plants is in-
fluenced by microbial metabolic products released into the soil.
Of great significance are certain fungi that infect roots and form mycor-
rhizae. These fungi can absorb and translocate phosphate and other essential
nutrients and make them available to plants. With a greater need for food for
an ever-growing population, increased attention should be given to the effects
of the rhizosphere on plant nutrition.


Nitrogen Fixation

As demands for fertilizer increase, and as the energy crisis becomes more
acute, greater attention must be given to microbial fixation of atmospheric
nitrogen. The emphasis should be on applying known technology, of which
legume inoculation to increase crop production is a good example. Basic







INTRODUCTION /
research on culture and ecology of both symbiotic and nonsymbiotic nitro-
gen-fixing microorganisms could lead to an increase in the world's supply of
edible protein. This would be of even greater significance if microorganisms
that fix nitrogen, or their nitrogen-fixing genes, could be transferred to micro-
organisms that can be established in nonleguminous crops, such as rice and
other cereals, so they could utilize nitrogen from the air. Cultivation of
free-living nitrogen-fixing blue-green algae that grow in nitrogen-deficient sub-
strates is another goal. The potential for development in these areas is great.


Microbial Insect Control Agents

In the search for safe, alternative methods of controlling insect pests, the
use of microorganisms that cause disease in insects offers distinct possibilities.
Insects, like humans, animals, and plants, are susceptible to microbial dis-
eases. Microbes that produce diseases in insects are termed entomopathogens.
In many cases they can significantly reduce natural populations of insects.
Safety, specificity, effectiveness, and cost are the decisive considerations in
the development of any insecticide. A number of entomopathogens fulfill
these criteria and are therefore potentially useful bioinsecticides. Some are
already being produced commercially, and more are in development.

Fuel and Energy

Most nations today are facing shortages of fuel and energy. Yet if develop-
ment is to proceed, increasing amounts of energy will be required. To meet
these growing requirements, attention must be directed to the development
of unconventional and renewable energy systems.
Microbial processes already help provide energy. In the countries of South
and Southeast Asia and in the People's Republic of China, for example, many
small farms and villages are using methane generators that utilize fermented
animal manure, human wastes, and other waste substances to produce "bio-
gas" for household cooking, lighting, and power. In some countries alcohol
produced by microbial fermentation is added to petroleum products to
supplement scarce fuel supplies. These processes that depend upon the solar-
produced biomass may hold unique promise for supplying some of the energy
requirements of less-developed nations. The microbiological conversion of
plant matter into fuel circumvents the millions of years required for plant
material to become fossil fuel through natural processes.


Waste Treatment and Utilization
A number of water and wastewater purification processes utilize microbes.
Many opportunities exist for waste utilization and recycling, including refeed-







MICROBIAL PROCESSES


ing of animal wastes; algal farming for fish culture and as a source of animal
feed and fermentable substrates; and the upgrading of cellulose wastes by
protein enrichment for use as fodder.


Cellulose Conversion

Cellulose, a renewable resource from agricultural and forestry products, is
a major component of many solid wastes and residues. Usually, cellulose is
bound to lignin. The lignocellulose complex is a substrate that must be chem-
ically degraded before the cellulose can be used in some commercial
processes.
Cellulose can be degraded by chemical or enzymatic hydrolysis to soluble
sugars. These sugars can then be used by microbes to form ethanol, butanol,
acetone, single-cell protein, methane, or other products of fermentation. In
some cases, cellulose can be converted directly into these products by fer-
mentation. The technology for refined cellulose degradation is readily avail-
able for recycling paper, cardboard, etc.
Biomass agriculture and forestry may hold great economic potential for
certain less-developed countries, particularly in tropical and subtropical re-
gions.


Antibiotics and Vaccines

Although approximately 4,000 antibiotics are known, most have no prac-
tical value because of their toxicity to human beings, lack of efficacy, or high
production cost. There are only about 50 widely used antibiotics. Extensive
use of antibiotics in medicine began in 1945 with penicillin. Currently, anti-
biotics are widely used in human and veterinary medicine, and to a lesser
extent in agriculture, where they are used to increase the weight of livestock
and poultry, to control plant diseases, and as insecticides. New antibiotics are
being sought and old ones are being modified to improve their properties.
Killed, attenuated, or living microorganisms, or their products, have been
used for many years to produce immunity against certain human diseases
such as smallpox, cholera, yellow fever, tetanus, and diphtheria. Additional
research is needed to improve these vaccines and to produce new ones. Spe-
cial emphasis should be placed on effective programs and delivery systems for
existing vaccines.


Pure Cultures for Microbial Processes

Microorganisms are an extremely important natural resource. Because of
the present and potential usefulness of beneficial microorganisms, it is essen-







INTRODUCTION 9

tial that their germ plasm be preserved, just as plant germ plasm is preserved
in seed banks and endangered animal life is protected in various ways.
Several outstanding culture collections of microorganisms exist today, and
they are essential to research and teaching in microbiology as well as com-
mercial microbial production.










Chapter 1


Raw Materials for Microbial Processes





Microorganisms, like all other forms of life, require water and nutrients for
growth, reproduction, and maintenance. In addition to suitable sources of
utilizable carbon, nitrogen, and sulfur, microbes generally require sodium,
potassium, phosphorus, iron, and other minerals. The major factor in select-
ing raw materials for microbial processes is the source of carbon.
Microbial processes have long been harnessed for the benefit of man in the
production of foods, medicines, and alcoholic beverages. Nature employs
microbes on a much grander scale to establish and maintain a balance among
the diverse forms of life on this planet. The underlying agents responsible for
the myriad syntheses, transformations, and other reactions caused by mi-
crobes are the enzymes-biological catalysts of high specificity and efficiency.
One important aim of science and technology has been to domesticate
beneficial microbes, especially for the transformation of raw materials to
worthwhile end products.
In general, most raw materials are naturally occurring substances from
which more useful materials can be produced. In this sense, microbes them-
selves may be considered raw materials suitable for further processing. The
use of microbes as single-cell protein (SCP) is an example (see Chapter 2). In
this report the discussion will be limited to major carbon sources found in
nature, formed mostly by plants through photosynthesis, which can be used
either for producing additional biomass (e.g., SCP) or for further transforma-
tions (e.g., alcohol).
In theory, any abundant carbon source might be employed for microbial
processes, including coal, petroleum, lignocellulose, starch, sugar, organic
acids, and even carbon dioxide. Some of these sources are currently used;
others, such as coal and carbon dioxide, present considerable technological
barriers. Coal would have to be converted first to a readily usable carbon base
(perhaps paraffin or methanol) because it is biologically inert and may con-
tain compounds potentially inhibitory or toxic to microbes. However, plant
biomass and, to a lesser extent, animal biomass, represent utilizable sources of
carbon for microbial processes. Well-known examples of microbial processes
based on these sources are the production of alcohol from grain and cheese
from milk.

10


'-I-I-----I--~i---






RAW MATERIALS FOR MICROBIAL PROCESSES


Although carbon dioxide is a form of carbon that can be assimilated by
some microorganisms, this raw material is utilized mainly by plants through
the mechanism of photosynthesis. Primary photosynthetic productivity
(growth of plants using solar energy) of the earth has been estimated to be
155 x 109 t* of material per year on a dry weight basis.
The distribution of plant biomass produced by photosynthesis is shown in
Table 1.1. Land-based plants account for 65 percent of the weight of the
biomass produced annually, even though they occupy only about 29 percent
of the area. The dominant yearly production of land-based biomass, approxi-
mately 42 percent, is produced as forest.
Although agricultural crops account for only 6 percent of the primary
photosynthetic productivity, they provide not only a vital portion of food for
man and animals, but other essentials such as structural materials, textiles,
and paper products as well. Agricultural raw materials are the most important
source of carbon for microbial conversion processes. The historical multi-
purpose use of agricultural crops has maintained a continued heavy depend-
ence on this source.
Most agricultural crops and residues are relatively free from toxic materials
and this, in addition to their availability, may have stimulated their use as a
raw material for microbial processes. Because of these advantages, along with
the real technological barriers to using other carbon sources, agricultural
crops and residues can be expected to retain their dominance as the carbon
source for microbial processes. But this must not preclude the further explor-
ation and exploitation of other sources, especially those indigenous to the
less-developed nations. In addition, based on their unique environments and
requirements, certain countries may have an opportunity to establish new
plants and practices for microbial processes.



Typical Raw Materials

Some typical raw materials and fermentation products used in developed
countries are listed in Table 1.2. Selected combinations of other materials are
used as substrates for various products. These raw materials provide carbon,
nitrogen, salts, trace elements, vitamins, and other requirements for the
processes; they are few in number because the conditions for large-scale mi-
crobial processes impose limitations on the materials that may serve as sub-
strates.
In general, the raw materials mentioned in Table 1.2 are traditionally used
for microbial processes because of their suitability for specific processes. But


*In this report t represents metric ton.







MICROBIAL PROCESSES


in another geographical area, or for new or different processes, one need not
be limited to what has been used in the past. Table 1.3 lists a variety of
important food crops grown in developing countries. These crops or their
residues may also be considered as raw materials for microbial processes.
A variety of other common waste materials derived from agricultural,
forest, and urban sources, may serve as substrates for microbial processes
(Table 1.4).

TABLE 1.1 Estimated Primary Photosynthetic Productivity of the Earth

Area Net Productivity
(total = 510 million km2) (total = 155.2 billion tons dry wt/yr)

Total Earth 100% 100%

Continents 29.2 64.6

Forests 9.8 41.6
Tropical Rain 3.3 21.9
Raingreen 1.5 7.3
Summer Green 1.4 4.5
Chaparral 0.3 0.7
Warm Temperate Mixed 1.0 3.2
Boreal (Northern) 2.4 3.9

Woodland 1.4 2.7

Dwarf and Scrub 5.1 1.5
Tundra 1.6 0.7
Desert Scrub 3.5 0.8

Grasslands 4.7 9.7
Tropical 2.9 6.8
Temperate 1.8 2.9

Desert (Extreme) 4.7 0
Dry 1.7 0
Ice 3.0 0

Cultivated Land 2.7 5.9

Freshwater 0.8 3.2
Swamp & Marsh 0.4 2.6
Lake & Stream 0.4 0.6

Oceans 70.8 35.4

Reefs & Estuaries 0.4 2.6
Continental Shelf 5.1 6.0
Open Ocean 65.1 26.7
Upwelling Zones 0.08 0.1


Source: James A. Bassham. 1975. Cellulose as a chemical and energy resource. In Cellu-
lose as a chemical and energy resource. New York: John Wiley and Sons.







RAW MATERIALS FOR MICROBIAL PROCESSES


TABLE 1.2 Typical Raw Materials
Countries


and Products in Industrialized


Raw Materials Products

Sulfite Waste Liquor Single-Cell Protein (SCP)
Ethanol SCP
Acetic Acid
Methanol SCP
Whey SCP
Lactic Acid
Paraffins SCP
Citric Acid
Amino Acids
Molasses Ethanol
Glutamic Acid


TABLE 1.3 Estimated Production of Major Food Crops in Developing
Countries*

Metric
Tons (in
Crop thousands) Percent

Paddy 186230 21.36
Cassava 103486 11.87
Wheat 95048 10.90
Maize 73328 8.41
Banana/Plaintain 55199 6.33
Coconuts 32664 3.75
Sorghum 31173 3.57
Yams, Taro, etc. 28777 3.30
Potatoes 26909 3.09
(Pulses)** 25997 (2.98)
Citrus 22040 2.53
Millet 21452 2.46
Barley 20775 2.38
Sweet Potatoes 17630 2.02
Soybeans 13842 1.59
Groundnuts 13502 1.55
Tomatoes 12755 1.46
Grapes 12720 1.46
Mangoes 12556 1.44
Watermelon 10436 1.20
Dry Beans 8537 0.98
Onions 6474 0.74
Percentage of Total Developing Country
Food Crop Production 94.39

*Developing market economies as defined in the FAO Production Year-
book (1977).
**Pulses-total legumes except soybeans and groundnuts.
Source: FAO Production Yearbook. New York: UNIPUB, 1977.







14 MICROBIAL PROCESSES
TABLE 1.4 Typical By-product Substrates for Use in
Microbial Processes in Developing Countries

Agricultural Other

Molasses Animal Manures
Maize Stover Sewage
Straw Municipal Garbage
Bran Paper Mill Effluent
Coffee Hulls Cannery Effluent
Cocoa Hulls Fishery Effluent
Coconut Hulls Slaughterhouse Effluent
Fruit Peels Milk-Processing Effluent
Fruit Leaves
Bagasse
Oilseed Cakes
Cotton Wastes
Tea Wastes
Bark
Sawdust


Of the lists of materials in Tables 1.2, 1.3, and 1.4, a few may hold special
promise for use in the microbial processes that occupy the bulk of this report.
The criteria for selecting these raw materials for research are their almost
year-round availability in large volume in many developing areas and their
ease of assimilation by microorganisms.
Among raw materials commonly used for microbial processes (Table 1.2),
molasses is probably the one most readily available for use as a substrate in
developing countries. Because it contains both easily assimilable sugars and
necessary micronutrients, it is a very useful substrate that is readily utilized
by a variety of microorganisms. However, it contains very little nitrogen.
Starch (or syrups produced from starch) is also a good substrate, and many
potential sources are available. These include the cereal crops (maize, rice,
wheat, etc.) and starchy tubers such as potato and cassava.
In addition to the crops that may be good sources of starch, a few of the
plentiful products and waste products listed in Tables 1.3 and 1.4 may merit
particular consideration for some of the microbial processes. Cassava, for
instance, may be a good choice as a substrate to produce ethanol, SCP, and
other economically valuable substances. This might be a better disposition of
the crop than its present widespread use for food, since its low protein-to-
calorie ratio makes it less than ideal nutritionally. Another promising raw
material is coffee-processing waste, which is produced in large amounts (4.5 t
of by-product for each t of dehulled coffee). It appears to be a good substrate
for the growth of various fungi and yeasts. Taro (Colocasia esculenta), though
a well-known staple food, has a more limited distribution than some of the
agricultural products mentioned above, existing as a commercial crop only in
Egypt, West Africa, Southeast Asia, and some Pacific and Caribbean Islands.






RAW MATERIALS FOR MICROBIAL PROCESSES


However, it, too, has potential as a substrate.
Domestic sewage or industrial wastes offer possible fermentation sub-
strates for algae or bacteria. Unless the supply is properly planned, however,
it cannot always be counted on to meet the demands of a large microbial
process.
The single most abundant potential source of carbon-in developing coun-
tries and elsewhere-is cellulose (see Chapter 8). This is a constituent of many
foods, fiber crops, agricultural residues, and wood and forest residues, some
of which are mentioned in Table 1.4.
Most of these selected substrates are rich in carbohydrates, but for certain
microbes they may have to be supplemented with sources of nitrogen, salts,
trace metals, and other requirements. Possible sources for some of these
supplements in developing countries may be whole yeast or distiller's dried
solubles from local alcoholic fermentations. In some countries meat or fish
by-products such as slaughterhouse wastes or gutting and canning residues
would be excellent supplements. Combinations of plants might also be used
to meet the nutritional requirements of producing organisms. For example,
cassava (carbohydrate) could be combined with soybeans (high nitrogen).


Underutilized Raw Materials

The materials so far discussed as possible substrates for microbial processes
are widely cultivated and available. But there are many less-known plants, or
plants that may be used only locally, that may be excellent candidates for
this purpose. An example is the winged bean (Psophocarpus tetragonolobus),
now becoming more popular as a food in Southeast Asia and West Africa
because of its unique combination of protein-rich and edible seeds, tubers,
and leaves.
Certain tropical plants, such as basella and amaranths, have not received
much attention as food sources, but they may give a greater yield than many
crops in extensive use and may also be useful as substrates. However, under-
developed raw materials selected for large-scale microbial processes will
probably have to meet the requirements discussed in the Introduction.
Plants can also be grown specifically for biomass as a fermentation sub-
strate. Plants selected for such use should grow and reproduce rapidly, con-
tain a low crystallinity cellulose and a low lignin content, and be easily
harvested and transported. Another desirable characteristic of plants for
biomass would be an ability to grow in ecological niches in which they will
not compete with or eclipse regular crops.
For instance, the buffalo gourd (Cucurbita foetidissima) does well in arid
conditions, and the salt bushes (Atriplex spp.) and tamarugo (Prosopis
tamarugo) are salt tolerant and might be introduced into countries with arid






MICROBIAL PROCESSES


and saline areas. Aquatic plants such as the reed (Phragmites communis),
cattails (Typha spp.), the papyrus reeds (Cyperus spp.), mat rush (Juncus
effusus), textile screw pine (Pandanus tectorus), and eel grass (Zostera ma-
rina) are examples of plants that grow in a saline aquatic environment.
The use of the water hyacinth (Eichornia crassipes) to treat domestic
sewage with large quantities of plant biomass produced during the process is
receiving increased attention because of the tremendous growth rate of this
plant-as much as 850 kg/ha/day of dry plant material has been reported.
This prolific growth has made the water hyacinth a troublesome weed in
certain tropical and semitropical areas, particularly the Nile Basin and south-
ern United States. Biomass from domestic waste treatment can be used to
produce biogas and fertilizer, feed, or a protein concentrate. At present,
domestic wastewater treatment using the water hyacinth is being demon-
strated in the United States.
Kelps and seaweeds are sources of carbohydrates other than cellulose.
These plants have the drawback of high water and salt content. Farming such
plants and harvesting them economically may also present special problems.
Countries with a limited supply of oil and natural gas are not likely to
consider petroleum hydrocarbons as a microbial substrate. But countries with
large oil and gas deposits also have a supply of methane, which may be used
as a carbon and hydrogen substrate for the growth of organisms for single-cell
protein.


Research Needs

In many developing countries there is need to establish basic information
about potential substrates for microbial processes by:

Systematic identification of resources;
Analysis of constituents and properties of promising individual sources;
Identification of scientific, technological, and institutional resources
and constraints; and
Determination of optimal process based on best use of substrate, eco-
nomic justification, available technical support, and unique area needs.


References and Suggested Reading
Bassham, J. A. 1975. Cellulose as a chemical and energy resource. In Cellulose as a
chemical and energy resource: Cellulose Conference Proceedings, held under the
auspices of the National Science Foundation, at the University of California, Berke-
ley, June 25-27. 1974, C. R. Wilke, ed., pp. 9-19. New York: John Wiley and Sons.
Birch, G. G.; Parker, K. J.; and Worgan, J. T., eds. 1976. Food from waste. London:
Applied Science Publishers Ltd.








RAW MATERIALS FOR MICROBIAL PROCESSES 17

Conference on Capturing the Sun through Bioconversion, Proceedings, sponsored by the
United States Energy Research Development Administration and others, Washington,
D.C., March 10-12, 1976. Washington, D.C.: Washington Center of Metropolitan
Studies.
Food and agriculture: readings from Scientific American. 1976. San Francisco: W. H.
Freeman and Company.
National Academy of Sciences. 1975. Underexploited tropical plants with promising
economic value. Report of an Ad Hoc Panel of the Advisory Committee on Tech-
nology Innovation, Board on Science and Technology for International Develop-
ment, Commission on International Relations. Washington, D.C.: National Academy
of Sciences.
1976. Making aquatic weeds useful: some perspectives for developing coun-
tries. Report of an Ad Hoc Panel of the Advisory Committee on Technology Innova-
tion, Board on Science and Technology for International Development, Commission
on International Relations. Washington, D.C.: National Academy of Sciences.
__- 1976. Renewable resources for industrial materials. Report of the Committee
on Renewable Resources for Industrial Materials, Board on Agriculture and Renew-
able Resources, Commission on Natural Resources. Washington, D.C.: National Acad-
emy of Sciences.
___ 1977. Methane generation from human, animal, and agricultural wastes. Report
of an Ad Hoc Panel of the Advisory Committee on Technology Innovation, Board on
Science and Technology for International Development, Commission on Inter-
national Relations. Washington, D.C.: National Academy of Scieiices.
Perlman, D. 1977. Fermentation industries, quo vadis? ChemTech 7:434-443.
Schlegel, H. G., and Barnea, J., eds. 1977. Mircrobial energy conversion. Oxford: Perga-
mon Press.
White, J. W., and McGrew, W., eds. 1977. Clean fuels from biomass and wastes. Proceed-
ings of the symposium held on January 25-28, 1977, at Orlando, Florida, sponsored
by the Institute of Gas Technology. Chicago: Institute of Gas Technology.
Wilke, C. R., ed. 1975. Cellulose as a chemical and energy resource: Cellulose Conference
Proceedings, held under the auspices of the National Science Foundation, at the
University of California, Berkeley, June 25-27, 1974. New York: John Wiley and
Sons.




