You are on page 1of 5

Anaerobe (1997) 3, 213217

REVIEW ARTICLE

Robert E. Hungate: Pioneer of Anaerobic Microbial


Ecology
King-Thom Chung1 and Marvin P. Bryant2
1

Department of Microbiology
and Molecular Cell Sciences, The
University of Memphis,
Memphis, TN 38152
2
Department of Animal Science,
University of Illinois, Urbana, IL
61801, U.S.A.
(Received 16 May 1997,
accepted 16 May 1997)

Introduction
Robert E. Hungate was the pioneer in studying rumen
and anaerobic microbiology. He developed the roll
tube anaerobic technique used to culture anaerobic
bacteria. His work not only contributes to the understanding of the complicated microbial ecosystem of
the rumen, but also paves the way for investigations of
vast number of anaerobic microorganisms, which are
an important part of general microbiology.
Robert E. Hungate was born on March 2, 1906 at
Cheney, Washington. His father was a biologist who
taught at the State Normal School (now Eastern
Washington State University) for almost 50 years. As a
child Hungate enjoyed outdoor activities, mounting
insects and skins of small animals and birds for
museum preparation. Young Robert graduated from
the Normal School (a 2-year college at that time) and
through his father developed a strong interest in the
plant and animal life of the Cheney area and of
Address correspondence to: Dr. King-Thom Chang.

1075-9964/97/040213 + 05 $25.00/0/an970109

Northern Idaho where the family camped at Priest


Lake. He taught grades 7 to 10 for a year as principal
of a three-teacher school at Wellpinit (19241925) and
for 2 years in the public school at Sprague (19251927).
At Sprague he became proficient at dancing the
Charleston, which he has enjoyed all his life. During
the summer of 1926 he worked for the U.S. Blister
Rust, eradicating currant and gooseberry plants, alternate hosts for the white pine blister rust, at Lamb
Creek on Priest Lake, and was called out for 34 days to
fight the bad fires of that summer. The next summer he
was a foreman of a blister rust crew on the Little North
Folk of the Coeur dalene River in Idaho.
He decided in 1927 to continue his education at
Stanford University, where he received his A. B.
magna cum laude with great distinction, in 1929, and
started graduate work in Biology. During this period
L. L. Burlingame gave him much advice, including
taking organic chemistry at the University of California, Berkeley during the summer of 1928, and was
responsible for his having an instructorship from
19301935, teaching the freshman biology course at
Stanford, together with Arthur Giese.
1997 Academic Press

214

K.-T. Chung and M. P. Bryant

In the summer of 1929, he worked a crew under


Frank Patty in the Sierras, studying the ecology of
Ribes plants. In the summer of 1930 he went to Ohio to
study means of eradicating the alternate host of wheat
rust, barberry bushes, planted as ornamentals in city
gardens. He showed that by cutting a healthy stem,
attaching a 5-inch rubber tube to the proximal end and
filling it with almost any poison solution, transpiration would create a suction tension that pulled the
solution down into the crown of the plant and kill it,
with no damage to other plants in vicinity. The
methods were suggested by his earlier success in
demonstrating the cohesive force of water by drawing
out a 45-ft U-tube in the stairwell of Jordan Hall at
Stanford.
In the spring of 1931 Hungate took C. B. Van Niels
first microbiology course at the Hopkins Marine
Station of Stanford at Pacific Grove. Van Niel (Kees), a
leading creative microbiologist of this century and the
third member of Delft SchoolBeijerinck, Kluyver, Van
Niel, exerted a profound influence on the only student
sitting by him and sketching illustrations and notes on
a yellow sheet, torn off for the student at the end of the
lecture. Van Niel had personally visited the great
microbiologist Beijerinck and Winogradsky, and
described his vivid recollections of Beijerinck who
showed him through his garden and cautioning him
not to step on this or that. Van Niel was so keyed up
by his visit with Winogradsky in Paris that he
afterward fainted on a Paris street. Also A. J. Kluyver,
Kees Delft professor, visited Pacific Grove. All these
experiences were exciting for the young student. Van
Niel also treasured meeting David Starr Jordan,
Stanfords first president.
The Delft School used enrichments to isolate bacteria, but Hungate said the isolates had to be enumerated and the rate of fermentation of pertinent compounds in the ecosystem had to be determined, thus
adding a new dimension to the study of microbial
habitats, among other contributions of Hungates life
career.
During Van Niels lectures he stressed, among other
things, the cellulose digesting capacities of many
bacteria. Hungate was teaching the freshman biology
course which included a laboratory study on the
protozoa in termites which in part nourished the
termites by digesting the cellulose on the wood which
they consumed. He proposed that maybe it was
bacteria instead of protozoa which digested the
cellulose. Van Niel agreed, and Hungate said he
would like to work on that as a thesis problem. Van
Niel agreed but after 6 months no positive results had
been obtained. Microscopic examination of the protozoa showed that their mass obviously greatly
exceeded that of the bacteria, and perhaps the slightly
acidic pH of the digesta somewhat inhibited the
bacteria. At this point the program could have been