Research Contacts

Carl-Goran Hed6n, Karolinska Institutet, Solnavagen 1, S-10401 Stockholm 60, Sweden.
Arthur E. Humphrey, School of Engineering, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, U.S.A.
F. K. E. Imrie, Tate and Lyle Ltd., Philip Lyle Memorial Research Laboratory, Univer-
sity of Reading, P. O. Box 68, Reading RG6 2BX, England.
David Perlman, School of Pharmacy, University of Wisconsin, Madison, Wisconsin
53706, U.S.A.
Steven R. Tannenbaum, Department of Nutrition and Food Science, Massachusetts Insti-
tute of Technology, Cambridge, Massachusetts 02139, U.S.A.
Noel D. Vietmeyer, National Academy of Sciences, 2101 Constitution Avenue, N.W.,
Washington, D.C. 20418, U.S.A.
Daniel Wang, Department of Nutrition and Food Science, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, U.S.A.
J. T. Worgan, National College of Food Technology, University of Reading, St. George's
Avenue, Weybridge, Surrey KT13 ODE, England.










Chapter 2


Food and Animal Feed






At least 25 percent of the world's population, approximately one billion
people, suffer from hunger and malnutrition. It is vital, therefore, that they
produce more food and improve their standard of nutrition. Food losses
should be decreased through low-cost methods of food preservation, since
standard modern methods of food processing such as canning, freezing, or
dehydration with artificial heat make the preserved food too expensive for
families on an income of US$200-$300 per year.*
Most cultures have traditionally used some form of microbial process to
preserve foods that would otherwise spoil. Some of these processes have also
contributed to increasing the nutritive value of the final product through the
increased production of essential nutrients or the synthesis of nutrients not
present in the original food. Cheeses are perhaps the most widespread and
best known of these foods. But there are many others, some of which are
largely confined to certain parts of the world and almost totally unknown
elsewhere. This section describes a number of these processes.
While it is recognized that food habits and customs are among the most
difficult to change, there have already been dramatic changes in some food
habits of developing countries. The spread of wheat flour bread throughout
much of the lowland tropics is an example of one such change, and it involves
a microbial process, yeast fermentation.
The purpose of this section is to bring to the attention of the reader a
number of lesser-known processes that preserve or enhance the nutritive value
of foods and beverages and that may merit introduction to areas where they
are unknown or invite more widespread use and improvement where they are
already used. None of the methods (except for large-scale single-cell protein
production of animal feeds) requires large investment in capital equipment or
plants.
These processes also illustrate the potential contribution of microbiology
toward diversifying the use of limited resources as well as the need to develop

*National Academy of Sciences. 1978. Postharvest Food Losses in Developing Coun-
tries. Washington, D.C.







FOOD AND ANIMAL FEED 19
trained microbiologists for a variety of research and development opportu-
nities. It must be stressed that food processing is always potentially hazard-
ous, and new methods or products should only be marketed after careful
investigation by qualified personnel.


Food Preservation

Three relatively low-cost methods of food preservation frequently carried
out in combination are: 1) salting; 2) sun or smoke drying; and 3) acid fer-
mentation.
Salt is one of the best low-cost chemicals for preserving fresh foods rang-
ing from vegetables to meats and fish. Added in concentrations of approxi-
mately 2-2.5 percent by weight to fresh vegetables, salt promotes an an-
aerobic lactic acid fermentation. The fermentation favors development of a
microbial flora that converts, for example, cucumbers to pickles and cabbage
to sauerkraut (see flow sheets). Pickling is the traditional method of providing
a winter supply of vegetables in much of Asia and Europe. There is less need
for extended storage in tropical areas where vegetables can be grown on a
year-round basis, but these methods may be valuable for taking advantage of
periodic surplus production and providing variety to the diet.
Korean kimchi, a staple in that country, consists of mixed vegetables
fermented by lactic acid bacteria. An inoculum is generally not required, as
the bacteria are ubiquitous. The salt concentration, coupled with anaerobic
conditions at ambient temperature, controls the development and sequence
of the lactic-acid-producing organisms. The combination of salt and acid leads
to products with excellent keeping quality.


Fish Sauce and Paste
Fish and shrimp are excellent protein foods, but they are highly perish-
able. Also, many fish caught in nets are too small to be sold commercially or
are species not generally consumed directly as fresh fish. Thus, low-cost
methods of preserving small and surplus fresh fish and shrimp are of great
importance in food distribution and consumption. Larger surplus fresh fish
are often salted, sun dried, and consumed as dried fish or as fish powder.
Large quantities of small fish are fermented to produce fish or shrimp
sauces and pastes in Southeast Asia. The basic procedure is to mix the freshly
netted small and trash fish with sea salt, in proportions that ensure that the
extracted fish juices contain about 20 percent salt in the final product. Such
high salt concentrations inhibit putrefaction. No inoculum is required: the
microorganisms in the gut of the fish and enzymes in the fish tissues control
hydrolysis (solubilization) of the fish proteins.







MICROBIAL PROCESSES


FLOW SHEET: Cucumber (Pickle) Fermentation

Fresh Cucumbers

Wash

Cover with 5% salt brine (add dill or other flavoring)

Cover fermentation container to avoid evaporation and to exclude air

Ferment for 1 or 2 weeks
(total acid 0.6-1.0% as lactic acid, pH 3.6-3.4)


FLOW SHEET: Cabbage (Sauerkraut) Fermentation


Fresh Cabbage*

Trim and clean

Remove core

Shred (to 2-5 mm width)

Add salt (about 2.25% by weight) and distribute evenly

Fill into fermentation containers
Salt and shredded cabbage can be mixed as fermentation container is filled.

Cover top of fermentation container and use a water seal to prevent entrance of air.
The seal must allow escape of CO2 gas produced during fermentation.

Ferment to acidity desired
For optimum keeping quality the acidity should be high (below pH 4), but
the sauerkraut can be consumed earlier if desired.




The fish sauces (nu'6c mam in Vietnam, patis in Indonesia and the Philip-
pines, and nampla in Thailand) are salty condiments adding some essential
amino acids and vitamins (mainly B-complex) to the diet. Similar processes in
which less protein hydrolysis occurs lead to fish or shrimp pastes. The pastes
may be mixed with cereals; ragi and millet are reported to be used for this
purpose. These products are of limited nutritional value (particularly for
infants)-despite their protein content-because of their high salt content.


*Or other green leafy vegetables.







FOOD AND ANIMAL FEED


Acid Milks, Yogurt, and Cereal
Fresh unpasteurized milk, if allowed to stand at ordinary temperature,
sours naturally because the streptococci and lactobacilli present convert milk
lactose to lactic acid. This results in a natural acid preservation of the nutri-
ents. The acid produced results in stable products resistant to putrefaction
and the development of food spoilage or some disease-producing organisms. It
is, however, susceptible to some fungi and especially to Geotrichum.
Modern yogurt processing involves inoculating milk with Streptococcus
thermophilus and Lactobacillus bulgaricus and incubating it at 450C. For the
production of Russian kefir, milk inoculated with kefir grains (consisting of a
lactobacillus and a yeast growing symbiotically) yield an acidified, carbonated
sour milk with a low alcohol content. Incubation is at room temperature.
Sour milks or yogurts boiled with ground whole wheat or bulgar wheat
and then sun dried yield extremely nutritious stable foods, which can be
stored for years without deterioration. This is the basic process for Egyptian
kishk and Greek trahana. Other ingredients such as spices, pepper, tomato,
onion, or garlic, and other vegetables may also be incorporated in the prod-
ucts, which are either consumed directly or used as a major protein ingredient
in soups.

Indian Idli (Dosai)
Indian idli is a nutritious, protein-rich acidic steamed bread popular in
South India. Its acidity makes it quite resistant to food spoilage and certain
disease-producing organisms.
In preparing idli, polished rice and dehulled black gram (Phaseolus mungo,
mung bean, a legume similar to split pea) are soaked separately during the
day. The proportions can be any combination from 1 to 3 parts rice to 1
part black gram. Since black gram is more expensive than rice, most poorer
people use higher proportions of polished rice.
In the evening, the soaked rice and black gram are ground separately with
a mortar and pestle. For idli, the rice is coarsely ground and the black gram
finely ground; for dosai, both are finely ground. Water, in a proportion twice
the weight of rice and black gram, and 1 percent salt (weight to volume) are
also added. The batter is incubated overnight. During this time the batter
becomes acidic (about pH 4.5) and it is leavened by carbon dioxide produced
by the principal fermenting microorganism Leuconostoc mesenteroides.
Streptococcus faecalis is also present and contributes to the acid content.
The leavened, acidified batter is steamed in small cups to produce the idli
cakes or fried like a pancake to yield dosai (Figure 2.1). Both are tasty,
nutritious foods.
This process of producing wholesome protein-rich food can be adapted to
other ingredients. Dehulled soybean can be substituted for black gram. Other
starchy cereals could be substituted for the rice.







MICROBIAL PROCESSES


African Acidic Porridges
Naturally fermented acidic porridges are staple foods in many parts of
Africa; for example, West African gari is prepared from an acid-fermented
cassava porridge. During the fermentation, at an optimum temperature of
350C, any cyanide-containing sugars present are hydrolyzed, removing the
cyanide. The cassava becomes acidic, and the characteristic pleasing flavor of
gari develops. The fermentation is usually complete in 3-4 days.
The principal microorganisms include Corynebacterium manihot, which
hydrolyzes the starch, producing lactic and formic acids-a process evolving
heat. As the product becomes more acidic (about pH 4.25), a yeast-like
fungus Geotrichum candidum (also found in camembert cheese) develops,
oxidizing the acid and producing the gari flavor.
Typically, the liquid is pressed from the ferment and the starchy residue is
either used directly as fufu or dried in a basket over a fire with continual
turning until it is converted to dry gelatinized granules, which can be stored
for later consumption.
Related acidic porridges are made by fermentation of millet and maize
(ogi), and mahewu (maize and wheat) and sorghum. The acidity protects the
products from food-spoilage organisms, thus providing a wholesome food that
keeps well in a relatively contaminated environment.


Limitations

The basic limitation to the introduction of these low-cost preservation
technologies to new locations or countries is cultural preference and taste.
This is particularly so in the case of fish, where, apart from the sauce and
paste processes in Southeast Asia, most people will only eat "conventional"
varieties. Fish, particularly in tropical conditions, spoils rapidly, and eating
spoiled fish can have dangerous consequences. Canning and mechanical refrig-
eration are the only widely accepted methods of fish preservation added since
ancient times, and both methods are too expensive for use by the poorest
sections of developing countries.
Where the products of fermentation technologies are acceptable, however,
there may be opportunities for increasing conservation of food resources
through more widespread use of such methods, particularly with better qual-
ity control. There may be an important role here for the microbiologist in
improving the techniques in ways appropriate to the local situation.
High salt content has excellent preservative action and is valuable as a
condiment, but it restricts the amount of food that can be consumed. Lower
salt contents, along with sufficient acid (pH 4.5), offer a satisfactory preserva-
tive action while permitting more consumption, thus contributing to better
nutrition.







FOOD AND ANIMAL FEED


FIGURE 2.1 Steamed Indian idli cakes or same dough fried as a pancake (dosai).
(Photograph courtesy of K. H. Steinkraus)


Generally, production of acidic cereal porridges like ogi and mahewu pro-
ceeds better at temperatures of about 500C, which are favorable for rapid
development of Lactobacillus delbrueckii. Fermentation time is shorter and
other undesirable microorganisms have less chance of developing.


Research Needs

Research on the improvement and popularizing of fermented foods should
be centered on:

Studying acidic fermentations for their reliability in areas where they
are not traditionally used; and
Undertaking socioeconomic research to determine if consumers will
accept the new products and, if not, how to encourage them to do so.







MICROBIAL PROCESSES


Improving Nutritional Value
Beers and wines make a valuable contribution to the proper nutrition of
people subsisting on low incomes in the developing world. The fermentation
processes involved raise the vitamin, protein, and, in some cases, the essential
amino acid content of starchy substrates such as cassava, rice, maize, millet,
sorghum, and other cereal grains.
Native maize or sorghum (kaffir) beers, indigenous rice wines, and alco-
holic rice pastes bear little physical resemblance to Western beers and wines.
The indigenous wines and beers are generally cloudy, opalescent, effervescent
beverages, because of their content of microorganisms and substrate residues.
All alcoholic beverages and foods provide a similar euphoria, depending on
their alcoholic content, but Western beers and wines often provide the con-
sumer with an excess of calories.
Most of the indigenous products provide essential nutrition to consumers
in the form of vitamins, protein, amino acids, and calories. In addition to the
food value in the basic ingredients, the microorganisms synthesize from these
ingredients essential amino acids, protein, and vitamins that are consumed
with the product. These organisms may also utilize a portion of the starch,
reducing total solids and increasing the percentage of protein in the product,
converting a low-protein food such as cassava to an acceptable staple in the
diet.
A few of these fermentation processes will be described, and similar or
related commodities can be produced wherever they might add valuable nutri-
tion to the diet.
Two basic processes are used. The first involves germination (malting) of
the grain, which produces enzymes (amylases) that transform a portion of the
starch to sugars (glucose and maltose). The sugars are then fermented by
yeasts such as Saccharomyces cerevisiae to ethyl alcohol. The yeast, however,
also grows and synthesizes amino acids, proteins, and vitamins from the grain
constituents. To retain all the nutrients in the beer or wine, it is essential that
the products not be clarified or filtered.
The second process involves a starch-digesting mold (Amylomyces rouxii)
and a yeast (Endomycopsis burtonii). The combination results in hydrolysis
of the starch to sugars, which are then fermented to alcohol.
An example of the first process is kaffir (sorghum) beer. Kaffir beer is an
alcoholic beverage with a pleasantly sour taste and the consistency of a thin
gruel. It is the traditional beverage of the Bantu people of South Africa, and
the alcohol content may vary from 1 to 8 percent. Kaffir beer is generally
made from kaffircorn (Sorghum caffrorum), malt, and unmalted kaffircom
meal. Maize or millet (Eleusine coracana) may be substituted for part or all of
the kaffircorn depending on the relative cost. Even cassava and plantains may
be used, though with these the procedure would not be the same as with
grain.







FOOD AND ANIMAL FEED


The kaffircorn grain is steeped for 6-36 hours. It is then drained and
placed in layers and germinated by periodic moistening for 4-6 days. Germi-
nation continues until the bud is about 2.5 cm long and the material is then
sun dried.
The essential steps are: mashing, souring, boiling, conversion, straining,
and alcoholic fermentation.
Mashing is carried out in hot (500C) water. Proportions of malted to
unmalted grains vary, but 1 : 4 is satisfactory. Approximately 4 liters of
water are added for about every 2 kg of grain. Souring begins immediately
due to the presence of lactobacilli (Lactobacillus delbrueckii), at a tem-
perature of 500C.
Souring is complete in 6-15 hours. Water is added and the mixture is
boiled. It is then cooled (to 400-600C) and more malt is added.
Conversion (starch hydrolysis) proceeds for 2 hours and then the mash is
cooled (250-300C). Yeasts present in the malt are responsible for the natural
fermentation, although Saccharomyces cerevisiae isolated from kaffir beer
can be inoculated. Kaffir beer is ready for consumption in 4-8 hours, while it
is still actively fermenting. The ethanol content is generally from 2 to 4
percent. The beer also contains from 0.3 to 0.6 percent lactic acid and from 4
to 10 percent solids. Production of acetic acid by Acetobacter species is the
principal cause of spoilage.
Daily consumption of 3 liters of kaffir beer, made from approximately
0.5 kg of grain, is not unusual for a workingman. The improvement in the
vitamin content of a diet that includes beer compared with a diet in which
the kaffir corn is consumed directly is shown in Table 2.1.

TABLE 2.1 Comparison of Diet with and without Maize Beer

Amount of Food Eaten (g)

Diet without Diet with Kaffir
Beer Beer

Food Item
Maize, wholemeal 350 137.5
Maize, 60% extraction 350 137.5
Maize beer 5 pints
(2840 ml)
Vegetables 130 130
Sweet potatoes 470 470
Kidney beans 30 30
Vitamin B, 0.002 0.00195
Riboflavin 0.00113 0.00232
Nicotinic acid 0.0117 0.0203
Calories 3016 2979

Source: B. S. Patt. 1964. Biological ennoblement: improvement of the nutritive value
of foods and dietary regimes by biological agencies. Food Technology (Chicago) 18:665.







MICROBIAL PROCESSES


The caloric content of the two diets is quite similar, only 37 calories being
lost in the diet containing beer. The most notable improvement is the doubl-
ing of the riboflavin and the near doubling of nicotinic acid in the diet
containing beer, because of synthesis of these vitamins during malting and
fermentation. Pellagra, which is relatively common in people subsisting on
maize, is never noted in those consuming usual amounts of kaffir beer.
An example of the second process is Indonesian tap6 ketan, which is
closely related to indigenous rice wine. It is a sweet-sour, alcoholic paste in
which a starch-digesting mold (Amylomyces rouxii) and at least one yeast
(Endomycopsis burtonii) hydrolyze steamed rice starch to maltose and glu-
cose and then produce ethanol and organic acids, which provide an attractive
flavor and aroma. Fermentation is complete in 2-3 days at 300C. If yeasts of
the genus Hansenula are present, the acids and ethanol are esterified, produc-
ing highly aromatic esters.
The inocula are obtainable in the markets of Indonesia as a product called
ragi (in Thailand, luk-paeng). Ragi is a white dried-rice flour cake about
2.5 cm in diameter (Figure 2.2) containing a variety of molds and yeasts,


FIGURE 2.2 Indonesian ragi cakes used for inoculum for tap6 ketan and tap6 ketella.
(Photograph courtesy of C. W. Hesseltine)






FOOD AND ANIMAL FEED


including those described above. Housewives prepare steam-soaked glutinous
rice, inoculate it with the powdered ragi, place the inoculated rice into earth-
enware jugs with added water and allow the mixture to ferment for 3-5 days.
The liquid portion is then consumed and additional water is added. Fermenta-
tion continues for 3-5 days, after which the liquid portion is again drunk.
This is repeated until all the rice is fermented. Any residual dregs are sun
dried and used as a type of ragi. Hence, there is no loss of nutrients.
Detailed studies have been made of the biochemical and nutritional
changes that occur during tape fermentations. Most of the starch is hydro-
lyzed to sugars, which, in turn, are fermented to ethanol and organic acids.
Lysine, the main limiting amino acid in rice, is selectively synthesized by the
microorganisms so that it increases by 15 percent. Thiamine, which is very
low in polished rice (0.04 mg/100 g), is increased threefold (to 0.12 mg/100 g)
by the action of the microorganisms. Up to 8 percent ethanol is produced;
this serves as calories for consumers. Also, it probably contributes to destruc-
tion of disease-producing and food-spoiling organisms that might be present
in the fermentation water.
Through the loss of total solids resulting from utilization of the starch, the
protein content of tape ketan is increased to as much as 16 percent (dry
basis) compared with 7-8 percent in rice.
The tape ketan process is a simple way of raising the protein quality in
starch substrates and also of producing thiamine, which may be deficient in
predominantly polished-rice diets.
Protein enhancement is all the more important in the case of tape ketella,
which is also produced in Indonesia. Tap6 ketella is a sweet-sour alcoholic
food made from cassava tubers. The tubers are peeled, steamed, and cut into
pieces about 5 x 5 cm. They are then carefully inoculated on all surfaces with
powdered ragi. A mold (Amylomyces rouxii) and yeasts of the genera Endo-
mycopsis or Hansenula, along with related types, overgrow the cassava, utiliz-
ing a portion of the starch for energy. Cassava contains as little as 1 or 2
percent protein and by itself is clearly unable to contribute to proper human
protein nutrition, even though it can provide sufficient calories. Consumption
of a portion of the cassava as tape ketella, which may contain 8 percent
protein, can have a beneficial effect on nutrition.


Limitations

In establishing these fermentations in areas of the world where they are
unknown, the proper cultures should be obtained from either culture collec-
tions or scientists who have done research on the products. Acceptance of
new foods will be more difficult. Technical studies must be accompanied by
socioeconomic studies of the potential role of these products in the particular
society.






MICROBIAL PROCESSES


Research Needs

Small-scale laboratory studies are needed in applying these processes in a
new environment.


Production of Meat-Like Flavors

Shoyu (soy sauce) and miso (soybean paste) are made by similar processes
from soybeans by using koji prepared with the molds Aspergillus soyae and
A. oryzae. The koji process for culturing microorganisms for various purposes
is described below. Both products are salty and are used to add flavor to
vegetables, fish, and meat. Miso exists in many colors and flavors and is a
paste, whereas shoyu is a liquid from which the insoluble solids have been
removed.
Koji is generally made from rice, although some forms involve the use of
barley or soybeans. The finished koji is added to soaked, pressure-cooked,
whole soybeans. When these products are mashed together, a salt-tolerant
yeast, Saccharomyces rouxii, and considerable amounts of salt (4-13 percent
weight to weight) are added. The salt is added for flavor and to retard growth
of toxin-forming bacteria. The mash is then placed in tanks made of concrete
or wood and containing several tons of substrate. The mash in the tanks is
allowed to ferment from a few days to a number of months, depending on
the type of miso desired. On completion of the fermentation, miso is either
ground into a uniform paste with the consistency of peanut butter or pack-
aged directly.
Traditionally, miso is used as a flavoring base for soup eaten at breakfast.
To this base, vegetables and seafood are added. Miso imparts a meat-like
flavor to the soup and is added to fish and meats before baking or broiling.
Currently, it is also being incorporated into sauces for pizza and spaghetti
and is used as an ingredient in some commercially prepared salad dressings.
Shoyu manufacture differs from miso in that wheat that has been cleaned,
roasted, and crushed is used in place of rice to make the koji. Whereas miso
requires the use of whole soybeans, modem shoyu manufacture utilizes de-
fatted soybean flakes, which are moistened and blended in a ratio of 55
percent soybean flakes to 45 percent crushed wheat. This mixture of wheat
and soybeans is inoculated with selected strains of a mold, Aspergillus oryzae
or A. soyae, and transformed to koji as it becomes overgrown with mold
(Figure 2.3). During molding the temperature is held below 400C, and about
3-4 days are required for completion of the process. At this time, about an
equal amount of brine is added to the koji and the mixture is placed in large
tanks and inoculated with a yeast, Saccharomyces rouxii, and a bacterium of
the Lactobacillus species. Depending on the temperature, the mash is allowed






FOOD AND ANIMAL FEED


B-- %-A -.
7--7 mm~~i.F I:~


FIGURE 2.3 First stage in traditional soybean fermentation. (Photograph courtesy of
K. H. Steinkraus)

to ferment without aeration for 6-9 months. When this fermentation is com-
pleted, the material is pumped to presses where the dark-brown liquid is
pressed out, pasteurized, and bottled. The press cake is used for cattle feed.
Shoyu contains a large amount of glutamic acid, salt at the level of about
18 g per 100 ml of liquid, reducing sugars, and alcohol. The nitrogen com-
pounds consist of about 40-50 percent amino acids, 40-50 percent peptides,
and less than 1 percent protein. In the Chinese soy sauce process more soy-
beans are added and less wheat. A chemical process is also used in which the
soybeans are hydrolyzed with acid. This is an inferior process in that the
product is harsh to the taste and is generally blended with fermented shoyu,
or the flavor is modified by the addition of various flavoring agents.
Because of the high salt content, pasteurization, and the addition of
preservatives, soy sauce can be kept for months without refrigeration. Similar
products can be made using barley, coconut, and even hydrolyzed yeast cells.
Shoyu is widely used both in the Orient and in Western countries. A modern
shoyu fermentation plant has been operating in the United States for several
years.


Limitations

The basic limitation of shoyu and miso-type products is shared with other
food products-cultural resistance to unconventional foods. In the case of
shoyu and miso, this is compounded by the difficulty of growing soybeans in
tropical countries and by their shortage or high price. The possibility of using







MICROBIAL PROCESSES


other, more readily available, legumes* should be investigated.
The process of making miso and soy sauce generally takes considerable
time. The technology is complicated and requires considerable experience and
training before an acceptable product can be produced. Although it can be
produced at the village level, manufacture on a large scale is more efficient
and usually yields a better and more uniform product.
Extreme care must be taken in selecting industrial strains of molds to
ensure that they are Aspergillus oryzae and not a closely related species, A.
flavus, which produces the highly toxic aflatoxin.


Research Needs

Research should be concentrated on:

Evaluating the potential of miso as a meat-flavoring agent in the local
preparation of foods;
Shortening fermentation time to reduce salt content to a minimum
level;
Evaluating the potential of other legumes and cereals such as sorghum,
millet, and corn to replace soybeans, rice, and wheat (research indicates that
corn might be used to make miso koji); and
Replacing the salt with another bacteriostat to make the products pala-
table for children, or using dehydration, boiling, and canning.


Koji Method of Producing Enzymes

Koji is the Japanese name for the solid-state culture of microorganisms on
rice, barley, wheat, soybeans, and other cereals. The microorganisms used are
the typical molds (Aspergillus oryzae, A. soyae, and species of Rhizopus and
Monascus).
The substrate is soaked in water, drained, heat-sterilized, cooled, and then
inoculated with spores of the appropriate koji mold. The inoculated substrate
is then placed in trays or in large shallow tanks in an incubator room. An
incubator is not always required. The mold is allowed to grow for 2 or 3 days,
with occasional turning of the material either mechanically or by hand. At
the end of the fermentation, the moist molded material is an excellent source
of enzymes.
Koji is used as a source of enzymes in the manufacture of shoyu, miso, and
sake (rice wine). For each of the above foods, special mold strains are used,

*See also National Academy of Sciences, The Winged Bean: A High-Protein Crop for the
Tropics, 1975, and Tropical Legumes: Resources for the Future, 1979, Washington, D.C.







FOOD AND ANIMAL FEED


and often two or three strains are combined. For example, one mold will
produce the desired enzyme for breaking down starch to sugar and another
will produce enzymes that hydrolyze proteins. Thus, the koji inoculum for
making sake will not necessarily be the same as that used for making soy
sauce.
The koji process can be modified by using different substrates and various
molds to produce enzyme preparations used in different industrial processes.
For example, a koji process is used to manufacture microbial rennet to make
curd in the cheese industry. It has also been used successfully to produce
coloring agents and in the continuous production of feed made from liquid
animal waste mixed with maize. A process has been reported that uses the
soybean residue from soybean milk manufacture to make a tempeh-like food
product (to be discussed in the next section).
The koji process has the following advantages:

The fermentation equipment can be as simple as, for example, wooden
trays.
Fermentation substrates can be low-cost. For instance, only broken and
damaged rice is used to make koji for the sake fermentation.
Energy requirements are low, since forced aeration is not required and
once the mold starts to grow it usually produces sufficient heat to warm the
air in the fermentation room to the proper temperature.
After inoculation, a pure culture fermentation is unnecessary.
Extraction of a product is simple, since it is not necessary to extract a
large volume of liquid, which could create a pollution problem after product
recovery.
Yields may be much greater with solid substrates than in liquid media.
Presumably, any product produced by fungi can be made by this
process after a suitable mold culture is found.


Limitations

The inoculum must be produced in large quantities and be as free of
contamination as possible.
The koji molds often generate excess heat, and cooling with fans is neces-
sary for maximum product formation.
The moisture and temperature of the fermenting substrate must be con-
trolled at the optimum levels required by the mold to produce the desired
product efficiently. Since molds require air for growth, the molding material
must be turned regularly and should not be too deep or dense. Thus, the
process requires trained personnel to ensure quality control in a nontradi-
tional production process.







MICROBIAL PROCESSES


Research Needs

Research should be concentrated on:

Improving molds for making koji by selection and mutation to produce
more of the product in a shorter period and to reduce the requirement for
aeration. Any enzyme used in the food industry can be manufactured by this
process if a suitable mold capable of producing the enzyme is available.
Testing in animals any fungus selected for food production to be sure it
does not form toxin.
Converting batch-type fermentation to continuous fermentation.


Indonesian Tempeh

Tempeh, a vegetarian meat analogue and source of vitamin B-12 (generally
lacking in vegetarian diets), is a product made in the East Indies by ferment-
ing soaked, partially cooked, dehulled soybeans (Figure 2.4).


FIGURE 2.4 Tempeh soybean cake: the soybeans are covered and knitted into a cake
by the mold mycelium. (Photograph courtesy of K. H. Steinkraus)


L-5i~:







FOOD AND ANIMAL FEED


In the tropics, the fungal fermentation with Rhizopus spp. is preceded by
a bacterial acid fermentation during soaking of the soybeans. This increases
the acidity of the beans (pH 5.0), which is favorable to subsequent growth of
the mold but inhibits many bacteria that could cause spoilage of the tempeh.
In temperate climates, the bacterial fermentation does not readily occur
and some researchers advise acidification of the beans during cooking by the
addition of 1 percent lactic acid or 0.5 percent acetic acid.
Optimum temperature for the fermentation is between 300C and 370C. At
such temperatures, fungal growth is completed in 24 hours or less. At temper-
atures below 300C, the fermentation may require 2 or 3 days. Fermentation
is complete when the beans are knitted into a compact cake by the mold
mycelium. The cake can then be sliced thin and fried in deep fat, or cut into
chunks and used in soups as a meat replacement.
The fresh tempeh has a pleasant, dough-like aroma. Tempeh is frequently
dipped in shrimp, fish, or soy sauces prior to or after deep-fat frying. The
taste of fried tempeh is bland, with a slightly nutty flavor acceptable to
nearly everyone.
Interestingly, it has been found that commercial tempeh shows vitamin
B-12 activity, produced by an unidentified bacterium that grows on the soy-
beans simultaneously with the mold. If the tempeh is made by inoculating
with pure mold, no vitamin B-12 is synthesized. Thus, tempeh prepared with
the mold and the bacterium can provide not only protein but also vitamin
B-12. This is extremely important for vegetarians, whose diet might otherwise
be deficient in this important vitamin.
In Indonesia soybeans are fermented in packets made from wilted banana
leaves, with the mold inoculum coming from a previous batch of uncooked
tempeh (Figures 2.5 and 2.6). A similar product, ontjom (oncom), is made in
the same region as tempeh, but with peanut press cake as the substrate and
Neurospora sitophila as the fermenting fungus. Peanut press cake is the
residue left after peanut oil has been removed, and it is high in protein. The
fermentation technology is similar to that for tempeh and the end product is
a pinkish-textured meat substitute with an almond or mincemeat flavor. It
is prepared in small factories (cottage industries) and eaten after deep-fat
frying or in soups.
Tempeh has been made using various cereals such as wheat, sometimes in
combination with soybeans, to make a product that tastes like bread or
popcorn. Tempeh, as well as some other fermented foods, has been shown to
contain antibiotic substances active against certain types of bacteria.
Research conducted on the nutritional value of tempeh shows that it is a
wholesome, nutritious food. It contains 42 percent protein, derived from the
soybeans and from the protein synthesized by the mold. Neither the tempeh
nor the ontjom fungus is known to produce any toxins, and they have been







MICROBIAL PROCESSES


FIGURE 2.5 Small packets of tempeh as sold in Indonesian markets. Wilted banana
leaves are used to cover the dehulled soybeans during fermentation. (Photograph courtesy
of K. H. Steinkraus)


consumed in the East Indies for hundreds of years. Some of the B-vitamins
such as riboflavin, B-12, and niacin are increased during the fermentation.
Feeding studies using rats indicate that the protein efficiency ratio (PER)
values are similar to those of soybeans.
Potentially, tempeh can be produced in many parts of the world without
elaborate equipment or extensive training. Since the food is bland, it can be
modified to suit local tastes by adding appropriate sauces and spices.







FOOD AND ANIMAL FEED


FIGURE 2.6 1. Tempeh mold inoculum grown on leaf. 2. Dehulled, partially cooked
soybeans. 3. Tempeh cake. 4. Sliced tempeh cake. (Photograph courtesy of K. H.
Steinkraus)

Limitations
Tempeh must be eaten within a day or two after fermentation unless it is
dried or steamed and refrigerated. When dehydrated, it will keep for months.
The technology is based on village-level processes, and large-scale equip-
ment for its production has not yet been developed.
It is important that only known and proven cultures of the mold be used.
Wild strains of the mold should not be used because they may contain a
toxin or may not produce an acceptable flavor.
Bacterial contamination and spoilage during fermentation can be a prob-
lem if the cooked beans are too moist or if they have not been acidified.
In general, tempeh is subject to the same limitations as miso and shoyu.








MICROBIAL PROCESSES


FLOW SHEET: Indonesian Tempeh Fermentation

Whole, clean soybeans

Soak overnight (or 24 hours) to hydrate soybeans and allow
bacterial fermentation and acidification*

Dehull by hand or by passing through machine to loosen hulls

Remove hulls by flotation on water

Boil cotyledons for 60 minutes

Drain and cool and allow surface moisture to evaporate

Inoculate with tempeh mold

Ferment small packets of inoculated soybeans wrapped in banana leaves
or in clean shallow covered pans

Incubate at a temperature of 300 350C until soybeans are completely
covered with mold mycelium (generally 24 to 36 hours)

Tempeh cakes can be sold on the market or used in home by slicing thin strips
and deep frying or cutting into chunks and cooking in soups



Research Needs


Research requirements include:

Establishing a small company or laboratory with microbiological know-
how to produce a dry, pure tempeh culture inoculum in small packages for
distribution at low cost. This is important for the production of tempeh in
small cottage industries that do not have technically trained people.
Standardizing the inoculum, as has been done with baker's yeast, to
yield a product that will produce a uniform tempeh under standard condi-
tions of time and temperature. The inoculum should have good keeping prop-
erties and should not require refrigeration.
Conducting research on the control of mold contamination in rural
environments.
Adapting simple fermentation equipment for tempeh production at the
local level in countries unfamiliar with its production.
Studying sociocultural aspects of introducing tempeh and tempeh-like
products to people unfamiliar with the product or with soybeans.


*In a temperate climate it may be necessary to add 0.5 percent vinegar during the cook-
ing to increase the acidity.







FOOD AND ANIMAL FEED


Single-Cell Protein Production
Single-cell protein (SCP) refers to the cells of yeasts, bacteria, fungi, and
algae grown for their protein content. Cells of these microorganisms also
contain carbohydrates, fats, vitamins, and minerals. SCP products are used
either for animal feed or human food. They are potentially very important
sources of amino acids, proteins, vitamins, and minerals that can be prepared
from otherwise inedible or low-quality waste material.


Yeasts
Yeasts in baked and fermented food products have a long history of hu-
man consumption. Dried brewer's yeast, a by-product of the brewing indus-
try, has an established use in animal feed formulations. It is also used in hu-
man nutrition as a "health food" dietary supplement.
In recent years SCP processes have been practiced on a commercial scale,
based on the growth of yeasts in deep-tank, agitated, and aerated cultures.
Examples of raw materials used in these processes and yeasts that utilize them
are molasses, Saccharomyces cerevisiae; n-paraffin hydrocarbons, Saccharo-
mycopsis lipolytica; and cheese whey, Kluyveromyces fragilis. The Symba
process, developed in Sweden, utilizes starchy wastes by combining two
yeasts, Saccharomycopsis fibuligera and Candida utilis.

Bacteria
Large-scale propagation of bacteria as a source of animal feed protein has
been considered only in the last decade. A large-scale (75,000 t) facility for
producing the methanol-utilizing bacterium Methylophilus methylotrophus is
being constructed in the United Kingdom, and a large pilot plant for growing
a stable mixed culture of methane-utilizing bacteria is being operated in The
Netherlands.
The conversion of cellulosic materials such as bagasse from sugar cane
processing to SCP by bacteria of the genera Cellulomonas and Alcaligenes has
been investigated on a laboratory and small pilot-plant scale at Louisiana
State University. Plans call for commercial-scale production. (The microbio-
logical utilization of cellulose is discussed in Chapter 8 of this report.)
Advantages claimed for bacteria over yeasts for production of SCP include
more rapid generation and a higher content of crude protein and certain
essential amino acids, particularly methionine. However, bacterial cells are
smaller than yeasts and may be more costly to harvest unless the cells can be
flocculated to give a higher solids slurry prior to centrifugation. Further
(apart from fermented milks and cheeses, which often contain as many bac-
teria as 5 x 109 per g of foodstuff), bacteria as such have had only a brief







MICROBIAL PROCESSES


history of use as either animal feed or human food. Food and drug regulatory
agencies in most countries will have to be convinced of the safety of bacterial
products before permitting their use.


Fungi

People have eaten higher fungi, particularly mushrooms, since ancient
times. Recently, a different method of growing fungal mycelium, including
mushroom mycelium, has been developed on a large pilot-plant or com-
mercial scale in deep-tank, aerated, and agitated cultures. Typical raw mate-
rials and organisms are shown in Table 2.2.
Problems encountered with production of fungal mycelium as a source of
SCP include slow growth rates and the consequent need to maintain sterile
conditions over an extended time to prevent overgrowth by bacterial and wild
yeast contaminants. This increases costs for fungal mycelium production. In
recent laboratory-scale studies, Chaetomium cellulolyticum, a thermo-tol-
erant cellulolytic fungus, has shown promise in the conversion of cellulose to
SCP.
Care must be taken to use strains of fungi that do not produce mycotoxins
that affect domestic livestock or human beings.


Algae

Algae are of interest as a source of SCP because they grow well in open
ponds and utilize carbon dioxide as a carbon source and sunlight as an energy
source for photosynthesis. Algae of the genera Chlorella and Scenedesmus
have been grown for food use in Japan. Spirulina species have been eaten for
many years by inhabitants of the northern shores of Lake Chad in Africa and
by the Aztec Indians in Mexico, where they are now being grown on a pilot-
plant scale in the alkaline waters of Lake Texcoco. Spirulina is a particularly


TABLE 2.2 Raw Materials Used in Growing Fungi Commercially

Raw Materials Fungal Species

Cane and beet molasses Agaricus campestris

Maize syrups, dexture (D-Glucose), Morchella esculenta, M. hortensis,
cheese whey, and canning wastes M. crassipes (Morel mushrooms)
Coffee-processing wastes Trichoderma spp.
Maize wet-milling waste Gliocladium spp.
Maize and pea-canning wastes Trichoderma reesei







FOOD AND ANIMAL FEED


attractive algal source of SCP because of its high nutritive value and because
the large cell filaments make it relatively easy to harvest by fine mesh nets or
filtration. Further discussions of algae are in the section on wastes in
Chapter 7 and in the NAS publication Underexploited Tropical Plants with
Promising Economic Value.