dropped, but Van Niels advice was to continue the


research if there is anything more to be done, so
Hungate continued to study a number of other aspects
of termite biology, including cultivation of cellulolytic
anaerobic bacteria, and a thesis titled Experiments on
the Nutrition of Zootermopsis was finished in 1935.
We now know that the failure to culture cellulolytic
bacteria from that termites gut was because that
genus as compared with other genera of termites
contained mainly protozoa rather than bacteria and
the culture medium and anaerobic techniques were
not adequate at that time. But the work had stimulated
him to continue the search for methods to culture
anaerobic cellulolytic bacteria.
Hungate joined the staff of the Department of
Zoology, at the University of Texas in 1935, as an
instructor. He first finished his work on the nitrogen
economy of the damp-wood termite gut but he soon
turned his attention to the problem of cellulose
digestion in cattle. In Texas, Hungate successfully
demonstrated that cellulolytic protozoa are found in
the rumen (the large first stomach of cattle). He
carefully demonstrated the cellulolytic activity and
also cultured the cellulolytic protozoa in an artificial
rumen. The literature indicated that it was highly
probable that cellulose was also digested by bacteria
in the rumen. He tested for them in the cultures of
rumen protozoa. He shook tubes of melted, somewhat
cooled agar media containing sterilized protozoan
cultures containing finely powdered cellulose inoculated with several dilutions of the protozoan culture,
solidified the medium and then incubated at 39C.
After several weeks, Hungate observed a colony in a
spherical clearing of cellulose from one of the higher
dilutions. This culture was isolated and later identified
as cellulolytic Clostridium cellobioparum an exciting
successful demonstration of cellulolytic bacteria in
pure culture.

The Hungate Method


Clostridium cellobioparum occurred in numbers too
small for it to be considered important in the rumen.
Further study was necessary to demonstrate the major
predominant cellulolytic bacteria in the rumen. Difficulty in seeing the clearings in the cellulose in agar
shake tubes led him to use roller tubes. His method
was simply to mechanically roll the tube horizontally
in shallow cold water as the agar solidified. This gave
a homogenous thin layer of solidified agar medium on
the inner surface of anaerobic tubes. By this method,
the bacterial colonies were kept separated and the
shape, size and color could be observed easily.
Cellulolytic ones gave a clearing if cellulose powder

Pioneer of Anaerobic Microbial Ecology


was included in the medium. Anaerobiosis was also
carefully observed. Since the rumen atmosphere contained 70% CO2; a CO2 atmosphere and 0.5%
NAHCO3 was selected as the chief buffer. This
necessitated flushing of culture tubes with O2-free
CO2. Initially this was achieved by bubbling through a
chromous acid solution. Later the O2-free CO2 was
achieved by passing CO2 from the regular CO2 tank
through a hot copper column reduced with
hydrogen.
After the tubes were filled with media, they were
closed with black rubber stoppers, which were impermeable to gases. A typical medium usually contained
three parts: one-third balanced salt solution, one-third
rumen fluid and one-third suspension of substrates
which could be HCL-treated absorbent cotton, or later
Whatman no.1 filter paper, wet ground in a pebble
mill to obtain a fine suspension. Of course, oxygen had
to be excluded and also, later, a reducing agent such as
cysteine or sodium sulfide was added to help reduce
any residue of oxygen and make the media chemically
reduced.
With the availability of the Hungate method, many
anaerobic bacteria including cellulolytic ones such as
Bacteroides succinogenes and Micromonospora propionici
from the rumen were isolated and studied. This
technique was spread to other laboratories, and over
the years scientists from many different countries
came to Hungates laboratory to study anaerobic
bacteria.