Limitations

SCP production is capital-intensive and, with the exception of algal pro-
duction by photosynthesis, energy-intensive. Processes that must be con-
ducted under sterile conditions require stainless-steel equipment that can be
cleaned and sterilized. They also require provisions for sterilizing the growth
medium and recovering the SCP product without introducing other microbial
contaminants, particularly human pathogens. Trained personnel are needed to
supervise and maintain quality control of production.
At present, SCP processes for the production of animal feed are the most
attractive, since conventional animal feedstuffs such as soybean meal and fish
meal must be imported to many tropical and subtropical countries at prevail-
ing international prices. In using SCP for animal feed, however, there is a large
loss of conversion efficiency as opposed to direct human use. For human
food applications, the use of microorganisms is limited to those such as
Saccharomyces cerevisiae and Candida utilis that are accepted by regulatory
and public health authorities as safe for human food use. Even those organ-
isms, if they are to be consumed as a significant portion of the protein in the
diet, must be processed further to reduce nucleic acid contents to below the
levels that could lead to kidney stone formation or gout.
To achieve economies of scale, an SCP production facility should have a
capacity of at least 50,000 t per year unless operated as a waste-treatment
facility in a food-processing plant. This implies that sufficient raw materials
will be available in close proximity to the SCP plant to meet these production
requirements.
For raw materials, carbohydrates such as sugars or sugar-containing by-
products, wastes, and starches are likely to be available in semitropical and
tropical countries in the quantities required for an economically viable scale
of production. In general, concentrations of utilizable carbohydrates in wastes
should be sufficiently high that handling of large volumes of dilute materials
is avoided.
A portion of the crude molasses produced from sugar cane operations
could be diverted to SCP production (yeasts), if a source of nitrogen were
added to provide a source of protein and vitamins for animal feeds, particu-
larly poultry rations.







MICROBIAL PROCESSES


Many cities in tropical and semitropical countries have breweries that pro-
duce residues of reasonably uniform composition throughout the year. In
addition to recovery of brewer's yeast, already widely used as animal feed, the
remaining liquid waste-after hydrolysis to simple sugars-can be used as a
potential carbon and energy source for SCP production. However, carbon-to-
nitrogen ratios of the material may have to be adjusted to favorable ranges for
yeast growth. In the case of starchy crops such as cassava, large quantities
must be available at one site to provide a sufficient source of raw material for
economic SCP production.
Coffee-processing wastes contain soluble carbohydrates and have a high
chemical oxygen demand (COD) and soluble solids content. Pilot-scale opera-
tions in Guatemala have shown that growing Trichoderma species in these
wastes reduces the COD considerably and yields an SCP product of interest
for use in animal feed.
Microorganisms require sources of nitrogen, phosphorus, and mineral
salts for growth, in addition to a carbon and energy source. The availability
of ammonium salts such as ammonium sulfate or diammonium phosphate
may be a problem in some countries. The same can be said for sources of
phosphorus. A feed-grade source of phosphoric acid or soluble phosphates
should be used because of the presence of arsenic and fluoride in crude
phosphates. Other minerals are usually present in the water supply.
To reduce contamination to a low level, the microorganism used should
multiply (grow) rapidly at an acid level of pH 4.5 or below. Operations under
these conditions will allow the use of clean, aseptic conditions without the
need for sterile facilities. SCP production (except from algae) requires aera-
tion to achieve suitable yields. Air should be filtered to remove contaminants.
Power costs for aeration, fluid handling, and steam for cleaning, recovery,
and drying the product can be significant factors in the total energy costs.
Water requirements for SCP production are considerable for both process-
ing and cooling. The growth of microorganisms produces heat, which must be
removed to maintain the growth temperature within the preferred range of
300-350C. Cooling water temperatures should be at least 100C lower than the
growth temperature. Location of an SCP plant near the ocean would permit
the use of seawater for cooling. Also, wastewater is produced from SCP
operations and must either be disposed of or, preferably, treated and recycled
in large operations.
Three SCP processes appear to have potential for further development.
These processes, although known, are not used in tropical and semitropical
regions to the extent that they might be. They are given in Table 2.3.
All of these processes can be carried out in either a batch or continuous
mode of operation. They should be operated under clean, aseptic conditions,
but they do not require tight control over sterility throughout the process.









TABLE 2.3 Production of SCP from Various Substrates

Substrate Organisms Conditions

Cane molasses Candida utilis Temperature: 300-34 C
C. tropicalis pH 4.0-4.5
Rhodotorula gracilis
R. pilimanae
R. rubra

Coffee wastes Candida spp. Temperature: 300-35C
Trichoderma spp. pH 2.0-4.0

Starchy materials, Symba processes: mixed Temperature 300-34C
especially cassava culture of Saccharo- pH 4.0-5.0
mycopsis fibuligera
and Candida utilis


Research Needs

In many countries there is need to:

Conduct feasibility studies to determine where there is an appropriate
mix of underutilized residue, technical competence, and need or economic
opportunity to use the SCP produced;
Conduct studies on the use of yeasts, e.g., genus Rhodotorula, which
have relatively rapid growth rates (2- to 2.5-hour generation times), tem-
perature over the range 280 to 340C and pH tolerance between 3.5 and 5.0;
Institute animal feeding studies using dried SCP as a component of
poultry and swine rations;
Investigate a wider range of thermotolerant organisms, particularly
yeasts, for their utility in producing SCP from coffee-processing wastes;
Evaluate nutritional and safety factors for animal feed applications of
certain thermotolerant fungi including Sporotrichum thermophile and Paecilo-
myces species that grow on cassava; and
Develop thermotolerant strains of microorganisms to reduce the
requirement for cooling water in semitropical and tropical regions.


References and Suggested Reading
Food Preservation
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No. 20. Lagos, Nigeria: Federal Institute of Industrial Research.
Morcos, S. R.; Hegasi, S. M.; and el-Damhougy, S. T. 1973. Fermented foods in common
use in Egypt I. The nutritive value of kishk. Journal of the Science of Food and
Agriculture 24:1153-1156.







MICROBIAL PROCESSES


Mukerjee, S. K.; Albury, M. N.; Pederson, C. S.; van Veen, A. G.; and Steinkraus, K. H.
1965. Role of Leuconostoc mesenteroides in leavening the batter of idli, a fermented
food of India. Applied Microbiology 13:227-231.
Pederson, C. S., and Albury, M. N. 1969. The Sauerkraut fermentation. New York
Agricultural Experiment Station Technical Bulletin 824. Geneva, New York: New
York State Agricultural Experiment Station.
Schweigert, F.; Van Berge, W. E. L.; Wiechers, S. G.; and de Wit, J. P. 1960. The produc-
tion of mahewu. Report No. 167. Pretoria, South Africa: Council for Science and
Industrial Research.
Stamer, J. R. 1975. Recent developments in the fermentation of sauerkraut. In Lactic
acid bacteria in beverages and food, J. G. Carr; C. V. Cutting; and C. S. Whiting, eds.,
pp. 267-280. New York: Academic Press.
Steinkraus, K. H.; van Veen, A. G.; and Thiebeau, D. P. 1967. Studies on idli-an Indian
fermented black gram-rice food. Food Technology (Chicago) 21:916-919.
van Veen, A. G. 1965. Fermented and dried seafood products in Southeast Asia. In Fish
as Food, G. Borgstrom, ed., Volume 3, pp. 227-250. New York: Academic Press.
S; Hackler, L. R.; Steinkraus, K. H.; and Mukerjee, S. K. 1967. Nutritive value of
idli, a fermented food of India. Journal of Food Science 32:339-341.

Improving Nutritional Value
Cronk, T. C.; Steinkraus, K. H.; Hackler, L. R.; and Mattick, L. R. 1977. Indonesian tape
ketan fermentation. Applied Environmental Microbiology 33:1067-1073.
Ellis, J. J.; Rhodes, L. J.; and Hesseltine, C. W. 1976. The genus Amylomyces. Mycologia
68:131-143.
Ko, S. D. 1972. Tape fermentation. Journal of Applied Microbiology 23:976-978.
Novellie, L. 1968. Kaffir beer brewing: ancient art and modern industry. Wallerstein
Laboratories Communications 31:17-32.
Platt, B. S. 1946. Fermentation and human nutrition. Proceedings of the Nutrition
Society 4:132-140.
1955. Some traditional alcoholic beverages and their importance in indigenous
African communities. Proceedings of the Nutrition Society 14:115-124.
_. 1964. Biological ennoblement: improvement of the nutritive value of foods and
dietary regimes by biological agencies. Food Technology (Chicago) 18:662-670.
; and Webb, R. A. February 1948. Microbiological protein and human nutrition.
Chemistry and Industry 7:88-90.
Schwartz, H. M. 1956. Kaffircorn malting and brewing studies. I. The kaffir beer brewing
industry in South America. Journal of the Science of Food and Agriculture
7:101-105.

Production of Meat-Like Flavors
Ebine, H. 1972. Miso. In Proceedings of the International Symposium on Conversion and
Manufacture of Foodstuffs by Microorganisms, pp. 127-139. Tokyo: Saikon Publish-
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Hesseltine, C. W., and Shibasaki, K. 1961. Miso III. Pure culture fermentation with
Saccharomyces rouxii. Applied Microbiology 9:515-518.
and Wang, H. L. 1967. Traditional fermented foods. Biotechnology and Bio-
engineering 9:275-288.
National Academy of Sciences. 1975. The winged bean: a high-protein crop for the
tropics. Report of an Ad Hoc Panel of the Advisory Committee on Technology
Innovation, of the Board on Science and Technology for International Development.
Washington, D.C.: National Academy of Sciences.
1979. Tropical legumes: resources for the future. Report of an Ad Hoc Panel of
the Advisory Committee on Technology Innovation, of the Board on Science and
Technology for International Development. Washington, D.C.: National Academy of
Sciences.







FOOD AND ANIMAL FEED


Shibasaki, K., and Hesseltine, C. W. 1962. Miso-fermentation. Economic Botany
16:180-195.
Shurtleff, W., and Aoyagi, A. 1977. The book of miso. Brookline, Massachusetts:
Autumn Press.
Yokotsuka, T. 1960. Aroma and flavor of Japanese soy sauce. Advances in Food Re-
search 10:75-134.
__. 1972. Shoyu. In Proceedings of the International Symposium on Conversion and
Manufacture of Foodstuffs by Microorganisms, pp. 117-125. Tokyo: Saikon Publish-
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Koji Method of Producing Enzymes
Hesseltime, C. W. 1972. Solid-state fermentations. Biotechnology and Bioengineering
14:517-532.
; Swain, E. W.; and Wang, H. L. 1976. Production of fungal spores as inoculum for
oriental fermented foods. Developments in Industrial Microbiology 17:101-115.
Nakano, M. 1972. Synopsis on the Japanese traditional fermented foodstuffs. In Waste
recovery by microorganisms, pp. 27-28. Kuala Lumpur: United Nations Educational,
Scientific, and Cultural Organization, distributed in the United States by UNIPUB,
New York.
Sakaguchi, K. 1972. Development of industrial microbiology in Japan. In Proceedings of
the International Symposium on Conversion and Manufacture of Foodstuffs by
Microorganisms, pp. 7-10. Tokyo: Saikon Publishing Company.
Indonesian Tempeh
Hesseltine, C. W. 1965. A millennium of fungi, food and fermentation. Mycologia
57:149-197.
Liem, I. T. H.; Steinkraus, K. H.; and Cronk, T. C. 1978. Production of vitamin B-12 in
tempeh-a fermented soybean food. Applied and Environmental Microbiology
34:773-776.
Roelofsen, P. A., and Talens, A. 1964. Changes in some B vitamins during molding of
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Science 29:224-226.
Steinkraus, K. H.; Bwee Hwa, Y.; Van Buren, J. P.; Provvidenti, M. I.; and Hand, D. B.
1960. Studies on tempeh-an Indonesian fermented soybean food. Food Research
26:777-788.
_ ; Van Buren, J. P.; Hackler, L. R.; and Hand, D. B. 1965. A pilot-plant process for
the production of dehydrated tempeh. Food Technology (Chicago) 19:63-68.
van Veen, A. G.; Graham, D. C. W.; and Steinkraus, K. H. 1968. Fermented peanut press
cake. Cereal Science Today 13:96-99.
Wang, H. L.; Swain, E. W.; and Hesseltine, C. W. 1975. Mass production of Rhizopus
oligosporus spores and their application in tempeh fermentation. Journal of Food
Science 40:168-170.
Single-Cell Protein Production
Aguirre, F.; Moldonado, O.; Rolz, C.; Menche, J. F.; Espinosa, R.; and Cabrera, S. 1976.
Protein from waste-growing fungi on coffee waste. ChemTech 6:636-642.
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Davis, P., ed. 1974. Single-cell protein. New York: Academic Press.
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44 MICROBIAL PROCESSES

Lewis, C. W. 1976. Energy requirements for single-cell protein production. Journal of
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National Academy of Sciences. 1975. Underexploited tropical plants with promising
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Food Engineering, May: 112-116.
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Massachusetts: Massachusetts Institute of Technology Press.
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Science and Nutrition 5:77-151.
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Sources of Cultures

Food Preservation
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A.

Improving Nutritional Value
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A.
Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether-
lands.
National Collection of Yeast Cultures, Lyttell Hall, Nutfield, Redhill, Surrey RH1 4HY,
England.

Production of Meat-Like Flavors
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A.
Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether-
lands.

Koji Method of Producing Enzymes
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A.







FOOD AND ANIMAL FEED 45

Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether-
lands.

Indonesian Tempeh
Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether-
lands.
American Type Culture Collection, 12301 Parklawn Drive, ockville, Maryland 20852,
U.S.A.

Single-cell Protein Production
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A.

Research Contacts
Food Preservation

O. Kandler, Botanisches Institut, Der Universitat Miinchen, 800 Miinchen 19, Menzinger
Strasse 67, Federal Republic of Germany.
Carl S. Pederson, Professor of Microbiology, Emeritus. Cornell University, Geneva, New
York 14456, U.S.A.
Keith H. Steinkraus, Department of Food Science and Technology, New York State
Agricultural Experiment Station, Geneva, New York 14456, U.S.A.
Reese Vaughn, Professor Emeritus, Department of Food Science and Technology, Uni-
versity of California, Davis, California 95616, U.S.A.

Improving Nutritional Value
Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University,
Peoria, Illinois 61604, U.S.A.
Keith H. Steinkraus, Department of Food Science and Technology, New York State
Agricultural Experiment Station, Geneva, New York 14456, U.S.A.

Production of Meat-Like Flavors
Hideo Ebine, National Food Research Institute, Ministry of Agriculture and Forestry,
Yatabe-machi, Ibaraki-ken, 300-31, Japan.
Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University,
Peoria, Illinois 61604, U.S.A.
Nakano Masahiro, Meiji Daigaku, Ikuta Kosha, Ikuta 5158, Kawasakishi, Kanagawa-ken
214, Japan.
Shinshu Miso Research Institute, Minamiagata-machi 1014, Nagano-shi 380, Japan.
William Shurtleff, New-Age Foods Study Center, 790 Los Palos Manor, Lafayette, Cali-
fornia 94549, U.S.A.
Tamotsu Yokotsuka, Kikkoman Shoyu Co., Ltd., 339 Noda, Noda-shi, Chiba-ken, Japan.

Koji Method of Producing Enzymes
Hideo Ebine, National Food Research Institute, Ministry of Agriculture and Forestry,
Yatabemachi, Ibaraki-ken, 300-31, Tokyo, Japan.
Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University,
Peoria, Illinois 61604, U.S.A.
William Shurtleff, New-Age Foods Study Center, 790 Los Palmos Manor, Lafayette,
California 94549, U.S.A.
Tamotsu Yokotsuka, Kikkoman Shoyu Co. Ltd., 339 Noda, Noda-shi, Chiba-ken, Japan.







46 MICROBIAL PROCESSES

Indonesian Tempeh
Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University,
Peoria, Illinois 61604, U.S.A.
Keith H. Steinkraus, Department of Food Science and Technology, New York State
Agricultural Experiment Station, Geneva, New York 14456, U.S.A.
A. G. van Veen, Division of Nutritional Sciences, Cornell University, Savage Hall, Ithaca,
New York 14853, U.S.A.

Single-Cell Protein Production
Allen I. Laskin, EXXON Research and Engineering Company, P.O. Box 45, Linden, New
Jersey 07036, U.S.A.
John H. Litchfield, Battelle Columbus Laboratories, 505 King Avenue, Columbus, Ohio
43201, U.S.A.
Jacques C. Senez, Centre National de la Recherche Scientifique, Laboratoire de Chemie
BactBrienne, 31 Chemin Joseph-Aiguier 13274, Marseille 2, France.
Steven R. Tannenbaum, Massachusetts Institute of Technology, Cambridge, Massachu-
setts 02139, U.S.A.










Chapter 3


Soil Microbes in

Plant Health and Nutrition





The parts of a plant above the ground can be compared to the tip of an
iceberg, in that the portion under the surface-the root system-is so exten-
sive. The root system is also very active metabolically and provides a continu-
ous source of food for soil microorganisms in the form of secretions of
organic compounds and sloughed-off dead cells and cell debris. Since the zone
where roots and soils meet is a special environment, it has been named the
rhizosphere (root zone). This zone comprises several poorly defined, hetero-
geneous regions in which microorganisms are particularly active (Table 3.1).
Although activity in the rhizosphere is of great importance to the plant, it
affects only a small fraction (about 5 percent) of the root surface. Some
microorganisms are loosely associated with roots, but others develop on the
root surface and many can invade root tissue, with effects that can be bene-
ficial or harmful. Certain soil-inhabiting microorganisms produce diseases of
great significance to agriculture and forestry. Others are beneficial-they part-
ly inhibit the growth of disease organisms or kill them. The vast majority of
the pathogens that infect roots are fungi, and they are exceptionally difficult
to control or eradicate.
In some cases, the invasion of roots by microorganisms is desirable. This is
true for the root-nodule bacteria of the genus Rhizobium that fix nitrogen, as
well as for mycorrhizal fungi, which assist roots in accumulating phosphate
and other essential minerals. Nitrogen fixation is the subject of Chapter 4 in
this report; mycorrhizal fungi are discussed in this chapter. However, it
should be emphasized that rhizosphere microorganisms can affect plant wel-
fare in a number of ways that are not yet well understood or easily con-
trolled. The processes of nutrient cycling, growth stimulation or inhibition,
and diseases are of great significance, but they are very complex population
effects rather than the result of simple interactions between roots and known
microorganisms. The rhizosphere is also influenced by external factors such as
soil moisture and even the intensity of light reaching the plant. No single
microorganism may be essential to the process, but the combined effect of
the rhizosphere population can be profound.







MICROBIAL PROCESSES


TABLE 3.1 Comparison of the Numbers of Various Groups of Organisms in the Rhizo-
sphere of Spring Wheat and in Control Soil

Numbers per g Numbers per g Approximate
in Rhizosphere in Control Rhizosphere:
Organisms Soil (X 10-6) Soil (X 10-6) Soil Ratio

Bacteria 1,200 53 23 : 1
Actinomycetes 46 7 7 1
Fungi 12 0.1 120 : 1
Protozoa 0.0024 0.001 2 1
Algae 0.005 0.027 0.2 : 1
Bacterial Groups
Ammonifiers 500 0.04 12,500 : 1
Gas-producing anaerobes 0.39 0.03 13 : 1
Anaerobes 12 6 2 :1
Denitrifiers 126 0.1 1,260 : 1
Aerobic cellulose decomposers 0.7 0.1 7 1
Anaerobic cellulose decomposers 0.009 0.003 3 1
Spore former 0.930 0.575 2 1
"Radiobacter" types 17 0.01 1,700 : 1
Azotobacter <0.001 <0.001

Adapted from: T. R. G. Gray, and S. T. Williams. 1975. Soil Microorganisms, New York:
Longman, p. 144.
The sum of the various interrelationships of rhizosphere microorganisms
and roots can benefit plant growth by influencing the availability of essential
nutrients, by producing plant growth regulators, and by suppressing root
pathogens.



Mineral Cycling by Soil Microorganisms

By decomposing plant and animal residues, soil microorganisms release
carbon, nitrogen, sulfur, phosphorus, and trace elements from organic mate-
rials in forms that can be absorbed by plants.
This process, known as mineralization, is the primary source of atmo-
spheric carbon dioxide. Without mineralization of organic carbon, the carbon
dioxide content of the air, which is essential for plant photosynthesis, would
be progressively reduced and plant production would ultimately cease. Main-
tenance of the carbon cycle, therefore, is one of the most important biolog-
ical processes on earth.
Microbial activities similar to those responsible for the carbon cycle also
transform soil nitrogen, sulfur, and phosphorus, and to a lesser extent are
instrumental in the conversion of other elements. Although particular atten-
tion has been directed to microbial transformations of nitrogen, plants also
have a nutritional need for sulfur. The microbial transformations of nitrogen
and sulfur are much alike because both elements can be oxidized and re-
duced. Sulfur reduction is necessary for the synthesis of sulfur-containing







SOIL MICROBES IN PLANT HEALTH AND NUTRITION 49
amino acids. Under anaerobic conditions, sulfur reduction may produce hy-
drogen sulfide, which can be harmful to plants. It accumulates in very wet
soils, like rice paddies, and can cause straighthead disease of rice and other
physiological plant disorders. At low concentrations, however, it can supply
the sulfur requirements of some plants.
A recent report indicates that oxidation of hydrogen sulfide by a bac-
terium in the genus Beggiatoa detoxified flooded rice soils. Beggiatoa species
may be significant in coastal marshes and estuaries as well as in rice paddies,
and their capacity to influence plant growth favorably deserves further study.
The oxidation of sulfur by bacteria of the genus Thiobacillus is also of poten-
tial significance in agriculture. The product of this transformation is sulfuric
acid, which can dissolve minerals that otherwise would not be available for
plant growth. Farmers who add elemental sulfur to rock phosphate find that
phosphorus is liberated more rapidly and in greater amounts than if the sulfur
is omitted. The explanation for this is that Thiobacillus species oxidize the
sulfur to sulfuric acid, which liberates the phosphorus from the insoluble rock
phosphate.
Microorganisms are also able to promote phosphorus solubilization by the
production of chelators, which form complexes with metal ions and increase
their solubility. Acid and chelate production can be easily demonstrated
under laboratory conditions, but little is known of the phosphate-dissolving
effectiveness of different types of microorganisms under natural soil condi-
tions. Solvent action by microorganisms is not restricted to a few species; it is
characteristic of many members of the rhizosphere population and can be
accomplished in part by plant roots as well. The microbial transformation of
nutrient elements other than those cited above is in some instances similar to,
and in other instances quite different from, the process just discussed. Un-
fortunately, much more is still to be learned about this subject.
Microorganisms require many of the same nutrient elements that are essen-
tial to plants for their growth. When nitrogen, sulfur, or phosphorus is in
short supply, the rhizosphere population will compete with roots for nourish-
ment. Because of their abundance, small size, and relatively large surface area,
and because they surround the absorbing part of the root, microbes will
absorb nutrients at the expense of the plant. Ultimately, plants will display
signs of nutrient deficiency and crop yields may decrease.
Barber and Martin (1976) have recently found that for barley, 10-20
percent of the photosynthate may be released from roots in nonsterile soil.
Less was released in sterile soil. The rhizosphere may exact a price in terms of
energy given up to the soil by the plant.
There has been speculation that in the rhizosphere oxygen consumption
occurs more rapidly than diffusion, so that anaerobic sites may form in places
in the root; such reduced conditions could be important in making ferrous
ions from ferric, for instance, which increases iron solubility. Wheat roots







50 MICROBIAL PROCESSES
have high populations of denitrifying bacteria, so oxygen-free conditions
must exist in their presence.
Microorganisms are prolific producers of vitamins, amino acids, hormones,
and other growth-regulating substances. Many bacteria and fungi isolated
from soil are able to synthesize compounds that provoke a growth response in
plant tissue. Some produce indoleacetic acid or gibberellins, which are hor-
mones that control plant growth, while others produce vitamins. Many may
also produce unidentified growth factors. Rhizosphere microorganisms are
variously credited with promoting increased rates of seed germination, root
elongation, root-hair development, nutrient uptake, and plant growth.


Inoculation

Root uptake of organic compounds has received more attention in the
U.S.S.R. than elsewhere, and Russian investigators claim rhizosphere micro-
organisms influence the quality as well as quantity of tissue produced by
plants. Although there are no experimental results that convincingly establish
that growth-promoting substances of microbial origin occur in the rhizo-
sphere, speculation persists that such compounds are synthesized in the vicin-
ity of roots and affect crop yields.
Various bacterial fertilizers have been marketed at different times, but
commercial preparations known as azotobacterin and phosphobacterin have
received the most attention. Azotobacterin is composed of cells of Azoto-
bacter chroococcum, a bacterium able, under some conditions, to fix atmos-
pheric nitrogen. Phosphobacterin contains the bacterium Bacillus mega-
terium var. phosphaticum, which mineralizes organic phosphorus compounds.
Russian scientists think that growth of these bacteria in soil will supply
plants with nitrogen and phosphorus, but this has not been proved. Treated
plants are favorably affected, but growth is not increased by more than 10
percent. Moreover, the effect is said to be due not to nitrogen fixation or
phosphorus solubilization, but to plant hormones.
Since the benefits are minimal and depend on conditions difficult to con-
trol in the field, and the results are unpredictable, bacterial fertilizers are not
recommended for general use.
Although the potential benefits of inoculating bacteria have not yet been
fully explored, it is questionable how much additional exploration may be
warranted. Present evidence is insufficient to justify the use of inoculants,
other than rhizobia for legumes, to increase crop yields, improve plant quality,
or control disease. A beneficial effect is even less likely when the microorga-
nism used as inoculum is a normal inhabitant of soil. The British soil micro-
biologist S. D. Garrett (1956) has described this problem as follows:

[Such] attempts to boost the population of an antagonistic organism by
inoculation alone have been doomed to failure from their inception, because







SOIL MICROBES IN PLANT HEALTH AND NUTRITION


they are in flagrant contradiction to the ecological axiom that population is a
reflection of the habitat, and that any change due to plant introduction
without change of the habitat must be a transient one.


Mycorrhizal Fungi

Most plants, both wild and cultivated, have roots infected with fungi that
increase nutrient and water uptake and may also protect the root from cer-
tain diseases. These infected roots are called mycorrhizae. Although the
mycorrhizal fungi probably increase uptake of all the essential elements, they
are usually most important in improving phosphorus nutrition. Phosphate is
generally present in the soil in low concentrations and it is also highly im-
mobile. Strands of fungal hyphae grow out from mycorrhizae and greatly
increase the volume of soil from which phosphorus is obtained. So mycor-
rhizal plants, in general, can grow and thrive in soils much lower in phosphate
and other essential nutrients than a comparable nonmycorrhizal plant. Many
plants are so dependent on mycorrhizal fungi for nutrient uptake that they
may starve if these fungi are absent. There are a number of types of mycor-
rhizae. The two that occur on the most economically important crops, the
endomycorrhizae and the ectomycorrhizae, are discussed below.


Endomycorrhizae of Crop Plants and Forest Trees

Endomycorrhizae of the vesicular-arbuscular (VA) type occur on nearly
all crop plants (plants in a few families such as the Cruciferae [cabbage,
mustard, etc.] and Chenopodiaceae [beets, spinach, etc.] may be nonmycor-
rhizal). They also occur on many trees in temperate regions and on the
majority of tree species native to the subtropics and tropics. VA mycorrhizal
fungi are present in almost all soils and they are not host-specific. Thus, the
same fungus producing VA mycorrhizae on trees will form mycorrhizae on
plants after land is cleared and planted to agricultural crops. The mycorrhizal
condition is normal for most plants, and absence or scarcity of mycorrhizal
fungi can greatly limit plant growth (Figure 3.1). Introduction of mycorrhizal
fungi to soil environments lacking or with low populations of such organisms,
such as biocide-treated soils, can enhance plant growth. VA mycorrhizae are
particularly important for many legumes in that they stimulate nodulation by
Rhizobium, thereby increasing nitrogen fixation. Improved phosphorus nutri-
tion of the mycorrhizal legume is responsible for increased nodulation.
VA mycorrhizal fungi survive in soil as resting spores. They obtain their
food from the plant roots and they are unable to grow independently in soil.
It is unlikely that they obtain much, if any, organic nutrient from soil.
VA fungi have not been grown in pure culture, which presents an obstacle
to artificial inoculation. However, these fungi produce the largest spores of






MICROBIAL PROCESSES


FIGURE 3.1 Endomycorrhizal (left) and nonmycorrhizal (right) peanut plants (ground-
nuts). (Photograph courtesy of J. W. Gerdemann)
any known fungi, some being 0.5 mm or more in diameter. The spores can be
easily extracted from the soil with sieves and then propagated on the roots of
living plants. The infected roots can also be used for inoculation. Heavily
infected palm roots collected in the wild have been used as a source of
inoculum. If field-collected inoculum is used, it is important that it be free of
dangerous pathogens.
There are situations in which inoculation with VA fungi is highly bene-
ficial. If soil is treated with steam or fumigants to kill pathogens, VA fungi
are also killed, and considerable time is required for them to become reestab-
lished naturally. The nutrient deficiencies and associated stunting that often
result may be prevented by inoculating the soil with VA fungi rather than by
applying excessive rates of fertilizer.
The greatest opportunity for the use of VA fungi is in soils low in available
phosphorus, which includes many untreated soils in tropical regions. There is
evidence as well that many of these soils also contain less than the optimum
number of spores of VA fungi. In such soils inoculation may enable the use of
inexpensive rock phosphate fertilizer instead of the more expensive super and
triple phosphates.
The major obstacle to greater use of VA fungi is the difficulty in obtaining
inoculum. However, there are several commercial companies interested in
producing pure inoculum, and it may become available in the near future. We
are now at the stage where different species of VA fungi should be tested on


aoptik I






SOIL MICROBES IN PLANT HEALTH AND NUTRITION


crop plants and forest trees and their effects on growth compared in the field
in untreated soils and in soils that have been sterilized.


Ectomycorrhizae of Forest Trees

Ectomycorrhiza is the second most common type of mycorrhizae. It
occurs on roots of pine, spruce, fir, larch, hemlock, willow, poplar, hickory,
pecan, oak, birch, beech, and eucalyptus (Figure 3.2). The fungi that form
ectomycorrhizae produce mushrooms and puffballs as their reproductive
structures (fruit bodies). In North America there are more than 2,100 species
of ectomycorrhizal fungi. The fungi are spread in nature by millions of micro-
scopic spores, finer than dust, which are released from fruiting bodies and
moved great distances by winds.
Many forest trees, such as pines, cannot grow beyond the first year with-
out an appreciable number of ectomycorrhizae. Ectomycorrhizae benefit
trees by: increasing nutrient and water absorption from soil; increasing the
tolerance of the tree to drought and extremes of soil conditions (acid levels,
toxins, etc.); increasing the length of the feeder root system; and protecting
the fine feeder roots from certain harmful soil fungi.
Ectomycorrhizal fungi cannot grow and reproduce unless they are in asso-
ciation with the roots of a tree host. These fungi obtain all their essential


FIGURE 3.2 Examples of pine ectomycorrhizae. Each different ectomycorrhiza is
formed by a different species of fungus. Each main root is approximately 3 cm long.
(Photograph courtesy of J. W. Gerdemann)







MICROBIAL PROCESSES


sugars, vitamins, amino acids, and other foods from their hosts. It is unlikely
that these fungi as a group are directly involved in any significant decomposi-
tion of forest litter.
Certain forest trees, then, must have ectomycorrhizae to survive and grow,
and the ectomycorrhizal fungi need their tree hosts to exist. This means that
the introduction of tree species as exotics into regions where the appropriate
mycorrhizal fungi are absent must be accompanied by the introduction of
their natural ectomycorrhizal fungi. In the past, this introduction has been
accomplished mainly by using soil collected from under healthy trees with
ectomycorrhizae, which is mixed into the upper layer of soil in nurseries. The
seedlings planted in this soil usually form abundant ectomycorrhizae in one
growing season, and they are then transplanted to the field. Unfortunately,
this method is not without risk, since pathogens can be present in the intro-
duced soils and cause serious damage to the trees. The logistics of transport-
ing large volumes of soil great distances is an added problem. By far the most
biologically sound method of correcting an ectomycorrhizal deficiency is by
the use of pure cultures of selected species of ectomycorrhizal fungi.
In recent years, techniques have been devised to inoculate soil with pure
vegetative and spore cultures of Pisolithus tinctorius in the United States and
to introduce spore cultures of Rhizopogon luteolus onto pine seed and into
soil in Australia. These two puffball-producing fungi form ectomycorrhizae
on many commercially important forest trees. Currently, research is being
done in the United States on the use of a commercially produced vegetative
inoculum of P. tinctorius, which should be available at economical prices on
the world market in the next few years. Thus far, P. tinctorius appears to
enhance growth more than other ectomycorrhizal fungi, and it can be used to
tailor-make seedlings to improve the performance of trees even in areas where
other ectomycorrhizal fungi are present.
Pines with Pisolithus ectomycorrhizae formed in nurseries and planted in
forest sites in the southern United States have not only survived, but have
grown to twice the heights of comparable pines with naturally occurring
ectomycorrhizae. On sites where it is difficult to establish pines, such as those
created by strip-mining for coal, the only trees capable of growing are often
those that have been inoculated with Pisolithus. The selection and use of
specific ectomycorrhizal fungi may well determine whether a productive
forest becomes established.
The most obvious research need on ectomycorrhizae in developing coun-
tries is to determine whether appropriate ectomycorrhizal fungi are present
prior to the establishment of forests of introduced species. If such forests are
already established or are accessible, then fruit bodies of ectomycorrhizal
fungi can be collected and the spores harvested. The puffball fungi usually
produce an abundance of easily extractable spores. For example, one fruit
body of P. tinctorius may contain 75 grams of spores, and there are more







SOIL MICROBES IN PLANT HEALTH AND NUTRITION


than one billion spores in a gram. Spores of P. tinctorius when kept dry and
cool have been stored for more than 4 years without losing their ability to
form ectomycorrhizae. The spores can be mixed into nursery soil and the
seed of the desired tree species planted. Moderate levels of fertility and at
least 2-4 percent organic matter should be maintained in the soil. Usually,
the seedlings will have adequate ectomycorrhizae in 6-7 months after seed
germination.
The production of vegetative cultures of ectomycorrhizal fungi requires
aseptic culture technique. This means that the substrate on which the fungi
are grown must be sterilized, usually by autoclaving, and maintained free of
other microbial contamination for at least several weeks, or until the fungus
has produced sufficient growth to overcome contamination. After it has been
leached with water, this inoculum can then be added to soil.
Some species of ectomycorrhizal fungi are more beneficial to tree growth
and development under different soil conditions than others. It is important
to select and test different species to determine which are best suited to
specific locations.


Biological Control of Soil-Borne Pathogens

Microorganisms that cause root diseases are sometimes suppressed by other
microorganisms in the soil. In many instances disease-causing organisms may
be present, but because of naturally occurring biological control, little or no
disease results. The prevalence of pathogens may be reduced by crop rotation
using a non-host crop, which often starves the pathogen and prevents it from
reproducing.
It is also possible to increase the level of organisms that are antagonistic to
soil-borne plant pathogens. This is generally done not by adding antagonistic
microorganisms directly to the soil, but by the use of various organic amend-
ments such as manure or plant residues. These amendments provide a source
of food for soil-borne microorganisms that can inhibit the development of
plant pathogens. Research is needed in order to exploit more fully the use of
various forms of organic matter to enhance this biological control of soil-
borne pathogens.
There have been many attempts to control pathogens in soil by the addi-
tion of specific microorganisms. In general, these attempts have failed to
increase the level of naturally occurring biological control. For example, soil
contains species of fungi that trap and feed on plant parasitic nematodes
(Figure 3.3). However, application of additional nematode-trapping fungi
failed to protect plants under field conditions. It is likely that unless the soil
is altered in some way, it naturally contains the maximum number of nema-
tode-trapping fungi that it can support. There is, however, hope that we may







MICROBIAL PROCESSES


FIGURE 3.3 A nematode-trapping fungus, Dactylella drechsleri, which captures prey
on adhesive knobs. (Photograph courtesy of D. Pramer)

be on the verge of a major advance in controlling soil-borne pathogens by
adding specific microorganisms or by altering the rhizosphere environment.
There are a few examples where disease has been reduced by applying a
hypovirulent strain or a mutant strain of a pathogen that is incapable of
producing disease. Such strains may prevent development of the pathogenic
strains. A nonpathogenic strain of the crown gall bacterium will thus protect
plants from attack by a pathogenic strain.
In soil, most pathogenic fungi must pass through the rhizosphere or must
live within this zone. Their success in colonizing or infecting plant roots
depends upon their ability to compete with other rhizosphere microorgan-
isms. The chemical and microbiological environment in this zone may be
altered slightly, but significantly, to effect changes in the inoculum potential
of pathogens, either by selections of plant genotypes that produce such
changes or by careful regulation of nitrification in soil.
Research on biological control should be highly encouraged, for it could
provide an alternative means of disease control to the use of expensive and
often dangerous pesticides.

References and Suggested Readings
Alexander, M. 1977. Introduction to soil microbiology. 2nd Edition. New York: John
Wiley and Sons.