Rumen Microbiology
Hungate stayed in Texas for about 10 years, undertaking numerous investigations, such as disproving
the viral etiology of mouse mammary carcinoma and
participating in the Alpha Chapter of the Society for
Applied Piscatology (SAP). He also carried a full load
of teaching (which he enjoyed profoundly all his
life).
At the end of the World War II in 1945, Hungate
accepted the offer to join the Bacteriology Department
at Washington State College and was soon busy
preparing lectures in Bacteriology! This was quite new
to him as he had taught zoology and an honors
program in general biology while at Texas. Research
continued to be focused on the microbial activities of
the rumen. This was and is important since rumen
microorganisms convert plant materials, including
cellulose, into nutrients for the animal, resulting in
milk, meat and other products used by mankind. This
process urgently needed study.
Hungate was one of the most devoted scientists
ever known who dedicated his effort to the under-

215

standing of this system. This is recognized as the


beginning of an era of rumen microbiology. Many
students came to work for their Masters and Ph.D.
degrees with him and most of them took some phase
of rumen microbiology as their thesis subject. Notable
graduate students included R. H. McBee, S. L. Adams,
R. L. Mylroie, Elizabeth R. Hall, Guy Anderson, D. W.
Fletcher, and Jose Gutierrez taking doctorate degrees,
and Marvin Bryant, Ed. Carroll, Leroy Maki, Wm. B.
Hardie, Julia Schmitz, John Hamlin, Larry Jayko and
Carolyn (Regier) Moomaw, who obtained their Masters degree with Hungate at Pullman. G. C. N. Jayasuriya worked as a postdoctoral associate. Some
interesting findings of this group include quantitative
measurement of the rumen fermentation, interpretation of legume bloating of rumen, continuous culture
fermentation, and isolation of methanogenic bacteria
from the rumen. In 1950, he was a Guggenheim Fellow
and spent a year at Cornell University.
After several offers from the University of California that he had declined, Hungate accepted the
appointment as Chairman of the Bacteriology Department, University of California, Davis, in 1956. He held
his chairmanship until 1962. He was also appointed
Bacteriologist in the Experimental Station. Work on
rumen protozoa and bacteria continued. Jose Gutierrez, Robert A. Mah, Dick Clarke and Brian Jarvis
worked with him on rumen protozoa. Many others
did their work there on rumen bacteria. Doctoral
studies included Paul H. Smith, Robert A. Mah, S. S.
Margherita, Patricia Grilione, W. L. Lester, James T.
Staley, John P. Robinson, Joan M. Macy, King-Thom
Chung, Kyoko Owaki, and Ida Yu. Masters degrees
were received by Abdur Rouf, Richard Martucci, and
Casey Caldwell. Postdoctorals and visiting scholars in
the Davis laboratory were Martin Knight, Douglas
Wright, Jose Gutierrez, Khaled-el-Shazly, R. T. J.
Clarke, R. J. Moir, Peter Nottingham, M. J. B. Paynter,
T. Bauchop, Brian Jarvis, Henry Blackburn, Jan Reichl,
Rudolf Prins, George Lanigan, Bernard Howard, Peter
Henry, Sten Sruve, Gerhard Gottschalk and Abdur
Rouf.
Hungates chief interest had long been analysis of
microbial ecosystems, starting from insects such as the
termites but extending to the bovine rumen. This wide
interest became possible with the collaboration of
many colleagues, graduate students, postdoctoral
scientists plus collaboration with other laboratories. A
notable example is Dr. Marvin P. Bryant, in Beltsville
and later at the University of Illinois, who took on an
extensive characterization of rumen microorganisms
and, in 3 years (19491951) with Hungate learnt all he
needed in methods and ideas to last a lifetime.
A concept was developed that a complete analysis
of an ecosystem involved not only identification of
microbial species and their activities, but also the
measurement and quantization of these activities. Not

216

K.-T. Chung and M. P. Bryant

only microbial activity in the individual species but


also that of groups comprise the beautiful metabolic
patterns of this unique microbial habitat concept.
Through Hungates efforts, the rumen is by far, the
best characterized ecosystem. This has been largely
accomplished by Hungate, with the graduate students
and colleagues influenced by him. The ecosystem was
described in a book The Rumen and Its Microbes which
was published by Academic Press in 1966. This book
provides the best indepth understanding of this
unique ecosystem.