SOIL MICROBES IN PLANT HEALTH AND NUTRITION 57

__ 1974. Microbial ecology. New York: John Wiley and Sons.
Baker, K. F., and Cook, R. J. 1974. Biological control of plant pathogens. San
Francisco: W. H. Freeman and Company.
Barber, D. A., and Martin, J. K. 1976. The release of organic substances by cereal roots
into soil. The New Phytologist 76:69-80.
Barber, D. S. 1968. Microorganisms and the inorganic nutrition of higher plants. Annual
Review of Plant Physiology 19:71-88.
Barron, G. L. 1977. The nematode-destroying fungi. Topics in mycobiology No. 1.
Guelph, Ontario: Canadian Biological Publications Ltd.
Brown, M. E. 1974. Seed and root bacterization. Annual Review of Phytopathology
12:181-197.
S; Hornby, D.; and Pearson, V. 1973. Microbial populations and nitrogen in soil
growing consecutive cereal crops infected with take-all. Journal of Soil Science
24(3):296-310.
Carson, E. W. 1974. The plant root and its environment. Charlottesville: The University
Press of Virginia.
Cook, R. J. 1977. Management of the associated microbiota. In Plant disease: an ad-
vanced treatise in how disease is managed. J. G. Horsfall and E. B. Cowling, eds.,
pp. 145-166. New York: Academic Press.
__ 1976. Interaction of soil-borne plant pathogens and other microorganisms: an
introduction. Soil Biology and Biochemistry 8:267.
Doetsch, R. N., and Cook, T. M. 1976. Introduction to bacteria and their ecobiology.
Baltimore, Maryland: University Park Press.
Garrett, S. D. 1956. Biology of root-infecting fungi, p. 11. New York: Cambridge Uni-
versity Press.
__ 1970. Pathogenic root-infecting fungi. Cambridge, England: Cambridge Univer-
sity Press.
Gerdemann, J. W. 1975. Vesicular-arbuscular mycorrhizae. In The development and
function of roots. J. G. Torrey and D. T. Clarkson, eds., pp. 575-591. New York:
Academic Press.
Gray, T. P., and D. Parkinson. 1968. The ecology of soil bacteria. Liverpool: Liverpool
University Press.
__ and Williams, S. T. 1975. Soil microorganisms. New York: Longman.
Harley, J. L. 1979. Proceedings of the soil-root interface symposium: London: Academic
Press.
Henis, Y., and Chet, I. 1975. Microbiological control of plant pathogens. Advances in
Applied Microbiology 19:85.
Hornby, D. 1978. Microbial antagonisms in the rhizosphere. Annals of Applied Biology
89:97-100.
Joshi, M. M., and Hollis, J. P. 1977. Interactions of Beggiatoa and rice plants: detoxifica-
tion of hydrogen sulfide in the rice rhizosphere. Science 197:179-180.
Kleinschmidt, G. D., and Gerdemann, J. W. 1972. Stunting of citrus seedlings in fumi-
gated nursery soils related to the absence of endomycorrhizae. Phytopathology
62:1447-1453.
Krasilnikov, N. A. 1958. Soil microorganisms and higher plants. Moscow: Academy of
Sciences USSR. English translation, by Y. Halperin, 1961. Jerusalem: Israel Program
for Scientific Translations, Ltd.
Marks, G. C., and Kozlowski, T. T. 1973. Ectomycorrhizae: their ecology and physiol-
ogy. New York: Academic Press.
Marx, D. H. 1977. The role of mycorrhizae in forest production. In Proceedings of the
TAPPI (Technical Association of the Pulp and Paper Industry) Annual Meeting,
February 14-16, 1977, held in Atlanta, Georgia, pp. 151-161. Atlanta: TAPPI.
Mosse, B. 1977. Plant growth responses to vesicular-arbuscular mycorrhiza: X. Responses
of stylosanthes and maize to inoculation in unsterile soils. New Phytologist
78:277-288.
1977. The role of mycorrhiza in legume nutrition on marginal soils. In Exploiting
the legume-rhizobium symbiosis in tropical agriculture: Proceedings of a workshop,







MICROBIAL PROCESSES


University of Hawaii, August 1976, College of Tropical Agriculture Miscellaneous
Publication No. 54, pp. 275-292. Honolulu: University of Hawaii.
Rovira, A. D.; Newman, F. I.; Bowen, H. J.; and Campbell, R. 1974. Quantitative assess-
ment of the rhizoplane microflora by direct microscopy. Soil Biology and Biochem-
istry 6:211-216.
Sanders, F. E.; Mosse, B.; and Tinker, P. B., eds. 1974. Endomycorrhizas: Proceedings.
Symposium on Endomycorrhiza. July, 1974, University of Leeds. New York: Aca-
demic Press.
Shipton, P. J. 1977. Monoculture and soilborne pathogens. Annual Review of Phyto-
pathology 15:387-407.
Smith, A. M. 1976. Availability of plant nutrients in reduced microsites in soil. Annual
Review of Phytopathology 14:53-73.
Tansey, M. R. 1977. Microbial facilitation of plant mineral nutrition. In Microorganisms
and minerals, E. D. Weinberg, ed., pp. 343-385. New York: Marcel Dekker, Inc.
United Nations Educational, Scientific, and Cultural Organization. 1969. Soil biology:
review on research. Natural Resources Research, Series No. 9. Paris: United Nations
Educational, Scientific, and Cultural Organization. Distributed in the United States
by UNIPUB. New York.

Research Contacts

Rhizosphere
Richard Bartha, Rhizosphere Group, Cook College, Rutgers University, New Brunswick,
New Jersey 08903, U.S.A.
J. P. Hollis, Department of Plant Pathology, Louisiana State University, Baton Rouge,
Louisiana 70803, U.S.A.
A. D. Rovira, Division of Soils, CSIRO, P.O. Box 2, Glen Osmond, Adelaide, South
Australia, 5064.

Endomycorrhizae
J. W. Gerdemann, Department of Plant Pathology, University of Illinois, Urbana, Illinois
61801, U.S.A.
John A. Menge, Department of Plant Pathology, University of California, Riverside,
California 92521, U.S.A.
Barbara Mosse, Rothamsted Experimental Station, Harpenden, Hertshire AL5 2JQ, Eng-
land
T. H. Nicolson, Department of Biological Sciences, University of Dundee, Dundee, DD1
4HN, Scotland

Ectomycorrhizae
Donald H. Marx, Director and Chief Plant Pathologist, Institute for Mycorrhizal Re-
search and Development, Forestry Sciences Laboratory, U.S. Department of Agri-
culture, Carlton Street, Athens, Georgia 30602, U.S.A.
Orson K. Miller, Jr., Department of Biology, Virginia Polytechnic Institute, Blacksburg,
Virginia 24061, U.S.A.
James M. Trappe, Forest Service, Forestry Sciences Laboratory, U.S. Department of
Agriculture, 3200 Jefferson Way, Corvallis, Oregon 97331, U.S.A.

Biological Control of Soil-Borne Pathogens
R. James Cook, Regional Cereal Disease Research Laboratory, U.S. Department of Agri-
culture, Washington State University, Pullman, Washington 99163, U.S.A.
Allen Kerr, Department of Plant Pathology, Waite Agricultural Institute, University of
Adelaide, Glen Osmond, Adelaide, South Australia, 5064.









Chapter 4


Nitrogen Fixation






Air is four-fifths nitrogen, yet it is the absence of this particular element
that most commonly limits food production. Neither man, animals, nor high-
er plants can use elemental nitrogen; it must first be "fixed," that is, com-
bined with other elements such as hydrogen, carbon, or oxygen before it can
be assimilated.
Certain bacteria and algae have the ability to utilize (fix) gaseous nitrogen
from the air. Some microorganisms work symbiotically in nodules on the
roots of plants, with the plant providing food and energy for the bacteria,
which, in turn, fix nitrogen from the air for their host. Other kinds of bac-
teria and algae work independently and fix nitrogen for their own use, but
these are often limited in their activity because of the lack of a dependable
energy supply.
Bacteria that fix nitrogen in nodules on the roots of leguminous plants are
called rhizobia (Figure 4.1). Other microorganisms that produce nodules on
certain nonleguminous plants are classified as Frankia spp. and are actino-
mycetes. Freshly crushed nodules from the same plant species also will induce
nodulation on these plants. Recently Callaham et al. (1978) have induced
nodulation in shrubs of sweet fern (Comptonia peregrina) with a pure culture
of Frankia.
Leguminous plants have been known for centuries to enrich soils, but the
reason was not understood until 1886 when two German scientists, Hellriegel
and Wilfarth, found that the bacteria in the nodules on the leguminous root
brought about nitrogen fixation.
Nitrogen-fixing microorganisms fix an estimated 175 million t of nitrogen
annually, or about 70 percent of our total supply. The remainder is produced
in chemical fertilizer factories. With the rising world population and the
declining supply of fossil fuels required to manufacture chemical nitrogen
fertilizer, it may be necessary to rely more on microorganisms to satisfy plant
needs for nitrogen. Some of the nitrogen-fixing systems involving micro-
organisms are described in the following sections.






MICROBIAL PROCESSES


SOYBEANS WITH NODULES

FIX AIR NITROGEN


A
vU

0


FIGURE 4.1 Rhizobia of the proper kind applied to leguminous seeds before planting
can induce nodules or nitrogen factories to form on the roots. These provide the plant
with usable nitrogen. The process of applying the rhizobia to seed is called inoculation.
(Photograph courtesy of Joe C. Burton)






NITROGEN FIXATION


Symbiotic Systems


Rhizobium-Leguminous Plant Associations

Of all the systems of biological nitrogen fixation, the Rhizobium-legumi-
nous plant association has been the most reliable. Legumes in association with
nodule bacteria fix at least 35 million t of nitrogen annually valued at several
billion U.S. dollars. Yet this beneficial association of nodule bacteria with
legumes has only been partially explored. Of the 13,000 known species of
legumes, only about 100 are grown commercially. Further, much of the seed
is planted without inoculations with the nodule bacteria. Nodulation, if it oc-
curs under these conditions, is by native soil rhizobia, which are often either
ineffective or too few in number to bring about effective nitrogen fixation.

Plant Groups and Rhizobium Species. Many soils do not contain the
proper nodule bacteria to bring about nitrogen fixation and successful growth
of legumes. In many cases when the bacteria are present, they are ineffec-
tive-that is, they produce nodules that provide little or no nitrogen. Farmers
can enhance nitrogen fixation by adding the proper nitrogen-fixing bacteria
to leguminous seeds before they are planted. Less than a kilogram of high-
quality inoculant, properly applied to legume seeds, can replace more than
100 kilograms of fertilizer nitrogen per hectare.
Certain groups of leguminous plants are nodulated by a single kind of
Rhizobium. The bacteria that nodulate each of these groups are often con-
sidered a species. All plants susceptible to nodulation by a Rhizobium species
constitute a "cross-inoculation" group. The Rhizobium species and their cor-
responding plant or cross-inoculation groups are given in Table 4.1.
Effective nitrogen-fixing nodules on some common legumes are shown in
Figure 4.2. These nodules are usually large and are often concentrated on the
primary root. In contrast, ineffective nodules are small, numerous, and scat-
tered over the root system (Figure 4.3).

Rhizobium-Host Interactions. Strains of rhizobia cannot be described as
effective or ineffective without specifying the exact species of legume host.
Strains of rhizobia that are good nitrogen-fixers in association with one host
are often worthless on another. There is voluminous literature on this point,
but so many of the leguminous plants cultured in the tropics and subtropics
are nodulated by the cowpea rhizobia that special mention is justified. The
cowpea cross-inoculation group encompasses numerous genera and species of
plants. Rhizobium strains effective on a wide spectrum of plants within this
group are scarce. Specific inocula containing two or three strains known to be
highly effective on the particular legume may be needed to assure good
yields.







MICROBIAL PROCESSES


TABLE 4.1 Rhizobium Species and Plants Nodulated


Rhizobium Species


Designated
R. meliloti



R. trifolii
R. leguminosarum



R. phaseoli


R. lupini

R. japonicum

Undesignated
Rhizobium sp.
(Cowpea type)





















Rhizobium sp.


Plants Nodulated


Medicago sativa (alfalfa)
Melilotus sp. (sweet clover)
Medicago sp. (burr and barrel medic annuals)
Trigonella foenum graecum (fenugreek)
Trifolium sp. cloverss)
Pisum sativum (garden and field peas)
Vicia faba (broad bean)
Lens esculenta (lentils)
Lathyrus sp. (peavine)
Phaseolus vulgaris (common, field, haricot, kidney, pinto,
snap beans, etc.)
P. coccineus (runner bean, scarlet runner)
Lupinus sp. (all lupins)
Ornithopus sativus (serradella)
Glycine max (soybean)


Vigna unguiculata cowpeaa)
Arachis hypogaea (peanut, groundnut)
Vigna radiata mungg bean)
Phaseolus lunatus (lima bean)
P. acutifolius (tepary bean)
Psophocarpus tetragonolobus (winged bean)
Sphenostylis sp. (African yam bean)
Pachyrhizus sp. (jicamus)
Centrosema sp. centroo)
Mucuna deeringiana (velvet bean)
Canavalia ensiformis (jack bean)
Lablab purpureus (hyacinth bean)
Phaseolus aconitifolius (moth bean)
Cyamopsis tetragonoloba (guar)
Voandzeia subterranea (Bambara groundnut)
Cajanus cajan (pigeon pea)
Desmodium sp.
Cassia sp.
Lespedeza sp.
Indigofera sp.
Crotalaria sp.
Pueraria sp.
Cicer arietinum (chick-pea, garbanzo)
Coronilla varia (crownvetch)
Onobrychis vicisefolia (sainfoin)
Leucaena leucocephala (ipil-ipil)
Petalostemum sp. (prairie clover)
Albizzia julibrissin
Lotus sp. trefoilss)
Anthyllis vulneraria (kidney vetch)
Sesbania sp.


Sources: R. E. Buchanan, and N. E. Gibbons, eds. 1974. Bergey'sManual ofDetermina-
tive Bacteriology. 8th edition. Baltimore: The Williams and Wilkins Co. E. B. Fred; I. L.
Baldwin; and E. McCoy. 1932. Root-Nodule Bacteria and Leguminous Plants. Madison:
University of Wisconsin Press.






NITROGEN FIXATION


NITROGEN FIXING NODULES


WINGED BEAN PEANUT


CHICKPEA


FIELD BEAN


FIGURE 4.2 Nodules or nitrogen factories on the roots of important food legumes:
winged bean, Psophocarpus tetragonolobus; peanut, Arachis hypogaea; chickpea, Cicer
arietinum; and field bean, Phaseolus vulgaris. (Photographs courtesy of Joe C. Burton)






MICROBIAL PROCESSES


SOYBEAN NODULES


INEFFECTIVE EFFECTIVE t


CLOVER NODULES


FIGURE 4.3 Rhizobia vary in their nitrogen-fixing abilities. Some are ineffective; they
produce nodules and use food that the plant provides, but fix little or no nitrogen.
Others are effective; these produce large nodules and fix appreciable amounts of nitrogen.
(Photographs courtesy of Joe C. Burton)







NITROGEN FIXATION


Nitrogen Fixed by Leguminous Plants. The amount of nitrogen fixed in
Rhizobium-leguminous plant associations varies with both the bacteria and
legumes as well as environmental factors. Forage legumes usually fix more
nitrogen than do grain legumes because carbohydrate requirements resulting
from seed development are small, whereas with grain legumes the developing
large seeds impose a large demand on the carbohydrate supply. The amounts
of nitrogen fixed are uncertain, because of the methods of measurement, but
relative quantities as related to host species give valid comparisons
(Table 4.2).
Rhizobium species in association with leguminous vegetables, in addition
to increasing plant protein, make the plants richer in vitamins and mineral
content by assisting general growth. Healthy, well-fed plants are more palat-
able and nutritious than plants suffering from lack of nitrogen.



Limitations

A major limitation to culture of leguminous crops is the lack of viable,
effective inocula for many of the legumes. Leguminous crops and soil condi-
tions vary with each location. Rhizobium strains should be selected for partic-
ular legumes and soil and climatic conditions. However, Rhizobium inocu-
lants are highly perishable and often lose viability before reaching their
destination.



TABLE 4.2 Nitrogen Fixed by Various Rhizobium-Legume Associations

Approximate Ranges
of Nitrogen Fixed
Plant (kg/ha/yr)

Alfalfa, Medicago sativa 100- 300
Sweet Clover, Melilotus sp. 125
Clover, Trifolium sp. 100-150
Cowpea, Vigna unguiculata 85
Faba bean, Vicia faba 240-325
Lentils, Lens sp. 100
Lupines, Lupinus sp. 150-200
Peanuts, Arachis hypogaea 50
Soybeans, Glycine max 60-80
Mung bean, Vigna radiata 55
Velvet bean, Mucuna pruriens 115
Pasture legumes, Desmodium sp., Lespedeza sp. 100-400
Sources: Adapted from R. C. Burns, and R. W. F. Hardy, 1975. Nitrogen fixation in
bacteria and higher plants. Berlin: Springer-Verlag; and W. S. Silver, and R. W. F. Hardy,
1976. Biological nitrogen fixation in forage and livestock systems. American Society of
Agronomy Special Publication No. 28. pp. 1-34.






MICROBIAL PROCESSES


A limitation to the legume symbiosis system, particularly with the grain
legumes, is the relatively short season of active nitrogen fixation in the
nodule, especially with plants such as field beans (Phaseolus vulgaris). Soy-
beans fix nitrogen a little longer. Nitrogen fixation with the soybean could be
doubled if the period of active nitrogen fixation in the nodule could be
increased by as little as 10 days (Hardy and Havelka, 1970).
A limitation to the symbiotic system in leguminous plants is the in-
adequacy of inoculants and inoculation methods to ensure a greater propor-
tion of nodules from the applied rhizobia when seeds are planted in soils
infested with highly infective rhizobia of poor nitrogen-fixing properties.

Research Needs
Rhizobium strains should be selected for the specific legume crops
being grown in each country. Strains should be selected for their nitrogen-
fixing ability and competitiveness under the prevailing soil and climatic con-
ditions.
Small-scale inoculant production methods should be studied. Effective
use of leguminous plants will depend both on effective Rhizobium strains and
a dependable delivery system that will help ensure a high production of nodules
by the inoculum rhizobia.
In devising delivery systems, consideration should be given to overcoming
an aggressive native population of both infective rhizobia and other micro-
organisms. The latter group of plant pathogens-as well as insects-may neces-
sitate separate application of inoculants and protective chemicals to the seeds.
New genera and species of legumes should be studied. Leguminous
plants such as the winged bean, Psophocarpus tetragonolobus, the climbing
lima, Phaseolus lunatus, the yam bean, Sphenostylis stenocarpa, and the
hyacinth bean, Lablab purpureus, all of which are climbers, can be very
productive under humid tropical conditions. Further, they will fix nitrogen for
months, providing the pods are gathered regularly and not allowed to mature
on the vine.


Frankia-Nonleguminous Plant Associations
Numerous nonleguminous trees and woody shrubs form root nodules and
fix atmospheric nitrogen under natural conditions. The organism responsible,
found in the nodules (called an endophyte), is an actinomycete (Frankia sp.)
and infection and nodule initiation have recently been achieved with the
cultured organism (Callaham et al, 1978). Crushed nodules from a growing
plant of the same species readily induce nodulation in most cases. Now that
the endophyte from one nonleguminous nodulating plant has been isolated
and cultivated, this can be done with others, thereby greatly facilitating cul-
ture of these nodulating nonleguminous plants. The same organism has now







NITROGEN FIXATION


been shown to nodulate A nus, Myrica, and Comptonia.
Nonleguminous trees and woody shrubs capable of fixing nitrogen com-
prise 145 species in 15 different genera of 7 plant families. Within such a large
group of plants, there is adaptability to a range of diverse soil and climatic
conditions. These plants often thrive in nitrogen-deficient eroded areas and
on sand dunes, barren slopes, and even arid soils. Certain species are pioneers.
They are the first to grow after glaciers have receded. Others make an early
appearance after volcanic eruptions and help develop soils from lava.
The hardiness of this group of plants is partially attributable to their
ability to fix nitrogen in association with Frankia sp. Many species also bene-
fit from association with external or ectotrophic mycorrhizae, and can thus
survive minimum phosphorus levels in the soil. The economic impact of the
nodulating nonlegume application is mainly in forestry rather than agricul-
tural crops. Trees of the birch family, especially Alnus sp., provide wood for
timber in many countries; the red alder (Alnus rubra Bong) (Figure 4.4) can
fix as much as 300 kg of nitrogen per ha per year.


FIGURE 4.4 Nodules on red alder, Alnus rubra Bong. Red alder can fix
as much as 300 kg nitrogen per ha per year (Table 4.3) when effectively
nodulated. (Photograph courtesy of H. J. Evans)







MICROBIAL PROCESSES


The nitrogen-fixing abilities of these nonleguminous trees and shrubs when
they are well nodulated are almost comparable to those of the Rhizobium-
leguminous plant associations, providing the stands are kept for several years.
Silvester (1977) has tabulated reports on nitrogen fixed by various species
(Table 4.3).
The unique abilities of these nodulating ndnleguminous trees and shrubs to
pioneer in new soils, increase fertility, and enrich the soil for growth of
economic crops should not be overlooked. Such plants can provide the base
for expansion of areas for food production, and they can help to restore soils
disrupted by the removal of coal and minerals.



Limitations

The nodulating nonleguminous trees and shrubs are long-term crops, mak-
ing them unsuitable when annual harvests of farm crops must be made for
subsistence. Recently a new plant (Datisca cannabina) was discovered, which
nodulates like Alnus, is not woody, and is propagated by seed. There may be
many others of this type.
Most known valuable species grow best in cool or temperate climates or at
high altitudes in the tropics. With further study, species well adapted to the
lowland tropics may also be discovered.
Seeds are very limited in availability.


TABLE 4.3 Nitrogen Fixed by Various Genera and Species of Nodulating Non-
leguminous Trees and Shrubs

Species Age-Years Nitrogen kg/ha/yr*

Alnus crispa 0-5 362
15-20 115
10-60 40
Alnus glutinosa 0-8 125
20 56-130
Alnus rubra 2-15 325
Casuarina equisetifolia 0-13 58
Ceanothus sp. 60
Coriaria arborea 14-25 129-192
Dryas drummondii 0-25 12
Hippophae rhamnoides 10-15 15
13-16 179
Myrica gale 3 9

*These are values reported in the literature; fixation rates vary widely with conditions
and should be treated as indicative estimates only, not as definitive rates.
Source: W. B. Silvester, 1977. Dinitrogen fixation by plant associations excluding le-
gumes. In A treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds. New
York: John Wiley and Sons.







NITROGEN FIXATION


Research Needs

Improved technology is needed for seed collection, production, and
handling for the more promising species.
Dependable laboratory-produced cultures of the endophytes specific to
all important species would be very helpful.
Surveys should be conducted to determine if nitrogen-fixing species of
these nodulating nonleguminous plants grow in the lowland tropics.
Plants should be evaluated for nitrogen-fixing ability as well as for their
value as human and animal food.

Azolla-Anabaena Associations
A small floating freshwater fern, Azolla pinnata invades lowland rice fields
in Indonesia, southern China, Vietnam, and other tropical areas. The upper
lobe of the Azolla leaflet contains a large leaf cavity inhabited by the blue-
green alga, Anabaena azolla. The symbiotic nature of the association is evi-
denced by two findings: 1) the algae in the leaf cavity have 15-20 percent
nitrogen-fixing cells (heterocysts) as compared to 5 percent in free-living
Anabaena species, and 2) the algae grow very poorly when taken from the
leaf cavity and placed on an inorganic medium with no combined nitrogen.
Growth is obtained in some cultures when the growth medium is supple-
mented with an organic compound in the form of 0.5 percent sugar (fruc-
tose). But in subsequent studies, nitrogenase activity of this isolated algae has
been only about half that of algae growing symbiotically in the leaf.
The Azolla-Anabaena association is literally a live floating nitrogen fac-
tory, using energy from photosynthesis to fix atmospheric nitrogen. Under
Indonesian environmental conditions, the Azolla-Anabaena association can
fix from 100 to 150 kg of nitrogen per ha per year in approximately 40-60 t
of biomass. It is important not to let the fern cover the water completely in
the rice paddies. Because the rice can be damaged from excessive shading of
the paddy water, 50 percent coverage is ideal. Nitrogen fixation occurs at
night as well as during the day, but the rate of fixation is lower in the dark.
Azolla pinnata plants floating on the water surface of irrigated rice paddies
are shown in Figure 4.5.
Azolla has been used extensively in Asia as a forage crop for duck and pig
feed, but its greatest potential appears to be as a green manure. On a dry-
weight basis it contains about 23.8 percent crude protein, 4.4 percent fat, 6.4
percent starch, and 9.5 percent fiber. Vietnam and Thailand have used Azolla
for years in their system of rice culture. Stocks of Azolla are kept during the
hot season for multiplication and distribution when cooler weather comes.
The stocks are then used to seed other paddies fertilized with ashes, urine,
and rotted manure. Azolla vegetation can double approximately every 5 days
under favorable conditions.








MICROBIAL PROCESSES


FIGURE 4.5 Small floating nitrogen factories on a flooded rice paddy. The small fern
Azolla pinnata harbors blue-green algae, Anabaena azolla, which fix nitrogen. Together
they provide nitrogen for a future crop. (Photograph courtesy of J. H. Becking)


`" ~~aprr.'y~i
iI_ Ifr~n;(
rF
:~IW--e ~lir~
1- ~U1






NITROGEN FIXATION 71
Nitrogen fixation by Anabaena azolla is apparently not adversely affected
by the level of combined nitrogen in the water because the organisms are ac-
tually sheltered in the leaflet cavity. The Azolla must die off and the nitrogen
must be mineralized before it becomes available to the plant. This happens
when the temperature rises (to as much as 400-450 C in Indonesia), and the
Azolla dies and settles to the bottom of the paddy. Nitrogen is released from
the decomposing cells and becomes available to the rice plants. The rice
plants then turn green and tillering (the production of multiple shoots from
the same plant) increases.


Limitations

Use of the Azolla-Anabaena association for food production is limited to
agricultural soils that can be flooded, and it is best adapted to rice culture.
Azolla could possibly be used, however, as a nitrogen source for other aquatic
plants such as taro (Colocasia esculenta) or water chestnut (Eleocharis dulcis).
In addition, the association may be beneficial in removing nutrients from
sewage treatment lagoons. Water, plenty of sunshine, and a temperature
regime that favors rhythmic self-destruction of the fern seem to be the most
important requirements for success.


Research Needs

More information is needed about nitrogen-fixation efficiencies of dif-
ferent Azolla-Anabaena strain combinations. It is possible that more efficient
nitrogen-fixing combinations can be discovered.
Good husbandry should be developed for using Azolla-Anabaena in rice
culture systems, as has been done in Indonesia and Vietnam. Technology for
using Azolla under different soil and climate conditions is needed, particu-
larly for temperate areas.
Experiments should be made to determine how best to grow Azolla
with rice to increase both nitrogen fixation and rice yields.


Asymbiotic Fixation


Blue-Green Algae
The blue-green algae are perhaps our most widespread group of nitrogen
fixers because they are present almost everywhere on land, in fresh water, and
in the sea. Regardless of environmental conditions (except at low pH), there
are almost always present some forms that fix atmospheric nitrogen. Blue-






MICROBIAL PROCESSES


green algae fix nitrogen in Antarctic waters as well as in hot springs. They
operate over the range of 00-600C.
One reason for their wide range of adaptability is that they include many
species, each of which may fix nitrogen under varying conditions. Nitrogen-
fixing species occur in the genera Anabaena, Aulosira, Cylindrospermum,
Gloeotricha, Tolypothrix, Calothrix, Nostoc, Haplosiphon, and others.
In rice culture, the blue-green algae can be depended on to provide nitro-
gen consistently. In long-term soil fertility experiments at the International
Rice Research Institute (IRRI) in the Philippines, 23 consecutive rice crops
have been harvested from soils ur fertilized with nitrogen, without any ap-
parent decline in soil nitrogen. TI.e algae replaced the nitrogen removed by
the rice crops.
Studies at the Agricultural Research Center in Giza, Egypt, have shown
that two blue-green algae, Tolypothrix tenuis and Aulosira fertilissima, fix
more nitrogen than other forms in that region. In a rotation with rice every
third year, it has been found advantageous to grow the effective algae and
inoculate the rice fields shortly after planting. Of the rice cultured, 10 percent
is now inoculated with a dried algal preparation of the two effective cultures
and this percentage is expected to increase rapidly in the future. Inocula are
also supplied to farmers in India by the Agricultural Research Institute in
New Delhi.
The importance of blue-green algae in rice paddies has long been recog-
nized. But these microorganisms also operate very well in desert regions; they
use the moisture of night dews during the early morning hours and fix nitro-
gen during this period of temporary activity. In the western United States
blue-green algae in crusts on the soil surface fix considerable amounts of
nitrogen per hectare per year. Nitrogenase activity (the activity of the enzyme
that splits molecular N2) ceases when the crusts become dry, but it is measur-
ably reactivated within 2 hours after crusts are moistened.
The importance of the role of the blue-green algae in fixing nitrogen was
not appreciated until the acetylene-reduction technique of measuring nitro-
gen fixation was developed. Now it is possible to study algal fixation in
streams and lakes and under various soil conditions, and the significance of
the blue-green algae in our world food production is becoming more evident.
Their main assets are 1) the wide range of adaptation to temperature and
moisture, and 2) their ability to respond quickly when environmental condi-
tions are suitable and to grow rapidly in paddy fields. The blue-green algae
fill ecological niches left by other systems of biological nitrogen fixation.

Limitations

From a management standpoint, knowledge of how to use the blue-green
algae effectively is meager. Nitrogen fixed by these microorganisms is not







NITROGEN FIXATION


readily available to food crops-the algal cells must decompose and the nitro-
gen must be mineralized. Although this may not be a problem in continuous
rice culture, it could be a major obstacle in other planting systems.
Algal growth in freshwater lakes is usually undesirable because they cause
the water to become stagnant. Technology on use of algal tissue as feed for
livestock or food for human consumption is needed. At present, its use is
chiefly as a green manure.


Research Needs

A survey should be made to determine the occurrence of good nitrogen-
fixing species.
Strains should be selected for environmental adaptation as well as nitro-
gen-fixing potential.
Technology related to husbandry and how to prepare, store, and dis-
tribute inoculants is needed.
Methods of culturing starter cultures and distributing dependable in-
ocula should be developed; biological and chemical methods of control will
result in better husbandry and more efficient handling of effective algae.
Information is needed on how to encourage the growth of desirable
organisms and discourage the growth of undesirable ones. The role of pred-
ators in reducing algal nitrogen fixation should be investigated and the use of
algae adapted to local soils.
Studies should be conducted on the use of algae for human food and
animal feed. The large biomass of algae could possibly provide high-quality
edible protein for animal consumption.

Free-Living Nitrogen-Fixing Bacteria
Free-living nitrogen fixers from at least 25 genera and many taxonomic
groups of bacteria are known. These organisms occur in diverse habitats; their
requirements for oxygen, a specific energy source, electron acceptors, and
other factors vary widely.
Fixation of significant amounts of nitrogen is dependent upon a suitable
carbon and energy supply. An adequate source of energy is one of the most
critical limiting factors to nitrogen fixation by free-living organisms. One of
three systems of obtaining energy may be utilized:

Energy may be obtained through breakdown of plant residues in soil.
Only rich, fertile soils harbor organic residues in amounts sufficient to pro-
vide significant energy. Clostridium, Klebsiella, and most Azotobacter species
rely on this source.






MICROBIAL PROCESSES


Certain bacteria are favored by root exudates of some plant genotypes
that are very efficient photosynthesizers. Exudates from the roots are selec-
tive energy sources for particular bacteria. This relationship is often referred
to as an "associative symbiosis"; a recent international symposium on nitro-
gen fixation recommended that it should be termed "biocoenosis." Azoto-
bacter paspali, Beijerinkia sp., and Spirillum lipoferum fit into this category.
(Recent work has suggested that Spirillum lipoferum should be reclassified
as two species of Azospirillum: A. lipoferum and A. brasilense [Krieg and
Tarrand, 1977].)
Other bacteria carry out photosynthesis themselves. But requirements
for growth are so restrictive that these microorganisms are not considered
highly important agronomically. Rhodospirillum rubrum and other photo-
synthetic bacteria are in this group.

The presence of nitrogen-fixing bacteria in the root zone does not assure
that they are actively fixing nitrogen; it does indicate the capability for
nitrogen fixation if there is sufficient energy and other conditions are present
for growth. If ammonia or nitrates are present in the soil, the organisms will
use these to produce new cells and will conserve energy in preference to
fixing nitrogen. Energy used for nitrogen fixation cannot be used in repro-
duction.
The real nitrogen contribution of free-living nitrogen fixers to the soil is
uncertain. With a mixed population under natural conditions, it is difficult to
assess the contributions of single species.
Clostridium, Klebsiella, and several Enterobacter species are credited with
substantial nitrogen fixation when energy-rich soils are flooded, but the iden-
tities of the major aerobic genera are uncertain. Azotobacter species, on the
other hand, prefer a moist, aerated environment, but they, too, are dependent
upon an adequate source of carbon. In both cases, large amounts of energy-
rich materials are required if significant amounts of nitrogen are to be fixed.
Nitrogen fixation efficiency is low. It takes the equivalent of about 50 kg of
sucrose for Azotobacter species to fix 1 kg of nitrogen at the oxygen concen-
trations found in air.
The inoculation of soils and seeds of nonleguminous plants with prepara-
tions of Azotobacter chroococcum has been practiced in Russia and India for
many years. Some types of Azotobacter have been credited with increasing
crop yields as a result of the nitrogen they fixed, but the low concentration
of cells in the soil could not have fixed appreciable amounts of nitrogen.
With the new highly sensitive techniques for measuring nitrogen fixation,
some doubt has been cast on the real contribution of free-living bacteria to
soil nitrogen. Bacterial inoculation experiments rarely show yield increases as
great as the 10 percent level required to attribute statistical significance to the
results. The greatest increases are on very fertile soil. There is little proba-






NITROGEN FIXATION


ability that any sound inoculation practice for free-living bacteria will be devel-
oped soon, and many of the reports of alleged growth stimulation are now
considered dubious.
In highly fertile soils, Azotobacter species are sometimes believed to pro-
duce growth factors and vitamins that are beneficial to vegetables. Benefits
from Azotobacter inoculation often are attributed to these growth factors
rather than to nitrogen fixation. The effect is manifested only in the highly
fertile garden soils used in vegetable production. The results of these studies
are still equivocal.
The discovery that the association of Bahia grass (Paspalum notatum)
with Azotobacter paspali in tropical soils resulted in nitrogen fixation stimu-
lated new interest in this field. Interest was heightened by the subsequent
finding by Dobereiner in Brazil that an associative symbiotic relationship
between Digitaria decumbens, cultivar "transvala," and the microorganism
Azospirillum lipoferum also brought about nitrogen fixation. It was reasoned
that tropical grasses with their more efficient 4-carbon photosynthetic cycle
could indeed provide the abundance of energy needed for fixation that was
lacking in other systems.
The enthusiasm has been dampened somewhat by the great variability that
has characterized field studies, and more study is needed to identify the
limiting factors and devise agronomic practices to bypass them. Large vari-
ations in growth cycles are observed with cereals, and they appear to fix
nitrogen only during the reproductive phase. Interactions of nitrogen fixa-
tion, nitrate assimilation, and denitrification raise the question whether the
nitrogen is being lost as rapidly as it is being fixed.
On the other hand, nitrate reductase-negative mutants of Azospirillum
species are now available that fix nitrogen in the presence of high levels of
nitrate. Much more study is needed to identify the factors important for
vigorous fixation and to reduce the high degree of variability.


Limitations

Too little is known of the physiology of this unique associative symbiosis
for it to be used effectively. In some cases, for instance, the organism may
enter the root cortex, but fail to proliferate enough to effect significant
nitrogen fixation. We need to know the reason for this.
To date, firm data have not been published to establish that Azospirillum
and free-living nitrogen fixers contribute substantial amounts of nitrogen and
increase crop yields under field conditions.
The conditions required for good inoculation trials are difficult to attain.
These conditions are: 1)low numbers of microorganisms already present in
the root zone with dinitrogen fixing capability; 2) inoculum able to compete







MICROBIAL PROCESSES


with established root-zone bacteria; 3) soil conditions that will support pro-
liferation of the organism to produce a large biomass of cells; 4) low levels of
available combined nitrogen in the soil; and 5) adequate substrate or plant
exudate to supply the energy required for fixation.


Research Needs

More knowledge is needed on the physiology of free-living, nitrogen-
fixing bacteria. Only a few strains of the microorganisms, and even fewer
genotypes of the host plants, have been studied. A report on nitrogen fixation
in wheat, Triticum aestivum, is of particular interest. Roots from lines con-
taining the 5-D chromosome were covered with a gelatinous material (prob-
ably a polysaccharide) which favored proliferation and nitrogen fixation by
gram-positive bacteria within the gelatinous layer on the roots in contrast to
wheat lines without this chromosome.
Mass screening for nitrogen-fixing activity of numerous grass genotypes
and strains of microorganisms should prove rewarding.
Greater emphasis should be placed on field studies. Acetylene-reduction
tests should be used when needed, but in field studies, increased yields and
higher quality are of greater significance than the amount of nitrogen fixed.
Attempts should be made to modify the rhizosphere bacterial com-
munity to allow the inoculum strain to become established.

References and Suggested Reading
Rhizobium-Leguminous Plant Associations
Brill, W. J. 1977. Biological nitrogen fixation. Scientific American 236:68-74.
Buchanan, R. E., and Gibbons, N. E., eds. 1974. Bergey's manual of determinative
bacteriology. 8th edition. Baltimore: The Williams and Wilkins Co.
Burns, R. C., and Hardy, R. W. F. 1975. Nitrogen fixation in bacteria and higher plants.
Berlin: Springer-Verlag.
Burris, R. H. 1975. The acetylene reduction technique. In Nitrogen fixation by free-
living microorganisms: International Biological Programme 6, pp. 249-257. Cam-
bridge, England: Cambridge University Press.
Burton, J. C. 1967. Rhizobium culture and use. In Microbial technology, H. J. Peppier,
ed., pp. 1-33. Huntington, New York: Robert E. Krieger Publishing Co.
Evans, H. J. 1969. How legumes fix nitrogen. In Crops grown-a century later, Agricul-
tural Experiment Station Bulletin No. 708, pp. 110-127. New Haven: Connecticut
Agricultural Experiment Station.
__ 1975. Enhancing biological nitrogen fixation: proceedings of a workshop held on
June 6, 1974. Sponsored by Energy Related Research and the Division of Biological
and Medical Sciences of the National Science Foundation. Washington, D.C.: U.S.
National Science Foundation.
Fred, E. B.; Baldwin, I. L.; and McCoy, E. 1932. Root-nodule bacteria and leguminous
plants. Madison: University of Wisconsin Press.
Hardy, R. W. F., and Havelka, U. D. 1970. Nitrogen fixation research, a key to world
food. Science 188:633-643.
Silver, W. S., and Hardy, R. W. F. 1976. Biological nitrogen fixation in forage and
livestock systems. American Society of Agronomy Special Publication No. 28,
pp. 1-34. Madison, Wisconsin: American Society of Agronomy.







NITROGEN FIXATION


Skinner, K. J. 1976. Nitrogen fixation-key to a brighter future for agriculture. Chemical
and Engineering News 54:22-35.

Frankia-Nonleguminous Plant Associations
Allen, E. K., and Allen, O. N. 1964. Non-leguminous plant symbiosis. In Microbiology
and soil fertility, 25th Annual Biology Colloquium, C. M. Gilmour and 0. N. Allen,
eds., pp. 77-106. Corvallis: Oregon State University Press.
Becking, J. H. 1977. Dinitrogen-fixing associations in higher plants other than legumes.
In A treatise on dinitrogen fixation, R. W. F. Hardy and W. Silver, eds., Section III:
Biology, pp. 185-276. New York: John Wiley and Sons.
Bond, G. 1974. Root-nodule symbiosis with actinomycete-like organisms. In The biology
of nitrogen fixation, A. Quispel, ed., pp. 342-378. Amsterdam: North-Holland Pub-
lishing Co.
Callaham, D.; Tredici, P. D.; and Torrey, J. G. 1978. Isolation and cultivation in vitro of
the actinomycete causing root nodulation in Comptonia. Science 199:899-902.
Silvester, W. B. 1977. Dinitrogen fixation by plant associations excluding legumes. In A
treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV:
Agronomy and ecology, pp. 141-190. New York: John Wiley and Sons.
Torrey, J. G. 1978. Nitrogen fixation by actinomycete-nodulated angiosperms. Bio-
Science 28:586-592.
Azolla-Anabaena Associations
Becking, J. H. 1975. Contribution of plant-algae associations. In Proceedings of the
International Symposium on Nitrogen Fixation, pp. 556-580. Pullman: Washington
State University Press.
Mague, T. H. 1977. Ecological aspects of dinitrogen fixation by blue-green algae. In
Treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV:
Agronomy and ecology, pp. 85-140. New York: John Wiley and Sons.
Moore, A. W. 1969. Azolla: biology and agronomic significance. Biological Review
35:35-37.
Peters, G. A. 1975. Studies on the Azolla: Anabaena symbiosis. In Proceedings of the
International Symposium on Nitrogen Fixation, W. E. Newton and C. J. Nyman, eds.,
pp. 592-610. Pullman: Washington State University Press.
- 1978. Blue-green algae and algal associations. BioScience 28:580-585.