Personal Caliber
With the stimulation of Hungate methods, the complete analysis of many anaerobic microbial ecosystems
became possible. There are so many habitats which are
free of oxygen in the world, such as human and
animal gastrointestinal tracts, sludges, sediments and
tundra and other environments with reduced oxygen.
Anaerobes constitute 90% or more of the total microbial population of each of these habitats. Without
Hungates anaerobic technology to make their microbial culture possible, our knowledge would be handicapped to only knowing the limited amounts of
aerobes and facultative anaerobes culturable by the
common aerobic media. One typical example is
Escherichia coli, which is not by any means a major
intestinal bacterium. But its name is so well known
that many have the misconception of forgetting the
anaerobes! Anaerobic technology has been extended
to other fields such as human intestinal microbiology;
this has made a great progress with a profound impact
on human health. Another example is the field of
methanogenesis. Hungate was interested in methanogenesis in the rumen for a long time. Hungates
laboratory at Washington State University was the
first to isolate methanogens using H2-CO2 as energy
source. His anaerobic technique is the most reliable
method for growing methane bacteria. Tremendous
progress in the field of methanogenesis including
molecular biology of methanogens is becoming possible. We should by no means forget to credit him for his
pioneering work.
In addition to his scientific contributions, Hungate
also generously served in various organizations. He
was a charter member of the Texas Branch of the
Society of American Bacteriologists. He is also a fellow
of the American Association for the Advancement of
Science (AAAS), the American Society of Zoologists,
the Northwest Scientific Association, the Society of
Protozoologists, the American Academy of Microbiology, American Academy of Arts and Sciences, and
the American Society for Microbiology. He has not

only been a leader, often elected to high position, but


has made innumerable other voluntary contributions
to various educational, scientific, and professional
activities of different organization. For example, he
was elected as President of the American Society for
Microbiology, 19711972, and was also active in many
national and international societies, to which he has
made significant input on many policies.
After retirement from Davis, he still carried on with
research and was active in many capacities. He had
the opportunities to work and travel in Africa, New
Zealand, Australia, Taiwan, China and to take part in
the Global Impacts of Applied Microbiology V meeting in Bangkok, International Symposium of Biogas,
Microalgae and Livestock Wastes in Taipei. He
assisted with the microbiology course at Wood Hole
and received an honorary doctorate from the University of Gottingen in 1989.
Dr. Hungate has been a modern pioneer of microbiology in the same genre as Koch and Pasteur.
How many biologists, let alone microbiologists, would
have been interested in a termites digestive system?
Or transferred this interest to a mammals rumen,
opening up a new universe of anaerobic microbiology? Microbiologists and, in fact, all scientists, are
inspired by his example of life-long self-instruction in
the most advanced current knowledge and its application to microbial ecosystems. All biologists are
inspired by his example as a doer. For example, he
was able to construct glass tubing up to 45 feet high to
measure the cohesive force of water. He made Warburg vessels with two side arms and a side pocket for
palladium black to absorb H2, and Warburg shakers
with manometers to a height of two meters in order to
accommodate the fermentation products from measurable quantities of cellulose.
His leisure activities reflected his capacity as a
doer. With his children, at Pullman, he constructed a
Baby Blue 16 foot outboard utility boat, from scratch!
The boat is as sound today as it was when launched 40
years ago. He has the unique talent of making things
work; something that everyone can learn from his life
to the benefit of their own scientific and personal
careers.
All who know Dr. Hungate and were fortunate to be
his doctoral students years ago in Davis, found him to
be a wonderful person. He has always taken care of
his students and all colleagues associated with him.
These admirable human characteristics have enabled
him to serve not only science, but also many other
organizations to which he has always contributed
generously giving his very best in all capacities.
His career was probably held back because he did
not allow his name to be included as a co-author of
many of his students papers and at Davis the number
of publications was of importance.

Pioneer of Anaerobic Microbial Ecology


Robert E. Hungate and Alice Wolcott were married
at Stanford on 3 February, 1933. They raised three
children, Robert, Dan and Harriet who are testimony
for their wonderful marriage. Over the years, Alice
assisted her husband in his professional efforts, as
well as enriching the life of the family beyond

217

measure. Due to the deterioration of his sight, Alice


also greatly helped with his writing and driving for
many years. The Hungates now live at Davis, California, near the campus where he carried out his final
academic research.