Blue-Green Algae
Burris, R. H. 1975. The acetylene-reduction technique. In Nitrogen fixation by free-
living microorganisms. International Biological Programme 6, pp. 249-257. Cam-
bridge, England: Cambridge University Press.
Dart, P. J., and Day, J. M. 1977. Non-symbiotic nitrogen fixation in soil. In Soil micro-
biology, N. Walker, ed., pp. 225-252. New York: John Wiley and Sons.
Fogg, G. E. 1971. Nitrogen fixation in lakes. In Plant and soil special volume: biological
nitrogen fixation in natural and agricultural habitats. Proceedings of the Technical
Meetings on Biological Nitrogen Fixation of the International Biological Program
(Section PP-N), Prague and Wageningen, 1970, T. A. Lie and E. G. Mulder, eds., pp.
393-401. The Hague: Martinus Nijhoff.
Hendrikkson, E. 1971. Algae nitrogen fixation in temperate regions. In Plant and soil
special volume: biological nitrogen fixation in natural and agricultural habitats. Pro-
ceedings of the Technical Meetings on Biological Nitrogen Fixation of the Inter-
national Biological Program (Section PP-N), Prague and Wageningen, 1970, T. A. Lie
and E. G. Mulder, eds., pp. 415-419. The Hague: Martinus Nijhoff.
Rinaudo, G.; Balandreau, J.; and Dommergues, Y. 1971. Algae and bacterial non-
symbiotic nitrogen fixation in paddy soils. In Plant and soil special volume: biological
nitrogen fixation in natural and agricultural habitats. Proceedings of the Technical
Meetings on Biological Nitrogen Fixation of the International Biological Program
(Section PP-N), Prague and Wageningen, 1970, T. A. Lie and E. G. Mulder, eds., pp.
471-479. The Hague: Martinus Nijhoff.







MICROBIAL PROCESSES


Stewart, W. D. P. 1966. Nitrogen fixation by free-living organisms. In Nitrogen fixation
in plants, pp. 68-83. London: Athlone Press. Distributed in the United States by
Humanities Press, Atlantic Highlands, New Jersey.
__ ed. 1976. Blue-green algae. Nitrogen-fixation by free-living micro-organisms. Inter-
national Biological Programme Series 6, pp. 129-229. Cambridge, England: Cambridge
University Press.

Free-Living Nitrogen-Fixing Bacteria
Barber, L. E.; Tjepkema, J. D.; Fussell, S. A.; and Evans, H. J. 1976. Acetylene reduction
(nitrogen fixation) associated with corn inoculated with Spirillum. Applied and Envi-
ronmental Microbiology 32:108-113.
Burris, R. H.; Albrecht, S. L.; and Okon, Y. 1978. Physiology and biochemistry of
Spirillum lipoferum. In Limitations and potentials for biological nitrogen fixation in
the tropics, VoL 10, Basic Life Sciences, Proceedings of a Conference on Limitations
and Potentials of Biological Nitrogen Fixation in the Tropics, Brasilia, Brazil,
Johanna Dobereiner, Robert H. Burris, Alexander Hollaender, Avilio A. Franco,
Carlos A. Neyra, and David Barry Scott, eds., pp. 303-315. New York: Plenum Press.
Dart, P. J., and Day, J. M. 1975. Nitrogen fixation in the field other than by nodules. In
Soil microbiology: a critical view, Norman Walker, ed., pp. 225-252. London: Butter-
worth's Scientific Publications.
Knowles, R. 1977. The significance of asymbiotic dinitrogen fixation by bacteria. In
A treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV:
Agronomy and ecology, pp. 33-84. New York: John Wiley and Sons.
Krieg, N. R., and Tarrand, J. J. 1977. Taxonomy of the root-associated nitrogen fixing
bacterium Spirillum lipoferum. In Limitations and potentials for biological nitrogen
fixation in the tropics, Vol 10, Basic Life Sciences, Proceedings of a Conference on
Limitations and Potentials of Biological Nitrogen Fixation in the Tropics. Brasilia,
Brazil. Johanna Dobereiner, Robert H. Burris, Alexander Hollaender, Avilio A.
Franco, Carlos A. Neyra and David Barry Scott, eds., pp. 317-333. New York:
Plenum Press.

Research Contacts

Rhizobium-Leguminous Plant Associations
R. H. Burris, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
53706, U.S.A.
R. A. Date, CSIRO, The Cunningham Laboratory, Mill Road, St. Lucia, Queensland,
Australia, 4067.
Deane F. Weber, Cell Culture and Nitrogen Fixation Laboratory, Beltsville Agricultural
Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A.

Frankia-Nonleguminous Plant Associations
J. H. Becking, Institute for Atomic Sciences in Agriculture, 6 Keyenbergseweg, Postbus
48, Wageningen, The Netherlands.
W. B. Silvester, Department of Biological Sciences, University of Waikato, Private Bag,
Hamilton, New Zealand.

Azolla-Anabaena Associations
Alan W. Moore, CSIRO, The Cunningham Laboratory, Mill Road, St. Lucia, Queensland,
Australia, 4067.
D. W. Rains, Plant Growth Laboratory, University of California, Davis, California 95616,
U.S.A.







NITROGEN FIXATION 79

Blue-Green Algae
T. M. Mague, Bigelow Laboratory for Ocean Sciences, McKown Point, West Boothbay
Harbor, Maine 04575, U.S.A.
W. D. P. Stewart, Department of Biological Sciences, University of Dundee, Dundee,
DD1 4HN, Scotland.
I. Watanabe, International Rice Research Institute, Los Bafios, The Philippines.

Free-Living Nitrogen-Fixing Bacteria
Lynn Barber, Department of Microbiology, Oregon State University, Corvallis, Oregon
97331, U.S.A.
R. H. Burris, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
53706, U.S.A.
Johanna Dobereiner, EMBRAPA, 23460 Seropedica, Rio de Janeiro, Brazil.
David H. Hubbell, Soil Science Department, University of Florida, Gainesville, Florida
32611, U.S.A.









Chapter 5


Microbial Insect Control Agents






There are more than 1,500 naturally occurring microorganisms or their
products that hold promise for the control of major insect pests. Microorgan-
isms that affect insects are termed entomopathogens. They may be used to
induce diseases in target insects or to suppress populations of insects directly
or in combination with chemical insecticides.
Energy-efficient pest-control approaches must be developed to reduce to a
minimum the use of factory-produced, toxic, broad-spectrum chemical insec-
ticides used in industrial countries in ever-increasing amounts. The microbial
approach can be applied to agricultural practices of both developing and
developed countries, and many of its techniques are ready for implementa-
tion. By adopting a systems approach to "integrated pest management," using
entomopathogens and other nonchemical factors for specific pests, develop-
ing countries have an important opportunity to bypass the traditional chem-
ical approach to insect control.
All types of microorganisms are represented among the potential microbial
control agents. As an example, nearly 100 species of bacteria and over 700
viruses have been isolated from arthropods and more are being discovered
each year. All classes of fungi are represented among the more than 750
known entomopathogenic fungi. Protozoa are also likely candidates as mi-
crobial control agents because many insects not attacked by other entomo-
pathogens are susceptible to at least one of the 300 known species of ento-
mophilic protozoa.




Development of Bioinsecticides

In planning an approach to the use of microbial control agents, the most
significant factors to be considered include production technology, safety and
specificity, and efficacy.







MICROBIAL INSECT CONTROL AGENTS 81
Production Technology

Entomopathogens are produced by fermentation methods (Figure 5.1), in
living insects (Figure 5.2), and in cell tissue cultures. Fermentation technol-
ogy is used for some bacteria and fungi, whereas living insects can be used for
the production of obligatory parasitic viruses and protozoa. Both processes
have been successfully used to produce commercial entomopathogenic prod-
ucts. For example, submerged fermentation is generally used for commercial
production of the entomogenous bacteria Bacillus thuringiensis and Bacillus
moritai and the fungi Beauveria bassiana and Entomophthora virulenta. Sur-
face fermentation is employed to produce pathogenic fungi, for example,
Nomuraea rileyi and Metarrhizium anisopliae, and a combination of both
surface and submerged techniques for Beauveria bassiana and Hirsutella
thompsonii. Living insects are exclusively used as substrates for the produc-
tion of the respective nucleopolyhedrosis viruses of Heliothis zea, Porthetria
dispar, and Hemerocampa pseudosugata. Cell tissue culture methods currently
produce only small quantities of viruses, but they are considered to be the
mass production method of the future.

Safety and Specificity
Entomopathogens are infectious, replicating living organisms that are a
natural part of our environment. Evidence that microbial insecticides pose
little human or environmental hazard has been demonstrated by laboratory
animal testing data developed to support federal pesticide registration. Never-
theless, safety cannot be absolutely guaranteed for all entomopathogens in
every living system, and it is important that potential hazards for new ento-
mopathogens be known prior to use. Informal guidance for evaluating the
specificity and risks in the use of microbial agents has been provided by reg-
ulatory agencies. Formal guidelines are now being developed by the U.S.
Environmental Protection Agency (EPA). Several baculoviruses have been
tested in living organisms with no evidence of toxic or pathogenic effects on
vertebrates or nontarget invertebrates.
Baculoviruses do not appear to replicate in vertebrate embryos or in cell
lines derived from birds, fishes, amphibians, or mammals. No deleterious
effects at normal field-use rates were reported in tests with such bacteria as
Bacillus thuringiensis, Bacillus popilliae, and Bacillus moritai. While allergens
are encountered among the fungi (Beauveria, Entomophthora, Hirsutella,
Metarrhizium, and Nomuraea species), results indicate that these fungi are not
toxic or infectious to vertebrates. Three entomopathogenic protozoa, one
from grasshoppers (Nosema locustae), one from mosquitoes (Nosema al-
gerae), and one from beetles (Mattesia trogodermae), have been evaluated
against nontarget organisms. Initial in vivo tests indicate no apparent risk to
vertebrates.








MICROBIAL PROCESSES


Growth factors


Packaging


FIGURE 5.1 Submerged fermentation pathway used to produce Bacillus thuringien-
sis. (Photograph courtesy of C. M. Ignoffo)


FIGURE 5.2 Scanning electron micrograph of entomocidal parasporal crystals
and spores of Bacillus thuringiensis. (Photograph courtesy of C. M. Ignoffo)







MICROBIAL INSECT CONTROL AGENTS


Efficacy

Entomopathogens have been used to control mites, beetles, and caterpillar
pests of agricultural crops, forests, and stored products with varying degrees
of success.
Microbial insecticides, like chemical insecticides, are usually sprayed or
dusted on crops. Entomopathogens may also be successfully introduced and
established in an ecosystem by other application methods to provide long-
term control of pest populations. For example, insects themselves can be used
to disseminate entomopathogens. Virus or fungus epizootics might be in-
duced in an insect population before crop-damaging proliferation takes place.
It may also be possible to manipulate the environment to create conditions in
which naturally occurring pathogens exert their greatest effect. Some of these
approaches may provide levels of control equal to or better than those cur-
rently obtained with chemical insecticides, but further research is needed to
exploit their potential.
The potential for substituting microbial control agents for chemical pesti-
cides can be deduced from the following examples in the United States shown
in Table 5.1.
Development of the use of entomopathogens or their by-products for
microbial control agents is underexploited. Safe, effective entomopathogens
formulated as microbial control agents are being developed by governmental
agencies and industry, and the commercial products are being effectively used
by growers. The newer agents have not been brought to their fullest potential.


TABLE 5.1 Potential Substitution of Chemical Pesticides by Microbial Control Agents
in the United States
Potential Replacement
of Chemical Pesticide
Disease Control Agent (t per year)
Cotton bollworm
and budworm
(Heliothis zea) Baculovirus heliothis >7,700

Citrus rust mite (Florida)
(Phyllocoptruta oleivora) Hirsutella thompsonii 1,800-7,200

Cabbage looper (Florida)
(Trichoplusia ni) Bacillus thuringiensis 450-1,400

Western-range
grasshoppers Nosema locustae 450- 900

Green peach aphid
on Maine potatoes
(Myzus persicae) Entomophthora ignobilis 90- 310







MICROBIAL PROCESSES


Bacteria


Many bacteria are associated with insects, most of which belong to the
families Pseudomonadaceae, Enterobacteriaceae, Lactobacillaceae, Micrococ-
caceae, and Bacillaceae (Table 5.2). Members of these families may be obli-
TABLE 5.2 Examples of Bacteria Pathogenic for Insects*


Bacteria


Insects


Pseudomonadaceae
*Pseudomonas aeruginosa
Pseudomonas septica

Vibrio leonardia


Enterobacteriaceae
Serratia marcescens and
*Escherichia coli
Enterobacter aerogenes

Proteus vulgaris, P. mirabilis,
and P. retigeri
*Salmonella schottmuelleri
var. alvei
*Salmonella enteritidis,
*Shigella dysenteriae

Lactobacillaceae
Diplococcus and Streptococcus
spp.

*Streptococcus faecalis

Micrococcaceae
*Micrococcus spp.





Bacillaceae
Bacillus thuringiensis
and B. cereus
Bacillus popilliae and
B. lentimorbus
Bacillus sphaericus
Bacillus larvae
Bacillus moritai
Clostridium novyi and
C. perfringens


Grasshoppers (Orthoptera)
Scarab beetles (Scarabaeidae), striped
ambrosia beetle (Tripodendron lineatum)
Greater wax moth (Galleria melonella),
European corn borer (Ostrinia nubilalis)

Varieties of butterfly, moth and skipper
(Lepidoptera)
Grasshoppers (Orthoptera), varieties of
butterfly, moth and skipper (Lepidoptera)
Grasshoppers (Orthoptera)

Honeybees (Apidae), greater wax moth
(Galleria melonella)
Greater wax moth (Galleria melonella)


Cockchafer (Melolontha melolontha),
silkworm (Bombyx mori), gypsy moth
(Lymantria dispar), processionary
moths (Thaumetopoeia spp.)
Greater wax moth (Galleria melonella)


Green June beetle (Cotinis nitida),
sawflies (Tenthredinidae), houseflies
(Muscidae), various Lepidoptera
including nun moth (Lymantria monacha),
European corn borer (Ostrinia nubilalis),
and cutworms (Noctuidae)

Varieties of butterfly and moth (Lepidoptera)

Scarab beetles (Scarabaeidae)

Mosquitoes (Culicidae)
Honeybees (Apidae)
Flies (Diptera)
Greater wax moth (Galleria melonella)


*Those asterisked may also be pathogenic for man.







MICROBIAL INSECT CONTROL AGENTS


gate or opportunistic entomopathogens, depending on their host association
in nature. Obligate entomopathogens are generally fastidious and are re-
stricted to growth in a living host insect. The occasional or opportunistic
pathogens are free living in nature, although they may commonly be found
associated with one or more hosts. About 100 bacteria have been reported as
entomopathogens, but only four (Bacillus thuringiensis, B. popilliae, B. lenti-
morbus, and B. sphaericus) have been closely examined as insect-control
agents. These species are sporeformers: the first two produce, in addition to
the spore, discrete crystalline inclusions within the sporulating cell; the last
two do not.


Production

Fermentation processes are employed in the production of B. thuringiensis
and B. sphaericus spores, whereas B. popilliae or B. lentimorbus spores are
produced exclusively in living insects. Two types of fermentation methods are
used for the production of B. thuringiensis and B. sphaericus: 1) surface or
semisolid, and 2) submerged. In surface fermentation, the B. thuringiensis
spores are inoculated on a semisolid medium composed of wheat bran, ex-
panded perlite, soybean meal, glucose, and inorganic salts. The bran is har-
vested after 36-48 hours and formulated to a product active against insects.
Submerged fermentation of B. thuringiensis (Figure 5.1) is carried out in a
liquid medium using standard fermentation protein sources (fish meal, soy
flour) and carbohydrate sources (molasses, sucrose). After sporulation of the
bacteria, the spent liquor is passed through a fine screen, the active ingredi-
ents are centrifuged from the medium, mixed with stabilizing agents, and
then packaged as either a powder or liquid. Similar materials may be used for
B. sphaericus.
For the in vivo production of B. popilliae and B. lentimorbus, third instar
Japanese beetle (Popillia japonica) larvae are infected with spores and in-
cubated in soil seeded with rye to feed the larvae. The infected larvae are
harvested 16-21 days after infection. The infected larvae are pulverized,
stabilizing agents are added, and the slurry is dried and packaged as a dry
powder. Living insects must be used to propagate these bacteria because
artificial culture methods that will support large-scale sporulation of the
E. popilliae and B. lentimorbus are not as yet available.

Safety and Specificity
B. thuringiensis, B. popilliae, B. lentimorbus, and B. sphaericus have been
subjected to many safety tests, with no harmful effects for animals or human
beings.
What impact these bacteria have on the environment when used as insecti-
cides is difficult to predict, because little data are available on persistence and







MICROBIAL PROCESSES


concentration of bacterial agents in the food chain. True parasites are ob-
viously dependent for existence on the population of their hosts.

Efficacy
B. thuringiensis (Figure 5.2) is one of the best-known and most widely
used microbial control agents. It is pathogenic for lepidopteran larvae (Figure
5.3) affecting more than 150 larval species that include some of the most
important economic pests listed in Table 5.3. Preparations of B. thuringiensis
can be mixed with a number of commercial insecticides, fungicides, and
various adhesives and wetting agents. Commercial products are applied to
field crops and stored commodities, employing the same methods used for
chemical pesticides.
B. popilliae and B. lentimorbus are pathogens of various beetles. When
ingested by beetle larvae, the bacteria invade the blood system, where they
proliferate and sporulate, causing death of the larvae. The mass of spores that
accumulates prior to death of the insect is ultimately released and survives for
extended periods in the soil. These spores may be eaten by newly hatched
beetle larvae and, upon germination and growth in the insect gut, begin the
infectious process again. The name given to this infection is "milky disease"
because the infected larvae take on a whitish or milky appearance. One appli-


FIGURE 5.3 Diseased larvae of the cabbage looper with symptoms of B. thuringiensis
poisoning. (Photograph courtesy of C.M. Ignoffo)







MICROBIAL INSECT CONTROL AGENTS


TABLE 5.3 Some Insect Pests Susceptible to Control with Preparations
of Bacillus thuringiensis


Insect Pest


Plants Affected


Cabbage looper
(Trichoplusia ni)
Imported cabbageworm
(Pieris rapae)
Tobacco hornworm
(Manduca sexta)
Tobacco budworm
(Heliothis irescens)
Tomato hornworm
(Manduca quinquemaculata)
Alfalfa caterpillar
(Colias eurytheme)
Gypsy moth
(Lymantria dispar)
European corn borer
(Ostrinia nubilalis)
Grape leaffolder
(Desmia funeralis)
Codling moth
(Laspeyresia pomonella)
Green cloverworm
(Plathypena seabra)
Orangedog
(Pailio cresphontes)
Range caterpillar
(Hemileuca oliviae)
Sugarcane borer
(Diatraea saccharalis)
Cotton bollworm
(Heliothis zea)
Spruce budworm
(Choristoneura fumiferana)
Indian meal moth
(Plodia interpunctella)


Broccoli, cabbage, cauliflower,
celery, lettuce, potato, melon
Broccoli, cabbage, cauliflower

Tobacco

Tobacco

Tomato

Alfalfa

Forest trees

Corn

Grape

Apples, pears

Soybeans

Citrus

Range grass

Sugarcane

Cotton

Forest trees

Stored grains


cation of these bacteria lasts many seasons, although the physical and chem-
ical properties of the soil as well as climatic conditions, agricultural practices,
and larval population density influence their effectiveness in nature over pro-
longed periods.
Several strains ofB. sphaericus have been isolated that are highly toxic and
specific to larvae of disease-carrying mosquitoes. The infectivity of B. sphaeri-
cus strains varies with the mosquito species; in general, larvae of the genus
Aedes are the least susceptible, while those of the genus Culex are the most
susceptible, at least at the current stages of strain development. Experi-
mentally, this bacterium has been produced commercially at prices competi-
tive with those of chemical insecticides. All isolates that have insecticidal
activity have not been fully characterized.




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